key: cord-022888-dnsdg04n
authors: nan
title: Poster Sessions
date: 2009-08-19
journal: Eur J Immunol
DOI: 10.1002/eji.200990224
sha: 
doc_id: 22888
cord_uid: dnsdg04n

No Abtract

The humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. PTX3, the prototype of long pentraxins, plays a nonredundant role in resistance against A. fumigatus lung infection. The model proposed suggests that upon binding, PTX3 facilitates recognition, phagocitosis and killing of A. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly Th1-oriented adaptive response. Actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop Th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. Both in vitro and in vivo defects can be rescued by the administration of recombinant PTX3, which does not show direct activity on fungal cells. Finally, PTX3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically.

In the present study, we investigated the mechanisms underlying the PTX3-mediated opsonic activity and the involvement of complement, complement receptors and Fcg receptors, by in vitro studies and genetic approaches in vivo. In vitro PTX3 amplified the complement-dependent effects on A. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. Accordingly, in the presence of PTX3-opsonised conidia, CD11b activation, internalization, recruitment to the phagocytic cup and CD11b-dependent phagocytosis were increased. As pentraxins interact with FcgReceptors, which in turn can control CD11b activation, the phagocytic assay was performed in the presence of FcgR blocking Abs. Data obtained strongly suggest that upon conidia opsonisation with PTX3, FcgRIIA/CD32 mediates inside-out activation of CD11b and consequently phagocytosis of C3b-opsonised conidia. In vivo phagocytosis experiments performed with C1q-and Fc common gamma chain-deficient mice and complement inhibitors support in vitro data. These data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, PTX3) and the cellular arm (FcgRs, CR3). Moreover, they confirm previous studies on the interaction between pentraxins and FcgRs and support the idea that pentraxins behave as predecessors of antibodies.

Innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. The humoral arm of the innate immunity includes soluble pattern-recognition receptors (PRRs) such as collectins, ficolins, complement components and pentraxins. The prototypic long pentraxin PTX3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. PTX3 binds selected microorganisms such as Aspergillus fumigatus and restores protective immunity against this pathogen in PTX3-/-mice.

Neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. A beneficial effect of breast feeding on newborn health is highly demonstrated. This protective effect is mediated by nutrients, immunomodulatory mediators (IFN-g, TNFa, or TGF-b), innate immunity factors (soluble CD14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. We thus hypothesized that milk may contain PTX3. We found high concentration of PTX3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( X 2 ng/ml). The presence of PTX3 in human colostrum seems to be due to the secretion of PTX3 by human mammary gland since we report the production of PTX3 by these cells. This PRR is also found in human milk cells (HMC), mainly in leukocytes, and penetrate into newborn tissus after suckling. Furthermore, human colostrum upregulated the PTX3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their PTX3 production after LPS injection. Collectively, these data demonstrate that newborn have three distinct ways of PTX3 supplying by breast feeding: (i) soluble PTX3 in colostrum (ii) HMC that can secrete PTX3 upon stimulation in the specific tissue, (iii) an increase of PTX3 production by immune cells in the presence of colostrum. Thus, soluble or cell-derived PTX3 may participate to the beneficial role of breast feeding on the newborn health.

A. M. Baru 1 , J. Stephani 2 , H. Wagner 2 , T. Sparwasser 1 1 Twincore, Institute for Infection Immunology, Hannover, Germany, 2 Technical University of Munich, Institute for Medical Microbiology, Immunology and Hygiene, Munich, Germany

Toll-like receptors (TLRs) represent the best characterized pattern recognition receptor family in mammalian species. The family currently comprise of 10 receptors in humans (TLR 1-10) and 12 in mice (TLR 1-9, 11-13). As transmembrane receptors, TLRs are expressed on the cell surface (TLR 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (TLR 3, 7, 8 and 9) . Toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. Bacterial DNA has been shown to possess immunomodulatory activity about a decade prior to the identification of CpG motifs. About 5 years later to this, Toll-like receptor 9 (TLR9) was identified and shown to be the receptor for unmethylated CpG DNA which is present mainly in non-vertebrate genome. Studies have defined potential role of TLR9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. Although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. One explanation for these discrepancies could be the fact that expression of human-TLR9 (huTLR9) is restricted to B-cells and plasmacytoid dendritic cells (pDCs) whereas murine-TLR9 (muTLR9) is also expressed on conventional dendritic cells (cDCs). Consequently, TLR9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. By employing bacterial artificial chromosome (BAC) technology, we generated transgenic mice with huTLR9 (henceforth called as huT9 mouse) integration in their genome under human epigenetic control. To avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto muTLR9 knock-out background. We expect that huT9-muTLR -/mice mimic the human specific expression pattern of TLR9, i. e. exclusively in B-cells and pDCs, allowing us to investigate detailed in vivo functions of huTLR9. By studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of TLR9 ligands in human immunotherapy.

The fractal analysis provides unique physical insights into the interactions between C1q and the PrP protein. If one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. If this is true, then surely, this is worth exploring further. Any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as Alzheimers is defintely worth the effort.

Interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. The main inducers of IL-12p40 are microbial components activating toll-like receptors with the magnitude of IL-12p40 induction depending on the specific TLR engaged. Differential induction of IL-12p40 upon TLR stimulation correlated with striking differences in the kinetics of NFkB activation. CpG-DNA strongly induces IL-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal IL-12p40 promoter region and (ii) to stimulate prolonged RelA activity.

Here we were interested in further changes in chromatin structure of the IL-12p40 promoter upon TLR triggering. We did not observe a change in DNA methylation, but using chormatin immunoprecipitation (ChIP) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. Acetylation of H4 showed a specific distribution pattern and occured mainly in regulatory elements within the IL-12p40 promoter, whereas acetylation of H3 took place over all regions analyzed. TLR tolerance has been reported to be associated with specific chromatin alterations. Methylation status of lysine residue 4 on H3 turned out to be important for the inhibition of gene expression upon repeated stimulation. Modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses.

A. D. Koepruelue 1 , W. Ellmeier 1 1 Medical University of Vienna, Institute of Immunology/Division of Immunobiology, Vienna, Austria

Macrophages are important in innate and acquired immunity. Failures are associated with inflammatory and autoimmune diseases. Understanding their stimulation is the basis for therapeutic targeting. Members of the Tec kinase family (Bmx, Btk, Itk, Rlk and Tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. Mutations in btk represent the source of human X-linked agammaglobulinemia (XLA). A mutation in the murine btk gene accounts for a similar syndrome, X-linked immunodeficiency (xid). Although the Tec family members Tec, Btk and Bmx are expressed in monocytes/macrophages, little is known about their function there. Tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or TLRs. Several studies in XLA or xid macrophages and in monocyte/macrophage cell lines implicated roles for Tec kinases in TLR signaling and as well as other macrophage effector functions like phagocytosis. Inspired by these findings, we aim to determine the role of Tec kinases in bone marrowderived macrophages (BMM), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. In a comprehensive functional analysis of TLR-mediated BMM activation from mice deficient for one or more of the Tec family members in vitro, we reveal which of the kinases play a role in which TLR pathway. Based on the results of this analysis, we set the goal to further study how Tec kinases regulate the respective signaling cascades. Our study will contribute insights into the role of Tec kinases in this important cell population of the innate immune system.

G. Lunazzi 1 , M. Buxadé 1 , J. Minguillón 1 , R. Berga 1 , J. Aramburu 1 , C. López-Rodríguez 1 1 Universitat Pompeu Fabra, Department of Experimental and Health Sciences (DCEXS), Barcelona, Spain NFAT5 is a transcription factor that regulates the expression of cytokines such as TNFa and lymphotoxin b in response to osmotic stress. In addition, NFAT5 participates in multiple processes not linked to the response to hypertonicity. In this regards, it has been recently reported that NFAT5 is required as a novel host factor that supports HIV replication in macrophages. Given the established connections between NFAT5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether NFAT5 could be activated by receptors for pathogens expressed in macrophages.

The activation of Toll-like receptors (TLRs) is central to innate immunity. Upon stimulation of TLRs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. As a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. We have analyzed whether NFAT5 is expressed in primary macrophages through the activation of different Toll-like receptors. Likewise, we have explored whether the activity of NFAT5 is induced during the response to TLRs. In addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of TLRs could interfere with the expression of NFAT5. Our work indicates that NFAT5 is a novel transcriptional regulator acting in response to the activation of TLRs. Our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. Objectives: Compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by NF-xB, a master switch of inflammation. Recent studies implicate some TLRs in tumor development or regression, and immune escape. However, mechanisms leading to tumor growth or apoptosis induced by TLR stimulation are not fully understood. Several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. We hypothesized that some TLRs can contribute to lung inflammation and tumor development in vitro and in vivo. Methods: TLR expression in lung cancer was assayed by immunohistochemistry or flow cytometry. NFxB activation was determined by western blot and nuclear translocation assay after TLR stimulation. Clonogenicity of stimulated cells was analyzed by colony assay. Transcriptomic analysis were performed by Taqman LDA technology. Tumor growth in vivo was analyzed in NOD/SCID mice after subcutaneously engraftment of human lung tumor cell lines.

We have observed that primary human lung tumors express TLR3, TLR4, TLR7 and TLR8 and that stimulation of these receptors in lung tumor cell lines by Poly I:C, LPS, Loxoribine or Poly U induces NFxB activation through atypical signaling pathway, with phosphorylation of IxBa without its degradation and nuclear translocation of p50 and p65 NFxB subunits. Interestingly, we observed that TLR3 stimulation induces apoptosis depending of the histological type of the tumor. On the contrary TLR4, TLR7 and TLR8 stimulation induces cell survival and increases clonogenicity. This is correlated with an up-regulation of Bcl-2 expression. Moreover, despite a common atypical activation of NFxB, our transcriptomic analysis revealed major differences in gene modulation after triggering of TLR3, TLR4, TLR7 and TLR8. Finally, in vivo TLR7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. Conclusions: Altogether, these data emphasize that TLR4, TLR7 or TLR8 triggering can directly favor tumor development whereas TLR3 signaling can induce tumor cell death. These data suggest that anticancer immunotherapy using TLR adjuvants should take into account the expression of these TLRs in lung tumor cells. Objective: Dasatinib (BMS-354825) is a small molecule Src/Abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and Philadelphia chromosome-positive acute lymphoblastic leukaemia. Members of the Src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. The purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (TLR) signalling, natural killer (NK) cell cytotoxicity as well as antigen-specific CD8 + and CD4 + T cell function. Methods: To analyse TLR signalling in vitro murine bone marrow derived (BMD) macrophages were stimulated with the TLR 4 ligand lipopolysaccaride (LPS) in the presence of dasatinib and tumour necrosis factor a (TNF-a) in the culture medium was measured. The response to TLR stimulation was also tested in vivo, dasatinib-treated mice were challenged with LPS and TNF-a in the serum was quantified. In addition, the clearance of the RMA-S cells, a MHC class I deficient thymoma sensitive to NK cell lysis, was analysed in mice undergoing dasatinib treatment.

To investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic CD4 + and CD8 + T cells specific for ovalbumin were utilised to measure antigen specific T cell proliferation. Endogenenous CD4 + and CD8 + T cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. Results: We show that dasatinib impairs: 1. Innate immune response; dasatinib treatment reduced the (a) production of TNF-a following TLR 4 stimulation of BMD macrophages in vitro, (b) production of TNF-a in vivo in response to LPS and (c) ability of NK cells to eliminate MHC class I deficient cells in vivo . 2. Adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic T cells, (b) endogenous antigen-specific helper T cell recall-responses and (c) T cell-mediated cytotoxic effector function. Conclusions: These findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock.

I. Zanoni 1 , R. Oatuni 1 , M. Collini 2 , M. Caccia 2 , P. Castagnoli 3 , G. Chirico 2 , F. Granucci 1 1 University of Milano-Bicocca, Biotechnology and Bioscience, Milan, Italy, 2 University of Milano-Bicocca, Physics, Milan, Italy, 3 Singapore Immunology Network (SIgN), Biomedical Sciences Institutes, IMMUNOS, Singapore, Singapore

The recognition of MAMPs by TLRs expressed on dendritic cells (DCs) plays an essential role for the regulation of the immune responses. By recruiting different combinations of adapter proteins, individual TLRs turn on signal transduction pathways leading to the activation of different transcription factors. Interleukin-2 (IL-2) is one of the molecules produced by DCs shortly after stimulation with different TLR agonists. Based on this observation and by analogy with the events following T-cell receptor (TCR) engagement leading to IL-2 production, we hypothesized that the stimulation of TLRs on DCs might lead to activation of the Ca2+/ calcineurin and NFAT pathway. We found that DC stimulation with LPS induces extracellular Ca2+ influxes, leading to calcineurin-dependent NFAT activation. The activation of this pathway was independent of TLR4 engagement, depending instead exclusively on CD14. We also found that LPS-induced NFAT activation in DCs was necessary for the efficient synthesis of cyclooxygenase-2 (COX-2) that, by generating prostaglandins (PGs), such as PGE2, regulates different DC functions including migration and polarization of T cell responses. Our findings reveal novel aspects of the molecular signaling triggered by LPS in DCs and define a new role for CD14. Given the essential involvement of CD14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 represents a major step towards the development of potential treatments with modes of action involving interference with CD14 functions. We have examined the interaction of CD55, a 80-kDa glycosyl-phosphatidylinositol (GPI)-anchored membrane protein, with the monocyte signalling receptor, CD14. Human monocytes were isolated from healthy adult donor's peripheral blood. This involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. The cells were labelled at saturation with anti-CD14 antibody coupled to biotin visualised by QD-525-streptavidin and anti-CD55 antibody coupled to allophycocyanin. The images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. Single Particle Fluorescence Imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. The images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional Gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. Changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that LPS promotes the interaction of CD55 and CD14, suggesting a new functional role of CD55 as a member of a multimeric LPS receptor complex.

L. Lundvall 1 , R.R. Schumann 1 1 Charité -Universitätsmedizin Berlin Campus Mitte, Institute for Microbiology and Hygiene, Berlin, Germany

Meningitis is a life-threatening disease mainly caused by bacteria and viruses. Bacterial components such as Lipopolysaccharide (LPS), lipoproteins or peptidoglycan breakdown products (i. e. MDP, mesoDAP) stimulate pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and the intracellular Nod-like receptors (NLRs) for an inflammatory response. We hypothesize that a synergistic effect of TLR-induced NF-xB activation and NLR-mediated caspase-1 induction leads to an increased release of mature IL-1b during bacterial meningitis in brain-derived cells. A mouse meningitis model with S. pneumoniae (D39) was established for assessing IL-1b induction during this disease. The murine RAW 264.7 cell line, the human astroglial U-87 MG and the murine microglial cell-line BV-2 were stimulated with the TLR4 ligand LPS, the TLR2 ligand Pam 2 Cys, the Nod2 ligand MDP, or the Nod1 ligand C12-iE-DAP, and, as control, ATP alone or in combination. We assessed IL-1b by ELISA and caspase-1 and pro-IL-1b expression by Western Blot. Furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by siRNA suppression of elements of the IL-1b induction pathway. S. pneumoniae (D39) infected mice showed a significant increase in IL-1b release after 24 hours. In vitro, an increase in IL-1b levels after costimulation with LPS or Pam 2 Cys, and MDP or C12-iE-DAP was observed in a dose-dependent manner. A synergistic enhancement of IL-1b by TLR-and NLR-ligands was observed in RAW cells, BV-2 cells, U-87 cells and primary astrocytes. Active caspase-1 (p10) was induced by MDP or C12-iE-DAP, corresponding with high IL-1b responses when LPS or Pam 2 Cys was added. SiRNA experiments show that a knock-down of Nod2 leads to a diminished IL-1b release after LPS-and MDP-stimulation. The precursor forms of IL-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. A synergistic enhancement between TLRs and NLRs in IL-1b release in brain-derived cells was observed. So a two-step stimulation seems necessary for the release of high levels of mature IL-1b by astrocytes and microglia. Bacteria containing both, TLR-and NLR-ligands thus have the potential to induce high levels of IL-1b which may contribute to disease pathology and may point to novel intervention strategies.

J. Rosenberg 1 1 

Toll-like Receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like (RLRs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . In the case of TLRs most sutides to date have delineated TLR expression and function on antigen presenting cells like Dendritic cellof this research. Extension of the profiling and presence of TLRs on cell characterized as adaptive immune cells such as T cells is the subject of this line of research.

Using a CD3 and CD28 activation model system -TLR4 presence on CD4+ cells is found in mouse T cells, human T cells and Jurkat cell lines. Following CD3/CD28 activation for 48 hours we have identified a small but distinct populationof TLR4+ cells. Further characterization indicates these cells to be CD4+CD25+ cells.

Further characterization of the expression and functional acitvity of the TLR4+ T cells indicates co-expression of TLR4 with MD-2 indicating a functional TLR4 receptor.

In addition LPS activiation did not lead to upregulation of TLR4 expression in T-cells.

The data indicate that TCR activation leads to TLR4 expression. The expression appears to be associated with CD4+CD25+ cells and refelecting an activated T cell phenotype which will be further characterized as perhaps related to Tregs or other Tcell subsets.

S. M. Lehmann 1 , D. Kaul 1 , C. Krüger 1 , F. Zipp 1 , R. Nitsch 2 , S. Lehnardt 1 1 Charité-Universitätsmedizin Berlin, Cecilie-Vogt-Clinic for Neurology, Berlin, Germany, 2 Charité-Universitätsmedizin Berlin, Institute for Cell Biology and Neurobiology, Berlin, Germany The innate immune system is the first line of defense against various pathogens and requires the expression of Toll-like receptors (TLRs). In macrophages, TLR7 plays a crucial role in immune responses elicited by GU-rich ssRNA (i. e. ssRNA40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). These compounds were initially described to activate mouse TLR7 (and human TLR8) and are potent immune response modifiers leading to important antiviral and antitumor activities. Microglia serve as the major innate immune cells in the central nervous system (CNS). Employing various techniques including PCR, in situ hybridization, and immunocytochemistry, we demonstrate that TLR7 is expressed in these cells. Incubation of microglia with all three of the above mentioned TLR7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as TNF-a and IL1-b in a dose-and time-dependent fashion. Analysis of wild type (WT) and TLR7 knock out (KO) microglia by real- Because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. Using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. Coronin-1 is an actinbinding protein that can associate with phagosomes and NADPH oxidase but its involvement in apoptosis was currently unknown. In coronin-1-transfected PLB985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by CD11b labeling. Concerning apoptosis, increased coronin-1 expression in DMF-differentiated PLB985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. Likewise, coronin-1 significantly decreased TRAIL-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or Bid truncation suggesting that coronin-1 interfered with mitochondria-related events. To validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (CF) patients were studied. Circulating neutrophils from CF patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. This was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. In addition, inflammatory neutrophils from CF patients lungs showed an intense coronin-1 immunolabeling. We concluded that coronin-1 could constitute a potential target in resolving inflammation.

P.-N. Hsu 1 1 National Taiwan University, Graduate Institute of Immunology, Taipei, Taiwan, Republic of China Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. Recent evidence indicates that the TNF-related apoptosis-inducing ligand (TRAIL) of the TNF ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated T cells. We show in this work that TRAIL can induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. We demonstrated that both cell models differentiate into osteoclast-like cells in the presence of TRAIL in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. The TRAIL-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. However, TRAIL-induced osteoclastogenesis is dependent on activation of NF-xB, ERK, and p38 MAP kinase. The TRAIL-induced osteoclastogenesis was significantly inhibited by treatment with TRAF-6 siRNA and TRAF-6 decoy peptide, indicating this pathway is TRAF-6 dependent. Thus, our data demonstrate that TRAIL induces osteoclast differentiation via direct engagement with the TRAIL death receptor through a signaling pathway distinct from apoptosis. Our results indicate that in addition to triggering apoptosis, TRAIL induces osteoclast differentiation. It provides a novel role for TRAIL in regulating osteoclast differentiation and in osteoimmunology.

Microglia are considered to be the local antigen presenting cells (APCs) of the central nervous system (CNS) which are thought to play a crucial role in local reactivation of autoreactive T cells during CNS autoimmunity e. g. in Multiple Sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) .

In this study we investigated if the anti-inflammatory nuclear transcription factor Peroxisome Proliferator-Activated Receptor gamma (PPARg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. Sustained Activation of PPARg both reduced microglial signalling via MHC molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules B7-H1 and B7-DC. Moreover, also production of pro-inflammatory cytokines like TNF-a and IL-6 was profoundly reduced if microglia were pre-treated with the PPARg-agonist Pioglitazone (Pio). In contrast to this, the lack of PPARg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that PPARg might play a cell-intrinsic role in controlling microglia immunogenicity. Importantly, if PPARg was activated in microglia, the capacity to prime ovalbumin-specific T cells was impaired. T cells primed by Pio-treated microglia produced reduced amounts of IL-2 and IFN-g which could not be overcome by restimulation with aCD3. This indicates that T cells primed by Pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. Furthermore, microglia were able to induce antigen-specific differentiation of naive CD4 T cells into T helper 17 (Th17) cells, which have been associated with autoimmune pathogenicity during EAE. However, if PPARg was activated, microglia were no longer able to induce Th17 differentiation.

In conclusion, activation of PPARg impairs microglial APC function leading to reduced activation of antigen-specific T cells and, in addition, inhibits the induction of Th17 cells. Therefore, activation of PPARg in microglia is a promising approach to limit local activation of autoreactive T cells in the CNS in CNS-autoimmune deseases. Bacterial lipopolysaccharide (LPS) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. However, once exposed to LPS, they become hyporesponsive to a subsequent endotoxin challenge. This phenomenon is defined as LPS desensitization or tolerance. Previous studies have identified some components of the biochemical pathways involved in negative modulation of LPS responses. In particular, it has been shown that the IL-1 receptor-related protein ST2 could be implicated in LPS tolerance. The natural ligand of ST2 was recently identified as Interleukin-33 (IL-33), a new member of the IL-1 family. In this study, we investigated whether IL-33 triggering of ST2 was able to induce LPS desensitization of mouse macrophages. We found that IL-33 actually enhances the LPS response of macrophages and does not induce LPS desensitization. We demonstrate that this IL-33 enhancing effect of LPS response is mediated by the ST2 receptor since it is not found in ST2 KO mice. The biochemical consequences of IL-33 pretreatment of mouse macrophages were investigated. Our results show that IL-33 increases the expression of the LPS receptor components myeloid differentiation protein-2 (MD2), CD14 and TLR-4 and the myeloid differentiation factor 88 (MyD88) adaptor molecule. In addition, IL-33 pretreatment of macrophages enhances the cytokine response to TLR-2 but not to TLR-3 ligands. Thus, IL-33 treatment preferentially affects the MyD88-dependent pathway activated by the TLR.

C. Klotz 1 , B. Lenz 1 , R. Lucius 1 , S. Hartmann 1 1 Humboldt-University Berlin, Molecular Parasitology, Berlin, Germany

Chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. Recently we showed that recombinant parasite cystatin (AvCystatin), derived from the model parasite Acanthocheilanema viteae, effectively abolished OVA-induced allergic airway responsiveness in a mouse model of asthma (Schnoeller et al., 2008) . The cystatin effect was blocked by the application of anti-IL-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. We hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive Interleukin-10 (IL-10).

The aim of the present study was to elucidate the molecular mechanisms by which AvCystatin induces IL-10 in primary macrophages. In vitro experiments with peritoneal macrophages from BALB/c mice confirmed specific and concentration dependent IL-10 production after AvCystatin stimulation. Application of specific inhibitors revealed that the IL-10 induction was p38 and ERK dependent, and inhibitor titration indicated a higher sensitivity towards p38. Western blotting experiments confirmed the phosphorylation of p38 and ERK in macrophages after AvCystatin stimulation. In addition, by using specific inhibitor and western blotting, we showed that AvCystatin induced IL-10 is also regulated by the phosphatidylinositol-3-kinase (PI3K) -Proteine kinase B (Akt) pathway. Further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. Hence, we conclude that AvCystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of IL-10 and possibly other regulatory markers. In general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. We screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. This screen revealed the impact of several novel kinases as well as kinases with previously established function. One of the top candidates identified to block endotoxininduced TNF-a secretion was Carkl, a gene with no previously described function. Subsequent biochemical analyses unequivocally revealed that Carkl is a phosphotransferase protein using Sedoheptulose as a phosphate acceptor and ATP as a donor. Sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. The product Sedoheptulose-7-Phosphate (S-7P) is also an intermediate metabolite of the pentose phosphate pathway (PPP) and so far was only known to be produced by condensation of Ribose-5P and Xylulose-5P via a transketolase reaction. To identify the molecular mechanism by which Carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. So far, our data favor a model where post-stimulatory downregulation, i. e. loss of Carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. Disentangling the signaling pathways responsible for the rapid regulation of Carkl unearthed NF-kB and p38/JNK but not ERK as driving forces. Counterbalancing endotoxin induced loss of Carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. Comparison of wild type and catalyticinactive forms of Carkl unveiled that most of the effects of Carkl on the inflammatory response were due to its phosphotransferase activity. Expression profiling using gene chip analysis further supported the concept that Carkl may represent a new key modulator of inflammatory processes. Taken together, detailed analyses to study the molecular function of Carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. In addition, connecting the PPP and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. In this context, the sedoheptulose kinase Carkl and its product S-7P may provide a novel basis for interfering with adverse immune responses.

T. Bosschaerts 1 , Y. Morias 1 , P. De Baetselier 1 , A. Beschin 1 1 VIB, CMIM, VUB Brussels, Brussels, Belgium

The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent M1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. We aim to identify mechanisms involved in regulation of M1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. Methods: We have scrutinized the phenotype and cellular origin of liver M1 in Trypanosoma brucei infected by FACS analysis and bone marrow transfer experiments. The contribution of different signaling pathways, including MyD88, IFNg, IL-10, CCR2 and NF-kB to the development and/or recruitment of pathogenic M1 in the liver was investigated using knock-out mice or by delivering IL-10 in infected mice. Results: We established that CD11b+Ly6C+CD11c+ TNF and iNOS producing DCs (TIP-DCs) represent the major M1 liver subpopulation. TIP-DCs differentiated in an IFNg/MyD88-dependent manner from CD11b+Ly6C+ inflammatory monocytes in the liver of infected mice. CCR2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to TIP-DCs in the inflamed liver. As a consequence, CCR2 KO mice experienced reduced pathogenic symptoms. On the other hand, the absence of IL-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to TIP-DCs, resulting in exacerbated pathogenicity and early death of the host. In addition, the therapeutic liver-specific delivery of IL-10 in T.brucei infected mice efficiently limited the differentiation and maturation to TIP-DCs, hereby limiting disease-associated pathogenicity. Finally, the absence of the NF-kB member p50 was associated with increased tissue injury associated with increased production of pathogenic TNF and NO by inflammatory monocytes, but not by TIP-DCs. Conclusion: Our data demonstrate that NF-kB p50 and IL-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic M1, in particular TIP-DCs. The inflammatory activity of liver M1 is controlled by IL-10 and/or p50 NF-kB at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic TIP-DCs, or their production of TNF and NO.

A. Popov 1 , J. Driesen 1 , Z. Abdullah 2 , A. Niño Castro 1 , T. Chakraborty 3 , M. Krönke 4 , O. Utermöhlen 4 , C. Wickenhauser 5 , J.L. Schultze 1 1 LIMES Institute, Laboratory for Genomics and Immunoregulation, University of Bonn, Bonn, Germany, 2 Institute of Molecular Medicine and Experimental Immunology, Bonn, Germany, 3 Institute of Medical Microbiology, University of Giessen, Giessen, Germany, 4 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany, 5 Institute for Pathology, University Clinic Leipzig, Leipzig, Germany

Dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. Here we report on a new role for the myeloid antigen presenting cells (APC) in granulomatous infections. Infection of myeloid DC and macrophages with Listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and CD25 and production of prostaglandin E2 (PGE 2 ) and interleukin 10. All these molecules are strictly dependent on autocrine TNF, released during infection, and are in concert suppressing T-cell responses; CD25, expressed by regulatory myeloid cells, acts as an IL-2 scavenger. Importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. Furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. Induction of regulatory and protective phenotype in macrophages and DC require at least two signals provided by TNF and either PGE 2 or TLR ligands. Transcriptional changes in human macrophages, infected by Mycobacterium tuberculosis, resemble the ones induced in DC during infection with L.monocytogenes. In fact, granuloma in patients with tuberculosis and listeriosis are enriched for CD25 + IDO + COX-2 + regulatory myeloid cells, whereas most effector cell populations, such as T cells, B cells and NK cells, are expelled from the granuloma. Of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. Altogether, our studies provide strong evidence that intracellular pathogens such as M.tuberculosis and L.monocytogenes induce a specific polarization of myeloid DC and macrophages characterized by a functional preponderance of inhibitory mechanisms. We postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. On one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated T cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination.

The interferon-gamma (IFN-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. Induction of IFN-g secretion by human T and NK cells through synergistic co-stimulation with interleukin 12 (IL-12) and IL-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with NK or T cells in the relevant experiments. The possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. To resolve this issue, we have determined the production of IFN-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (ELISPOT) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of IL-12 and IL-18 or with macrophage-colony stimulating factor (M-CSF) were able to produce IFN-g when further stimulated with a combination of IL-12 and IL-18. In addition, naturally activated alveolar macrophages immediately secreted IFN-g upon treatment with IL-12 and IL-18. Therefore, human macrophages in addition to lymphoid cells contribute to the IFN-g response, providing another link between the innate and acquired immune response.

A. J. Denzel 1 , M. Rodriguez Gomez 2 , M. Niedermeier 2 , Y. Talke 2 , N. Göbel 2 , K. Schmidbauer 2 , M. Mack 2 1 Unversity Hospital Regensburg, Internal Medicine II, Regensburg, Germany, 2 University Hospital Regensburg, Regensburg, Germany

We have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of IL-4 and IL-6. Activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin Fc-receptors. We now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. Following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. Peripheral blood basophils are markedly reduced during the first hours after restimulation. In the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. Depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. Our datas show that basophils are recruited to draining lymph nodes during a memory response. TNF-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including Mycobacterium tuberculosis. It is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. Three TNF-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against TNF-a (adalimumab and infliximab); the other is a soluble TNF receptor/Fc fusion protein (etanercept) . TNF-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. We studied the effects of TNF-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. All three drugs had an inhibitory effect on IFN-g-induced phagosome maturation in PMA-differentiated human THP-1 cells infected with M. bovis BCG, the avirulent M. tuberculosis H37Ra strain and the virulent M. tuberculosis H37Rv strain. Adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (MDM) in the presence or absence of IFN-g. Macrophages secreted TNF-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with IFN-g. Treatment of infected macrophages with TNF-a increased maturation of mycobacteria-containing phagosomes. These results suggest a role for TNF-a in activating phagosome maturation and highlight a novel mechanism through which TNF-a blockade can affect the host innate immune response to mycobacteria.

Z. G. Dobreva 1 , L.D. Miteva 1 , S.A. Stanilova 1 1 Trakia University, Faculty of Medicine, Molecular Biology, Immunology and Genetics, Stara Zagora, Bulgaria IL-23 is a heterodimeric cytokine composed of a p19 subunit associated with the IL-12/23p40 subunit. Like IL-12, IL-23 is expressed by the activated antigenpresenting cells and both cytokines induce IFN-gamma secretion by different T cell subsets. The proper balance between IL-12p40-related cytokines controls the appearance of normal Th 1 and pathological Th 17 mediated immune responses. In this study, we examined the dynamics of inducible IL-12p40 and IL-23p19 mRNA expression and protein production in purified human monocytes and how JNK and p38 MAPKs inhibitors influenced IL-12p40 and IL-23 production. The cytokines' quantity determination was performed by ELISA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for mRNA transcripts detection. Results were calculated in fold increase compared with gene expression in nonstimulated monocytes. IL-23p19 gene expression was higher than those observed for IL-12p40 gene at all time-points. The level of IL-23p19 mRNA increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for C3bgp and 22.7 fold for LPS). C3bgp and LPS triggered IL-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. The higher level of IL-12p40 gene expression was detected at 6 th h in LPS compared to C3bgp stimulated monocytes (8.1 vs. 4.9 fold). However, IL-12p40 and IL-23 protein production was increased in the highest level after C3bgp stimulation. The inhibition of p38 led to the statistical significant augmentation of C3bgp stimulated IL-12p40 production. The inhibition of the same MAP kinase enhanced LPS stimulated IL-12p40 production without significant difference. The inhibition of JNK and p38 MAPKs significantly decreased C3bgp stimulated IL-23 production from human monocytic cells.In summary, the present study demonstrates the different time-course and ability of C3bgp and LPS to induce the expression of IL-12p40 and IL-23p19 mRNAs in purified human monocytes. We showed that inhibition of p38 MAPK down regulated IL-23 and upregulated IL-12p40 production in stimulated monocytes. We concluded that in human monocytes p38 MAP kinase activation has an opposite effect on the IL-12p40 and IL-23p19 expression. Neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, T and B cells. For instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (moDC). Indeed, upon interaction with neutrophils, moDC up-regulate the expression of costimulatory molecules, such as CD83, CD86 and CD40, and secrete IL-12, thus acquiring the ability to induce proliferation and Th1 polarization of naï ve T cells. In order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. In human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC), the latter further divided in three different subsets based on the expression of CD1c, BDCA-3, and CD16. By analyzing different chronic inflammatory pathologies, such as Crohn's disease, psoriasis and Sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mDC) with characteristics resembling the CD16+ subset of mDC. In order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and CD16+ DC were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. Here we show that neutrophils modulate different effector functions of CD16+ DC, including their survival and their ability to produce IL-12p70. Besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. Chitin is a linear polymer of N-acetyl-D-glucosamine (GlcNAc) residues present in human pathogenic fungi or nematodes. Chitotriosidases (ChT) and acetic mammalian chitinase (AMCase) have been identified as the only functional chitinases in mammalians. The expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. AMCase is considered as predominant chitinases in mice while ChT is regarded as major chitinases in humans. Interestingly, ChT is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. Although, AMCase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of ChT in humans is virtually unknown.

To evaluate the physiological function of ChT we have characterised the substrate specificity of human ChT and the mode of substrate cleavage by analysing ChTproduced fragments of chitosan, a close but water soluble derivate of chitin. Degradation products of chitosan have been investigated by gel electrophoresis and MALDI-TOF mass spectroscopy. Moreover, the application of a computer-based model of ChT activity revealed the mode of substrate cleavage. We found that ChT is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. In further studies we could show that those ChT-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines TNF-a, IL-6, IL-8 and MCP-1 involving the transcription factor NFxB. Moreover, these stimulated monocytes/macrophages revealed an increase of ChT expression indicating an autocrine positive feed-back regulation. Our data suggest that human ChT is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments.

M. Hasenberg 1 , S. Wolke 2 , A. Brakhage 2 , M. Gunzer 1 1 Otto-von-Guericke Universität, Institut für Molekulare und Klinische Immunologie, Magdeburg, Germany, 2 Hans-Knöll-Institut, Abteilung für Molekulare und Angewandte Mikrobiologie, Jena, Germany

Since their discovery in 2004 nucleic extracellular traps (NETs) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. Recently it was found by us and others that neutrophil granulocytes release NETs also upon contact to the filamentous fungus Aspergillus fumigatus. In the present study we aimed to characterize this process in more detail focusing on the kinetics of NET-formation as well as clarifying the responsible cell-biological mechanisms. By the use of several microscopic techniques (Scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of NET like structures after coincubation of A. fumigatus germlings and freshly isolated murine or human PMN. The analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of NETs up to 3 hours later. Moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. Therefore we added the NADPH-oxidase inhibitor DPI to the cell coincubation and found clearly reduced NET formation. By fluorescence staining of reactive oxygen species we could demonstrate that ROS are released prior to NET detection. Interestingly, our data currently suggest that in contrast to other pathogens investigated so far, NETs are not directly toxic to fungal elements. Whether and how NETs control growth of A. fumigatus currently remains open.

To summarize our data, we found rapid NET formation as a commonly observed immune response of neutrophil granulocytes contacting A. fumigatus. Consistent with studies on different pathogens this mechanism seems to be ROS-dependent, however not toxic for the fungus. Thus, in the future we will have to clarify whether NET-formation really occurs in vivo and how NETs can control the outgrowth of A. fumigatus at sites of infection. Production of type I interferons (IFN-I, mainly IFN-a and IFN-b) is a hallmark of innate immune responses to all classes of pathogens. When viral infection spreads to lymphoid organs, the majority of systemic IFN-I is produced by a specialized 'interferon-producing cell' (IPC) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pDC). It is unclear whether production of systemic IFN-I is generally attributable to pDC irrespective of the nature of the infecting pathogen. We have addressed this question by studying infections of mice with the intracellular bacterium Listeria monocytogenes. Protective innate immunity against this pathogen is weakened by IFN-I activity.

In mice infected with L. monocytogenes systemic IFN-I was amplified via IFN-b, the IFN-I receptor (IFNAR) and transcription factor interferon regulatory factor 7 (IRF7), a molecular circuitry usually characterisitic of non-pDC producers. Synthesis of serum IFN-I did not require TLR9. In contrast, in vitro differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA. Consistent with the assumption that pDC are not the producers of systemic IFN-I, conditional ablation of the IFN-I receptor in mice showed that most systemic IFN-I is produced by myeloid cells. Furthermore, results obtained with FACS-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pDC is responsible for bulk IFN-I synthesis. The amount of IFN-I produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. Based on these data we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.

T. Naessens 1 , S. Vander Beken 1 , P. Bogaert 1 , J. Grooten 1 1 Ghent University, Biomedical Molecular Biology, Zwijnaarde (Ghent), Belgium

Introduction: Although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. Recent insights in monocyte and macrophage heterogeneity render the picture even more complex. In addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. In this study we focused on the dynamics and function of resident alveolar macrophages (rAM) during and after allergic bronchial inflammation. Strategy: We used an ovalbumin (OVA)-alum based mouse model of allergic asthma and an OVA-Complete Freund's Adjuvant (CFA) based mouse model of hypersensitivity pneumonitis, constituting a Th1-driven immunological counterpart of the Th2-driven experimental asthma. rAM were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. As complementary approach, rAM and elicited alveolar macrophages (eAM) were distinguished using CD45 bone marrow chimeric mice. Combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited AM populations in the course of Th1-and Th2-driven allergic airway inflammation. Results: During the acute phase of the allergic response, isolated rAM and eAM showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. In both types of allergic inflammation, microsphere-tagged CD45.1 + rAM remained constant in cell number for the first 2 days of chronic OVA-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of OVA-exposure. As a consequence, following the clearance of inflammation, inflammation-experienced rAM replaced the initial rAM population. Strikingly, in both types of allergic inflammation, this secondary rAM population showed a markedly altered responsiveness to LPS stimulation. This involved macrophage activation markers and NF-kB inducible inflammatory genes. However, especially genes induced by IFN-beta showed strongly increased expression in secondary rAM as opposed to their near lack of induction in primary rAM. This switch from an IFN-beta deficient to an IFN-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection.

E. Schlecker 1 , A. Stojanovic 1 , A. Cerwenka 1 1 German Cancer Research Center, Innate Immunity, Heidelberg, Germany

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that expand during cancer, inflammation and infection. These cells play a critical role in suppressing T cell responses. The exact nature and function of MDSC remain unclear. Here we show that a subpopulation of MDSC (Gr-1 + CD11b + F4/80 + ) isolated from RMA-S tumor-bearing mice did not suppress but rather activated NK cells to produce IFN-g. Additionally, NK cells eliminated this subpopulation both in vitro and in vivo. In order to identify molecules and pathways that might be involved in MDSC accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of MDSC subpopulations was performed using whole genome microarrays. Understanding the reciprocal interaction of MDSC with NK cell could improve the efficiency of cancer immunotherapy.

G. Solinas 1 , F. Marchesi 1 , M. Fabbri 1 , S. Schiarea 2 , C. Chiabrando 2 , A. Mantovani 1,3 , P. Allavena 1 1 Istituto Clinico Humanitas, Rozzano, Italy, 2 Istituto Mario Negri, Milano, Italy, 3 Università di Milano, Milano, Italy Experimental and clinical evidence has highlighted that tumor-associated macrophages (TAM) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. Macrophage population is generally divided into two distinct subsets: M1 and M2. M1 macrophages act as a first line of defence against pathogens whereas M2 cells participate in wound repair and maintenance of tissue integrity.

In the tumour micro-environment TAM interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior.

To investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. In selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. The phenotype analysis of tumor-conditioned macrophages (TC-macro) demonstrated high expression of the mannose receptor, CD16, CD68 and low levels of MHC class II. TC-macro produced IL-10, IL-6, TNF but not IL-12, even after LPS stimulation. Moreover, TC-macro produced a panel of chemokines including CCL2, CXCL8, CCL17 and CXCL10. The transcriptional profile of TC-macro revealed that several genes in line with an M2 polarization are highly expressed. The nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( G 100.000 KDa). IL-3 and IL-6 were not detectable in tumor supernatants whereas M-CSF was present at low levels. By mass spectrometric techniques, we surprisingly found that the tumor-derived M-CSF had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. The characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. Genomic effects of glucocorticoid hormone (GC) are exerted by glucocorticoid receptor (GR)-mediated changes of gene expression. This is relatively timeconsuming process, needing hours to develop. In contrast, non-genomic effects may occur within minutes. GCs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. Moreover, many cells and cell lines of haemopoetic origin are sensitive to GC-induced apoptosis. Recent findings indicate, that non-genomic GC effects mediated by mitochondrial GR may have important function in generating pro-apoptotic signals.

We aimed the investigation of non-genomic GC effects on in vitro cultured RBL-2H3 rat mast cell line. We demonstrate that GR nuclear translocation begins within 5 minutes and completes after 30 minutes in DX treated RBL-2H3 cells. Since genomic effects occur in the nucleus through gene expression changes, we considered GC effects within 5 minutes as non-genomic. Studying GC-caused apoptosis, RBL-2H3 cells proved to be GC-resistant and no mitochondrial GR translocation neither impaired mitochondrial function could be observed upon GC treatment.

In further experiments we used RBL-2H3 cells sensitized with anti-DNP (dinitrophenyl) IgE and DNP-conjugated bovine serum albumin was used for stimulation. 5 minutes of DX treatment inhibited Ca 2+ -signaling in antigen stimulated RBL-2H3 cells in the concentration range of 100nM -10mM. Moreover, 5 minutes of DX treatment altered the tyrosine phosphorylation pattern of RBL-2H3 cells. DX treatment alone caused slight increase in tyrosine phosphorylation, while DX treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. The tyrosine kinase Syk plays indispensable role in regulating mast cell activation through the Fc[epsilon] receptor I. Our immunoprecipitation studies show, that DX treatment results in decreased Syk phosphorylation in both resting and activated cells. This finding raises the possibility, that Syk phosphorylation thus kinase activity may be directly or indirectly regulated by GCs via non-genomic pathway. Taken together, our experiments along with the clinical experiences suggest that GCs rapidly influence mast cell activation via a non-genomic pathway, too. The elucidation of the exact signal transduction mechanisms behind rapid GC effects need further experiments. High mobility Group Box 1 (HMGB1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. Notably, HMGB1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . Extracellular HMGB1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. Interestingly, HMGB1 exerts antibacterial functions in human adenoid and testis (2) . Recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (NETs), made of DNA fibers, nuclear proteins (histones) and granule proteins. It has been shown that histones on NETs have an anti-microbial activity (3). We asked whether HMGB1 from neutrophils is a component of NETs and whether it has a function in NETs. We purified human primary neutrophils from peripheral blood of healthy volunteers on Ficoll gradients. To induce NET formation, we stimulated cells for 40 or 120 minutes with 25 nM phorbol ester (PMA), 100 ng/ml interleukin 8 (IL-8), or 100 ng/ml LPS. The presence of NETs was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (MPO) and against a DNA-histone H2A-histone H2B complex. DNA was stained with Hoechst. Using a polyclonal antibody we found HMGB1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of NETs induced by all stimuli. ELISA assays revealed that HMGB1 is not present in the supernatants of activated neutrophils, confirming its binding to NETs.

In conclusion, we found that HMGB1 localizes on NETs. We hypothesize that NET-bound HMGB1 might exert a direct antimicrobial function, or that NETs might concentrate HMGB1 locally to recruit macrophages to the site of infection. These receptors were present on the mast cell surface. Incubation (37°C, 3 h) of HLMC with VEGF-A, VEGF-B, VEGF-C, VEGF-D and Placental Growth Factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-VEGFR-1 and anti-VEGFR-2 antibodies. These data indicate that human mast cells represent both a source and a target of VEGFs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors.

Macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as Mycobacterium tuberculosis. A very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. Whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the IFN-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. These include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. Mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. We hypothesize that the very early steps of pulmonary infection directs the outcome of disease. Therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. Preliminary data show that M. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. Cells harboring mycobacteria are found during early and late stages of infection. Both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. Neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of IL-18. Altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. Human CD1 molecules are dedicated to lipid presentation to T cells and are implicated in inflammatory and auto-immune responses. The CD1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. Our previous studies have demonstrated that Ii associated with CD1a and cholesterol-dependent lipid rafts impact on CD1a surface expression and CD1a-restricted T cell response. Bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated DCs acquire the ability to stimulate CD1-restricted autoreactive T cells. This mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. Sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by CD1 group I molecules. The functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by CD1a molecules could impact in such pathologies. We have thus conducted a preliminary study to understand the implication of CD1 molecules in multiple sclerosis. We first analyzed CD1 expression on monocytes from 16 MS patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. Results obtained in this preliminary study demonstrate that CD1a was not expressed on MS patient monocytes, while the other members of the CD1 family were expressed. Moreover MS sera and plasma induced an earlier and more rapid dendritic cell differentiation than AB sera. These preliminary results confirm our hypothesis that CD1 molecule expression is modified in MS and also reveal that serum from patients with MS modifies lipid-antigen presenting cells. Further studies should contribute to define precise mechanisms involved in lipid presentation by CD1 molecules in this context.

C. Ohnmacht 1 , D. Voehringer 1 1 Ludwig-Maximilians-Universität Munich, Institute for Immunology, Munich, Germany

Basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. However, their development and in vivo functions are largely unknown. Here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth Nippostrongylus brasiliensis. For this purpose, BrdU incorporation experiments and in situ fluorescence microscopy of IL-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. Our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. N. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. Basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. Activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of N. brasiliensis in the absence of Th2 cells. These results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1beta and G-CSF as well as reduced levels of anti-inflammatory TGF-beta. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. Objectives: To investigate the existence of systemic inflammatory response to subchronic oral warfarin (WF) consumation in rats. Methods: Dark Agouti (DA) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. Oxidative activity (cytochemical NBT reduction) and myeloperoxidase (MPO) intracellular content of peripheral blood neutrophils, plasma levels of IL-6 and TNF-a (ELISA) and superoxide dismutase (SOD) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. Changes in prothrombin time (PT), as basic biological warfarin activity was determined as well. Results: Significantly increased PT was noted at the lower WF dose, with tremendous rise after the higher dose. Increase of PMA-stimulated neutrophil NBT reduction capacity (neutrophil priming) was noted at both WF doses, while increase in MPO intracellular content was noted at the higher WF dose solely. Warfarin consumation resulted in no changes in plasma levels of IL-6 and TNF-a. Significant decrease in the SOD activity was detected in red blood cell lysates at both WF doses, suggesting systemic oxidative activity. Conclusion: Increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. Absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. This work is funded by Serbian Ministry of Science and Technological Development (grant 143038). Objectives: Although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. We have recently reported that immune complexes (IC) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. In disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with IC. Here we address the consequences of IC opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, Mph1), and during resolution of inflammation (type-2 macrophages, Mph2). Methods: Mph1 / 2 were generated by culturing CD14 + human monocytes for 6 days in the presence of GM-CSF or M-CSF, respectively. Phagocytosis by Mph1 / 2 of IC opsonised and unopsonised neutrophils was assessed by flow cytometry. After phagocytosis Mph1 / 2 were stimulated with LPS and secreted IL-6, IL-8, IL-10, IL-12p40 and TNF were quantified by ELISA. Results: Mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas Mph1 are only poorly phagocytic. Opsonisation with IC leads to enhanced neutrophil uptake by both Mph1 and Mph2 which is specifically inhibited in the presence of a blocking mAb for macrophage FcyRII. Uptake of IC opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. In both macrophage subsets IL-6, IL-12 and TNF production is suppressed while IL-10 secretion is increased. In contrast, engagement of macrophage FcyR with IC alone induces the release of pro-inflammatory cytokines. Conclusion: Our data demonstrate that IC opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of IC opsonisation. We suggest that IC present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. Excessive production of reactive oxygen species (ROS) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. On the other hand, intracellular heat shock proteins (HSP) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. In this work, correlation analysis was applied to analyze relationship between ROS production and intracellular HSP70 in neutrophils of elderly people. Neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). Intracellular ROS and HSP70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (Invitrogen) and anti-HSP70 antibody (BRM-22, Sigma), respectively. Intracellular level of HSP70 was also estimated in neutrophils after heat shock (HS) performed at 43°C for 10 min. Extracellular ROS production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. A positive correlation was determined for intracellular ROS level and zymosan-mediated extracellular ROS release although the dynamics of ROS release at 1-15 min time range varied within the group. The correlation was unaffected by HS of neutrophils performed for 1 min at 42°C, although this short heat treatment decreased significantly ROS release. There was no correlation between basal intracellular HSP70 (HSP70 basal ) and ROS level, both intracellular and extracellular. At the same time increased HSP70 level immediately after HS (HSP70 (0 min)) correlated negatively with intracellular ROS (initial and after HS). The HSP70 increase value (HSP70 (0 min) -HSP70 basal ) correlated negatively also with intracellular ROS and extracellular ROS release in response to zymosan; and the correlation with ROS level became lower when HSP70 increase was registered in 60 min after HS (HSP70 (60 min) -HSP70 basal ).

Thus it was found that within this age group the alteration in HSP70 induced by HS in neutrophils but not basal HSP70 itself is the parameter associated negatively with both spontaneous ROS level and ROS production in response to activating action of zymosan. This work is supported by ISTC grant #3303.

D. Goyeneche-Patiño 1 , Z. Orinska 1 , F. Mirghomizadeh 1 , S. Bulfone-Paus 1 1 Forschungszentrum Borstel, Borstel, Germany Several studies have shown different roles of mast cells (MC) in innate and adaptative immune responses. In fact, crosstalk between CD8 + T cells and MC has shown to induce multiple genes implicated in the signaling of specific programs such as Type 1 IFN. Two novel genes, Receptor Transporter Protein (RTP4) and Virus Inhibitory Protein, Endoplasmic Reticulum-associated, Interferon-inducible (Viperin) are IFN inducible and were found to be over-expressed in CHIP analysis. The aim of this study is to characterize the expression and protein production of RTP4 and Viperin in mast cells after TLR ligand stimulation in mice lacking of IFNRa and the adapter proteins MyD88 and TRIF. Bone marrow derived mast cells (BMMC) from WT, IFNRa -/-, MyDD8 -/and TRIF -/mice, were exposed to TLR ligands (LPS, pIC, CpG, P(dA-dT) and New Castle Disease Virus (NDV)) during 8 and 48 h. mRNA and protein extraction were performed for further qRT-PCR and SDS PAGE analysis for RTP4 and Viperin. Intracellular stimulation of TLR was performed transfecting cells with nucleic acids using Lipofectamine 2000. Stimulation of WT cells with pIC, PdA-dT and NDV showed an increased expression of Viperin and RTP4 in comparison to control cells (untreated). The same trend was observed for MC from the TRIF and MyD88 knockout mice. In contrast, in the IFNRa deficient mice, expression of genes and protein production was abrogated to the same levels of WT untreated cells. Lipofectamine stimulation does not increase the expression/production of the genes. Direct stimulation of the well recognized viral sensors TLR 3 and 9, as well as, infection of mast cells with NDV (RNA virus) induce the expression of RTP4 and Viperin. The findings suggest that activation of MC with the ulterior expression of genes is Type I IFN dependent. In contrast, the adaptor proteins MyD88 and TRIF and the pathways that they represent are not relevant in the expression of RTP4 and Viperin. These findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of MC in innate immune responses. In recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in Drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. We have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (APCs) to activate lymphocytes; but inflammatory macrophages do not express c-Myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-Myc. We studied some phenomenons suggested to be Myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. Finally, using lentiviral vectors, we over-expressed exogenous c-Myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy.

G. Germano 1 , E. Erba 2 , R. Frapolli 2 , M. D'Incalci 2 , A. Anselmo 1 , S. Pesce 1 , P. Allavena 1 , A. Mantovani 1, 3 1 Humanitas Clinical Institute, Rozzano, Italy, 2 Mario Negri Institute, Milan, Italy, 3 Institute of General Pathology, University of Milan, Milan, Italy

Several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. Inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (TAM) as well as tumor cells, and are attractive target of novel anti-tumor therapies. Trabectedin (ET-743) is a natural product derived from the marine tunicate Ectenascidia Turbinate, it binds the minor groove of DNA, affects transcriptional factor activity and blocks cell cycle. This novel anti-tumor agent is currently used in Phase II studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. We previously demonstrated that Trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. We tested the effect of Trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. Trabectedin significantly reduces CCL2, CXCL8 and the inflammatory protein Pentraxin3 (PTX3) either at transcriptional and protein level, especially after TNFa/IL1b stimulation. Down-regulation of CCL2, CXCL8, PTX3 and also of IL6 and VEGF were confirmed in primary cultures of liposarcoma.

According to the previous in vitro data we now show in a mouse model , using the fibrosarcoma MNMCAI , that Trabectedin treatment selectively affects monocytes in the blood and bone marrow. Moreover Trabectedin treatment strongly reduces the number of macrophages and of CD31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. Overall these results suggest a possible triple role of Trabectedin. Besides its direct cytotoxic effect on tumor cells, Trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. As the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of Trabectedin makes it an attractive candidate in inflammation-associated tumors. Interleukin-4 (IL-4) is a key cytokine of the T helper 2 cell response. IL-4 has been found to be a major regulator of immunoglobulin class switching to IgE and has important functions in the regulation of allergic diseases. Here, the onset of the IL-4 production after birth was investigated in equine neonates. The form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. IL-4 producing cells were measured in peripheral blood mononuclear cells (PBMC) of neonates and foals by flow cytometric analysis. At day 3-6 after birth, a small population of IL-4 producing cells was observed in the absence of any stimuli. The IL-4 + population was not detectable at 6 or 12 weeks of age. Other cytokine producing cells (IFN-g, IL-10) were not detected using these conditions. The stimulation of neonatal PBMC with PMA and ionomycin did not alter the IL-4 + cell population. Phenotyping of the neonatal IL-4 + cells showed that they were IgE + /MHCII -/CD4cells. The occurrence of CD4 + IL-4 producing cells after PMA stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. Magnetic cell sorting of the IgE + /MHCIIcells identified them as basophils. Previous work has shown that foals do not produce endogenous IgE for at least six months of life. IgE bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. Here, the stimulation of neonatal PBMC with anti-IgE induced the secretion of IL-4 at day 5 after birth. Neonatal PBMC collected before colostrum uptake did not produce IL-4 in response to anti-IgE. In summary, equine neonates provide a model to investigate IgE mediated IL-4 responses after birth. The transfer of maternal IgE from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal IL-4 response mediated by the mare's accumulated acquired immunity to allergens. 

S. Schmechel 1 , D. Voehringer 1 1 Ludwig-Maximilians-University Munich, Institute for Immunology, Munich, Germany

Macrophages display broad phenotypic heterogeneity depending on their microenvironment. The initial inflammatory response to Th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a Stat6-dependent manner when stimulated with the Th2 cytokines IL-4 or IL-13. Alternatively activated macrophages (AAM) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. Furthermore, it has been shown that AAM suppress the proliferation of T cells by a yet to be determined mechanism. Currently there is still very limited information about the phenotype, migration and function of AAM. We began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a Stat6-dependent manner. To this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and Stat6-deficient mice and infected these chimeras with the helminth Nippostrongylus brasiliensis to determine whether lack of Stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. The highest turnover of macrophages was found in the peritoneum, irrespective of Stat6 expression. No major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on Stat6 expression in macrophages. We could further confirm that in vitro generated AAM from wild-type but not from Stat6-deficient mice have a strong inhibitory effect on T cell proliferation. We are now trying to identify the mechanism(s) by which T cell proliferation is inhibited. Furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. We have previously shown that AAM can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. To identify potential AAM-derived eosinophil chemotactic factors we currently compare the gene expression profile of IL-4 exposed macrophages from wild-type and Stat6-deficient mice. Candidate genes will be expressed using retroviral transfections of Stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. Macrophages are an essential component of leukocytes infiltration in the tumor. They are identified as tumor associated macrophages (TAMs). These cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. The aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines.

We tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. We have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. In the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. The following human malignant cell lines were tested: A549, HT29, HCT116, SW60, MCF7, MDA-MB231, Jurkat and HL60. Results: Our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. Macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. Down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. Macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. The up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins.

The results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to M1, and the TAMs isolated from the malignant effusions similar to M2.

Among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating MS4A4A and MS4A6A: highest trascriptional level after 18h of stimulation with 10-6M Dexamethasone. MS4A murine genes are differently expressed respect to the human counterpart and only the homologs of MS4A6A (MS4A6b, 6c and 6d) have a similar regulation. Finally, EGFP-tagged MS4A4A, MS4A6A, and MS4A7 expressed in CHO cells showed that all molecules traffic to the cell membrane. Though the biological functions of these MS4A proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized MS4A members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. During inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. These signaling circuitries are even more interfaced during infections in which microbial agonists activate Toll-like (TLR), RIG-like (RLR) and NOD-like (NLR) receptors. On the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (MCP-1)/CCL2 and the GPCR ligand fMLP or the TLR4 agonist lipopolysaccharide (LPS) on monocytes. In fact, the bacterial tripeptide fMLP, but not the cytokines IL-1b or IFN-g, significantly and dose-dependently synergized with CCL2 in monocyte chemotaxis. Furthermore, LPS rapidly induced the expression of interleukin-8/CXCL8, but not of the CCL2 receptor CCR2 in monocytic cells. In turn, the induced CXCL8 synergized with CCL2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by CXCR1/CXCR2, because CXCL8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to CCL2 intact. These data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. Objectives: In recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. Most data were collected on endothelial cells and on thrombocytes. However, there are only few data on vesicles derived of neutrophilic granulocytes (PMN), and most of these investigations applied only pharmacological agents. Our aim is to investigate PMN-derived cell-free particles and their possible role in bacterial killing Methods: Preparation of PMN and investigation of bacterial killing by our semi-automatic method was described by Rada et al. (Blood, 2004) . Cell-free vesicles were prepared after co-incubation of human PMNs with different activating agents for 20 min at 37°C with gentle shaking, followed by spinning down of PMNs for 5 min, at 4°C and 500g. The supernatant was sedimented at 15000g for 10 min, 4°C, and we used the sedimented fraction for our investigations. Formation of particles was followed by fluorescent and electron microscopic assays. The amount of particles was estimated with flow cytometer and by their protein content.

We observed that upon co-incubation of PMNs with S. aureus, opsonized by mixed human serum, PMNs produce a well detectable amount of vesicles. Omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. Production of particles could be inhibited with diphenyl-iodonium (DPI), cytochalasin B (CB) or with azide treatment. Treating PMNs with DNAse or withdrawing glucose during co-incubation had no effect on vesicle formation. In killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. This antibacterial activity could be inhibited by DPI, CB, azide treatment or by withdrawing glucose from the medium during the killing assay. However, treatment of the microvesicles with DNAse had no effect on their antibacterial capacity. For long, CD56 has been used for the detection and identification of natural killer (NK) cells. Recently, the presence of a minor subset of CD56 low CD33+ blood monocytes (MO) in healthy individuals and the increase in CD16+CD56+ blood MO in patients with inflammation has been reported. The functional activity of human CD56+ blood MO has been studied in vitro but not tested ex vivo so far. Healthy people living permanently in malaria endemic areas are exposed to Plasmodium infection, and we hypothesized that blood MO of these individuals could be activated and display increased CD56 expression. We tested if this phenotypic expression was associated with detectable changes in the MO anti-parasitic activity. The MO phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. Myeloid cell markers included CD33 and activation markers such as HLA-DR and TREM-1. Percentages of blood MO involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given MO phenotype and phagocytosis activity was then looked for, using SPSS ® and STATVIEW ® softwares. Our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (P X 0.0001) in the total number of circulating CD56 low MO present in the blood samples of healthy malaria exposed Asian individuals living in Thailand. According to the density of surface antigen determined by Fluorescence Intensity (FI), the decrease in CD33 and the concomitant increase in HLA-DR expressions indicated that in this malaria endemic area, blood MO were mature and highly activated by comparison with surface markers of MO from malaria naï ve donors. The relative levels of CD56+ blood MO were associated with the percentages of membrane-bound IFN-g present at the MO surface.

In conclusion, (i)-A subset of CD33+ blood MO expressed increased levels of CD56 on MO of healthy malaria exposed individuals; (ii)-Blood MO with activated (HLA-DR+) and mature (TREM-1+) phenotypes were present in these healthy individuals;

(iii)-Increased expression of HLA-DR and CD56 on CD14 high MO was associated with a high phagocytosis activity. Introduction: Adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. For instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. The present work focuses on the effects of leptin on the reactivity of M1-and M2-polarized human macrophages. Methods: Monocytes were isolated from the peripheral blood by magnetic cell sorting. Polarization to M1 and M2 macrophages was induced by culture in the presence of MCSF or GMCSF respectively. Polarized cells were characterized by flow cytometry, stimulated with LPS and response assessed by characterization of cytokine profiles via cytometric bead array (CBA). Results: Culture of monocytes in the presence of MCSF or GMCSF induced two different phenotypes. Cells cultured in the presence of GMCSF represented the M1 type and were CD14 negative but CD80 and MHCII positive and produced high levels of IL-8, TNFalpha and IL-6 following LPS stimulation. Culture in the presence of MCSF resulted in induction of the M2 phenotype. These cells were CD14 positive with intermediate expression of CD80 and MHCII expression and produced high levels of IL-10, IL-6 and IL-8 following LPS stimulation. Interestingly, already baseline IL-8 production was high in these cells. Stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. However, if leptin was present in cultures stimulated with LPS, the induction of cytokine production was significantly reduced in both, M1-and M2-polarized cells as compared to cells stimulated with LPS alone. Summary: Whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the TLR4 ligand LPS. Thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in Crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens.

Methods: HL60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to PMA (50 ng/ml) treatment for 48h. Morphological evaluation was performed with conventional microscopy and electron microscopy. Immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. A colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ROS). Results : Besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the PMA-treated fibronectinadherent HL60 cells expressed antigen receptors CD14, TLR2, TLR4 and CD68 , and displayed enhanced phagocytic activity and production of ROS. Expression of CD13, CD33 and CD15 was also maintained however the cells were HLA-DR and CD1a negative. Conclusion: We describe the enhanced ability of fibronectin-adherent HL60 cells to differentiate into macrophages in response to PMA. HL60 may provide a functional model for macrophage differentiation. Above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies.

A. Aporta 1 , N. Ferrer 2 , A. Gómez 2 , J. Gonzalo 2 , A. Arbués 2 , A. Anel 1 , C. Martín 2 , J. Pardo 1 , Apoptosis, Immunity and Cancer 1 University of Zaragoza, Molecular and Cellular Biochemistry and Biology, Zaragoza, Spain, 2 University of Zaragoza, Mycobacterium Genetics, Zaragoza, Spain

Mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. It has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. It has been proposed that M. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. However the biological relevance of this process is not clear. Our group has generated SO2, a M. tuberculosis phoP mutant strain that was shown (Perez et al 2001) to be more attenuated than the present attenuated vaccine strain BCG and conferred protective immunity against M. tuberculosis infection in mice and guinea pigs (Martin et al 2006) . In the present study, we compare the time course and phenotype of cell death induced by SO2, BCG and wild type M. tuberculosis on the murine macrophage cell line J774 and on bone marrow derived mouse macrophages. Our results indicate that wild type M. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. Of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. In order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. Experiments are ongoing with macrophages derived from TRL2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phoP M. tuberculosis strain. Objectives: VIP is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. Type 1 receptor for VIP (VIPR1) gene is highly conserved through species and, in humans, is highly polymorphic. VIPR1 has been reported to be down-modulated in cells of the immune system after activation. An association of some SNPs with some autoimmune diseases has also been reported. In this study we have investigated the correlation between these SNPs and gene expression in monocytes exposed to LPS. Methods: Monocytes from 53 blood donors were separated from PBMC and stimulated with LPS. Total RNA was reverse transcribed and the level of VIPR1 in untreated or LPS-stimulated monocytes was measured by real-time RT-PCR and protein expression. Protein level was measured by Western blot and densitometric analysis.

The kinetic of expression of VIPR1 after 3, 6, 9 and 12 h of exposure to LPS was firstly analysed in monocytes from five individuals. There were two kinetics: one in which a reasonable high levels ( G 50 %) of mRNA was maintained trough time and a second one in which the decrease of mRNA was pronounced. The experiments were repeated using monocytes from 53 donors that had been typed for the relevant VIPR1 SNPs. The down-regulation of VIPR1 correlates with the presence of a T at rs896 mapping in the 3'-end of the gene (p= 0.004). The VIPR1 protein level was decreased about 40 % in monocytes of 3 subjects typed as T/T at rs896 whereas 3 subjects typed as C/C at rs896 maintained a high level of expression after 9h of LPS treatment.

The data show that different haplotypes of the VIPR1 gene correlate with a different kinetics of gene expression in activated monocytes. A possible consequence of these data is that the anti-inflammatory properties of VIP governed by the VIPR1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases.

J. Oujezdska 1 , T. Vavrochova 1 , D. Filipp 1 , Immunobiology 1 Institute of Molecular Genetics AS CR, Immunobiology, Prague, Czech Republic

Phagocytes which appear in early mouse development (E7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. Recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, YS-derived phagocytes. In addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. This origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. The aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. Here we report the temporal and spatial expression of Toll-like receptors (TLRs) and CD14 in the early mouse embryo (ME). FACS analysis of cell suspension prepared from 10.5 day ME showed that about 0.7-1 % of cells were positive for CD11b. These cells exclusively were also positive for CD14, TLR2, and CD45 antigens. Using qPCR and flow cytometry we show that TLRs and other TIR domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. Reciprocal matings between wild type and MHCII-EGFP knock-in mice revealed that while maternallyderived MHCII + macrophages are present in the embryo from early developmental stages (E7,5), embryo-derived MHCII + macrophages start to appear in the embryo around day 13. Multicolor FACS analysis of CD11b, CD45, CD14, F4/80, TLR2, TLR4, c-kit and MHCII surface markers revealed differential expression of TLR2 and c-kit on embryonal phagocyte subpopulations. Moreover, the microarray analysis of CD11b + TLR2 + cells isolated from the E10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. These molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. These results are first to characterize the regulated expression of TLRs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. Humans may be exposed to a variety of mycobacteria ranging from environmental or BCG vaccine to more pathogenic mycobacteria. Only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. Interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. IL12/IL23-dependent IFNg pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. Due to the high rate of consanguineous marriages in the North African countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. In Tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated BCG infection (BCG-osis). Among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. Using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several IFNg pathway genes, other candidate genes are being investigated for the 2 other patients.

In the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including MHC and NRAMP1. We did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: TLR2 is the principal mediator of macrophage activation in response to mycobacteria through NFKb pro-inflammatory signaling pathway and DC-SIGN is the major receptor of M. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory IL-10 cytokine. Using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in Tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of SNP and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis.

Host inflammatory responses play a major role in granuloma formation and control of the infection. Unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. IFNg therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant M. tuberculosis strains. Francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. Research into Francisella has expanded over recent years due to its designation as a potential biological warfare agent. Several species of Francisella exist and have varying degrees of pathogenicity. F. tularensis live vaccine strain (LVS) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. However, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. F. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. Therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of F. tularensis would decrease and antibiotic treatment may be more effective. A flow cytometric assay was developed to measure bacterial uptake. This method used a FITC labelled anti-F. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. A series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (MHS) and on ex-vivo mouse lung tissue to determine whether uptake of F. tularensis LVS could be altered. The presented data shows that several inhibitors work efficiently to reduce LVS uptake by up to 70-80 % in both the in vitro and ex vivo assays. However, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. In addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against F. tularensis. It may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã Crown Copyright. Dstl, 2009. Hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. It is also known that extracellular Hsp70 have pronounced immunomodulatory properties. To study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface Hsp70 on reactive oxygen species (ROS) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). Neutrophils were isolated from human peripheral blood by using a standard protocol. Respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. For the experiments human recombinant Hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface Hsp70 were used. Exogenous Hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. As the control we registered amplitude of oxidative burst in samples supplemented with PBS or live mouse thymocytes having no Hsp70 on their surface. Results demonstrating effect of exogenous Hsp70 on phagocytosis-induced ROS release from human peripheral blood neutrophils have been obtained. It was demonstrated marked dose-dependent inhibiting action of exogenous Hsp70 on amplitude of respiratory burst. The cells expressing surface Hsp70 impacted on ROS production in this model similarly. The results of chemiluminescence analysis demonstrated that zymosan induced ROS production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. The effect was more pronounced for increased amount of thymocytes added to the samples. Thus, immunomodulatory effects of exogenous Hsp70 might be caused by influence of the protein on ROS release from phagocytes. We suppose that the registered effects are connected with ability of Hsp70 to inhibit activity of NADP-oxidase -the key enzyme for ROS production during respiratory burst.

Results: We recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. No substantial changes were detectable in the number of circulating monocytes. In contrast we observed a clear expansion of CD14/CD86 and CD14/CD163 double positive subsets. This event was transient; it abated at the later time point suggesting a causal relationship to the treatment. It correlated with sensitivity to the treatment. In fact we observed that in the responder patients the expansion of the CD14/86 subset was clear in the first weeks of treatment and decreased there after. In contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. All the patients had an initial expansion of the CD14/163 subset. In the responder patients this population was still present at the time of surgery. The immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative M2 polarization. Conclusion: These data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors.

M. Dimitrijević 1 , I. Pilipović 1 , S. Stanojević 1 , K. Mitić 1 , K. Radojević 1 , V. Pešić 2 , G. Leposavić 1,2 1 Institute of Virology, Vaccines and Sera "Torlak", Immunology Research Centre "Branislav Janković", Belgrade, Serbia, 2 Faculty of Pharmacy, University of Belgrade, Department of Physiology, Belgrade, Serbia

The primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (NO) and hydrogen peroxide (H 2 O 2 ) production and phagocytic activity of these cells. In addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. To address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both NO and H 2 O 2 production and phagocytic activity of these cells. Using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. Furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. In vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated NO production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide H 2 O 2 production. An increase in H 2 O 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. Chronic propranolol treatment in vivo led to increased NO and H 2 O 2 production by peritoneal macrophages. Furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). In conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on H Objectives: Monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: CD14 ++ CD16 -('classical') and CD14 + CD16 + ('pro-inflammatory') monocytes. A central monocyte function is cytokine production in response to Toll-like receptor (TLR) ligation. The CD14 + CD16 + monocytes display higher TLR2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the CD14 ++ CD16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. Newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. Studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. Thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following TLR2 stimulation. Methods: Cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for CD14 and CD16 and intracellular IL-12p70 and TNF expression. The Mann-Whitney U-test was used to evaluate differences between groups. Results: A significantly higher percentage of neonatal monocytes were positive for IL-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. GeoMFI of IL-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. In both newborns and adults, IL-12p70 (% positive cells and GeoMFI) was significantly higher for CD14 + CD16 + cells than for CD14 ++ CD16cells, unstimulated and stimulated. Regarding TNF, neonatal and adult monocytes did not differ in unstimulated cultures, however GeoMFI of TNF was significantly higher in neonatal monocytes after stimulation. Whereas the TNF response following stimulation was similar between the adult monocyte subsets, in newborns the CD14 ++ CD16cells were positive for TNF to a significantly higher extent than the CD14 + CD16 + cells.

In particular the TNF response to TLR2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. Notably, in newborns the CD14 ++ CD16monocytes were positive to a higher extent for TNF following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. Objective: Chronic granulomatous disease (CGD) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. CGD generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating CGD may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features.

We report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. There was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. The family history was unremarkable, and the parents were unconsanguineous. Physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. A large frontol mass with diffuse peripheral vasogenic edema was discovered on MRI. Subfalcine herniation was noted secondary to mass effect. CGD was suspected and the analysis with flow cytometric dihydrorhodamine assay (DHR assay), for functional analysis of neutrophils was compatible with the diagnosis of CGD and no bimodal histogram pattern spesific for X-CGD was found in the mother and sister. After the diagnosis of CGD, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. Aspergillus fumigates grew from the culture; liposomal amphotericin B and voriconazole were started; which were found to be sensitive to the cultured species. In addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. After 2 months, control MRI showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. The screening of the other family members with DHR assay demonstrated that one of his sisters had also CGD and phenotype was autosomal recessive. Mutaton analysis in "hot spot" in NCF1 gene concerns the well-known GT deletion in the second exon of NCF1 gene both at the patient and his sister. Results: This was an atypical clinical presentation of CGD in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. We documented a good response to the combination of IFN-g, liposomal amphotericin B and voriconazole after surgery. Conclusion: CGD should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. Recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. Therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. To test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (CCI) model in Sprague-Dawley rats. Animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after CCI. Levels of TNF-a, IL-1b, IL-6, IL-10 and IL-1 receptor antagonist were analyzed in brain tissue using real time RT-PCR at 4 and 72 hours after treatment. Immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. Data were analyzed with the Mann-Whitney U test and a two-tailed p X 0 .05 was considered significant. All cytokines were highly up-regulated 4 hours after CCI but no differences between the groups were observed at this time point. Three days after trauma the levels of IL-10 were significantly lower in the ubiquitin treated animals, whereas the levels of IL-6 and TNF-a were higher when compared to the placebo group. Interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. The infiltration of neutropils was not affected by ubiquitin treatment. Here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. Macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (PTB) patients. Efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (PTB) patients. There is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (TPP), total antioxidant activity (TAA), malondialdehyde (MDA), and oxidative stress index (OSI) in PTB patients during chemotherapy with or without micronutrient supplementation. The present study was designed to assess the levels of neopterin, TPP, TAA, MDA, and OSI during chemotherapy with (C+M) or without (C-M) micronutrient supplementation using ELISA and spectrophotometric methods. Thirty-eight (38) newly diagnosed PTB patients and forty non-PTB apparently healthy subjects volunteered to participate in this study. Twenty of the PTB patients were on anti-tuberculosis drugs supplemented with micronutrients (C+M) while 18 were treated with anti-tuberculosis drug alone (C-M) for a period of four weeks. The levels of neopterin (p=0.02), TPP (p=0.00), OSI (p = 0.00), MDA (p = 0.00) were significantly raised but TAA (p = 0.01) was significantly reduced in PTB patients compared with controls. The levels of MDA (p = 0.04), neopterin (p=0.00) and TPP (p=0.00) were significantly reduced in C+M after two weeks of treatment compared with baseline values before commencement of treatment. The levels of TPP (p=0.00), MDA (p=0.00), neopterin (p=0.02), OSI (p=0.00) were significantly reduced while TAA (p=0.01) was significantly raised in C+M after 4 weeks of treatment compared with the baseline concentrations. In C-M, only MDA showed significant decreased after 4 weeks of treatment when compared with the baseline values. Plasma level of neopterin, TPP, OSI and MDA declined faster in C+M than C-M. Therefore, micronutrient supplementation of PTB drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. This will improve deranged macrophage activation and reduce oxidative stress indices in PTB patients.

A. P. Aguas 1 , E.M. Cunha 1 , M.J. Oliveira 1 1 ICBAS, University of Porto, Anatomy, Porto, Portugal

The acute in vivo intake of mercury (Hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of Hg by scanning electron microscopy coupled with x-ray elemental microanalysis (SEM-XEM). The intracellular distribution of Hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. BALB/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. In some mice, before the Hg injection, peritoneal phagocytes were activacted by ip injection of BSA. Pre-injections with a selenium (Se) salt were also performed in order to study the putative modulatory role of Se on Hg intake by phagocytes. Peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with PBS, they were cytospinned, fixed with formaldehyde, and processed for observation by SEM-XEM. Five min after the Hg injection more than half of the mouse phagocytes were positive for Hg. A higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). Phagocyte activation enhanced the number of Hg particles seen inside the phagocytes. Pre-injection of the peritoneal cavity of mice with Se resulted in finding that more than half of the Hg intracellular particles were coupled with Se. Subcellular topography of Hg particles showed that they were presented in individual small cytoplasmic vesicles. We conclude that Hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. Hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. This study was supported by a grant from FCT, Portugal. Mast cells play central roles in allergic inflammatory reactions and innate immunity. SWAP-70 is a Rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. In B cells and mast cells SWAP-70 regulates F-actin cytoskeletal rearrangements, cell polarisation and cell migration. (Pearce et al., 2006; Sivalenka and Jessberger, 2004) . Swap-70-/-bone marrow derived mast cells (BMMC) are specifically impaired in FceRI-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (Gross et al., 2002; Sivalenka and Jessberger, 2004; Sivalenka et al., 2008) . Crucial regulators of these processes are members of the Rho family of small GTPases such as Rac1 and RhoA. SWAP-70 interacts with Rac1 in vitro and preferentially binds the active GTP-bound Rac1. SWAP-70 supports the increase of active Rac1 in vitro by a yet to be defined mechanism (Shinohara et al., 2002) . In this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between SWAP-70 and Rac1. It was found that fulllength SWAP-70 preferentially binds to constitutively active Rac1 (Rac1Q61L) but not to its dominant negative form (Rac1T17N). Binding assays with SWAP-70 truncated mutants showed interaction of SWAP-70's N-terminus with GTPgS Rac1 or Rac1 depleted of guanine nucleotide, whereas SWAP-70 central or C-terminal regions do not bind to any form of Rac1. Preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire SWAP-70 sequence, mapped the Rac1 binding site near the N-terminus of SWAP-70. Full-length SWAP-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of Rho GTPases. Elucidating the molecular interactions of SWAP-70 with Rho GTPases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express SWAP-70.

V. C. Barbosa 1 , C. D. Polli 1 , M.C. Roque-Barreira 1 , M.C. Jamur 1 , C. Oliver 1 , G. Pereira-da-Silva Mast cells are essential cells in IgE-associated immune responses. FceRI crosslinking induces mast cell degranulation and release of proinflammatory mediators. We have previously shown that the lectin ArtinM induces mast cell activation but the mechanisms involved in this activity remain unknown. Objective: The present study was undertaken to further characterize the ability of ArtinM to activate mast cells. Methods: RBL-2H3 cells were sensitized with IgE anti-TNP and stimulated with DNP 48 -HSA or ArtinM. ArtinM binding to RBL-2H3 cells was assessed by flow cytometry. Mast cell degranulation was determined by measurements of released b-hexosaminidase activity. Microplate binding assays were utilized to assess ArtinM binding to IgE. To investigate FceRI recognition by the lectin, western blots of cell lysates were stained with biotinylated ArtinM and BE's antibodies specific for fceRI b-subunit. Intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. MCP-1 and TGF-b levels released by mast cells were measured by ELISA. Results: ArtinM binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of IgE. The release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of D-mannose, suggesting that mast cell degranulation induced by ArtinM might be the result of interactions between the lectin CRDs and glycosylated components on the cell surface, like FceRI or IgE. Indeed, it was observed that the lectin bound to IgE in a dose-dependent manner and recognized the FceRI b subunit in western blot analysis. Exposure to ArtinM resulted also in phosphorylation of intracellular proteins, MCP-1 release and TGF-b production. Significant increases in these activities were observed upon sensitization with IgE. Conclusions: These results suggest that ArtinM may bind to glycans of the high affinity IgE receptor and/or of the IgE (bound to FceRI) and that such interactions would be implicated in its ability to activate and degranulate mast cells. In view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. The adhesion receptor L-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. It is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. A distinct feature is the exclusive presentation of L-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. This topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. Other adhesion molecules such as P-selectin glycoprotein ligand 1 (PSGL-1), b1 and b7-integrins also share this special phenotype. Taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. Despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown.

In this study we identified the highly conserved transmembrane domain of L-selectin to regulate microvillus receptor positioning and adhesion under flow. Taking advantage of the inverse surface expression pattern of CD44 (cell body) compared to L-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. Transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. In turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. Thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. Objectives: Macrophages are one of the principal effector cells involved in the innate immunity response. They kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as IL-1b, IL-18 and TNF-a. Herpes Simplex virus 1 (HSV-1) is an enveloped DNA virus that infects mostly oral mucosa and sensory neurons. Innate immunity responses activated by HSV1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. IL-18 and TNF-a are cytokines produced by macrophages that contain known anti-HSV properties. The objective of this study was to characterise the secretome of human primary macrophages infected with HSV1. Methods: Human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. Macrophages were left untreated or primed with poly(I:C) (10ug/ml), a mimetic of double-stranded RNA, after which cells were left uninfected or infected with HSV-1 for 18 h. After this, cell culture supernatants were collected, concentrated and proteins purified. The secreted proteins were digested into peptides, identified and quantified using iTRAQ (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanoLC-MS/MS. The raw MS/MS data was analysed using ProteinPilot 2.0 software. Results: In the first iTRAQ experiment over 300 human proteins were identified in the HSV1 infected cell supernatants. From these proteins 119 had at least 3 fold increase after poly(I:C) + HSV1 infection compared to the uninfected cells. HSV1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: iTRAQ labelling showed a total of 2.7 fold increase in the protein amount in the poly(I:C) + HSV1 infected cell supernatant and a 2.6 fold increase in the HSV1 infected cell supernatant when compared to the uninfected cell supernatant. Amongst the upregulated proteins there were known inflammatory proteins: chemokine (C-X-C motif) ligand 10, IL-6, TNF-a induced protein 6, Complement factor B, Galectin-1 and MxA. At present, further experiments are on-going for more detailed analysis of the HSV1 infected macrophage secretome.

H. P. Prakash 1,2 1 German Cancer Research Centre, Translational Immunology, Heidelberg, Germany, 2 Max Planck Institute for Infection Biology, Molecular Biology, Berlin, Germany Chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, CHD and COPD. Chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (IAPs). The significance of cellular inhibitor of apoptosis protein-1 (cIAP-1) and X-linked Inhibitor of Apoptosis proteins ( XIAP) in Chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and XIAP knockout (KO) mice using a novel non-invasive intra-tracheal infection method.

In contrast to wildtype, IAP knockout mice failed to clear the infection from their lung. Wildtype mice responded to infection with a strong inflammatory response in the lung. In contrast, the recruitment of monocytes and macrophages was reduced in IAP KO mice compared to wildtype mice. The concentration of Interferon gamma (IFN-g) was increased whereasTumor Necrosis Factor (TNFa) was dysregulated in the lungs of infected IAP KO mice compared to infected wildtype mice.

Ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that IAPs are required for innate immune responses of these cells. Our findings thus suggest a new immunoregulatory role of IAPs in C.pneumonaie pulmaonry infections.

Methods: Human monocytes were purified from venous blood of normal volunteers by Ficoll density gradient centrifugation. hrGal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mM lactose or sacarose, was assessed by flow cytometry and confocal microscopy. In transwell systems, assays were performed using hrGal3, laminin or fibronectin immobilized or not on the filters. These were added to wells containing soluble hrGal3 or RPMI and monocytes (1x10 5 ) were added into each insert. When necessary, hrGal3 was pre-incubated with 10mM lactose or sacarose. MCP-1 (100ng/ml) was used as positive control.

We observed that hrGal-3 binds to the surface of human monocytes through its CRD, since this interaction can be inhibited by lactose. We corroborated some data of literature that hrGal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. When we evaluated the participation of the ECM laminin and fibronectin in monocyte migration induced by hrGal-3, we observed that the association between these glycoproteins and hrGal-3 resulted in a 60 % increase in the number of migrating cells. Both N-and C-terminal domains of hrGal-3 are involved in the association between laminin or fibronectin and hrGal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively Conclusions: Our results showed that hrGal-3 induces monocyte migration by haptotaxis, through the interactions established between both N-and C-terminal domains of the lectin and ECM glycoproteins, laminin and fibronectin. In a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. Their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. Recent studies have shown that monocyte heterogeneity is conserved in humans and mice. The different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. In order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hESC).Culturing of embryoid bodies for 3 weeks in the presence of BMP4,VEGF and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of CD14+CD45+ cells. The sorted CD14+CD45+ cells were further cultured for 7 -10 days in the presence of M-CSF and gave rise to a homogenous population of adherent mature macrophages. Embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. While virtually all embryonic-derived macrophages expressed the LPS-receptor CD14, M-CSF receptor CD115 and the scavenger-receptor CD36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the CD14 and CD16 (FcgammaRIII) : the CD14lowCD16-and CD14+ CD16+. Trancscriptional, phenotypic and functional assays suggest the alternative (M2) polarization of CD14+CD16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, M2 -related gene expression).The exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. Collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny.

B.-S. Choi 1 , P. Kropf 1 1 Imperial College London, Immunology Department, London, United Kingdom

The balance between T helper (Th) 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. We have recently shown that arginase-induced L-arginine depletion results in local suppression of antigen-specific T cell responses in nonhealing leishmaniasis. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, supplementation with L-arginine restored T cell effector functions and resulted in reduced lesions size and parasite load. However, despite the efficient production of IFN-g by CD4 + T cells at the site of infection and despite the reduced pathology, the mice did not heal. We hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to IFN-g mediated signals. To test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. In addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (iNOS) to kill the intracellular parasites. Vg9Vd2 T lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. The phosphoantigen isopentenyl pyrophosphate (IPP) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. Previous work has shown that a molecular complex homologous to mitochondrial ATP synthase (ecto-F1-ATPase) is expressed on many cell types and is a possible specific ligand for the Vg9Vd2 TCR. The present study aims at understanding the role of F1-ATPase in antigen regognition. Using video microscopy calcium imaging in single Vg9Vd2 T lymphocytes, we can now show that the T cell response to IPP requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface F1-ATPase. Purified F1-ATPase immobilized on polystyrene beads can partly replace the need for cell-cell contact. IPP in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in T cell activation assays clearly shows that it is not recognized as such on tumors. However, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. One of these, a derivative of IPP, is barely able to stimulate Vg9Vd2 cells in the absence of APCs, as opposed to the non-nucleotidic antigen IPP. However it can bind stably to F1-ATPase. Thus the F1-ATPase complex acts as a presenting structure for nucleotide phosphoantigens. Altogether, our data suggest that Vg9Vd2 T cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. Surface Plasmon Resonance was used to analyse the molecular interaction between TCR and F1-ATPase. By using purified F1-ATPase and peptides derived from Vg9Vd2 TCR sequences, interaction sites between F1-ATPase and TCR were identified on both ligands. Based on these findings a generalized model for Vg9Vd2 T cell activation is proposed.

Ligands for the cytotoxic lymphocyte activating receptor NKG2D are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by NKG2D(+) lymphocytes. A key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific T cells. A second key issue is the scope and scale of events that follow NKG2D activation in vivo. By transiently overexpressing the NKG2D ligand Rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident Vgamma5Vdelta1 gamma-delta T cells and Langerhans cells (LC), that were swiftly followed by epithelial infiltration of unconventional alpha-beta T cells. These data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of NKG2D ligands is sufficient to provoke a series of multicomponent immunological changes. The effects on LC, which lack NKG2D and presumably respond to changes initiated by local gamma-delta T cells, are particularly interesting. Ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to NKG2D ligands in vivo.

To assess the scope of this response, ovalbumin was applied to the skin concomitant with Rae1 induction. The primary systemic Th2 response is increased by concomitant responses to a stress antigen. We will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. In addition, the many ligands available to the NKG2D receptor suggest that different ones may play unique roles. A novel NKG2D-ligand, H60c, is uniquely expressed in mouse skin. When the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of Rae-1 induction. Such studies may help resolve a long-standing puzzle over the pleiotropy of NKG2D ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels.

A.-S. Invariant natural killer T (iNKT) cells are a distinct lineage of T lymphocytes that co-express a highly conserved ab T cell receptor (TCR) along with typical surface receptors for natural killer (NK) cells. These lymphocytes recognize glycolipid antigens presented by the non-classical class I molecule CD1d. iNKT cells are characterized by their capacity to produce rapidly large amounts of both Th1 (IFN-g, TNF) and Th2 (IL-4, IL-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. Converging studies in mouse models suggest that iNKT cells can prevent the development of type 1 diabetes. The frequency of iNKT cells is lower in non-obese diabetic mice (NOD mice). Manipulation of iNKT cells, either by increasing their frequency or by stimulating them with agonists such as a-GalCer, inhibits diabetes onset in NOD mice. Recently, a new population of CD4 -NK1.1 -iNKT cells producing high levels of the pro-inflammatory cytokine IL-17 has been identified (iNKT17 cells). Given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of iNKT17 cells in type 1 diabetes. Interestingly, NOD mice exhibit a higher frequency of iNKT cells producing IL-17 as compared to C57BL/6 mice. This increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. As previously described in normal mice, iNKT17 cells present in NOD mice were mainly CD4 -NK1.1 -, express the ROR-g transcription factor and IL-23 receptor, both molecules being usually associated with Th17 commitment. We are currently analyzing, using co-transfer experiments, whether these iNKT17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in NOD mice.

J. S. Dodd 1 , R. Muir 1 , S.S. Affendi 1 , P.J. Openshaw 1 1 Imperial College London, Respiratory Medicine, London, United Kingdom Natural killer T (NKT) cells are a heterogeneous population of innate T cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. Upon activation with their cognate glycolipid antigen presented by CD1d molecules, activated NKT cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. Consequently, NKT cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. Respiratory syncytial virus (RSV) is a common cold virus of the family Paramyxoviridae. It is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. Despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. It is known that CD1d-deficient mice with poor NKT cell responses have inefficient induction of CD8 T cells and reduced clearance of RSV, perhaps because of IFN-g release by activated NKT cells. We now show that activation of lung NKT cells with intranasal aGalCer during RSV infection of mice boosts Th2 immunity (increasing IL-5 and IL-10), promoting pulmonary eosinophilia and ablating CD8 T cell recruitment. By contrast, intraperitonal injection of aGalCer enhances NK cell recruitment and boosts pulmonary CD8 T cell activity (as measured by CD25 expression), increasing IFN-g production in the airway and lung and inhibiting viral replication. Effects on illness (as measured by weight loss) were similarly distinct: intranasal aGalCer induced early (d4) weight loss independent of conventional T cells, whereas intraperitonal aGalCer enhanced late (d7) weight loss by a CD8 T cell dependent mechanism. Therefore, NKT cells stimulated by aGalCer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. In general, neonatal conventional T cells, especially CD4 + ab T cells, are regarded as immature or T H 2 biased. Vg9 + Vd2 + T cells are unconventional lymphocytes: they are MHC-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP)) or aminobisphosphonates (e. g. zoledronate) in adults. Until now, little is known on the functional reactivity of neonatal Vg9 + Vd2 + T cells towards these activators. Because IL-23 is preferentially secreted by neonatal dendritic cells (DC) upon TLR stimulation, we investigated the potential costimulatory effect of this cytokine on HMB-PP and zoledronate-treated neonatal Vg9 + Vd2 + T cells. Herein, we observed that zoledronate induced neonatal Vg9 + Vd2 + T cell proliferation and IFN-g production in cord blood mononuclear cells (CBMC) cultures. Other T H 1-like cytokines like TNF-a and GM-CSF were also produced upon this stimulation, but less than IFN-g, while T H 2-like cytokines such as IL-4 and IL-5 were not induced. Addition of IL-23 to zoledronate selectively costimulated IFN-g production from neonatal Vg9 + Vd2 + T cells. Furthermore, zoledronate/IL-23 treatment resulted in neonatal Vg9 + Vd2 + T cells expressing high levels of the cytotoxic mediators perforin and granzyme A. Zoledronate induced the expression of the receptor for IL-23 (IL-23R) and the transcription factor T-bet, which is known to be important for the production of IFN-g in gd T cells. In addition, costimulation with IL-23 resulted in a further increase of T-bet expression in neonatal Vg9 + Vd2 + T cells. These changes in the expression of IL-23R and T-bet likely contribute to the observed selective IFN-g response towards zoledronate/IL-23 treatment. Of note, in contrast to adult peripheral blood Vg9 + Vd2 + T cells, HMB-PP had no or only a minor effect on the functional reactivity of neonatal Vg9 + Vd2 + T cells. Altogether, these observations show that neonatal Vg9 + Vd2 + T cells are functionally active and that this T cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when DC-derived IL-23 is produced in response to microbial stimuli. The evasion of antigen presentation is a feature common to herpesviruses. One of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral E3 ligases such as HHV8 encoded K3, K5 or mHV68 encoded mK3. These viral genes represent homologues of the March family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. Ubiquitination targets surface molecules to the lysosome via the multivesicular body (MVB), a structure which also has an important role in the budding of many viruses. We investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral E3 ligases manipulate them. We discovered that both the cellular March and viral E3 ligases ubiquitinate CD1 molecules. However, whereas viral molecules inhibit CD1-antigen presentation, the March molecules are essential for the recirculation and function of the long-lived and lysosome-resistant CD1 molecules. In contrast MHC class II was only targeted by cellular and not by viral E3 ligases. Furthermore CD1 molecules could be found in viral particles as a result of ubiquitination, presumably via the MVB. Thus, virally expressed and cellular E3 ligases have opposite effects, despite their homology. How this is achieved is a matter of active investigation. Gamma delta (gd) T cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. Our previous study revealed that Vd2 negative ( neg ) gd T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. In order to investigate the anti-tumor effects of Vd2 neg clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of Vd2 neg clones, in contrast to Vd2 + cells, prevented the development of HT29 tumors. Vd2 neg clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein (MIP)-1d and monocyte chemoattractant protein (MCP)-4. More importantly, a systemic intraperitoneal (i. p.) treatment with Vd2 neg clones delayed the growth of HT29 subcutaneous (s. c.) tumors. The effect of in vivo gd T cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. gd T cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since Vd2 neg clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific Vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. More recently, we generated HT29 cells expressing the luciferase and realized orthotopic injection of HT29-luc cells. Progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. Intraepithelial lymphocytes (IEL) compose large, oligoclonal, tissue-associated repertoires of non-MHC-restricted T cells that play key roles in immunosurveillance. It is commonly considered that the characteristic IEL repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating IEL function. However, no such molecules have been identified. Here we characterise Skint1, currently the only known determinant of a canonical IEL compartment, that is selectively required for Vg5Vd1 + dendritic epidermal T cell (DETC) development. We show that both peripheral and thymic Skint1 expression is essential for full DETC development. Its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects DETC, nor affects any other T cells. Unexpectedly, however, Skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. Mouse genetic models allow powerful insight into Skint1 function; for example, we demonstrate that the constitutive expression of wild-type Skint1 fully restores DETC development in a Skint1 mutant mouse, but does not rescue normal DETC function. Thus, Skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated T cell compartments, and how normal versus dysregulated tissues may be demarcated. Marginal zone (MZ) B cells are strategically localized in the MZ of the spleen. Since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. Besides being able to rapidly secrete antibodies, MZ B cells may also act as professional antigen presenting cells (APCs). They are known to express high levels of CD1d which is the presenting molecule for NKT cells which are also located in the MZ. Therefore we hypothesised that MZ B cells may be efficient activators of NKT cells.

To test this hypothesis, we used freshly sorted splenic MZ B cells (CD19 + CD21 hi CD23 lo CD11c -) and splenic conventional dendritic cells (cDCs) (CD11c hi CD8a +/-CD11b +/-B220 -) from WT and CD1d -/mice as APCs for NKT cells from Va14-Ja18 transgenic or WT mice. The APCs were treated with agalactosylceramide (aGalCer) or heat killed (HK) Listeria monocytogenes or Salmonella typhimurium. Both MZ B cells and cDCs proved to be highly efficient APCs for priming of NKT cells and induced robust proliferation. In contrast, other populations of B cells failed to activate NKT cells. We showed, using CD1d -/mice as well as blocking antibodies to ICOSL, that proliferation of NKT cells depends on TCR/CD1d and in case of MZ B cells, also on ICOS/ICOSL interactions. Importantly, APCs primed with HK bacteria were not able to induce NKT cell proliferation. Interestingly, MZ B cells exclusively induced production of IL-4 by NKT cells. In contrast, cDCs mostly induced production of IFN-g and IL-4 producing cells were scarce under these conditions. Cytokine production by NKT cells proved to be independent of TCR signalling, but dependent on ICOS/ICOSL interactions when MZ B cells were used as APCs, and GITR-dependent when cDCs were used. Taken together, our data suggest that both MZ B cells as well as cDC act as professional APCs for NKT cells. Notably, the nature of APCs appears to be critical for polarization of the immune response: MZ B-cell-primed NKT cells induce cytokine milieu fostering a T H 2 response, whereas cDC-primed NKT cells rather favor a T H 1 response. Objectives: IL-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. SLE and RA) and the allergic disease atopic eczema. In mice, injections of IL-18 give rise to an early polyclonal isotype switched antibody response which is absent in iNKT cell deficient (CD1d -/-) mice. We set out to investigate the activated B cells in IL-18 injected mice and how these are regulated by iNKT cells. Methods: Mice received daily i. p. injections of IL-18 (2 mg) for 10 days and the antibody response in serum was monitored using ELISA. The B cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. Results: Mice injected with IL-18 developed self reactive (anti-PC and anti-DNA) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. SLE and atopic eczema. The antibody producing cells formed CD138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. Surprisingly, the antibody response induced by IL-18 was increased in iNKT cell deficient (CD1d -/-) mice, in contrast to published data. An increased response to IL-18 was also observed in Ja281 -/mice, which lack the a-chain of the TCR used by iNKT cells, and thus our data suggest that iNKT cells inhibit antibody producing cells in IL-18 induced antibody responses. Further characterization of the recruitment of B cells in IL-18 injected mice revealed a marked expansion of the Marginal Zone B cell (MZB) population in the spleen, suggesting an important role for MZBs in the IL-18 induced autoreactive antibody response. MZBs are innate-type B cells that express high levels of CD1d, are prone to autoantibody production and often involved in early immune responses. The IL-18 induced antibody response in MZB deficient (CD19 -/-) mice was either decreased (IgG) or delayed (IgE), supporting the importance of MZBs in IL-18 induced antibody responses.

We conclude that the role for iNKT cells in IL-18 induced antibody responses is to inhibit the production of autoreactive antibodes from MZBs in extrafollicular foci. Objectives: Amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan Entamoeba histolytica. There are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ALA). Interestingly, only a small proportion of E. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. So far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (IFN-g). However, information is lacking about the sources of early IFN-g production as well as the amoeba antigens involved in this activation process. Methods: Using a recently developed C57BL/6 mouse model for ALA, the contribution of natural killer T (NKT) cells for protection against amoebic disease was investigated. Applying NKT cells and dendritic cells as antigen-presenting cells from various KO-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by NKT cells was analysed. Results: NKT cells were found to play a key role in the defense against ALA. Specific activation of NKT cells by a-galactosylceramide (a-GalCer) induced significant protection, whereas Jalpha 18-/-and CD1d-/-mice lacking iNKT as well as dNKT cells suffered from more severe abscess formation. A lipopeptidophosphoglycan, which is present in large quantities on the surfcae of E. histolytica trophozoites (EhLPPG), was identified as a major amoeba antigen that activates NKT cells resulting in the production of IFN-g, but not of IL-4. Moreover, IFN-g production required the presentation of EhLPPG by CD1d and signaling through the TLR receptor cascade in combination with a simultaneous secretion of IL-12. Similar to a-GalCer application, treatment of mice with purified EhLPPG significantly reduced the severity of ALA in amoeba-infected mice.

Our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of E. histolytica-infected individuals do not develop amoebic disease. A few years ago, we have observed a significant expansion of circulating effector gamma delta T cells following cytomegalovirus (CMV) infection in kidney transplant recipients (KTR). These unconventional T cells display TCR dependent cytotoxicity against both CMV-infected cells and carcinoma cells. In the present study, an extensive phenotyping of gamma-delta T cells allowed us to demonstrate an over-expression of CD16 in CMV-infected individuals. CD16 is the FcgammaRIIIA, a Natural Killer cell marker usually absent on conventional T cells. We found that 71.9 ± 15.9 % of gamma-delta T cells from 21 CMV-infected KTR expressed CD16, when compared with only 19.8 ± 16.7% in 11 non CMV-infected KTR (p X 0.0005). Similarly, 51.9 ± 25.7 % of gamma-delta T cells from 13 CMV-seropositive blood donors expressed CD16 compared to 27.1 ± 25.1 % in 15 CMV-seronegative donors (p X 0.01). CD16+ gamma-delta T cell lines generated from CMV-infected individuals were able to produce IFN-g (a potent anti-viral cytokine) in a CD16-dependent manner when activated by CMV/IgG immune complexes. This production greatly increased in the presence of IL-12 and IFN-alpha, two cytokines highly produced during CMV-infection. The supernatants of gamma-delta T cells activated with agonist anti-CD16 mAb inhibited CMV replication in vitro and this effect was abrogated in the presence of a blocking anti-IFN-g antibody. CMV/IgG immune complexes were also able to induce the expression of the cytotoxicity marker CD107a on CD16+ gamma-delta T cell lines. CD16 is well-known to mediate antibody-dependant cellular cytotoxicity (ADCC), especially in Natural Killer cells. Accordingly, we demonstrated that CD16+ gamma delta T cell lines could make ADCC against the Daudi lymphoma cell line and the A431 skin carcinoma cell line pre-incubated either with rituximab (anti-CD20) or cetuximab (anti-EGFR), respectively. In contrast, no ADDC could be observed against CMV-infected fibroblasts pre-incubated with polyclonal anti-CMV IgG (Cytogam), probably because Cytogam weakly stained infected cells. These data reveal a new CD16-dependent anti-CMV function of gamma-delta T cells through recognition of immune complexes and secretion of IFNg. Moreover, they demonstrate that these cells are able to kill through ADCC lymphoma and skin carcinoma cells, two tumour types frequently encountered in KTR. Dendritic epidermal T cells are a prototypic population of intraepithelial gd T cells in the mouse skin. Found in the basal layer of epidermis and in close contact with Langerhan's cells and keratinocytes DETC facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd T cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-MHC restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. By using gd T cell knock-out mice Tcrd-/-, Ovalbumin transgenic K5mOva mice and a skin grafting model we aimed to elucidate the role of gd-DETC in adaptive immune responses associated with elimination of foreign antigen presented in the skin.We show that in the absence of gd T cells in the skin there is a decrease in rejection of Ovalbumin expressing skin grafts compared to wildtype mice. We show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. However frequency of primed host animals is reduced in Tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting DETC enhance cross presentation of classical MHC bound antigens in the skin. Considering the incapability of gd T cells to recognize peptide antigens in the context of MHC we plan to dissect the relationship between DETC and professional antigen presenting cells in the skin. Understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional APC function in skin by DETC and potentially other epithelia by intraepithelial gd T cells and can be useful in designing therapies to epithelial infections and malignancies.

We demonstrate a rapid and HMB-PP-dependent crosstalk between gd T cells and autologous monocytes that resulted in the production of inflammatory mediators including IL-6, IFN-g, TNF-a, OSM, CCL2, CXCL8, CXCL10, and TRAIL. Moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory DCs with antigen-presenting functions. These cells expressed CD40, CD86, HLA-DR, and DC-SIGN, and lost CD14, CCR2, CCR5, and CXCR4. Addition of further microbial stimuli (LPS, peptidoglycan) induced CCR7 and enabled these inflammatory DCs to trigger antigenspecific CD4 + effector ab T cells expressing IFN-g and/or IL-17. Importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from PD patients and translated directly to episodes of acute PD-associated bacterial peritonitis, where Vg9/Vd2 T cell numbers and soluble inflammatory mediators were elevated in patients infected with HMB-PP-producing pathogens. Conclusion: Our findings suggest a direct link between invading pathogens, microbe-responsive gd T cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. The mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of NKT cell manipulation remains unpredictable. There is growing evidence that the NKT cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of NKT cellmediated immunosuppression. We examined the differential ability of NKT cell subsets from the thymus and liver to prevent type I diabetes when transferred into prediabetic NOD mice. The transfer of abTCR+DN thymocytes (a population enriched for NKT cells) has previously provided robust protection against TID development; however it has not been formally shown that NKT cells are solely responsible for the protection. Our study found that while the transfer of thymic DN NKT cells can prevent TID and severe insulitis in NOD mice, not all NKT cell subsets show the same tolerogenic capabilities. These findings both formally demonstrate the disease-preventing effects of NKT cell transfer in NOD mice and provide further evidence that NKT cells are a functionally heterogeneous population. Objective: Vg9/Vd2 T cells constitute a minor T cell population in human blood that expands specifically and rapidly in response to the microbial metabolite HMB-PP. Our previous microarray studies showed that Vg9/Vd2 T cells stimulated with HMB-PP in the presence of IL-21 express markers associated with a possible follicular B cell helper function. We therefore investigated in more detail whether and how HMB-PP and IL-21 regulate expression of the B cell attracting chemokine CXCL13/BCA-1, its receptor CXCR5, and co-stimulatory molecules involved in B cell help.

Purified peripheral Vg9/Vd2 T cells were co-cultured with autologous monocytes or B cells (as feeder cells) for up to 4 days with and without HMB-PP, in the absence or presence of IL-2 or IL-21, or in medium alone. Cells were analysed by flow cytometry and immunofluorescence microscopy. Results: High levels of CXCL13 protein were detected in co-culture supernatants only when both IL-21 and HMB-PP were provided, implying an IL-21-dependent and TCR-dependent expression. Vg9/Vd2 T cells were confirmed as producers of CXCL13 by flow cytometry and immunofluorescence. Under the same conditions, activated Vg9/Vd2 T cells expressed CD27, CD28, CD40L, CD70, ICOS and OX40. In contrast, neither CXCR5 nor CCR7 changed markedly by IL-21 stimulation of peripheral Vg9/Vd2 T cells. Conclusion: Our findings confirm on the protein level that stimulation of Vg9/Vd2 T cells with HMB-PP and IL-21 induces markers typically associated with follicular B helper T (T FH ) cells. These data suggest that gd T cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. Ongoing analyses of gd T cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings.

Y. Emoto 1 , M. Emoto 1 1 Gunma University School of Health Sciences, Department of Laboratory Sciences, Maebashi, Japan Invariant (i) natural killer (NK)T cells become undetectable after stimulation with a-galactosylceramide (a-GalCer) or interleukin (IL)-12. Although downmodulation of surface T cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after a-GalCer stimulation, it is unclear whether this holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C or by block of de-novo synthesis of these molecules, and to examine the role of IL-12 in disappearance of iNKT cells after a-GalCer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mRNA expression of TCR/NKR-P1C by iNKT cells after stimulation with a-GalCer or IL-12, and influence of IL-12 neutralization on down-modulation of sTCR/sNKR-P1C expression by iNKT cells after a-GalCer stimulation were examined. The s/cTCR + s/cNKR-P1C + iNKT cells became undetectable after in-vivo administration of a-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C + iNKT cells became undetectable after in-vivo administration of IL-12, s/cTCR + iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by a-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. In contrast, cTCR + cNKR-P1C + sTCR -sNKR-P1C -iNKT cells and cNKR-P1C + sNKR-P1C -iNKT cells were detectable after in-vitro stimulation with a-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signaling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after a-GalCer stimulation by down-modulating not only sNKR-P1C but also sTCR. The fetus and infant are highly susceptible to viral infections. A number of viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared to later life. It is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd T cells are unconventional T cells that can react rapidly upon activation and show MHC-unrestricted activity. Herein, we show that upon CMV infection in utero, fetal gd T cells expand and become differentiated. The response was restricted to Vg9-gd T cells, irrespective of their Vd chain expression. Differentiated gd T cells expressed high levels of IFN-g, transcription factors T-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. In addition, congenital CMV-infection induced a highly restricted complementary-determining region 3 d1 (CDR3d1) and CDR3d2 repertoire, with a striking enrichment in a specific germline-encoded CDR3d1 sequence. Differentiated gd T cells and the enriched CDR3d1 sequence were detected as early as after 21 weeks of gestation. Our results indicate that functional fetal gd T cell responses can be generated during development in utero and suggest that this T cell subset could participate in anti-viral defense in early life.

Results: Spectratyping showed only in-frame selection for Vd1-Jd1 and VgI-Jg1.3/2.3 rearrangements in TCRgd thymocytes and to a lesser extent in TCRgd CB cells. In contrast, clear in-frame Vd2-Jd1 and Vg9-Jg1.2 selection was seen in PB TCRgd cells. Detailed analysis of the CDR3 motifs revealed selection determinants in both Vg9-Jg1.2 (canonical length and CDR3 motif) and Vd2-Jd1 (minimal CDR3 length in combination with an invariant T nucleotide) rearrangements. Upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve TCRgd thymocytes (˚4) and TCRgd CB cells (6-7) to TCRgd PB cells (˚10 or more). No increase was seen between CB and PB TCRgd T cells within the first year of life, suggesting that peripheral proliferation occurs later in life.

Our results indicate that the human peripheral TCRgd repertoire is shaped by (antigenic) selection and proliferation processes. Moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. Background: Natural Killer (NK) T cells have been implied in the regulation of disease in the non obese diabetic (NOD) mouse model of type 1 diabetes (T1D).

We have previously shown that transgenic expression of a CD1d-restricted, Va3.2-Vb9 TCR in NOD mice lead to an increase in CD1d-restricted type II NKT cells (24abNKT cells), and prevention of the development of T1D in the transgenic mice. In this study we have investigated the requirements and underlying mechanism of disease protection by type II NKT cells in a disease transfer model.

To investigate the mode of regulation by 24abNKT cells, we explored a disease transfer model into NOD.scid mice using transgenic diabetogenic BDC2.5 CD4+ T cells, in the presence or absence of selected cells from 24abNKT cell transgenic mice. Results: In 24ab transgenic mice a high frequency of activated transgenic NKT cells was found in the pancreas of the protected mice. In this organ, 24abNKT cells expressed a high level of CXCR3 and a low level of CCR7 and CD62L, a pattern similar to that observed in T cells homing to inflammatory tissues. Adoptive transfer of CD4+ BDC2.5 T cells into NOD.scid recipients rapidly induced onset of diabetes. Using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with BDC2.5 CD4+ cells resulted in the prevention of diabetes development. The protection from disease required a minor CD4+ subset of 24ab+ NKT cells, but was independent of CD25+ T regulatory cells. Analogs of alpha galactosylceramide (a-GalCer) that may modulate the strong activation of iNKT and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. A new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by CD1d and recognized by iNKT. The structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the O glycosidic linkeage with the ceramide by a N. In this basic structure, substitutions in the cyclohexane ring with OH in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the N linkeage conform a series of analogs that have been analyzed in their capacity to stimulate iNKT cells. Proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that iNKT cells are specifically stimulated by some of the analogs tested. In particular, the active compound HS44, induces in vitro iNKT cell expansion and IFNg and IL-4 secretion in a similar fashion but less potently than a-GalCer. Dose response assays show a bias towards a Th2 profile response after recognition by NKT cells, more similar to the response induced by OCH. The degree of structural similarity of the cyclitol ceramides with a-GalCer parallels their cellular activities. These data open the way towards the development of a new class of a-GalCer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential iNKT cell sphingolipid agonists that can modulate the immune response due NKT cell activation. Objectives: Invariant Natural Killer (iNK) T cells represent an innate lymphocyte subset with important modulatory functions. In the presence of pathogens or tumors, iNKT cells play an adjuvant function that boosts T cell immunity through cytokine secretion and DC maturation. In steady-state conditions, i.e. in the absence of pathogens, iNKT cells acquire a regulatory function that promotes T cell tolerance and prevents autoimmune disease. Our aim was to assess the mechanism of action of iNKT cells in the steady state and, specifically, to test the hypothesis that iNKT cells promote immune tolerance through modulation of DCs. Methods: To assess the direct influence of regulatory iNKT cells on DC maturation in resting conditions, we derived murine iNKT cell lines in vitro and, after staining with aGalCer-loaded CD1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid DCs in the absence of any other maturation signals. We analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of iNKT cell-modulated DCs (iNKT-DCs).

The cell-cell interaction with iNKT cells provoked dramatic phenotypical changes on immature DCs that acquired the cardinal features of tolerogenic DCs such as intermediate levels of MHC class II and co-stimulatory molecules expression and high secretion of IL-10 with no release of pro-inflammatory cytokines. Most importantly, iNKT-DCs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory Tr1 cells and immune tolerance in vivo. DCs, simultaneously stimulated with iNKT cells and through toll-like receptor (LPS) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. Conclusion: It is still mysterious how iNKT cells can play a dual role and either boost T cell immunity or promote immune tolerance. Our results suggest that the same mechanism could underlie both iNKT cell functions. In the presence of pathogen-driven maturation signals, the iNKT cell-modulation of DCs favors their acquisition of a pro-inflammatory phenotype and function. On the contrary, if iNKT cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature DCs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. Th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. The Th17 immune response is characterized by the secretion of IL-17A and IL-17F. The IL-17 locus encodes the highly conserved IL-17A and IL-17F cytokines that are syntenic in 44kB distance to each other. Besides CD4 + Th17 and NKT cells, approximately 50 % of the IL17A producers are gd T-cells. Like CD4 + Th17 cells, IL-17 producing gd Tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. Our findings show a difference between the IL-17 production of gd T cells in the peripheral system and mucosal epithelia. Mucosal gd T-cells generally do not produce Th17 cytokines. In the periphery, we define novel subsets of gd T-cells that can produce either IL-17 or IFN-g. Combined with the well known classification of IL-17 producing gd T-cells along the markers CD27 and CD122, our data point at specialized functions of the different gd T cell subsets depending on their location and origin. Functional studies are currently carried out in order to address the role of the different gd T-cell subsets for Th17-type immune responses in vivo. In this context, the potential redundancy of IL-17A and IL-17F may complicate the analysis. So far, most studies were carried out with IL-17A single-deficient or IL-17F single-deficient mice. To further clarify these issues, we will have to address the above mentioned findings in IL-17A and IL-17F double-deficient mice. Several subsets of gd Tregs have been described and intensively studied, but the potential regulatory role of innate T cells in controlling immune responses remains unclear. Lymphocytes expressing gd TCR are involved in both innate and adaptive immune responses. Vg9Vgd2 T cells, which represent a major peripheral blood gd T-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.Here we report that TGF-b1 and IL-15 differentiate in vitro a subset of gd T lymphocytes with regulatory functions (Vd2 Tregs) in the presence of specific antigen stimulation. These cells express the forkhead/winged helix transcription factor (FOXP3) and, similarly to ab Tregs, suppress the proliferation of anti-CD3/anti-CD28 stimulated-PBMC. Detailed knowledge about the phenotype and functionality of Vd2 Tregs will improve our understanding of the role of gd T cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-GalCer) has the potential to activate invariant (i) NKT cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. Although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-GalCer. Here we show that treatment with a-GalCer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following Listeria monocytogenes infection. Liver infiltration of granulocytes and g/d T cells was accelerated by a-GalCer treatment. Granulocyte and g/d T cell depletion exacerbated listeriosis in a-GalCer-treated mice, and this effect was more pronounced in granulocyte than in g/d T cell depletion. Although secretion of GM-CSF and IL-17 was detected among the NKT cell population in the liver and bone marrow immediately after a-GalCer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mAb. Yet, in parallel to the numerical increase of granulocytes expressing CD11b in the liver following a-GalCer treatment, numbers of cells lacking CD11b diminished in the bone marrow. In addition, respiratory burst in granulocytes was enhanced by a-GalCer treatment. Our results indicate that a-GalCer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d T cells, into the liver. We also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. Objectives: The aim of this work is to evaluate whether phenotypic and functional features of Vgamma9/Vdelta2 T cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in CLL. Methods: Eighty seven previously untreated CLL patients were evaluated for in vitro Vgamma9/Vdelta2 T cells expansion upon stimulation with Zoledronic acid (ZA) and interleukin-2 (IL-2). Gammadelta T cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. The mutational status of the tumor immunoglobulin heavy chain variable region (IgVH) was analyzed by DNA sequencing. The activity of the Mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of Mev pathway metabolites. Results: Proliferation of gammadelta T cells was observed in 43 patients (49 %) (responders, R), whereas 44 patients (51 %) were non-responders (NR). Vgamma9/Vdelta2 T-cell subset distribution was well balanced in R patients, whereas effectors subsets [i. e., effector memory (TEM), and terminally differentiated effector memory (TEMRA)] were largely predominant in NR patients. TEMRA of NR patients mainly expressed the inhibitory receptor ILT2, whereas TEMRA of R patients had an higher expression of the costimulatory molecule NKG2D. The proliferative response of Vgamma9/Vdelta2 T cells was significantly associated with IgVH mutational status, which is a well known prognostic factor in CLL. Indeed, 82 % of R patients were M, whereas 77 % of UM patients were NR (p X 0.001).

Given this association, we evaluated the activity of the Mev pathway in tumor cells of M and UM patients. The pathway was more active in tumor cells of UM than M patients, suggesting that the former can more easily engage gammadelta T cells and drive their differentiation into functionally exhausted T EMRA . Given the association between the R/NR status and the IgVH mutational status we also analyzed the independent prognostic impact of R/NR status in multivariate Cox analysis. NR patients had a significantly shorter time to first treatment thus pointing to R/NR status as an independent prognostic factor. Conclusion: These data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in CLL patients. The studies reported here were undertaken to ascertain and delineate the ability of Kupffer cells to regulate the response of iNKT cells to biliary obstruction. Methods: C57BL/6 mice were not treated or rendered Kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. To clarify the factors that elicit iNKT cell activity, additional mice were administered anti-IL-12 p40 (clone R2-10F6; ATCC) or anti-Cd-1d (clone 1B1) monoclonal antibody (mAb) prior to surgery. Midline laparotomies were performed; the common bile duct was ligated twice and divided. Sham-operated animals served as controls. Blood and liver samples were collected at periodic intervals post-surgery. The hepatic lymphoid population was purified and characterized by flow cytometry.

The NKT cell population was increased significantly in the livers of control, but not Kupffer cell-depleted, mice at 18 hours post-BDL. The response of iNKT cells was diminished in mice pretreated with mAb specific for IL-12p40, a component of both IL-12 and IL-23; pretreatment with anti-CD1d mAb had no effect. IL-12Rb-deficient mice also exhibited a marked increase in hepatic iNKT cells following BDL suggesting that IL-12 was not a critical factor. This suggestion is supported by the increased expression of IL-23p19 and IL-12p40 (but not IL-12p35) mRNAs by Kupffer cells purified from the livers of BDL animals. These findings imply that IL-23 production by Kupffer cells promotes the response of hepatic iNKT cells to biliary obstruction. Objectives: P-glycoprotein (Pgp or ABCB1) is a member of the ABC family of transporter proteins which are characterized by their ability to pump molecules across membranes in an ATP-dependent manner. Although Pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. Our work primarily focuses on gd T cells that complement and regulate the activities of ab T cells, particularly in tissues. We have recently described functional subsets of gd cells based on CD27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing IL-17. This study investigates the role of Pgp in gd cells with specific reference to these recently-identified CD27-defined subsets. Methods: Pgp activity was measured based on the expulsion of Rhodamine 123. Cells were incubated with Rho followed by a period in the presence or absence of the Pgp inhibitor Cyclosporine-A. Cell populations were identified using monoclonal antibodies and flow cytometry. Percentages of subpopulations were compared by ANOVA, statistical results are shown as P values that were calculated using a Newman-Keuls multiple comparison post-hoc test.

Results: Up to 40 % of intraepithelial lymphocytes (IELs) from the small intestine are TCRgd + . Of these, virtually all displayed Pgp activity. Indeed, Pgp activity was generally higher in TCRgd + than TCRab + IELs. In the thymus, Pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. By contrast, in peripheral lymph nodes, mesenteric lymph nodes and Peyer's Patches, 40-60 % of gd 27+ cells were positive for Pgp activity, although their gd 27counterparts remained largely negative (p X 0.01).

Conclusion: This study demonstrates that subsets of gd cells display different levels of Pgp activity depending on their location in the body and their expression of the newly identified functional marker CD27. As Pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. Objectives: An effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. In this context, the Vgamma9Vdelta2 T cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. The activation of Vgamma9Vdelta2 T cells by aminobisphosphonate drugs such as zoledronic acid (ZOL) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (DCs) and B lymphocytes. The aim of this work was to evaluate the ability of activated Vgamma9Vdelta2 T lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. Methods: Peripheral mononuclear cells (PBMC) and purified Vgamma9Vdelta2 T cells from healthy donors were stimulated with different compounds (ZOL, IPP) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. Briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and Elisa tests.

We showed that soluble factors released by ZOL-stimulated Vgamma9Vdelta2 T cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. Proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated Vgamma9Vdelta2 T cells. Moreover, MCP-2 depletion by neutralizing Ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. Altogether, these data show a Vgamma9Vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of Vgamma9Vdelta2 Tlymphocytes in orchestrating the immune response. Conclusion: An immune modulating strategy targeting Vgamma9Vdelta2 T cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. Objectives: The aim of this study was to analyse the activity of Vg9Vd2 T lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. Human Vg9Vd2 T cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. Interestingly, drugs already in clinical use, such as Zoledronic Acid, are able to promptly activate Vg9Vd2T cells through an indirect mechanism involving the block of Farnesyl Pyrophosphate synthase of the mevalonate cycle. The Vg9Vd2 T cell activation by Zoledronic Acid results cytokines and chemokines synthesis and cytotoxic activity. Glioma are tumors arising from glia in the central nervous system. Unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. Methods: In order to analyse the activation of Vg9Vd2 T cells and their effects on the viability of glioma cells, we expanded in vitro Vg9Vd2T cells from PBMCs of healthy donors by using phosphoantigen stimulation and tested the ability of Vg9Vd2T cell lines to kill three different glioma cell lines (T70, U251, U373) by cytokinic/cytotoxic mechanism by flow cytometry. Results: Our results demonstrated that Vg9Vd2T cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. Moreover, we analysed whether Zoledronic Acid treatment could improve the susceptibility of glioma cells to Vg9Vd2 T lines. We showed that Zoledronic Acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of Vg9Vd2 T lines. Conclusions: Altogether, our results suggest that the induction of a strong antitumor response in vitro of Vg9Vd2 T cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. Viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. Natural killer T (NKT) cells are a population of regulatory T lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. Using a mouse model of chronic viral infection in the skin, in which Human papillomavirus (HPV) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for NKT cells in abrogating protective immunity to viral antigens in cutaneous tissue. We show that local HPV-E7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of CD1d-restricted NKT cells. This NKT infiltrate is required to maintain local HPV-E7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. The local suppressive environment evident in E7-expressing transplanted skin is dependent on interactions between populations of CD1d-expressing CD11c+/F480+ myeloid cells and NKT cells. Removal of either donor-resident or host-infiltrating NKT cells is sufficient to break immune suppression and allow E7 graft rejection. Dissecting the suppressive properties of NKT cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of NKT cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. NKT cells were expanded from total PBMCs from healthy donors by treatment with IL-2 and a-GalCer. Expression of CD1a, CD1d and the costimulatory molecules CD86 and HLA-DR, was established by flow cytometry. RNA was quantified by Real Time-PCR. Functional assays were performed by analysis of NKTs cytokine production (IFN-g, IL-4) and cytotoxicity against treated-iDCs. Results: iDCs stimulated with olive pollen lipids up-regulated CD1d expression on the cell surface in comparison with control cells. In contrast CD1a expression was decreased. CD86 and HLA-DR slightly increased, indicating certain grade of maturation. The amount of CD1D mRNA was higher in treated cells than in control cells. By contrast, there was less transcription of CD1A, CD1B and CD1C genes than in control cells. NKT cells efficiently killed treated iDCs as "in vitro" cytotoxic killing assays showed. IFN-g producing cells increased slightly in response to treated iDCs compared to unstimulated cells, but the number of IL-4 producing cells was not modified. Similar results were obtained using monocytes as antigen-presenting cells.

Conclusions: iDCs treated with lipidic extracts from olive pollen up-regulate the expression of CD1d on the cell surface. In addition, NKT cells are able to recognize iDCs and monocytes treated with lipids from pollen, producing IFN-g and cytotoxicity. All these data suggest that NKT cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. Outline: In humans, 0.5-10 % of circulating lymphocytes express a Vg9Vd2 T cell receptor, yet strikingly little is known about the function and properties of such unconventional T cells. We performed cDNA microarrays to find Vg9-enriched genes compared to conventional MHC-restricted CD8 + ab T cells, and found reciprocal enrichment of Nectin-like adhesion molecules IGSF4 & CRTAM in gd and ab T cells respectively. Because IGSF4 binds to CRTAM, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. Interestingly, previous studies show that activated NK, NKT and CD8 + ab T cells express CRTAM, and that engagement of IGSF4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. Our data extends this to the prospect of cytolytic immunoregulatory interactions between T cells mediated by IGSF4/CRTAM. We therefore sought to answer: 1. What is the function of IGSF4/CRTAM on gd T cells? 2. How is the IGSF4-CRTAM axis regulated in T cells? Results and conclusions: Flow cytometry showed IGSF4 enrichment on resting gd T cells, with expression also detected on˚10 % of ab T cells. The properties of those cells are being examined. However, IGSF4 generally correlates with markers of activation/antigen experience such as CD45RO. Thus, IGSF4 cells may comprise activated-yet-resting/pseudo-memory unconventional T cells and memory-effector conventional T cells. Stimulating Vg9 + T cells in vitro led to rapid CRTAM induction, resulting in the majority of cells co-expressing both IGSF4 and CRTAM within 48 hours. However, engagement of IGSF4 by CRTAM or vice versa is not sufficient to induce cytotoxicity, as stable CHO cell transfectants expressing either molecules were not specifically lysed by PBMC in vitro, compared to efficient and parallel targeting of MICA + cells. Instead, our current experiments address the possibility that CRTAM-IGSF4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as MICA-NKG2D. This may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that CRTAM-IGSF4 interactions are sufficient for CD8 T cells to kill gd T cells and/or vice versa. Instead, CRTAM-IGSF4 interactions may set the threshold for cytotoxic immune-surveillance responses. T Cell Receptor (TCR) is a multisubunit complex in which the invariant subunit CD3z is a 16 KDa transmembrane protein indispensable for coupling antigen recognition by TCR to diverse signal transduction pathways. Approximately 3-6 % of human peripheral blood lymphocytes express the gd TCR and the majority of these cells express the Vd2 TCR variable segment associated with the Vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. These compounds trigger Vg9Vd2 T cells without antigen presentation. In vitro stimulated Vg9Vd2 T cells with antigens are able to produce IFN-g and TNF-a and exert a powerful cytotoxic activity against infected cells as HIV-infected cells. However, during HIV infection a marked decrease of Vg9Vd2 T cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. Aim of the present work was to study the mechanisms of Vg9Vd2 T cell anergy observed in HIV+ patients. To this aim, CD3z expression and IFN-g production by Vg9Vd2 T cells from HIV+ and HIV-subjects were analyzed. We show that Vg9Vd2 T cells from HIV-infected patients expressed lower level of CD3z compared with healthy donors. A direct correlation between CD3z expression and IFN-g production capability by Vg9Vd2 T cell was found. However, PKC activation by PMA is able to restore CD3z expression and IFN-g production. Our findings may contribute to clarify the molecular mechanisms of Vg9Vd2 T cell anergy found in HIV+ patients and have implication in the design of effective immune-based therapies.

L. Abeler-Dörner 1 , M. Swamy 1 , S.L. Clarke 1 , A. Hayday 1 1 King's College London, Immunobiology, London, United Kingdom Gut intraepithelial lymphocytes (IEL) constitute one of the largest T cell compartments in mice and in man. Their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance.

Intestinal IEL comprise many unconventional T cells including TCRgd cells and TCRab CD8aa or CD8 -CD4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. Unlike conventional T cells, IEL might initiate an immune response rather than simply being late effector cells. It is therefore important to elucidate the "immunological information flow" in the gut. To this end, this project characterizes different subsets of IEL and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo.

In the past, it has been notoriously difficult to study IEL ex vivo. To solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. The cells are initially activated by plate-coated aCD3 antibody and a cytokine cocktail and maintained further in medium containing low levels of IL-2. After a resting period, the cells can be restimulated in vitro. In this new system, we studied responses of different IEL subsets to stimulation via TCR, NKG2D and cytokine receptors, either alone or in coculture with epithelial cells. As readouts we monitored proliferation, cytokine secretion (IFNg, IL-6) and expression of activating and costimulatory molecules. Reactivation in response to various stimuli could already be observed after 6 hours. The in vitro data set forms the basis for analysing IEL responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. The characterization of IEL responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. Objective: Behcet's Disease (BD) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. Increased expression of CD94 in BD patients suggested that NK receptors may play a pathogenic or regulatory role in the pathogenesis. Considering the regulatory functions of NKG2 molecules in heterodimer with CD94, we screened the presence of these receptors on T cell subsets in BD.

The expression of NKG2 A/C/D molecules on gd and CD8+ T cells were analyzed in 17 active and 9 inactive patients with BD and 21 healthy controls. Expression of NKG2 molecules was evaluated on CD8+, gd T and CD56+ NK cells by using flow-cytometry. Results: gd T cells were increased in patients with BD compared to controls (4.4 vs. 2.8 %, p=0.001). In addition to the increase of gd T cells, increased expression of activating NKG2C molecules was also observed on gd T cells (20 % vs. 11 %, p= 0.01). NKG2A expression on gd T cells was found to be higher than NKG2C expression in patients and controls; but NKG2A expression on the T cells was not statistically different in both groups (33.5 vs. 40 %). NKG2D receptors were present on most of the gd T cells in both groups. However these activating molecules on CD8+ cells were decreased in patients with BD compared to controls ( Revlimid is a therapeutic agent used to treat myelodysplastic syndrome (MDS), a group of haematological disorders characterised by ineffective haematopoiesis. The mechanism of action for Revlimid is poorly understood, but there has been increasing interest in the strong association reported between MDS and defects within the immunoregulatory NKT cell compartment. Indeed, some studies now suggest an important outcome of Revlimid treatment is the restoration of normal cytokine production by NKT cell levels and an increase in their overall numbers. We have conducted the most thorough study to date of the NKT cell compartment of MDS patients treated with Revlimid/Ancestim and can report that MDS patients had normal NKT cell levels prior to treatment, and no significant increase as a result of Revlimid/Ancestim treatment. Furthermore, NKT cells from MDS patients produced high levels of Th1 and Th2 cytokines when stimulated with PMA/ ionomycin and the proportion of NKT cells capable of cytokine production did not increase significantly after Revlimid/Ancestim treatment. These are highly significant findings given the recent emphasis on NKT cells as a potential therapeutic target for MDS. Our study provides an extensive analysis of the impact of Revlimid/ Ancestim treatment on the NKT cell compartment and sheds new light on the role of NKT cells in MDS and the mechanism of Revlimid immunomodulation. Objectives: Human gd T cells are potent killers of a variety of tumour cell lines, and mice lacking gd T cells suffer from high incidence of experimentally-induced tumours. However, the molecular mechanisms mediating tumour cell recognition by gd T lymphocytes remain largely unknown. We aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd T cells for tumour cytolysis. As immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for Vg9/Vd2+ lymphocytes.

We have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using Vg9/Vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two Vg9/Vd2+ resistant ("non-target") cell lines (under 20 % death) for cDNA microarray analysis. We compared the differential expression in pairs of tumour cell lines of identical origin: the Burkitt's lymphoma cell lines Daudi (target) vs Raji (non-target), and the pre-B cell leukemia cell lines RCH-ACV (target) vs 697 (non-target), and validated the results by RT-qPCR quantification. Results: We identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ULBP1, IFITM1 and PRAME, for example, are up-regulated, whereas CD274 and CLEC2D are down-regulated in target cell lines. As these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. The expression of these candidate genes was studied by RT-qPCR in a broader panel of cell lines and primary biopsies. We are currently testing, in functional assays based on RNA interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd T cells.

The comparison between the transcriptomes of Vg9/Vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd T cells. Mice and humans are the only species in which phenotype and function of iNKT cells have been properly described. Our aims are to directly identify this cell population and to investigate CD1d, in the rat. Mice and rats have very similar CD1d and iNKT TCR genes, with the exception of the VA14 gene segment, which is a multimember gene family in the rat. Novel monoclonal antibodies with nearly identical binding capacities to mouse and rat CD1d revealed a very similar pattern of CD1d distribution, and could inhibit cytokine production after aGalCer stimulation of primary cells in both species. Response to aGalCer was studied in five different rat strains, showing big inter strain differences. Notably, IFN-g and IL-4 production was 10-100 fold lower in the best responder rat strain (F344) compared to mouse (C57/BL6). Since NKRP1A (rat homologue of mouse NK1.1) and TCR are not appropriate markers for rat iNKT, CD1d oligomers where tested for binding to iNKT-TCR transduced cells. Newly generated aGalCer loaded rat CD1d dimers, recognized rat iNKT TCR and, although less efficiently, bound to mouse iNKT TCR. However, mouse CD1d aGalCer dimers did not bind to rat iNKT TCR. aGalCer loaded rat CD1d dimers were then used to stain primary intrahepatic lymphocytes. But, although mouse iNKT cells were stained to some extent, the identification of a discrete population in the rat was not possible. The reasons behind could be: that the avidity of the dimers for the TCR is not high enough to stain primary cells and/or that the frequencies are so low that the detection by FACS analysis is difficult. In order to clarify these issues we currently produce and test rat CD1d tetramers. Burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. This disease is endemic in tropical regions, especially Thailand and Northern Australia, and has a serious outcome for many infected individuals. B. pseudomallei is an intracellular bacterium and many B. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. In addition to this, no vaccine is currently available to prevent the disease. Human g9d2 T cells are involved in the immune response to infection with a number of intracellular pathogens including Brucella suis and Mycobacterium tuberculosis. g9d2 T cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. Phosphoantigens cause expansion and activation of g9d2 T cells during infection with intracellular pathogens including Fransicella tularensis and M. tuberculosis. Analogues of natural phosphoantigens have been developed to manipulate g9d2 T cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of Hepatitis C virus. We aimed to determine in vitro whether enhancing gd T cell responses in human blood using the synthetic phosphoantigen Picostim could reduce growth of intracellular B. pseudomallei in the human monocytic cell line THP-1. A significant (p X 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of PBMCs cultured with Picostim+IL-2 in comparison with PBMCs cultured with IL-2 or media alone. Picostim+IL-2 caused significant expansion and activation of gd T cells following culture of PBMCs for 10-14 days. Purified gd T cells stimulated with Picostim were able to reduce intracellular B. pseudomallei numbers 100-fold. This data demonstrates that PBMCs, stimulated with the synthetic phosphoantigen Picostim+IL-2, reduced growth of intracellular B. pseudomallei in a gd T cell-dependent manner. Objectives: Vgamma9/Vdelta2 (gd) T cells play a major role in innate immunity against microbes, stressed and tumor cells. They represent less than 5 % of peripheral blood lymphocytes (PBL), but can be expanded in vitro by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC).The purposes of this study are: 1) to determine whether DC generated from multiple myeloma (MM) patients are as effective as their normal counterparts in the ability to activate gd T cells; 2) to evaluate whether gd T cells can exert immunoadjuvant activity on DC generated from MM patients and primed with tumor-specific antigens (survivin-SV); 3) to establish whether the same issues could be solved using a simplified protocol of DC generation.

1) DC were generated from CD14 + cells of healthy donors/MM patients; immatureDC on day 6 were induced to fully mature by incubation for 18 hours with TNFa + IL-1b + PGE2 in the presence or absence of 5 mM ZA. After 7 days of co-culture DC:PBL, percentages and total counts of gd T cells were determined by flow cytometry; 2) iDC generated from CD14 + cells of HLA-A*0201 + healthy donors/patients were pulsed with SV-peptide and stimulated for 18 hours with TNFa + IL-1b + PGE2 in the presence or absence of 5 mM ZA; after 2 rounds of autologous T cells stimulation by DC, the frequency of SV-specific CD8 + T cells was determined by SVpentamers staining; 3) the same experiments were performed both with DC generated following a standard protocol and a 48h protocol (DC Fast Objective: Depletion of or deficiency in gd T cells aggravate colitis in different animal models. Additionally, reconstitution of mice with syngeneic gd T cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd T cells in intestinal inflammation. Therefore, we asked whether human gd T cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or Crohn's disease. Hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd T cells were determined in vitro. Methods: Human gd T cells were isolated from whole blood of healthy donors by MACS technology. The proliferation was determined by [6-3 H]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via CFSE fluorescence intensity. The cytokine profile was determined by ELISA from culture supernatants as well as by flow cytometry intracellularly. Finally, the in vitro characteristics of gd T cells were compared to those of CD4 + CD25 + regulatory T cells (Treg).

Human peripheral gd T cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. While the proliferation of gd T cells and Treg cells is comparable, the suppression of gd T cells on responder cell proliferation is even stronger than the suppression by Treg cells though gd T cells being Foxp3 negative. Additionally, gd T cells are strong producers for TGF-b, particularly by the Vd1 subset. Conclusion: Human peripheral gd T cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. Their suppressive activity is even superior to Treg cells and might be due to strong TGF-b secretion. For application of human gd T cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. There are previous descriptions of gamma-delta T lymphocytes (GD) from Behçet's disease patients (BD) but, in most of cases, they are incomplete or contradictory. It has been suggested that NKG2D on GD is involved in BD lesions through interaction with MICA molecules. Furthermore GDCD8+ have been recently proposed as a new regulatory T subset (Treg).

Objectives: To study GD phenotype in BD active (BDA) (n=8) and inactive (BDNA) (n=20), versus healthy controls (HC) (n=30) and patients with recurrent oral ulcerations (RU) (n=14). To determine GD cytokine profile and surface markers Treg-related in BD (n=9) and HC (n=11). Methods: We obtained mononuclear cells from peripheral blood (PBMC). We determined by flow cytometry: -Surface expression of: GD TCR, Vdelta1, Vdelta2, CD8alpha, CD8beta, NKG2D, NKG2A and CD103.

-Intracellular expression of CTLA-4, and Foxp3.

-Intracellular expression of IL-2, IL-4, IFNgamma, IL-10 and TGFbeta after PBMC polyclonal stimulation. We used two tailed test for means comparison (Mann-Whitney U or Student's t test).

-Vdelta2+ cells were significantly increased in RU. Vdelta1+ and GDCD8+ lymphocytes were significantly increased in BD versus RU and HC.

-The mean fluorescence intensity of NKG2D was slightly increased in GD from BDA. -NKG2A expression by GDCD8+ was not different in BD versus HC.

-Most of GDCD8+ presented CD8alpha-alpha homodimers in BD and HC and were negative for CD103, Foxp3 and CTLA-4. GDCD8+ and GDCD8-subsets were (in BD and HC): -High IFNgamma-producers without differences.

-Low IL-2-producers: IL2+ cells were lower in GDCD8+ than in GDCD8-.

-Low IL-10-producers: IL10+ cells were lower in GDCD8+ than in GDCD8-.

-Low TGFbeta-producers: TGFbeta+ cells were lower in GDCD8+ than in GDCD8--Very low producers of IL4 in most of cases.

The hallmark in BD was the increase of GDCD8/Vdelta1+. This subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to NKG2A in intraepithelial intestinal lymphocytes from celiac patients. We did not find a cytokine profile or a phenotype T-reg-related for GDCD8+, except a lower percentage of IL-2+ cells than in the GDCD8-subset. GDCD8+ from BD did not show significant differences versus HC. Natural killer T (NKT) cells comprise a highly heterogeneous subset of T lymphocytes that co-express a T cell receptor (TCR) and NK cells markers such as CD56 in humans. A subgroup, the invariant NKT cells (iNKT), expresses the Va24Vb11 TCR rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. Objectives: To establish a method to growth cord blood-derived NKT cells (CD3 + CD56 + ), in order to evaluate their phenotypic characteristics and the TCRVb repertoire. Methods: Mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with IFN-g (50 ng/ml), anti-CD3 (25 ng/ml) and IL-2 (500 UI/ml). These cells were cultured for 21 days and the expanded CD3 + CD56 + cells were isolated by immunomagnetic methods. Surface markers were determined by flow cytometry. Total RNA was extracted from the purified CD3 + C56 + cell suspension using Trizol ® reagent and mRNA expression of twenty TCRVb gene families was measured by semiquantitative RT-PCR. Statistical analyses were performed using Mann-Whitney U test and one-way ANOVA, a P value of X 0,05 was considered significant. Results: We could significantly expand cord blood CD3 + CD56 + NKT cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). Table 1 shows the percentage (mean±SD,n=10) of phenotypic markers in CD3 + CD56 + cells at baseline (day 0) and after 21 days of culture. Expression of mRNA for the Vb families studied was confirmed in each individual cell culture with a significant high expression of Vb4 and Vb8 families (p X 0,001). Conclusion: Our results show that cord blood-derived NKT cells are mainly CD8 + and CD94 + subsets, similar to peripheral blood NKT cell with a low percent of iNKT cells. Additionally, we confirm a diverse TCR Vb repertoire with a significant expression of the Vb4 and Vb8 families in these cells.

L. Marischen 1 , D. Wesch 1 , P. Rosenstiel 2 , A. Till 2 , D. Kabelitz 1 1 Institute of Immunology, Kiel, Germany, 2 Institute of Clinical Molecular Biology, Kiel, Germany gd T cells account for a minority of T cells in human blood, but represent the majority of intraepithelial T cells in the intestinal tract. Due to their ability to respond rapidly and in an MHC-independent fashion to particular antigens by cytokine production, gd T cells are considered as a link between innate and adaptive immunity. In addition, the expression of distinct pattern recognition receptors such as Toll-like (TLR) and Nod-like receptors (NLR) are characteristic for cells of the innate immune response. Recent reports have demonstrated the TLR expression in human and murine gd T cells. Here we provide evidence also for a gd T cell responsiveness to muramyl dipeptide (MDP), the putative ligand of the NLR family member NOD2. Peripheral blood mononuclear cells (PBMCs) containing gd T cells as well as freshly isolated gd T cells were stimulated via the gd T cell receptor in the absence or presence of MDP and analyzed for proliferation and IFNg-production. While the proliferation of gd T cells within PBMCs was decreased, IFNg-production was increased after costimulation with MDP compared to the stimulation with a non-activating DD-stereoisomer of the ligand (MDPi). The enhanced IFNg production of PBMCs after costimulation was mediated mainly by gd T cells as shown by intracellular flow cytometric staining. With regard to the IFNg-production after co-stimulation with MDP vs. MDPi, freshly isolated gd T cells from different healthy blood donors can be divided into responder and non-responder. Responder gd T cells showed a significant increase of the IFNg-production due to MDP-stimulation, whereas IFNg-production was not influenced in non-responder gd T cells. In further experiments, as first approach to explain the different reactivity patterns of gd T cells, it is planned to analyze the polymorphisms of the NOD2 gene in various donors. Taken together, our preliminary data indicate that gd T cells are a major source of IFNg-producing cells among PBMCs when challenged with specific antigens plus MDP, and support the role of gd T-cells as an important team player in the early immune response against bacteria. Objectives & methods: An increasing of gamma-delta T cells during acute P. vivax infection and convalescent period has been reported. Moreover, the activation of gamma-delta T cells leads to the inhibition of blood stage P. falciparum parasites in vitro. To determine the killing mechanisms of P. vivax parasites by gammadelta T cells comparing with what has been found in P. falciparum, the gamma-delta T cells were enriched by Isopentenylpyrophosphate (IPP) from naïve PBMC. Different number of gamma-delta T cells and normal PBMC were incubated with intact of P. vivax parasites and protein extract of P. vivax parasites, recombinant PvMSP1 19 and PvAMA1 proteins. Gamma-delta T cells was daily determined the cytokine and granzyme intracellular releasing by Flow cytometry until day 5 culturing.

Results: Among the enriched gamma-delta T cells, the percentage of cells expressing CD69 + and CD25 + was elevated after co-culturing with intact and the proteins of P. vivax parasites. The overall gamma-delta T cells showed proliferation at day 3 after the co-cultivation. Moreover, the gamma-delta T cells expressing IFNgamma + and CD107a + (lysosomal associated membrane proteins: LAMP-1) elevated from the first day of PBMC collection after co-culturing with the intact and P. vivax antigens. This level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition.

Our results showed the activation of gamma-delta T cells during P. vivax infection in vitro. This suggests that gamma-delta T cells could be stimulated by P. vivax parasites and these actively activated gamma-delta T cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. This study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. Objectives: Several evidence suggest that invariant NKT cells (iNKT) connect innate and acquired immune system. They are able to produce both Th1 and Th2 cytokines after stimulation. Atopic dermatitis (AD) is a chronic inflammatory skin disease. Th1-like and Th2-like cytokines have been implicated in the pathogenesis of AD, but there are controversial data on their role in AD.

The frequency and absolute number of iNKT cells in mononuclear cells (PBMCs) of peripheral blood of patients with atopic dermatitis (AD) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-CD3 and monoclonal antibody specific for the CDR3 loop of the invariant TCR a chain of iNKT cells (clone:6B11). Furthermore, after PMA/ionomycin stimulation for 4 hours, intracellular IFNg and IL-4 cytokines were detected in CD4+CD8-, CD4-CD8-(DN), CD4-CD8+ and CD4+CD8+ subsets of iNKT cells by five colour flow cytometry in patients with AD (n=10) and healthy controls (n=10). Results: Both frequency and absolute number of iNKT cells were significantly lower in patients with AD (P X 0.01) compared to healthy controls. The frequency of DN subpopulation was significantly lower in AD patient (P X 0.01). There was a positive correlation between the frequency of DN cells and iNKT cells both in AD patients (R=0.726 and P X 0.001) and healthy controls (R=0.693 and P X 0.001). In the intracellular IFNg level there were no significant difference in any of the iNKT subsets of AD patients, however the intracellular IL-4 level was significantly higher in DN subpopulation of iNKT cells of AD patients compared to healthy controls (P X 0.05).

The frequency, the number of iNKT cells and the cytokine producing capacity of the CD4/CD8 iNKT subsets are different in peripheral blood obtained from AD patients compared to healthy controls. Our result suggest that the DN iNKT cell subset can serve as a source of IL-4 that promotes the Th2 differentiation in AD patients and might play a role in the pathogenesis of this disease. Introduction: Intrahepatic immune cells (IHIC) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. Aims: In the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of IHIC. These cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and DNase. Results: The mechanical disruption yielded viable IHIC in considerably greater numbers than those obtained following enzymatic digestion. The IHIC isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which B, T, natural killer (NK), NK T cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). The IHIC obtained following enzymatic digestion contained markedly lower numbers of NK T cells (1.8 %) . The B, T and NK T cells among IHIC isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin A and alpha-galactosylceramide respectively) and produced immunoglobulin M and interferon-gamma. Conclusions: Thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent IHIC for various types of investigation. Nature Killer T cells (NKT) are a special T cell population with co-expresses NK and T cell surface markers. Murine NKT cells include CD4 + NKT and CD4 -CD8 -NKT cells. NK1.1 + NKT cells may release large amounts of IL-2, IL-4, IFN-g and IL-10 after they are activated. It has been reported that a-Galactorsykeramide (a-Galcer), a glycolipid, may induce proliferation of NKT cells with the role of immune regulation by stimulating mouse spleen cells. This study demonstrated that superantigen staphylococcal enterotoxin B (SEB) , a kind of peptide, can activate the NKT cells with the function of immune tolerance. The response ability of SEB-activating effect cells to ConA, LPS and IL-2 had significantly decreased compared with that of normal lymphocytes. The effect cells exerted an inhibitory effect for the response of normal lymphocytes to ConA and IL-2. There was a significantly increase in the percent of CD8 + NK1.1 + and TcRVb8 + NK1.1 + NKT cells identified from the SEB-activated cells. Based on the cell distribution detected in the upper part of the FACS picture, expression of CD69 molecule existed in 68.95 % of the cells from large-scale selection. The percent of CD8 + NK1.1 + and TcRVb8 + NK1.1 + NKT cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. Under a light microscope at x400 magnification, the SEB-activating lymphocytes in size were larger than not only the ConA-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. There were a few granules seen in cytoplasm. The value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. The differentiation pathway of the SEB-activating CD8 + and TcRVb8 + NKT cells was not relative to a NK source. They were produced directly from T cell population and were considered as a subsets of T lymphocytes. Our results suggest that the superantigen SEB can act on the CD8 + NKT cell and TcRVb8 + NKT cells. And the two NKT cell subsets may play a critical role in SEB mediated tolerance. gd T cells in the intestinal intraepithelial compartment (gd iIEL) show an intrinsic activated phenotype. We hypothesised that their T cell receptor gd (TCRgd) is implicated in the activation of gd iIEL. Because the TCR gd ligands in mice are not well described, monoclonal antibodies (mAb) directed against the gd TCR, like the clone GL3 which binds the d subunit of TCR gd, are important tools to specifically activate gd T cells. Using cytometric Indo-1AM measurement, we could detect calcium flux of intestinal and peripheral gd T cells from TCRd-H2BeGFP reporter mice. Stimulation with anti-gd clone GL3 or anti-CD3 clone 2C11 elicited activation of gd T cells suggesting that TCR gd and CD3 molecules in gd T cells are functional and signalling competent.

Next, using ELISA and Cytometric Bead Array, we found that iIEL stimulated with plate bound GL3 in vitro produced CCL4, IFNg and TNFa. Therefore, we were interested whether the CCL4 production of gd iIEL influenced the homing of CCR5 cells such as lamina propria (LP) CD4 + FoxP3 + cells (Tregs). To test this, WT mice were i. p. treated with GL3 mAb and LP Tregs were analysed by cytometry at various time points post inoculation. We found similar frequencies of LP Tregs population but a slight decrease in CCR5 + Tregs. However, when we compared WT and Tcrd -/mice, we found both lower percentages of total LP Tregs and of LP CCR5 + Tregs in Tcrd -/mice compared to WT mice.

In conclusion, our data suggest that intraepithelial activation of gd T cell may directly or indirectly induce changes in the iIEL and lamina propria (LP) lymphocyte compartment and influence the CCR5 expression and the homeostasis of LP Treg. The ability of NKT cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different NKT cell subsets. Diversity in cytokine production by NKT cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation.

Here, we investigated cytokine production by murine NKT cell subsets in vivo under conditions where minimal manipulation of the cells was required. To this end, we examined IL-4 production in G4 reporter strains in which DNA encoding green fluorescent protein (GFP) was inserted into the first exon of the IL-4 gene. In the absence of any manipulation GFP was expressed from the Il-4 locus in populations of immature thymic NKT cells (predominantly CD4+CD44loTCRhi cells on a BALB/c background, and CD4+CD44loNK1.1-on a C57BL/6 background) and some splenic NKT cells, with overall numbers of GFP+ cells in both tissues decreasing with age. After i. v. administration of the NKT cell ligand a-galactosylceramide, IL-4 production was induced predominantly in CD4+ NKT cell subsets of the liver and spleen, and after i. n. administration, in CD4+ NKT cells of the airways. Spontaneous and a-GalCer-induced expression from the Il-4 locus occurred in the absence of STAT6 signalling, and did not require initial exposure to IL-4 protein from other sources in the host. Diversification in cytokine expression by NKT cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. Interleukin-17 (IL-17) plays an important role in neutrophil recruitment. Herein, we investigated the role of IL-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (CLP). Methods: Adult C57BL/6 (WT) and IL-17 receptor gene-deficient (IL-17R KO) mice were subjected to non severe (NS-CLP) sepsis. Intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. The ability of IL-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. The means of different treatments were compared by analysis of variance (ANOVA), followed by Bonferroni's t test and the survival rate by the Mantel-Cox log rank test. Results: It was observed that IL-17R KO mice, subjected to NS-CLP sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to WT. As a consequence, the mice showed an increased mortality rate. Moreover, IL-17 induced neutrophil migration in vivo and in vitro. Besides, we demonstrated that neutrophils harvested from IL-17R KO mice already show reduced microbiocidal activity, compared with WT, suggesting a physiological role of IL-17receptor signaling in the microbiocidal activity of neutrophils. Furthermore, WT neutrophils treated with IL-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. Conclusion: During NS-CLP besides the importance in recruit neutrophils to focus of infection, IL-17 also enhances the microbiocidal activity of neutrophils. Therefore, our results demonstrated that IL-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. Objectives: Members of the Toll-interleukin-1 receptor (TIR) family are important for host defense, inflammation, and immune regulation. Their canonical signaling pathway involves adaptor proteins and IL-1R associated kinases to activate NFxB and p38 mitogen-activated protein kinase. The IL-33-induced signal transduction in mast cells is poorly understood. In this work we studied the signal transduction of IL-33 in different mast cell subsets. Methods: Different mast cells subsets (HMC-1, human CBMCs and murine BMMCs) were stimulated with IL-33. The resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-Kit, pErk1/2, pAKT, pNFxB, p38 and pJNK). Additionally, we studied the signal transduction of IL-33 in IL-33R transfected HEK293T cells. Results: We found, that a TIR family member, IL-33R, transactivates the receptor tyrosine kinase c-Kit in mast cells and that IL-33-induced cytokine production depends on c-Kit transactivation. IL-33R and IL-1R accessory protein (IL-1RAcP) form a physical complex with c-Kit. Thereby the complexation is dependent on the activity of c-Kit. Conclusion: These results show for the first time that the biological function of an IL-1R family member is dependent on the presence of an activated receptor tyrosine kinase. Furthermore, these results reveal that certain IL-33-induced signaling pathways and effector functions are dependent on activated c-Kit and could therefore explain the effects of IL-33 in mast cells in absence of IgeR activation. (1) . We now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo TNFmediated programmed cell death. Independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to TNF, presumably due to the participation in the Fenton reaction of the Fe atom present in the protoporphyrin IX ring. Once exposed in vitro to free heme, a sustained c-jun N-terminal kinase (JNK) activation was observed in hepatocytes in response to TNF, an effect that promotes further free radicals production. Pharmacologic or genetic (shRNA) inhibition of JNK in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants N-acetylcystein (NAC) or Butylated hydroxyanisole (BHA). Expression of the heme catabolyzing enzyme heme oxygnease-1 (HO-1) in hepatocytes affords protection against heme sensitization to TNF cytotoxicity. Recombinant adenovirus mediated HO-1 expression in the liver suppresses TNFmediated hepatocyte apoptosis and prevents the lethal outcome of Plasmodium infection in mice. In conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo TNF-mediated cytotoxic effect, critically involved in the outcome Plasmodium infection. The multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. Responsiveness to chemotactic ligands is mediated by G protein-coupled receptors (GPCRs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. Recent evidence indicates that, in addition to playing a regulatory role in GPCR desensitization and internalization, beta-arrestins may contribute to GPCR signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. On this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the Rap1 small GTPase, recently emerged as pivotal regulator of integrin function. The analysis of KC the (Keratinocyte-derived Chemokine) Rap1 activation profile in RBL (Rat Basophilic Leukemia) cells expressing mCXCR2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. RNA interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of Rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in Rap1 activation and that the oscillations in the formation of Rap1-GTP are regulated by different molecular mechanisms. In order to elucidate the GEFs and GAPs involved in the GTPase regulation we are at present down-regulating the expression of C3G (Rap1GEF) and Spa1 (Rap1GAP): preliminary results suggest that Spa-1 has probably a role in the early activation peak. Since this oscillatory chemokine-induced Rap1 activation is present on other myeloid cell lines (HL60, 32D) and fresh PMN's we are also translating our research to these more appropriated cells. Interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. In order to find SH3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different SH3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. The cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. Aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. As a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. Here we present novel insights into the role of the Ig-like domain of the GM-CSFRa in GM-CSF binding. Deletion of the Ig-like domain abolished direct GM-CSF binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. The results indicate a previously unrecognized role for the Ig-like domain of GM-CSFRa. Furthermore, we address a longstanding controversy in the field of GM-CSF, IL-3 and IL-5 receptor biology, by performing a systematic study of the role of N-glycosylation upon on the bc, and related murine b IL-3 , in ligand-binding and receptor activation. These data demonstrate definitively that N-glycosylation does not play a role in mediating ligand-binding or receptor activation. These findings clearly establish that the determined human bc structures lacking glycosylation at Asn 328 are biologically relevant conformers of the human bc ectodomain.

Our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the GM-CSF: GM-CSFRa: hbc receptor complex. Reference: (1) MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules acting as key regulators of immune cell differentiation and innate immune responses. miRNA-146 expression is induced by activation of the Toll-like/interleukin-1 receptor pathway (TIRpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. Since TNFa also up-regulates the expression of miRNA-146a, we decided to explore whether this miRNA is involved in the regulation of apoptosis. To this end, we used the HeLa human epithelial cell line as a model system for TNFa signaling. Following TNFa and cycloheximide (CHX) treatment miRNA-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (CASP8/3). In line with this, miRNA-146a conferred enhanced protection against TNFa-induced DNA fragmentation and mitochondrial potential drop-down. Our results demonstrate that miRNA-146a is a regulator of receptor-mediated apoptosis. Similar to the TIR-pathway, miRNA-146a seems to be part of a negative feedback mechanism of the TNFa signaling cascade. Ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by miRNA-146a. Furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. Objectives: The aim of this study was to evaluate the role of single nucleotide polymorphisms (SNPs) located in IL-15, IL-7R a-chain and IL-15R a -chain genes in HIV disease progression. Methods: We studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (LTNP). We analyzed 2 SNPs in the IL-15 gene, 5 SNPs in the IL-7R gene and 3 SNPs in the IL-15R gene.

In univariate analysis, we found an association between the presence of at least one mutated A allele in IL-15R aa 182 and a higher possibility of being LTNP ( 

Our study suggests that genetic polymorphisms located in IL-7R and IL-15R genes can influence the rate of disease progression in HIV+ patients, especially when a combination of aplotypes is present. Mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of CD4 and CD8 T cells homeostasis. Aims: Mono-ADP-ribosyltransferases (ARTs) are GPI-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an ADPribose moiety from extracellular NAD+ to specific arginine residues of target proteins. In this study, we report that human tumor necrosis factor (TNF) is ADPribosylated by ART1, and that ADP-ribosylation affects both the release of TNF from cells and its cytolytic action. Methods: Transcription of ART1 in human leukocytes was analyzed by RT-PCR. ADP-ribosylation of TNF was detected by monitoring the incorporation of ADPribose from labeled NAD. Release of TNF from transfected HEK cells was monitored by ELISA. Binding of TNF to TNF receptors was analyzed by Biacore. TNF cytotoxicity was monitored by flow cytometry. The ADP-ribosylation site on TNF was analyzed by LC/MS mass spectrometry. Results: We identified ART1 transcripts by RT-PCR analysis in human blood leukocytes. Soluble ART1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase C (PI-PLC), ADP-ribosylated recombinant human TNF in vitro. Co-transfection of HEK293 cells with ART1 and TNF resulted in modification of TNF at at least 2 distinct sites, i. e. one within the TNF ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by TNFa Converting Enzyme (TACE). Analysis of modified recombinant TNF by mass spectrometry provided evidence that the TNF ectodomain is ADP-ribosylated at R32, a site that has previously been implicated in binding to TNFR2. Binding assays indicated that ADP-ribosylation inhibited binding of TNF to its receptors. Importantly, modified TNF was less potent at inducing cell death in the human T cell lymphoma line KIT225 than wildtype TNF. Furthermore, cell surface ADP-ribosylation of HEK cells co-transfected with TNF and ART1 resulted in reduced release of TNF into the supernatant. Conclusions: ADP-ribosylation of TNF or other cell surface proteins interferes with the biology of TNF signals by at least two distinct mechanisms. ADPribosylation of TNF blocks binding to its receptors, thereby inhibiting TNF-mediated cytotoxicity. Additionally, ADP-ribosylation of TNF or another protein on the surface of TNF-producing cells inhibits the proteolytic release of TNF.

NONINFLAMMATORY CHRONIC PELVIC PAIN SYNDROME : IMMUNOLOGICAL STUDY IN EJACULATE G. N. Drannik 1 , T.V.Poroshina 1 1 Institut of Urology AMSci of Ukraine, Laboratory Immunology, Kyiv, Ukraine

Chronic prostatitis (CP) is a disease which likely is associated with abnormalities in local immune responses. Secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. The aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. Methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and 11 control men were assessed for SLPI, TNF-alpha, IL-8 and free TGF-b1 in ejaculate by ELISAs using STAT-FAX 303 Plus. A 3 to 5 day sexual abstinence period was required from the subjects before semen collection. After liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees C°until assayed. The materials were processed after the standard programmes for statistical analysis.The study was approved by the local ethics committee.

The SLPI concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p X 0.001) in seminal fluid, in comparison with the healthy control subjects.

The TNF-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p X 0.05). The IL-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p X 0.05) in seminal fluid. Free TGF-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory CP/CPPS TGF-b1 concentrations were 36.2 ± 6.2 pg/ml. Conclusion: Ejaculate's SLPI, TNF-alpha, IL-8 and free TGF-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome.

M. R. Marrakchi 1 , E. A. Elgaaeid 1 1 Faculté des Sciences de Tunis, Biology, Tunisie, Tunisia

Ulcerative colitis (UC) and Crohn's disease (CD), collectively referred to the inflammatory bowel disease (IBD), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. The excessive production of either Th1 or Th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of IBD. Studies have identified a gene that encodes the NOD2/CARD15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to CD. Indead, it has been suggested that high rates of ASCA (Saccharomyces cerevisiae ) in absence of pANCA (perinuclear ANCA: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of CD and that the important rise of pANCA was more frequent at UC . In a sample of Tunisian patients, we examined the contribution of NOD2/CARD15 gene in CD. We performed a cases /controls study upon 75 CD patients and 90 healthy controls. This study suggests that in Northen Tunisian population, 3020insC mutation in NOD2/CARD15 gene is a prevalent mutation leading to the typical Crohn's disease including ileal location, stricturing and penetrating clinical types and ASCA expression. Since conflicting results were obtained on IL-10 polymorphisms as risk factor for IBD, the aim of our study was also to explore anti-inflammatory IL-10 cytokine genetic profile in patients with IBD. We examined the contribution of IL-10 gene promoter polymorphisms (-627 and -1117) to Crohn's disease (CD) phenotype, and the possible genetic epistasis between these polymorphisms and CARD15/NOD2 gene mutations in CD presentation and location. In Tunisian population, the 3020insC insertion in NOD2/CARD15 gene is a marker of susceptibility to CD, while the A allele at position -627 in the IL-10 promoter increases the risk of CD ileal location and severe disease presentation. A genetic epistasis between IL-10 gene polymorphisms and CARD15/NOD2 gene mutation was suggested.

In conclusion, genetic and serologic markers might be useful in defining patien Gangliosides were shown to inhibit the IL-2-dependent proliferation of T-cells, implying that gangliosides interfere with one or more of the IL-2-driven events. It is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of IL-2 molecule by ganglioside. But gangliosides apparently can also form complexes with IL-2R; such complexes influence on the signal transduction through IL-2R. This effect of gangliosides may lead to the failure of this pathway. Unfortunately, the biological and structural aspects of this problem are poorly understood. In this study we propose possible modes of interactions between exogenous gangliosides and IL-2R subunits. In our work we use IL-2-dependent cytotoxic T-cell murine line CTLL-2. Two different approaches for study of possible interaction types between exogenous gangliosides and IL-2R subunits were applied: antibody staining of IL-2R subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 I-Dcp-GM1 followed by immunoprecipitation of IL-2R subunits. The fluorescence intensity of the antibody-labeled IL-2R a-subunit substantially decreases after the treatment of cells with gangliosides. It has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside GM1, and by 10.7 % after incubation with GM2. Labeling of the cells with antibodies to the IL-2R b-subunit results in a less significant fluorescence decrease after cells incubation either with GM1 (2.8 %) or with GM2 (5.9 %).

To determine the mode of this impressive masking influence of ganglioside GM1, photoaffinity cross-linking has been used. In our modification this method could identify is interaction of GM1 with subunits of IL-2R occur with or without incorporation exogenous ganglioside into plasma membrane. Electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of IL-2R, but not for IL-2R a-subunit. These results demonstrate that exogenous ganglioside GM1 can interact with a-and bsubunits of IL-2R in different modes. Interaction of IL-2R b-subunit with ganglioside GM1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit.

A. Respa 1 , J. Bukur 1 , S. Purpose: Loss of interferon (IFN)-g inducibility of HLA class I antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. This kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. Methods: Phospho-specific Western blot analyses were performed to verify the functionality of the different IFN-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and HLA class I cell surface antigens, quantitative real time-PCR experiments to confirm the absence of JAK2 and presence of pathway relevant molecules as well as, genomic PCR and chromosome typing technique to prove the deletion of JAK2. Results: Different IFN-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong IFN-g inducibility.

Resistance to IFN-g treatment was associated in one melanoma cell line with the lack of JAK2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other IFN-g signal transduction components like STAT1 and JAK1 were not affected. JAK2 blockade by two JAK2-specific inhibitors resulted in reduced levels of HLA class I surface antigens. Conclusion: Structural abnormalities of JAK2 leading to a lack of JAK2 expression are associated with loss of IFN-g inducibility as well as reduced constitutive HLA class I surface expression. In addition the JAK2 inhibition modulates the expression of the HLA class I antigen processing components. of renal cell carcinomas (RCCs) are associated with the size, grade, T, N, M, stage and death of RCCs patients. The genotypes were compared with those of a random sample of 506 controls of the Spanish population. Methods: Two IL18 polymorphisms located on the IL-18 promoter region, SNPs -607 A/C (rs1946518) and -137 G/C (rs187238) were genotyped using TaqMan SNP Genotyping Assays.

The functional IL-18 gene polymorphisms studied do not influence the susceptibility to RCCs in the Spanish populations (IL-18 -607 p=0,318. IL18 -137 p=0,740) but may contribute to disease onset and aggressiveness: The genotype IL-18 -607 CC genotype, which is associated with higher IL-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), T (p=0,001), M (p=0,012) and stage (p=0,002). The influence of the IL-18 -103 GG genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), T (p=0,026) and stage (p=0,011). The multivariate analysis with Cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas IL-18 polimorphism can not be considered as independent prognosis factors.

Our results suggest that the polymorphism variants from the IL-18 gene (IL-18 -607 and -137) may be associated with an worse prognosis of RCC. High levels of IL-18 production can play an important role in grow, invasion and metastasis of renal cancer. Interleukin-18 (IL-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. The most prominent biologic property of IL-18 is its ability to induce the production of IFN-gamma in presence of IL-12. Moreover, it stimulates the expression of TNF-a and IL-l, enhances the differentiation of T cells to the Thl and impairs the synthesis of the anti-inflammatory cytokine IL-l0. Then it seems that IL-18 has a crucial role in immunity against Brucella infection. Since the expression of IL-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different IL-18 gene polymorph isms and brucellosis. Methods: A total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. All individuals were genotyped for six IL-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

The distribution of alleles for IL-18 polymorphisms at position -137 G/+113T/+127C (correlated with high production of IL-18), codon 35/3C and -656G/-607C (correlated with higher production of IL-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). Discussion: As data revealed genotypes that have correlation with higher production of IL-18 are more frequent in the controls than the patients. Then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of IL-18 at the onset of infection and it leads to more IFNgamma production and control Brucella infection before appearing brucellosis.

Abstract withdrawn by author Objectives: A dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (IBD) pathogenesis. To dissect the influence of these cytokines, specifically interferon gamma (IFNg), on the inflammatory response and colitis we have created a cell specific IFNg receptor 2 (IFNgR2) mouse mutant.

We have generated a mouse line carrying a conditional IFNgR2 allele using the 2 loxP 2-Flp recognition target (FRT) approach. A targeting vector with 2 loxP sites flanking exons 4-6 and 2 FRT sites flanking the neomycin resistance cassette was generated. After confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with Flp deleter mice. Cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific Cre expressing mouse lines. Functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total STAT1 following IFNg stimulation. Results: Successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. Functional inactivation of the receptor with no STAT1 activation following IFNg stimulation has been demonstrated in the complete knock out mice. Furthermore, flox/flox mice retain full responsiveness to IFNg. Breeding with LysM Cre and CD4 Cre mice will been completed to create a cell specific deletion of the IFNg receptor in macrophages/granulocytes and T cells respectively. The specificity of this deletion will be confirmed through cell sorting and functional assays.

In order to determine the influence of IFNg on a specific cell type a conditional gene targeting approach has been utilised. This has allowed the generation of conditional mice mutants with deletion of IFNgR2 on macrophages and T cells. This has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. Moreover, the ability to cross the conditional mice with additional Cre lines will enable the analysis of more cell types in the future.

A. Gonzalez 1 , R. Carretero 2 , P. Saenz-Lopez 2 , J. Cantón 2 , J. Carretero 2 , F. Ruiz-Cabello 2 , L.M. Torres 1 , cts-143 1 Hospital Universitario Virgen de las Nieves, Departamento de Ginecología, Granada, Spain, 2 Hospital Universitario Virgen de las Nieves, Departamento de Análisis Clí nicos, Granada, Spain

Objetives: Cervical cancer is almost associated with infection by human papillomavirus (HPV). However, only a subset of infected women will ever develop the malignancy. IFNG dinucleotide (CA) repeat polymorphism is responsible for genetic differences in interferon-gamma production. Our objective was to investigated the relationship between IFGN polymorphism and cervical intraepithelial neoplasia (CIN) Methods: we have studied nineteen women with CIN and 130 normal women control. DNA was extracted from blood samples, and was genotyped for functional microsatellite (CA) repeats in the first intron of IFN-gamma gene. Results: Heterozygosis in (CA)12 allele of IFGN is significant more frequent in healthy control than in CIN patient (p=0,030) in contrast homozygosis for (CA)12 allele did not show significant differences between both population. Analysis of relation between this polymorphism and the CIN stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). Conclusions: Our study suggest that IFNG (CA)12 allele may influence in CIN risk and progression. IFNG is associated with HPV clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. Further studies of the role of IFNG and other cytokines may contribute to the understanding of CIN promotions and progression.

Introduction: Macrophages play a fundamental role in controlling of Brucella infection, mainly through the secretion of cytokines such IFN-y and TNF-a. Interleukin-15 (IL-15), a Th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of IFN-y. As the cytokine production is under the genetic control, we decided to investigate the association between IL-15 single-nucleotide polymorphisms (SNPs) and susceptibility to brucellosis in Iranian patients. Methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. All individuals were genotyped for IL-15 (C267T, G367A, C13687A and A14035T) polymorph isms using PCR-RFLP. Results: At position 267, the distribution of C allele and CC genotype were significantly lower in the patients than the controls (P=0.025 and P=0.042, respectively). At position 13687, the distribution of C allele and AA genotype were significantly higher and lower in the patients than the controls, respectively (P=0.050, P=0.015). Discussion: As shown the frequency of CC genotype and C allele at position 267 were higher in healthy controls than the patients. Hence, our study provides evidence that presences of CC genotype and/or C allele are significantly associated with susceptibility to brucellosis. The frequency of AA genotype at position 13687 is lower in patients than the healthy controls and the frequency of C allele at position 13687 is higher in patients than the healthy controls. Hence, our study provides evidence that presences of C allele and lack of AA genotype are significantly associated with susceptibility to brucellosis. Objectives: Increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. Our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, MCF-7 and SKBR-3, as well as their ability to respond to several cytokines and to the G-1 GPR30 agonist.

The MCF-7 and SKBR-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: IL-2Ra, IL-2Rg, IL-3/IL-5/GM-CSFR, IL-4/IL-13R, IL-5Ra, IL-6Ra, IL-6R (gp130), IL-7Ra, IL-10R, TNFR I, TNFR II, IFNgRa, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5. Cells were incubated with IL-10, IFN-g, TNF-a and TNF-b (for which both cell lines displayed receptors) alone and with the GPR30 agonist. Cytosolic Ca 2+ concentrations [Ca 2+ ] i were measured by the microfluorimetric technique. Results: Different cytokine receptors phenotypes emerged for the two cell lines. The less well differentiated SKBR-3 cells were found positive for a larger number of receptors than the MCF-7 cells. Both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. Pretreatment of MCF-7 cells with IL-10 reduced the calcium response to G-1, while pretreatment with IFN-g and TNF-a potentiated the calcium response to G-1. TNF-b had no effect on MCF-7 G-1 stimulation. No direct effect on basal [Ca 2+ ] i stimulation could be noticed when administering the cytokines alone. In SKBR-3 cells, pretreatment with IL-10 or TNF-b had no effect on basal [Ca 2+ ] i and did not significantly alter the calcium response to G-1, while pretreatment with IFN-g induced calcium oscillations and potentiated the calcium response to G-1. Pretreatment with TNF-a produced calcium oscillations and reduced the response to G-1.

Conclusions: MCF-7 and SKBR-3 cell lines express distinctive cytokine receptor phenotypes. Furthermore, their ability to respond to cytokines in terms of modulating the GPR30 stimulation proved to be different. The susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. Results: In initial analyses of the early infection phase we identified splenic pDCs expressing IFNb/YFP. Furthermore, we show for the first time in vivo that these IFNb producing pDCs are not directly infected with MCMV and that not only cDCs but also CD8a -pDCs expressed GFP as a marker for MCMV infection. We observed that at early time points equal frequencies of CD8a -pDCs and cDCs were infected with MCMV, whereas after 24h p. i. the frequency of infected cDCs wins over that of the pDCs. Conclusions: With this experimental system we are able to visualize the IFNb response vs. the infection status of MCMV in vivo on a single-cell level. From our initial results we can conclude that infected cells in the spleen induce IFNb production in noninfected pDCs which initiate the antiviral immune response in this organ.

Recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (LCMV) carrying the vesicular stomatitis virus (VSV) envelope gene (rLCMV/VSV-G). Since interferon (IFN) signaling is known to be crucial for adaptive immune responses against wildtype (wt) LCMV and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rLCMV/VSV-G infection. Mice lacking both, IFN type I and II receptors generated potent virus-specific CD8 T cells and neutralizing antibody responses against rLCMV/VSV-G, even exceeding the respective responses in wild type animals. In further contrast to wt LCMV infection, IFN type I and II signaling was dispensable for rLCMV/VSV-G control. rLCMV/VSV-G infection of RAG1-deficient mice (lacking mature T and B cells) resulted in persistent levels of circulating viral RNA that was solely detectable by qRT-PCR but not by classical measures of infectivity. Overt viremia was only found in mice lacking both RAG1 and IFN type I receptor (AR). Thus, viral attenuation for vaccine use was found to considerably relax the IFN-dependence of adaptive immune responses and virus control. This redundancy of IFN and adaptive immune responses in rLCMV/VSV-G control provides a better understanding of the attenuation of this vector. It furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of CD8 T cell and antibody responses, which may have implications for vaccine development. Objectives: The class of Type I interferons (IFNs) consist of multiple IFNas and a single IFNb subtype. Although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. While in general the first IFN to be produced is IFNb, the cell types responsible for its initial production remain unclear. To assess the cellular sources of IFNb and its role for the outcome in bacterial infections we use an IFNb reporter-knockin mouse model, in which yellow fluorescent protein (YFP) is expressed from a bicistronic mRNA linked by an internal ribosomal entry site (IRES) to the endogenous IFNb mRNA. Methods: To induce a Type I interferon response we intravenously injected the TLR agonists poly(I:C) and CpG 1668, respectively, or infected reporter mice or bone marrow derived macrophages (BMDMs) or dendritic cells (BMDCs) with the facultative intracellular pathogen Listeria monocytogenes. The spatiotemporal tracking of the IFNb response was done using FACS analysis and immunofluorescent microscopy. Results: After poly(I:C) injection in vivo a small subpopulation of IFNb + CD8a + dendritic cells relocalize from the red pulp via the marginal zone to the T cell areas of the spleen whereas IFNb + activated pDCs were localized in the splenic white pulp at the T/B-cell interface after CpG administration. In vitro infection of BMDMs, BMDCs and FLT3-L derived DCs with Listeria monocytogenes resulted in a low frequency of IFNb + cells depending on the Listeria strain used and multiplicity of infection with BMDMs being the most potent producers of IFNb. In vivo mostly activated F4/80 + macrophages were accountable for the IFNb expression. Simultanous visualization of the cellular state of infection shows that IFNb + BMDMs carry a higher bacterial load then IFNbcells.

The cellular detection of IFNb expression reveals a remarkably low frequency of IFNb-producing cells in response to distinct PAMPs or the infection with intracellular bacteria. This hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. Additional data will be presented further resolving the timecourse of the IFNb response vs. the state of infection after bacterial challenge. The spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens.

We investigated the role of the spleen in the generation of antigen-specific cytotoxic CD8 T cell (CTL) responses in a systemic infection with recombinant Adenovirus expressing ovalbumin (AdOVA). Although Adenovirus mainly infects the liver, the CTL response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific CTL response upon systemic challenge with AdOVA. Additionally, dendritic cells (DC), macrophages and an intact splenic structure were mandatory for the induction of an efficient CTL response. We detected AdOVA specific CTL responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. Furthermore, we asked how Toll-like receptor (TLR) ligands influence AdOVA-specific CTL responses. TLR ligands as "danger signals" are generally known to exert immune stimulatory effects. Importantly, we observed that systemic administration of single-stranded RNA (ssRNA) prior to AdOVA infection inhibited the generation of antigen-specific CTL responses in a TLR7 and type I interferon (IFN) dependent manner. ssRNA injection induced the production of type I IFNs as detected in sera and supernatants of splenocytes isolated from wild-type mice. Experiments performed in IFNbeta reporter mice revealed that splenic plasmacytoid DCs represented the cellular source of type I IFNs.

Additionally, splenic CD11c+ DCs purified from ssRNA pretreated mice showed a reduced capacity to cross-prime OVA-specific CD8 T cells upon AdOVA challenge.

In vivo pre-activation of endogenous OVA-specific CD4 T cells as well as an adoptive transfer of in vitro activated transgenic OVA-specific CD4 T cells prevented the ssRNA mediated suppression of the CTL activity. We assume that DCs preactivated by systemic ssRNA were impaired in their ability to activate naive CD8 T cells in response to an AdOVA infection due to an impaired CD4 T cell help. Taken together, we show that type I IFNs cannot only stimulate, but also inhibit the induction of CTL responses in the spleen. Within hours after infection many viruses induce early type I interferon (IFN) responses that can confer protection against lethal infection. Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus (VACV) strain generated by more than 500 passages in chicken embryo fibroblasts. MVA stimulates systemic IFN responses, whereas VACV-induced cytokine milieus are dominated by IL-12. To study the impact of virus-triggered IFN on the induction of T cell responses, we used recombinant MVA-GP33 and VACV-GP33 expressing the GP33 epitope of lymphocytic choriomeningitis virus. For adoptive transfer experiments transgenic mice expressing the p14 T cell receptor recognizing the GP33 epitope were intercrossed with mice devoid of the IFN receptor (IFNAR -/-) to obtain p14IFNAR -/mice. Upon adoptive co-transfer of p14IFNAR -/and p14IFNAR wt/wt T cells and subsequent challenge with MVA-GP33, a massive expansion of p14IFNAR wt/wt T cells was observed, whereas p14IFNAR -/-T cells expanded less efficiently. In contrast, challenge with VACV-GP33 induced a rather similar expansion of IFNAR competent and IFNAR deficient p14 T cells. In the absence of IFNAR-triggering T cell expansion was associated with reduced proliferation capacity and increased apoptosis.

To study the impact of IFNAR-signaling on the expansion of endogenous T cells, conditional mice with a T cell-specific IFNAR-ablation were infected with MVA. Interestingly, in those mice the virus-specific T cells showed a reduced expansion compared to T cells of WT mice. Additionally, we found that IFNAR-triggering of dendritic cells but not of macrophages further supported specific T cell expansion. Thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific T cells. To delineate H5N1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. Blocking of specific signaling pathways revealed that H5N1 induced an exceptionally NF-kB dependent antiviral response. IRF3 is essential part of this interferon-response of human endothelia. Furthermore, we identified HMGA1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by H5N1. Finally, NFATC4 was found to be a transcriptional regulator for specifically H5N1-induced genes. We therefore describe for the first time defined signaling patterns specifically activated by H5N1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. Objectives: To study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. Methods: To dissect the contribution of the unique viral antagonist NS1, delta-NS1 influenza virus (a recombinant influenza virus lacking the NS1 gene) was compared side by side with wild type influenza virus in vivo. Mice infected with influenza virus were sacrificed at different time points after infection. To study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by Multiplex ELISA and quantitative PCR (qPCR). To determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (DC) migration to the draining lymph nodes was quantitated in infected mice. Further, functional in vitro and in vivo assays in infected animals with influenza-OVA viruses were used to determine whether antigen-bearing migratory DCs were capable of priming transgenic T cells.

To investigate the effect of NS1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. Strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including IFNs, cytokines, and chemokines occurred. This burst preceded the robust DC migration and T cell activation in the lymph nodes. Virus-loaded DCs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered T cell priming in vivo. A direct comparison of delta-NS1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the NS1 protein.

We demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza NS1 protein. Thus, we propose that the virally encoded NS1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. Keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. In addition, keratinocytes actively participate in immune response. Viral double-stranded RNA (dsRNA) is the most important viral structure involved in activation of innate immune response. Intracellular detection of dsRNA triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis.

Here we have used subcellular proteomics to characterise dsRNA-induced human keratinocytes. Cells were transfected with a mimetic of dsRNA, poly(I:C), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. These proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. We show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsRNA stimulation. In mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. We show that cytokeratin 18 is cleaved during poly(I:C) stimulation and fragments are solely localized in mitochondria. Similar degradation of cytokeratin 18 is seen in EMCV-and VSV-infected keratinocytes. Cytokeratin fragmentation after dsRNA stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. In addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsRNA-stimulation and during viral infection. Viral dsRNA also induced transient phosphorylation of 14-3-3 target proteins. Thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections.

I. Wessels 1 , D. Fleischer 1 , L. Rink 1 , P. Uciechowski 1 1 Institute of Immunology, RWTH Aachen University Hospital, Aachen, Germany

Objectives: The proinflammatory cytokine interleukin (IL)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. However, the molecular regulation of IL-1b expression is not elucidated, yet. It is known that the IL-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing IL-1b when stimulated. B-cells which are IL-1b non-producers reveal an inaccessible promoter structure. In this study the chromatin structure of the IL-1b promoter and the impact of methylation on IL-1b expression were examined in a cellular monocytic differentiation model. Methods: Promyeloid HL-60 cells were differentiated into monocytic cells after dihydroxyvitamine D 3 treatment. The monocytic phenotype was confirmed by flow cytometry. The IL-1b promoter was analyzed using the chromatin accessibility by real time PCR (CHART) assay. To test the influence of methylation, cells were treated with 5-Aza-2-deoxycytodine (AZA) and changes in IL-1b expression were measured by PCR, ELISA and Western Blot analyses. Results: In contrast to undifferentiated HL-60 cells, differentiated cells displayed upregulation of CD14 antigen and acquired the ability to express IL-1b. By comparing the accessibilities of IL-1b promoter we detected that the IL-1b promoter was not accessible in undifferentiated HL-60 cells but highly accessible in differentiated monocytic cells. The accessibilities of differentiated cells were comparable to that observed in primary monocytes. LPS stimulation did not affect promoter accessibility in promyeloic and monocytic HL-60 cells, demonstrating that the chromatin remodeling of the IL-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. Demethylation via AZA led to the induction of IL-1b expression in both undifferentiated and differentiated cells which could be increased after LPS stimulation. Conclusion: Two independent mechanisms involved in the regulation of IL-1b expression were found. Our data indicate that the IL-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. As a second mechanism, IL-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells.

A. Holweg 1 , G. Wetzel 1 , H. Arnold 1 , A. Gessner 1 1 Microbiological Institute-Institute for Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen, Erlangen, Germany

The p65 family members of interferon (IFN) inducible GTPases also known as guanylate binding proteins (GBPs) are among the most abundantly induced transcripts after stimulation with IFN-g. Although the stimulatory capacities of the Toll like receptor ligands LPS and CpG, cytokines like IFN-b and IL-1b and the intracellular pathogens Listeria monocytogenes and Toxoplasma gondii have been described to induce their expression, the function of GBPs during bacterial infections is still ill defined. Here we report for the first time the massive induction of murine GBPs in two independent in vivo models of pneumonia (infection with Streptococcus pneumoniae and Pseudomonas aeruginosa). A strong and rapid induction of mGBP2, 3, 4, 5 and 6 mRNA after intratracheal infection was detected by realtime PCR analysis of bronchoalveolar lavage (BAL) cells. Using newly generated antibodies in Western blots and fluorescence microscopy, we identified macrophages as the main producers of mGBPs in BAL samples. Although the signaling cascade involved in the upregulation of mGBPs after IFN-g stimulation has been extensively studied, the mechanisms responsible for mGBP induction upon bacterial stimuli are unclear. In this study we could show that the induction of mGBPs upon infection is abrogated in IFN type I/ type II receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. In contrast, the TLR adaptor molecule MyD88 was found to be dispensable arguing for a TRIF-mediated interferon production subsequentially resulting in enhanced GBP expression. Based on these findings our future experiments will address the functional role of GBPs in innate and adaptive immune responses against extracellular bacteria. (2)). Activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. Clearance of replicating virus from the CNS required MCMV specific CD8 + T lymphocytes whose effectors functions still remain incompletely defined (J. Immunol. 2008 Aug 1;181 (3)). Humoral immunity appeared to also play a role in the control of MCMV infection in developing brain. Infected newborn mice treated with MCMV-specific antibodies had lower viral titers in the CNS, significantly less CNS inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (J. Virol. 2008 Dec; 82 (24) ). Conclusion: Peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse CNS followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. Passive immunization of infected newborn mice reduces MCMV-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during CMV infection of developing CNS. Chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. Tumors enhance the accumulation of myeloid derived suppressor cells (MDSC), which contribute to tumor escape, immune tolerance and immune suppression. Previously, we have shown that tumor-derived inflammatory cytokines, such as IL-1b in the tumor microenvironment can induce a massive accumulation of MDSC in the spleen of tumor bearing mice and induce T cell suppression. In this work, we describe a novel polymorphnuclear MDSC subpopulation -Inflammatory MDSC (InfMDSC) which accumulates in the BM and spleen of mice bearing inflammatory 4T1 breast cancer cells over expressing IL-1b (4T1/IL-1b) tumor cells, but rarely in untransfected 4T1 cells. Secretion of IL-1b from tumor cells is crucial for InfMDSC generation and accumulation. InfMDSC have the ability to suppress NK cell activity via reduction of the NK activating receptor NKG2D in vivo, and in a cell-cell contact dependent manner in vitro. Inflammation up-regulates IL-4Ra expression on the cell surface, which correlates with tumor growth and induction of suppression on NK cells. Our data suggest that tumor derived inflammation enhances a specific MDSC subset which has the ability induce suppression of NK cells, and perhaps can serve as a new chemotherapy target. Objectives: LPS constitutes a main target of innate immune recognition of Gram-negative bacteria and other LPS carrying pathogens. Cytokine production after in vitro stimulation of whole blood with LPS is used as an expression of individual LPS reactivity. We assess genome-wide data to analysed the association to LPS induced cytokine response, and replicated the top findings in two independent cohorts. Materials and methods: We used 130 healthy Caucasian blood donors as discovery samples, and replicated SNPs from Affymetrix -Genome-Wide Human SNP Array 5.0 having p X 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from Illumina and real-time PCR respectively. In all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of IL-6, IL-8, IL-10, TNF-Alfa and IL1-ra with R&D ® assays on Luminex platform.. The association analysis was performed with Wald statistical test assuming an additive model.

The discovery sample statistical analysis revealed 150 Snps with p X 1,0*10 -4 . To identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. A combined analysis revealed 10 Snps with p X 9,1*10 -5 .These results are not genome wide significant. The 10 SNPs showing nominal association to LPS cytokine response are being analysed in a replication cohort Conclusion: We find 10 nominal associated SNPs between LPS stimulated cytokines in blood samples of healthy Caucasian blood donors. The importance of these SNPs are to be determined in a replication cohort. Adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. Adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. The physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. Heat shock proteins, such as Hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive.

Here, we investigated the regulatory effects of Hsp60 on adipocytes. For the first time we could show a Hsp60-stimulated release of the proinflammatory cytokines IL-6, CXCL1 and MCP-1 in a time-and concentration-dependent manner from murine 3T3-L1 adipocytes. Analyses of Hsp60-signalling in these adipocytes revealed that members of the MAPK-family (ERK1/2, p38) and the transcription factor NFkB are involved in Hsp60-mediated induction of the mediators IL-6, CXCL1 and MCP-1.

Binding-studies with fluorescence-labelled Hsp60 demonstrated that the interaction of Hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. Hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mM. Binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of Hsp60-binding. Further analyses to characterize Hsp60-receptor structures on adipocytes revealed the presence of Toll-like receptor (TLR)4 on adipocytes. TLR4 has been found to be expressed on macrophages and to interact with Hsp60, therefore suggesting TLR4 as a potential receptor candidate for Hsp60 on adipocytes. In order to identify the responsible binding-epitope of Hsp60 we investigated the effect of specific antibodies directed against different epitopes of the Hsp60-molecule. Incubation with antibodies directed against the N-terminus of Hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the Hsp60-binding to adipocytes (47-80 %) indicating that the N-terminal region of Hsp60 is involved in receptor binding. Our experiments demonstrate that Hsp60 stimulates the release of proinflammatory adipocyte mediators. The findings implicate that the Hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. Legionella pneumophila, a Gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as Legionnaires' disease. Classically, type I IFNs (IFNa/b) have been associated with antiviral immunity. IFNa/b signal through the IFNa/b receptor (IFNAR) leading to the induction of hundreds of IFN-stimulated genes (ISGs), many of which have anti-microbial activities. Recently, it was demonstrated that IFNa/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic DNA.

Here we show by RNAi that L. pneumophila infected host cells produced IFNb dependent on IRF3. We observed enhanced L. pneumophila replication in mouse macrophages lacking IFNAR and human cells after IRF3 knock-down, suggesting that endogenously produced IFNb activates a cell-autonomous defence against Legionella. Moreover, IFNb treatment restricts Legionella replication in human and murine host cells. IFNa/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of ER markers nor fusion of the Legionella-containing vacuole with the lysosome. Moreover, IFNa/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. We thus hypothesize the crucial involvement of antibacterially acting ISGs. Ongoing studies focus on the role of IFN-induced immunity-related GTPases (IRGs). Mesenchymal stem cells (MSCs) are identified by their capacity to differentiate into connective tissue cell types. Mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. Several authors have reported the expression of alfa-smooth muscle actin (a-SM actin) by MSC. This protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. This activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of MSC. In this work, we study the presence of alfa-SM actin in human MSC by flow cytometry and immunoflurescence. We also study the contractility of these cells under the effect of different cytokines. Human bone marrow samples were obtained from bone marrow aspirates. Bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in Opti-mem culture medium with 3 % of fetal calf serum at 37°C and 5 % CO2. Bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. Cells grew adherent, with a fibroblastic morphology and expressed CD10, CD29, CD73, CD21 and STRO-1 and were negative for CD45. Intracellular staining was performed for alfa-SM actin. Cell contractility was measured with the collagen gel contraction assay. Alfa-smooth muscle actin was detected in almost 100 % of MSC. Interleukin-2, IFN-gamma and TNF (Th1 cytokines) increased MSC contractility, whereas IL-10 (a Th2 cytokine) decreased MSC contractility. By immunofluorescence, we observed that IL-2, IFN-gamma and TNF increased the incorporation of alfa-SM actin into the stress fibers of MSC, whereas IL-10 decreased that incorporation. Our results suggest that Th1 and Th2 cytokines regulate MSC physiology by acting on their contractility. Aims: Thapsigargin (TG) is a sesquiterpene lactone (SL) of guaianolide type isolated from the Mediterranean plant Thapsia garganica L. It is widely recognized as an inhibitor of sarco-endoplasmic reticulum Ca 2+ -ATPase leading to elevation of intracellular calcium. This activity is shared by trilobolide (TB), a SL from Laser trilobum (L.) Borkh. TG has been shown to possess prospective immunotherapeutic properties. It kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. The aim of our work was to investigate possible immunostimulatory potential of TG and TB. Methods: The effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (PECs), and human peripheral blood mononuclear cells (hPBMCs). They were cultured in density of 2 × 10 6 /mL in complete RPMI-1640 medium. Supernatant levels of IFN-g were determined by ELISA. Production of NO by animal PECs was assayed using Griess reagent. Possible contamination of the samples with LPS was excluded using the chromogenic Limulus amoebocyte assay. Results: We have found that both TG and TB at as low doses as 40 nM and 400 nM induce IFN-g secretion in rat PECs and hPBMCs, respectively. The concentration of IFN-g produced by the highest dose of the agents (10 mM) at the 24-h culture interval reached the values of approximately 3 ng/mL and 2 ng/mL in rat PECs and hPBMCs, respectively. The increase was apparent within the interval of 2-6 h in rat PECs. The 24-h interval was optimal for accumulation of IFN-g in cultures of hPBMCs. Only modestly enhanced secretion of IFN-g was observed in mouse PECs. Production of IFN-g was found to depend on activation of NF-xB and MAP kinases p38 and ERK1/2. It was not suppressed by the calcium chelating agents BAPTA-AM and TMB-8. The TG-and TB-induced IFN-g production was associated with activation of the high-output production of NO. Conclusions: Sesquiterpene lactones TG and TB are potent inducers of IFN-g in animal and human cells. The effect is independent of their SERCA inhibitory potential. Underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. Acknowledgements: The work was supported by the grant GACR 305/07/0061. Pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated Ser/Thr-Pro protein motifs and catalyzes the cis/trans isomerization of peptides. Mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are Pin1 substrates and targeting sites. Recent data show that Pin1 interacts with APO-BEC3G (A3G). The Pin1/A3G interaction results in a reduced A3G expression and a diminished A3G-mediated restriction of HIV. Two single nucleotide polymorphisms (SNPs) in the promoter region of the Pin1 gene (-842 G/C and -667 T/C) modulate PIN1 expression; in particular, the -842 GC genotype or CC haplotype are associated with reduced protein levels (Neurobiol. Aging, 28; 69-74, 2007) .

The -842 C/G and -667 T/C polymorphisms in the promoter of Pin1 gene as well as Pin1 protein levels were analyzed in 30 Exposed Seronegative Individuals (ESN), heterosexual partners of HIV-infected patients; 40 HIV-infected patients (HIV) and 40 Healthy Controls (HC). The genotype and allele distributions of the -842 SNP was skewed in ESN (genotype: p= 0.008; allele: p= 0.013). In particular ESN showed a significantly lower frequency of the -842 GG genotype compared to HIV and HC (p=0.017 and p=0.019, respectively) and consequently a lower G allele frequency (p=0.026 and p=0.028, respectively). No significant differences were found for the -667 SNP. These SNPs are in linkage disequilibrium and combine to form haplotypes. Conclusions: Our findings support the role of HIV viral replication as the most important promoter of immune activation and prove the importance of ART in reducing immune activation and viral replication even in a sub-Saharan African environment, where patients are exposed to an abundance of other infectious agents. Our data further indicates that HIV replication rather than host genetics is the key determinator of circulating cytokine levels among the studied HIV infected participants. Aims: Acyclic nucleoside phosphonates (ANPs) are synthetic analogues of natural nucleoside monophosphates. They have proved to be outstanding antivirals inhibiting replication of both DNA-viruses and retroviruses. Tenofovir, 9-(R)- [2-(phosphonomethoxy) propyl]adenine [(R)-PMPA] is broadly used for therapy of AIDS, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) has been approved for the treatment of hepatitis B. The aim of our work was to investigate possible interactions of ANPs with production of cytokines and nitric oxide (NO) implicated in antiviral defence mechanisms.

The immunobiological effects of ANPs were screened in vitro using mouse resident peritoneal cells. They were cultured in density of 2 × 10 6 /mL in complete RPMI-1640 medium. Secretion of cytokines was determined after the 5-h culture by ELISA. Production of NO was assayed at the interval of 24 h using Griess reagent. Approximately 300 compounds belonging to several distinct ANP groups were included in the study: a) PMEA derivatives, b) PMEDAP i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (HPMPA), and HPMPDAP derivatives, d) guanidino analogues of PMPDAP, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. Possible presence of LPS in the stock solutions of the samples was checked and excluded using the chromogenic Limulus amoebocyte assay.

Results: Approximately 30 compounds were found to activate the secretion of anti-HIV effective chemokines RANTES and MIP-1a and cytokines TNF-a and IL-10. Although these ANPs did not stimulate biosynthesis of NO on their own, they substantially augmented production of NO triggered by IFN-g. No clear-cut relationship between the chemical structure and biological effects of ANPs was observed. However, the most effective proved to be the N 6 -cycloalkyl derivatives of PME-DAP. The effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mM. The remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. The effects were found to depend on activation of NF-kB. Conclusions: It may be suggested that ANPs represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. Acknowledgements. The work was supported by the grant 1M0508. Host related immune factors in childhood chronic hepatitis B and change of initial profile with IFN-a treatment needs to be clarified. Sixteen patients were included, and 10 million units of IFN-a treatment three times a week for 6 months was initiated. Before and after treatment: Percentages of the IL-2 and IFN-g in CD4+ T cells were assessed to determine intracellular T helper cell 1 (Th1) type cytokine expression. Similarly, percentages of intracellular IL-2 and IFN-g were detected to verify cytotoxic T cell 1 (Tc1) type cytokine expression in CD8+ T cells. Percentage of Th2 and Tc2 type cytokine expression, (IL-4 and IL-13) were determined in CD4+ and CD8+ T cells, respectively. Six (50 %) of these were evaluated as having no response and the other half with partial/complete response. All patients had higher percentages of Th2 cells with respect to healthy controls before treatment. Tc percentages, both Tc1 and Tc2, were significantly different between these groups, being higher in the patient group. When values of no responder group were compared with healthy controls, IL-4 expression was higher and the percentages of Th1 type cells were significantly low. IL-13 expression in Th and Tc cells decreased after treatment in the unresponsive group. Intracellular cytokine profiles of treatment responders and normal controls were not different. This has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. 

The frequency (29,8 %) of high concentration of IgG anti-oxLDL antibodies in patients with HCV infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). The immune response was significant but it was not assosiated with the viral load. It is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of HCV-infection.

Abstract withdrawn by author

T. Schwandt 1 , F. Juengerkes 1 , B. Schumak 1 , G. Gielen 1 , J. Kalff 2 , P. Knolle 1 , B. Holzmann 3 , A. Limmer 1 1 University Hospital Bonn, Institute of molecular medicine and experimental immunology, Bonn, Germany, 2 University Hospital Bonn, Department of surgery, Bonn, Germany, 3 Department of Surgery, TU Munich, Munich, Germany

Bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. Patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity.

Here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. To mimic abdominal surgery, mice were subjected to intestinal manipulation (IM). To study septic conditions, mice underwent colon ascendens stent peritonitis (CASP) or received E.coli intravenously. We monitored the distribution of gut-derived bacteria by in vivo imaging (Xenogen) and additional microbiological assays and determined antigen-specific CTL responses towards subsequent infection with recombinant adenoviruses (AV). We detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent CASP or were injected i. v. with E.coli. In addition, mice infected systemically with AV lacked an antigen-specific CTL response, whereas the CTL responses of locally AV infected mice were not affected. In contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to IM or received E.coli by injection into the hepatic portal vein. Here, the CTL response was not impaired. Depletion experiments imply that Kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. Our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. We suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. Failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. Objectives: Varicella-zoster virus (VZV) is one of the most frequent pathogens for which a vaccine is available. Tropism of VZV for skin is the most obvious manifestation of VZV infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. The striking difference in the clinical outcome of infection by rush inducing circulating virulent VZV strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. Therefore, we comparatively investigated the impact of VZV clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (iDCs) and epithelial gd T cells. Methods: Skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. Phenotypic changes of cutaneous DCs and monocyte-derived DCs after VZV infection were investigated by flow cytometry. The cytokine profile of VZV-infected DCs and epithelial gd T cells was determined through ELISA. Results: We observed that innate lymphocytes recognize DCs, which infiltrate herpes zoster lesions, after infection with VZV. Strikingly, only the vaccine but not VZV clinical isolates could license the bidirectional crosstalk between iDCs and gd T cells resulting in release of IFN-g and IL-12, the signature cytokines of antiviral immune responses. This is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite.

We describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. This result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair DC instruction in order to allow stimulation of powerful antiviral immune responses.

A. Jafarzadeh 1 1 Rafsanjan University of Medical Sciences, Rafsanjan, Iran, Islamic Republic of Objective: It has been reported that the cagA + H. pylori strains induce more severe gastric mucosal inflammation. The aim of this study was to investigate the association of the H. pylori virulence factor CagA with serum levels of IL-17 and IL-23 in H. pylori-infected duodenal (DU) patients and H. pylori-infected asymptomatic (AS) carriers. Methods: Totally, 45 H. pylori-infected DU patients (23 patients were positive for anti-CagA antibody and 22 patients were negative for anti-CagA antibody), 30 H. pylori-infected AS carriers (15 subjects were positive for anti-CagA antibody and 15 subjects were negative for anti-cagA antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. A blood sample was obtained from all participants and the serum levels of IL-17 and IL-23 was measured by ELISA method.

The mean serum levels of IL-17 in total DU patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total AS subjects (5.19 pg/ml ± 3.75, p X 0.001) and healthy control group (3.55 pg/ml ± 3.76, p X 0.0001). In DU group, it was found that the mean serum levels of IL-17 in subjects with positive test for anti-CagA (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-CagA (7.65 pg/ml ± 4.74; p X 0.05). The mean serum levels of IL-23 in DU (8.66 pg/ml ± 8.41) and AS groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, P X 0.02 and P X 0.03, respectively). However, no significant difference was observed for mean serum levels of IL-23 between DU and AS groups. Moreover, in both DU and AS groups the mean serum levels of IL-23 was not significantly differ in subjects with positive test for anti-CagA and those were negative for anti-cagA antibody.

The results of the present study showed higher serum concentrations of IL-17 and IL-23 in H. pylori-infected subjects as compared with control group.

In DU group the expression of IL-17 influenced by the bacterial CagA factor.

A. Aral 1,2 , A. Atak 1 1 Gazi University Faculty of Medicine, Department of Immunology, Ankara, Turkey, 2 Gazi University Institution of Health Sciences, Department of Immunology, Ankara, Turkey

Objective: EBV is a human herpesvirus, which infects human B lymphocytes latently and immortalizes the cells due to transformation. EBV infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (IM) in adolescence. In immunodefective patients, the virus may lead to malignancies like Burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. The virus can also cause latent infections. Cytokine production in response to EBV infection differs according to the phase of the infection. Neopterin, IFN-g and IL-6 levels are elevated in acute EBV infection in vitro; but in chronic phase, particularly, IL-6 elevation could not be detected. TNF-a enhances the effects of IFN-g on neopterin synthesis while neopterin enhances the secretion of TNF-a via various stimuli. IFN-g levels are elevated particularly in the acute phase of IM. Since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. IFN-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. Increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity.

Method: In order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via EBV. Neopterin, IFN-g, TNF-a and IL-6 levels have been measured with EIA kits in culture supernatants. Results: Neopterin levels increased dependent on time and independent of EBV transformation. In EBV-transformated cell culture TNF-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. Likely TNF, IFN-g levels also increased at the 1st week and started to decrease at the 3rd week. IL-6 reached to its peak at the 3rd week. Conclusion: According to these results, neopterin levels, which increased dependent on time but independent of EBV transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation.

V. Jurisic 1 , M. Jurisic 2 1 University of Kragujevac, School of Medicine, Kragujevac, Serbia, 2 University of Belgrade, School of Dentistry, Belgrade, Serbia TNF-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. We investigated TNF concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. Further, we estimated the role of cyst wall on production of TNF-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. The concentrations of TNF-alpha in the cystic fluids were measured by ELISA, while proteins analyzed after separation by two-dimensional gel electrophoresis. The presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for CD3, CD20 and CD68. TNF-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. Higher concentration of TNF-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (Mann-Whitney U-test, p X 0.05) in radicular cysts. No correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of TNF-alpha. Objectives: Interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. In susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. We will study prolactin role in defensive reaction of immune system to bacterial infection. As a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. Our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system.

The aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery.

1) Drawings blood from 20 patients and 20 healthy donors. Blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. Status of patient had to meet these conditions: a) The presence of systematic inflammatory response syndrome (SIRS) according to standard clinical and laboratory criteria. b) Positive hemoculture or determination of septic focus with demonstration of microbiologic source.

2) Detection of the gene expression of prolactin and TLR-2 in CD14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by RT-PCR at the level of mRNA and flow cytometry at the level of intracellular protein Results: We have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. These differences were at both levels of expression, mRNA and protein.

Conclusion: Septic statuses change TLR-2 and peripheral prolactin expression in CD14+ monocytes. The function of interferon (IFN)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of MHC-class I antigens. The experiments performed here challenge this existing paradigm of i-proteasome function. We demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under IFN-induced oxidative stress. IFNs up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. I-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. Enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. Thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. Background: Revived by the description of new functions, B cells are considered to be essential in the genesis of autoimmune diseases. In strong support of this interpretation, BAFF would play a key role in their physiology. However, the correlation between circulating BAFF levels and disease activity is not clearly established. Conflicting results concerning levels of BAFF and B-cell repopulation after rituximab treatment have been reported. In fact, basal serum levels of BAFF reported in literature vary according to the antibodies (Ab) used in the ELISA and the mode of calculation. The aim of this study was to understand these variations. Material and methods: Different anti-BAFF Abs were tested to verify whether they recognize glycosylated BAFF purified from U937 culture supernatant. Sera from patients with autoimmune diseases were also tested. A Western-blot analysis of sera was performed to evaluate anti-BAFF Abs specificity and the best combination of anti-BAFF Abs validated for ELISA. Then, different commercial BAFF ELISA-quantification kits were compared to our "in-house" ELISA. Results: Unexpectedly, the binding of some anti-BAFF Ab was restricted to glycosylated BAFF. However, both glycosylated and non-glycosylated forms of BAFF were found in sera from patients with autoimmune diseases. Most of the kits commercially designed to quantify BAFF suffer from some limitations. Some sera are indeed positive with a kit and negative with another and vice versa. Furthermore, there seems that rheumatoid factors (RF) interfere and correlate with the optical density of the anti-BAFF ELISA.

Conflicting results concerning levels of BAFF and disease activity or auto-Abs titers should be reconsidered in light of the glycosylation status of BAFF. (Table-2 ). When TB household contacts and healthy controls were compared, CFP10 and ESAT6 seemed to be more useful than TST in TB contacts for displaying LTB (Table-2) . Although CFP10 spot numbers were much more than ESAT6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(P: 0,069)( Table-3 ). Both ESAT6 and CFP10 spots were prone to decline during the follow up period.

[ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by HCV.

To gain insight into some features of the innate immune response activated in vivo in the context of acute HCV replication, we analysed cytokine and chemokine levels in serum samples from chronic HCV patients undergoing liver transplantation. Luminex multiplex assays and ELISAs were used to quantitate serum levels of G 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. The increase in serum HCV RNA levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including IFN-gamma, TNF-alpha, IP-10, IL-6 and IL-10. Notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary HCV infection. High-magnitude elevations in systemic type 1 IFN levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by HCV similar to that observed in in vitro studies.

We suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute HCV infection. To test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (HAE). The hemagglutinin of the pandemic virus A/Hong Kong/1/68 (H3N2) (HK) differs from its putative avian precursor by 7 amino acid substitutions. We generated the complete recombinant virus rHK and its HA variants with amino acid reversions back to the ancestral avian sequence (rHK-5aa-I62R, N81D, K92N, S193N, G144A, Human (2-6); rHK-R2-L226Q, S228G and rHK-7aa-I62R, N81D, K92N, S193N, G144A, L226Q, S228G, Avian (2-3)). Among these variants, the double mutant rHK-R2 and the seven mutant (rHK-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions L226Q and S228G.We infected HAE cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. Virus titers were determined in MDCK cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.Concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. Many cytokines, including T-cell-attracting chemokines such as IP-10 and RANTES, were induced to similar levels by different viruses. However, some mediators were induced significantly stronger by avian-like viruses rHK-R2 and rHK-7aa as compared to rHK and rHK-5aa. In particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as G-CSF and IL-8, shedded adhesion molecules (CD25, VCAM-1, ICAM-1), and pro-apoptotic factors (TRAIL). Remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. Whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rHK and rHK-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. These data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. Further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. 

A total of 98 newborn infants were enrolled in the study. Forty-nine newborn infants (Group I), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of PCT, IL-10, and neutrophilic CD64 levels, Procalcitonin and IL-10 were measured by ELISA technique while, neutrophilic CD64 by single colour flowcytometric technique. Of these 49 "infected" infants, 16 had positive blood culture (Subgroup IA: Culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (Subgroup IB: Culture-negative sepsis). Another 49 newborn infants classified as control group (Group II) . Results: Sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for PCT, IL-10, CD64, and CRP. IL-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/mL. For PCT, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/mL. Neutrophilic CD64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. Combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. We found that the best combination was IL-10 and neutrophilic CD64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and NPV 86 %. The combination of IL-10 and CRP had high sensitivity and moderate specificity, 93 % and 79 %, respectively. Conclusions: IL-10 and neutrophilic CD64 levels determination can be used as good tests for early detection of neonatal sepsis. Procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. Objectives: The assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. Inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the NOD-like receptors (NLR). The NLR family members NALP3, NAIP5 or Ipaf and the adaptor ASC are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of IL-1b and IL-18. Recent studies have demonstrated that TLR-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. Autophagosome biogenesis and maturation requires activation of the class III PI3K, Vps34 and can be inhibited with the PI3K inhibitors wortmannin and 3 methyladenine (3MA). In contrast, activation of Akt, via class I PI3K, results in inhibition of autophagy. The aim of this study was to determine the nature of this inflammasome and whether autophagy influences IL-1b secretion in dendritic cells. Methods: Murine bone marrow-derived dendritic cells (BMDM) were treated with TLR ligands in combination with 3MA, wortmannin or an Akt inhibitor. Supernatants were analysed for IL-1b by ELISA. Results: 3MA enhanced IL-1b secretion by BMDC treated with the TLR3 and TLR4 ligands poly(I:C) and LPS, but not with any other TLR ligands tested. Similar results were obtained using wortmannin. This increase in IL-1b secretion was greatly reduced in BMDC from NALP3 -/mice compared to wild type C57/Bl6 controls. Treatment with the Akt inhibitor had no effect on LPS-induced IL-1b secretion by BMDC. TLR-dependent secretion of IL-1a was also enhanced by treatment with 3MA. Conclusions: These data demonstrate that IL-1b secretion by BMDC in response to treatment with PI3K inhibitors, in combination with LPS or poly(I:C), is dependent on the NALP3 inflammasome. This response is limited to TLR3 and TLR4 agonists. Inhibition of Akt had no effect on LPS-induced IL-1b production, suggesting that the effect of wortmannin and 3MA on inflammasome activation is not dependent on the inhibition of Akt activation by Class I PI3K. Objectives: In the last few years, several evidences have shown the modulation of Toll-like receptors (TLRs) by G-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of TLR2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (S1P) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the S1P anti-atherogenic effects. Since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of TLR-S1P receptors crosstalk on the expression of adhesion molecules in endothelial cells. Methods: For the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of TLR ligands and S1P, and later analyzed by flow cytometry. A pharmacological analysis of the S1P receptor subtype involved in the response was also performed. Results: Data from flow cytometry experiments revealed that ICAM-1 expression was increased following LPS and S1P concomitant stimulation in both venous and arterial cells, suggesting that TLR4 and S1P receptors cooperate in the expression of ICAM-1. Conversely, no cooperation was observed when TLR2 ligands were used. In order to elucidate which S1P receptor subtype was involved in the increase of ICAM-1 expression, we used a pharmacological approach with S1P receptor inhibitors and pertussis toxin. Results showed differences between arterial and venous cells. While in arterial cells elevated ICAM-1 after LPS and S1P challenge was significantly reduced by blocking S1P receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of S1P receptors 2 and 3, which suggest that S1P receptor 1 might be involved in the effect. Conclusions: Altogether these data demonstrate that TLR4 and S1P receptors can interact to increase adhesion molecules such as ICAM-1 in human endothelial cells, and the S1P receptor subtype involved in the effect differs between arterial and venous cells. 15 with SSc without PAH) and a pool of 12 sera of healthy controls (HC) were tested. Results: In 1 dimension immunoblot, serum IgG from SSc patients, patients with IPAH and HC tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human VSMC protein extract with qualitative and quantitative differences between groups, respectively. In 2 dimension immunoblot, IgG of pools of patients with iPAH, IgG of pools of patients with SSc with or without PAH, and IgG of a pool of HC recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. Twenty one protein spots were recognized by more than 80 % of IgG of pools of sera in each group of patients and not by IgG of HC. Twenty seven protein spots were recognized by the great majority of IgG of pools of patients with a higher intensity than IgG of pools of HC. Identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise.

We have identified anti-VSMC Abs in the serum of patients with idiopathic and SSc-associated PAH. These Abs bind to cytoskeleton, oxidative stress and cell cycle antigens. Objectives: This study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species.

Methods: This rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes.

Results: This rat model is adequate to study the regeneration of transplanted lymph nodes. Lymph node fragmentation seems to affect transplant regeneration negatively. An immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. However, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. Conclusion: Lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. Acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. This disease causes lifelong disability due to chronic swelling and increased risk of infections. It currently lacks an effective treatment. Methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). Blood specimens were collected before hyperbaric oxygen treatment and the serum levels of ICAM-1 and VCAM-1 were assesed by an enzyme immunoassay (ELISA). Results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). Reference value for ICAM-1 was 130-299 ng/ml. Vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. Conclusions: Our study revealed a high rate of patients (40 %) having increasd levels of ICAM-1. High ICAM-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). Mean values were found 735,5 ng/ml and 400 ng/ml accordingly. Those findings verify the positive correlation between ICAM-1 and inflammatory diseases and tissue damage but not for VCAM-1. Colorectal cancer (CRC) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. Tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. Angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. Inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of CRC carcinoma potentially through repression of tumour angiogenesis. We investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of CRC: 16 cases of ulcerative colitis (URC), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. Immunohisto- Objectives: To determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of CD18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (CEC). Methods: They were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (CEC). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group Study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). Samples of outlying veined blood were extracted to the 24 hours. The separation of leukocytes was made in peripheral blood, to determine the expression of CD18 in such. In all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. Results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. The intensity of the expression was classified in 3 degrees: Degree 0. Without expression. Degree 1. Weak; Degree 2. Moderate; Degree 3. Intense. In the Group Control: Most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. The Placebo group: mixed pattern, degree 1-2. Group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: Degree 2-3. The percentage of cells that expressed CD 18 was greater in the group study (40 mg. oral pravastatina).

The preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression CD18 answer induced by the CEC; It seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. Several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases ADAM10 and ADAM17. We recently identified transmembrane chemokines, junctional adhesion molecule-A (JAM-A), and members of the proteoglycan family as novel substrates for these proteases. Here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble JAM-A and proteoglycan ectodomains. This release is enhanced by treatment with the proinflammatory cytokines IFNg and TNFa. The enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. Both, constitutive and induced release required the presence of ADAM17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of ADAM17. These data suggest that the proinflammatory cytokines IFNg and TNFa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells.

To investigate the physiological relevance of this induced shedding, mice were treated systemically with IFNg/TNFa leading to increased presence of soluble JAM-A in the blood serum. Both cytokines also stimulated JAM-A release from excised murine aortas with was associated with enhanced ADAM17 activity in the tissue.

In the presence of the ADAM17 inhibitor induction of JAM-A release was suppressed.

In cultured epithelial cell lines enhanced shedding of JAM-A or proteoglycans was not associated with increased mRNA or surface expression of ADAMs but rather with increased activity of cellular ADAM17 as shown by means of a synthetic substrate assay. Our study demonstrates that the proinflammatory cytokines IFNg/ TNFa upregulate ADAM17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings.

rium, and the haplotype VEGF-460/ VEGF+405 is associated with RCC risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). On the other hand, the polymorphism VEGF -2578 A/C is not associated with RCC risk and clinical parameters. Our results shed a new light to the knowledge on the association between VEGF polymorphism and RCC risk and development. These data could help to improve our understanding of the RCC pathogenesis and disease progression. Pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. Therefore, pten is one of the main antagonists of the PI3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. Here we investigated the role of pten in collagen induced arthritis. We show that conditional deletion of pten under the LysM promoter (LysMCrePten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (CIA). Histological analysis of CIA, LysMCrePten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. Total anti-collagen antibodies, however, as well as anti-collagen IgGs were identical in both groups. Upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of IL-6 as well as IL-8 levels. Furthermore, pten deficient macrophages and dendritic cells showed reduced induction of IL-6 as well as IL-12 and IL-23 mRNA upon stimulation with various TLR-ligands. Since these cytokines play an important role in the induction of pathogenic Th-17 T cells, we measured Th-17 cytokines in lymph nodes after immunisation with collagen. Although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of IL-17 and a strong reduction of IL-22 mRNA in the draining lymph node of immunized LysMCrePten flox/compared to wild-type mice. One of the mechanisms through which IL-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. In this context, particularly important is the production of IL-1ra, whose expression is induced by LPS in human neutrophils and monocytes and significantly potentiated by the presence of IL-10. Based on our previous observation that support a direct role of IL-10-activated STAT3 in the enhancement of IL-1ra transcription induced by LPS, we plan to characterize the transcriptional activators recruited to the IL-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of LPS-treated cells to IL-10. Quantitative chromatin immunoprecipitation (ChIP) studies were employed to examine the in vivo binding of transcriptional activators to the IL-1ra promoter. Crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after IL-10 addition with different antibodies and immunoprecipitated DNA was analyzed by quantitative real-time PCR for the presence of target sequence located in the IL-1ra promoter. ChIP assays showed that the Pol II recruitment to the IL-1ra promoter induced by LPS is significantly increased by IL-10, further strengthening the concept that the rapid enhancement of LPSinduced IL-1ra gene expression by IL-10 initially occurs by targeting transcriptional events. As expected, real-time PCR of anti-STAT3 immunoprecipitated DNA showed statistically significant levels of STAT3 binding to the IL-1ra promoter only in cells stimulated with LPS in the presence of IL-10. Surprisingly, anti-p65 and anti-p50 ChIP assays revealed enrichment of both p65 and p50 recruitment to the IL-1ra promoter when IL-10 was added to LPS-stimulated cells, suggesting that IL-10 enhances the recruitment of NF-kB to the IL-1ra promoter. Interestingly, when NF-kB is recruited to this promoter in LPS + IL-10-treated cells, the overall NF-kB nuclear translocation (analyzed by Western blot) and DNA binding activity (detected by EMSA analysis) were not modified with respect to cells stimulated with LPS alone. The enrichment of NF-kB at the IL-1ra promoter site is dependent on IL-10-activated STAT3, since it is greatly reduced when STAT3 activation by IL-10 is impaired. The molecular mechanism through which IL-10-activated STAT3 promotes the recruitment of NF-kB to the IL-1ra promoter is currently under investigation. Major components of mast cell secretory granules are proteases. We could recently report that intracellular stored mast cell-produced cytokines regulate MC protease activities and provided evidence that IL-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mMCP-2). We examined the mechanisms underlying the repression of mMCP-2 gene expression. Our data show that the "repressor" effects of IL-15 on mMCP-2 promoter activity are still operating on the mMCP-2 591 bp long minimal promoter. Moreover, IL-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors C/EBPb and YY1. Furthermore, chromatin immunoprecipitation revealed that IL-15 promoted specific reciprocal recruitment of C/EBPb but not YY1 to the mMCP-2 promoter. Finally, IL-15 deficient mast cells display a predominantly non-CpG methylated pattern of the mMCP-2. Thus, we proposed that the expression of mMCP-2 and possibly other immunoregulatory genes may be regulated by IL-15 through epigenetic modification and by balancing the content and binding of C/ EBPb and YY1 in mast cells.

I. Nagy 1 , K. Filkor 1 , A. Vörös 1 , L. Kemény 2 , A. Szász 1 1 BZAKA, Baygen, Szeged, Hungary, 2 University of Szeged, Department of Dermatology and Allergology, Szeged, Hungary

MicroRNAs (miRNAs) are evolutionary conserved small non-coding RNAs that act as key regulators of gene expression at post-transcriptional level by targeting mRNAs for translational repression and/or degradation. miRNAs have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. During the last years several studies highlighted that miRNAs play critical role in the differentiation and function of the adaptive as well as innate immunity. Little is known however, about the differential regulation of miRNAs following the activation of Pattern Recognition Receptors. In order to tackle this issue, we treated HaCaT keratinocytes with Staphylococcus aureus-derived peptidoglycan (PGN) once or repeatedly, the latter mimicking persistent infection. After appropriate treatments we first analyzed the expression profile of miRNAs miR-203, miR-146a and miR-155, which are known to participate in immune processes of the skin. Repeated PGN-treatment significantly decreased miR-203 expression; in contrast, PGN re-stimulation had no further effect on miR-146a and miR-155 expression. Next, we investigated the correlation between the expression of miR-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (SOCS-3). Although the gene-expression profile of neither p63 nor SOCS-3 changed, we found that the expression of miR-203 reversibly correlates with both p63 and SOCS-3 protein expression, a phenotype that we verified by two independent protein analysis methods (Western blotting and immunofluorescent labeling). Importantly, transfection of HaCaT cells with anti-miR-203 prior to PGN-treatment completely abolished both p63 and SOCS-3 down-regulation, revealing the involvement of miR-203 in PGN-induced transcriptional regulation. Finally, methylation-specific PCR experiments unravelled the role of DNAmethylation in regulating miR-203 expression upon PGN-treatment. Taken together, our results strongly suggest that sets of miRNAs may be differentially regulated during persistent infection.

Results: TGFb1+/-had a lower incidence and burden of benign papillomas when compared to TGFb1+/+ animals. However, more SCC developed in the TGFb1+/-mice. After acute and chronic promotion, TGFb1+/-skin showed a reduced proliferative response with no increase in epidermal TGFb1 or nuclear p-Smad2 compared to TGFb1+/+ mice. TPA-induced PKCa activity as well as phosphorylation of specific PKC substrates in keratinocytes correlated with TGFb1 gene dosage. Further, pharmacological inhibition of ALK5 suppressed TPA-mediated PKCa activation suggesting that physiological levels of TGFb1 are required for maximal activation of PKC-dependent mitogenic responses. Even though the TPA-induced inflammatory response was greater in TGFb1+/-skin, TGFb1+/+ papillomas had more tumor infiltrating neutrophils. TPA-induced proinflammatory gene expression was sustained in TGFb1+/-skin and primary keratinocytes but it was elevated in v-ras Ha -transduced TGFb1+/+ but not TGFb1+/-keratinocytes, indicating that TGFb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. Despite this differential proliferative and inflammatory response to TPA and enhanced papilloma formation in the TGFb1 +/+ mice, there was no increase in conversion to SCC in this genotype. Conclusions: TGFb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate PKC activation in skin, yet retains a suppressive function for malignant conversion. Background: Proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (RA). In the present study we studied the relationship between survivin and urokinase (uPA), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. Material and methods: Levels of survivin and uPA were measured in the paired blood and synovial fluid samples of 132 patients with RA, using ELISA and compared to controls with non-inflammatory joint diseases. The ability of uPA to induce survivin and requirement of uPA receptor (uPAR) was studied in primary synovial fibroblasts and PBMC of RA patients, human monocytic (THP-1) and fibroblast (MRC-5) cell lines employing antibodies against uPAR, siRNA technique, and synthetic inhibitors of intracellular pathways. The ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. Results: In the present material of 132 RA patients and 82 controls the levels of survivin correlated to urokinase (uPA) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. We found that 30/132 RA patients had high circulating levels of survivin. These patients had erosive arthritis and were characterized by high levels of uPA. In vitro studies showed that uPA induced survivin in leukocytes and this process was dependent on signaling through uPA receptor. In turn, survivin was required for expression of uPAR. Additionally, survivin was essential for uPA production in MRC-5 and synovial fibroblasts. Down-regulation of survivin with siRNA was followed by significantly reducion of uPA synthesis. Finally, treatment with downregulation of survivin by siRNA in vivo efficiently abrogated uPA-induced arthritis in mice model.

These findings indicate that survivin is an essential mediator of arthritogenic properties of uPA regulating its synthesis in synovial fibroblasts and uPAR expression in leukocytes. Close correlation between survivin and uPA in patients with RA supports the improtance of these interaction for the pathogenesis of arthritis. Upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type B (ITPKB) phosphorylates Inositol (1, 4, 5) trisphosphate (Ins(1,4,5)P 3 ), a calcium-mobilizing second messenger with pleiotropic effects. ITPKB inactivation leads to severe T cell deficiency, altered thymo-independent B cell responses and neutrophil hyperactivation. We here report that ITPKB-deficient (ITPKB -/-) mice also display profound alterations in mast cell development and function. Indeed, while mast cell number, c-kit and FcepsilonRI expression were comparable in ITPKB-deficient and proficient mice, ITPKB -/mast cells were almost completely devoid of granules. This phenotype could be partially reversed upon treatment with sodium cromoglycate. Nevertheless, FcepsilonRI or c-kit activation on mast cells led to increased Ca 2+ responses and to stronger ERK phosphorylations. However, ITPKB -/mice displayed an attenuated sensitivity to IgE-mediated passive systemic anaphylaxis, correlated to the absence of FcepsilonRI-dependant histamine release and to downregulation of H1 and H2 receptor expression due to high basal histamine concentrations. Production of neosynthesized mediators remained normal. Finally, ITPKB deficiency also severely impaired SCF-induced mast cell differentiation in vitro. Taken together these results demonstrate that ITPKB is a key regulator of mast cell activation. ITPKB antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. The large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. IL-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. IL-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. Caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. We have used a subset of the TRC lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. We were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. After the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of IL-1b by THP-1 cells after a 24h challenge with PFA-fixed E. coli, as measured by ELISA. Knockdown levels were analyzed by qRT-PCR for the most significant candidates to validate the phenotypes observed. Exon array analysis are being performed to identify possible targets of the most significant Splicing factor candidates obtained by the shRNA screening in order to dissect their mechanism of action in the regulation of the Inflammasome and IL-1b secretion. Tissue transglutaminase (TG2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. We have previously described the key role of TG2 in cystic fibrosis (CF), a genetic disease characterised by chronic lung infections and inflammation. In CF, mutation on the CFTR gene results in an increased TG2 expression and activity leading to functional sequestration of the anti-inflammatory PPARg and increase of the classic parameters of inflammation.

Here we tested whether in vivo inhibition of TG2 can reverse inflammation in chronic inflammatory diseases. To assess the importance of TG2 not just in CF but in chronic inflammatory diseases in general, we injected cystamine, a potent TG2 inhibitor, in a transgenic mouse model CF and in the TAZ10 transgenic mice that spontaneously develop autoimmune thyroiditis. Intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the CF model, where it decreased TG expression and activity. The treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional PPARg. Interestingly, cystamine injections could also block inflammation in the TAZ10 TCR transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of TG2 in generating inflammation in two very different pathologies. This work underlines the critical role of TG2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. Angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. Angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. We have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated DC, A-DC) display potent angiogenic activity in vivo which is mediated by the release of biologically active VEGF-A.

Here, we investigates the molecular mechanisms leading to VEGF-A secretion in LPS+PGE 2 stimulated A-DC. Preliminary results indicate no accumulation of HIF-1alpha in A-DC, therefore suggesting that VEGF-A is induced by a non-classical, HIF-1alpha independent pathway. In addition, we found that VEGF-A secretion depends on the activation of MAPK p38 but not ERK1/2 or JNK. Inhibitor studies, nascent transcript analysis and Polimerase II recruitment on the promoter show that the induction of VEGF-A is largely due to new transcription and not to changes in mRNA stability. Chromatin immunoprecipitation studies aimed at the characterization of the modifications of VEGF-A regulatory regions in A-DC and at the identification of transcription factors bound to VEGF-A promoters are being performed. This will possibly allow the description of novel transcription factors involved in VEGF-A activation in A-DC. Wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. They are best known for their role in embryonic development and tissue homeostasis. Deregulation of Wnt signaling has been shown to promote carcinogenesis. Recently we identified Wnt signaling to be involved in the regulation of inflammatory processes: Wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines IL-12 and IFN-gamma. To gain deeper insights into Wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous Wnt homologs. We monitored Wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by Real-Time PCR and measured the secretion of inflammatory cytokines by ELISA. Exogenous Wnt5a -but not Wnt3a -was able to induce cytokine expression in primary macrophages. In infection experiments Wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. In contrast, addition of Wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. These data corroborate our previous findings and further support the notion that TLR/NF-kappaB and Wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected Infection of immunocompetent mammals with T. gondii induces a chronic infection of the brain. T. gondii cysts persist in neurons and escape elimination by the immune system. In immunodeficient individuals, the infection can be reactivated resulting in a lethal Toxoplasma encephalitis (TE). In TE, the parasite is primarily controlled by infiltrating T and B cells. Also brain resident cells may contribute to control of the disease and However, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic TE are largely unknown.

In a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of TE. In the present study, we analyzed the function of neuronal gp130 in TE. After infection with T. gondii, mice lacking neuronal gp130 (Synapsin-Cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. Death of Synapsin-Cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. Additionally, TE of Synapsin-Cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. In vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with T. gondii or stimulation with TNF as compared to gp130 expressing neurons. Interestingly, the intracerebral parasitic burden was not increased in Synapsin-Cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function.

T. Objectives: Persistent production of TNFa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. Targeting TNFa with neutralising antibodies or TNF receptor fusion proteins is often, but not always, an effective therapy. High serum concentrations of TNFa, IL-1b, IL-6 and IL-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of IL-4, IL-10 and TGFb are reduced. This suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. Thus, understanding the homeostatic control of TNFa by TGFb1 further may generate more effective therapies. As TNFa mRNA 3' untranslated region (UTR) contains an AU-rich element (ARE), which targets mRNAs for degradation, we wished to test whether TGFb1 suppresses TNFa protein production by upregulating the RNAbinding protein FXR1, which can bind to TNFa mRNA and inhibit translation. Methods: Using RAW 264.7 cells and mouse bone-marrow derived macrophages stimulated with LPS and TGFb1, we assessed mRNA expression by Q-PCR and TNFa protein expression by flow cytometry. The 3'UTR of TNFa mRNA was isolated and inserted into a luciferase reporter vector on a constitutive promoter. Transfected RAW cells were treated with LPS and TGFb1 and luciferase expression was quantified. Cells treated with LPS and TGFb1 were also examined for FXR1 expression using PCR and western blot. Following FXR1 knockdown using siRNA, the influence of TGFb1 and LPS on TNFa protein production was examined by flow cytometry. Results: We find that while TNFa mRNA expression remains constant, LPS induced TNFa protein expression is suppressed by TGFb1. Using the luciferase-TNF-3'UTR vector we show that TGFb1 targets the 3'UTR of TNFa. Furthermore, TGFb1 and IL-10 both upregulate FXR1 mRNA and protein; and treatment with TGFb1 and LPS can synergistically upregulate mRNA expression, more than TGFb1 alone. Following siRNA inhibition of FXR1, TGFb1 can no longer inhibit LPS-induced TNFa production. CoMTb up-regulated MMP-1 and MMP-3 secretion from SAECs, NHBEs and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. Interleukin-17 augmented CoMTb-stimulated up-regulation of MMP-1 and MMP-3 secretion from SAECs and fibroblasts in a synergistic manner. In contrast, Interleukin-17 down-regulated MMP-9 secretion from SAECs by 50 %. Interleukin-22 up-regulated MMP-1 and MMP-3 secretion from fibroblasts but not from SAECs. TIMP1 secretion from SAECs was enhanced by Interleukin-17 but there was no effect of Interleukin-22. MMP up-regulation by Interleukin-17 and CoMTb was inhibited by the Pi3Kinase inhibitor LY294002 and on western analysis AkT (Protein Kinase B) was phosphorylated at 30 minutes. Chemical inhibition of the P110d isoform of PI3Kinase with IC87114 abrogated the IL-17 and CoMTB driven secretion of MMP-3 from the small airway epithelial cells. Chemical inhibition of the tumour suppressor phosphatase, PTEN (phosphatase and tensin analogue on chromosome 10) accentuated MMP-3 secretion. These inhibitory effects were confirmed with siRNA. MMP-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. In summary, Interleukin-17 and Interleukin-22 drive transcription dependent MMP-1 and MMP-3 secretion from airway epithelial cells and fibroblasts. Interleukin-17 also increases TIMP but down-regulates MMP-9 gene expression and secretion. This may contribute to the matrix degrading phenotype in tuberculosis. The PI3Kinase pathway is central in Interleukin-17 driven tissue destruction in the context of M. tuberculosis infection.

V. Delgado-Maroto 1 , L.S. Moreira 2 , E. Gonzalez-Rey 2 , M. Delgado 1 1 Institute of Parasitology and Biomedicine Lopez-Neyra , CSIC, Granada, Spain, 2 University of Seville, Sevilla, Spain

Objectives: Atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. Over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. Atherosclerosis will probably become the most common cause of death within 15 years. One of the initial hallmarks of the disease is the uptake of oxLDL particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. T cells undergo activation after interacting with foam cells, which process and present local antigens including oxLDL, generating a T helper 1 response. Cholesterol metabolism is regulated by factors such as PPARg1 (proliferator activated receptor g), SRB1 (a class B scavenger receptor), CD36 or ABCA1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. Expression of these factors depends on intracellular cAMP. Adrenomedullin (AM), Urocortin (UCN) and Vasoactive intestinal peptide (VIP) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. They are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit Th1 immune response. Their mechanism of action involves binding to GPCR and adenylate cyclase activation with subsequent increase of cAMP in the cell, which is recognized as an anti-inflammatory response. Methods: We investigate AM/UCN/VIP effect on bone marrow-derived macrophages stimulated with oxLDL. We determine the levels of PPARg1, SRB1, CD36, and ABCA1. We also analyze the cholesterol metabolism of oxLDL-stimulated macrophages after neuropeptides incubation using Oil Red O staining of lipids drops and tritium labelled cholesterol. Objective: Endovascular Aortic Repair (EVAR) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. However up to 60 % develop a Systemic Inflammatory Response Syndrome (SIRS), resulting in prolonged convalescence. As yet there is no satisfactory explanation to this severe response. Previous studies have shown a high level of IL-6 in the mural thrombus lining the aneurysm. The thrombus is manipulated during the procedure, but whether or not it is the source of circulating IL-6 and/or other cytokines during and after the operation is unknown. Methods: Quantitative analysis of the pro-inflammatory cytokines IL-6, TNF-a, IL-1b, IL-8 and IL-12, and the anti-inflammatory cytokine IL-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma IL-23 was performed using the Luminex platform. The cytokine levels were compared to the clinical response, in terms of SIRS. Results: EVAR induced the production of IL-6 and IL-10, and in some cases, of IL-23. The maximal plasma levels of IL-6 and IL-10 were found at 24 hours and of IL-23 between 48-72 hours. Modest plasma levels of IL-8 were also observed, with maximal production at various time points (4-24 hours). By contrast, production of TNF-a, IL-1b and IL-12 did not occur to a significant extent, while production of IL-17 occurred sporadically. Although our preliminary data indicate that SIRS is associated with enhanced cytokine responses, the production of IL-6, IL-8, IL-23 and IL-10 also took place in patients who did not develop SIRS. Conclusion: EVAR is associated with the sequential production of IL-6, IL-10, IL-8 and IL-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of SIRS; but the production seems to be exaggerated in patients developing SIRS. Further studies involving 165 patients are in progress, and will clarify this. We hypothesize that SIRS is elicited by IL-6, activated by manipulation of the mural thrombus. To reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. IL-33 is a novel IL-1 cytokine family member that is expressed as an intracellular precursor (pro-IL-33) and is thought to be cleaved by Caspase-1 to yield a mature bioactive form of the molecule (mat-IL-33). To date however, evidence of cell-associated proteolytic processing and Caspase-1 dependent secretion of mat-IL-33 has not been reported. Here we show that pro-IL-33 but not mat-IL-33 is released from UVB-irradiated keratinocytes. We demonstrate binding of pro-IL-33 to the IL-33R and also IL-33R-dependent bioactivity of pro-IL-33 on mast cells. We propose a previously unrecognized role for pro-IL-33 as a pro-inflammatory mediator and suggest a direct link between UVB-mediated epithelial cell damage and cutaneous mast cell activation. We have previously shown that induction of ER stress and TLR signalling synergistically enhance IL-23 p19 mRNA expression in myeloid cells, and markedly increase secretion of IL-23, but not IL-12, by dendritic cells. The aim of this study is to investigate the mechanism of this synergy. We examined the IL-23 promoter for potential binding sites for ER stress induced transcription factors and identified a putative site for CHOP10. Chromatin immunoprecipitation (ChIP) assays using anti-CHOP10 and isotype control Mab were performed using nuclear lysates from U937 cells and IL-23 promoter DNA measured by qPCR. CHOP10 binding on the IL-23 promoter was detected following stimulation of U937 cells with LPS or TP alone, but this was significantly enhanced when ER stress and TLR stimuli were combined. IL-23 promoter DNA was not detectable following ChIP with the isotype control antibody. To confirm the role of CHOP10 in IL-23 gene transcription, U937 cells expressing shRNA's specific for CHOP10 or non-specific gene target were tested for their ability to express IL-23 following TLR and ER stress stimulation. U937 expressing three independent shRNA targets for CHOP10 exhibited significant reductions in IL-23p19 mRNA (up to 87 % reduction of the response to LPS+TP) compared to U937 expressing a control shRNA. CHOP10 shRNA expression did not affect the expression of other LPSresponsive genes, including IL-1, IL-8, CCL3 and SOD2.

To identify if ER stress induction of IL-23 mediated by CHOP10 expression plays a role in a more physiological setting, we examined the role of CHOP10 in the induction of IL-23p19 gene expression following Chlamydia trachomatis (CT) infection. Infection of U937 cells with live but not g-irradiated CT induced expression of ER stress response genes, including CHOP10. U937 infected with live CT exhibited increased IL-23p19 mRNA expression compared to U937 infected with nonviable bacteria. CHOP10 silencing significantly reduced the ability of live CT to induce IL-23p19mRNA, confirming the important role of CHOP10 in this response. These data suggest that ER stress induction of CHOP10 could contribute significantly to the pathogenesis of diseases in which IL-23 plays an major role, through induction of IL-17 and IL-22 producing cells.

The clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. Here, we show that Gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. Gadd45b expression was inducible in different in vitro and in vivo models of negative selection. Strikingly, only TCR-ligating peptides resulting in negative selection induced Gadd45b expression, while positively selecting ligands or dexamethasone, a TCR-independent apoptosis agonist, failed to do so. Expression of Gadd45b maintained a sustained activation of p38 kinase and thereby promoted TCR-mediated apoptosis. In contrast, thymocytes from Gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. Interestingly, we observed a switch to positive selection in Gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. Moreover, markers of positive selection as CD5 and CD69 were elevated on GADD45b-deficient thymocytes. Thus, we provide evidence that Gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. These results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. Di Santo 1 1 Institut Pasteur, Paris, France, 2 Monash University, Victoria, Australia > The thymus represents the ''cradle'' for T cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. Although IL-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic IL-7 niche remains poorly characterized in vivo. > Using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (YFP) is under control of IL-7 promoter, we identify a subset of thymic epithelial cells (TECs) that co-express YFP and high levels of Il7 transcripts (IL-7 hi cells). IL-7 hi TECs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (Ccl19, Ccl25, Cxcl12) and cytokines (Il15) that are critical for normal thymopoiesis. In the adult thymus, IL-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature TECs. Interestingly, the frequency of IL-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining IL-7 levels. Conversely, the frequency of IL-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in T cell development. This augment indicates that thymocyte-TEC crosstalk may condition IL-7-expression by TECs. > Together, our temporal-spatial analysis of IL-7-expressing cells in the thymus suggests that thymic IL-7 levels are dynamically regulated under distinct physiological conditions. This novel IL-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic IL-7 expression in vivo. Two lines of evidence have recently demonstrated that the pre-B cell receptor (pre-BCR) is associated with autoimmunity, through its surrogate-light-chain (SLC) components l5 and VpreB. It has been shown that pre-BCRs are polyreactive for several self-antigens. The polyreactivity of the pre-BCR induces pre-BCR signaling and activation. Furthermore, in human a self-reactive B cell subset was identified that co-expresses immunoglobulin light chain (Ig LC) and the SLC components. These VpreB + LC + B cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like SLE and RA. To elucidate the contribution of pre-BCR components to the development and activation of autoreactive B cells, we have recently generated a SLC transgenic (SLC-Tg) mouse model in which all B cells express SLC proteins. SLC-Tg mice exhibit spontaneous IgM + plasma cell development. Moreover, aging SLC-Tg mice have elevated anti-nucleosome IgM levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. Nevertheless, VpreB + LC + B cells may induce pathology when self-tolerance mechanisms fail. To test this hypothesis, SLC-Tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) Em-Bcl2-Tg mice with B cell-specific overexpression of the anti-apoptotic protein Bcl2 and (ii) FcgRIIb-deficient mice. Both in young SLC-Tg;Em-Bcl2-Tg double Tg mice and in SLC-Tg;FcgRIIb -/mice spontaneous germinal center (GC) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control Em-Bcl2-Tg and FcgRIIb -/mice. In SLC-Tg;FcgRIIb -/mice, numbers of splenic IgG2 plasma cells and serum IgG2 levels were˚5-10 fold increased. Importantly, serum from young SLC-Tg;Em-Bcl2-Tg and SLC-Tg;FcgRIIb -/mice contained high titers of IgG auto-antibodies in 2/3 and 6/6 cases, respectively. These values were increased when compared with control groups: 1/6 in FcgRIIb -/and 0/6 in Bcl2-Tg. Finally, we found that the collagen induced arthritis (CIA) was significantly enhanced in SLC-Tg;FcgRIIb -/mice, compared to FcgRIIb -/mice. Taken together, these findings demonstrate the SLC components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in RA. T cells are generated from progenitor cells that enter the thymus from bone marrow via blood. These progenitor cells once within the thymus have little selfrenewal capacity. Differentiation from hematopoietic stem cells to early T lineage cells proceeds through a series of intermediate precursor populations. However, it is largely unknown to what extent these cell populations contribute to T cell development in the presence of other precursor populations and how the earliest intrathymic T cell progenitors are generated from extrathymic precursors.

To assess the relative contribution of potential precursors to T lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. Thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological T cell precursor or many. We showed that, under such competitive conditions, T lineage progenitors are confined to the CD27 + CD135 + fraction of bone marrow cells. Notably, T lineage reconstitution was not restricted to either CD117 hi cells, representing multipotent progenitors, or CD127 + cells, representing common lymphoid progenitors, both of which contributed to T lineage differentiation with different kinetics. In conclusion, our data suggest that multiple physiological extrathymic T cell precursors exist, which are able to compensate for the loss of depleted populations. Thus, our findings may have implications for devising strategies for improved T lineage reconstitution after hematopoietic stem cell transplantation.

Background: Previous results from our group have demonstrated EphB2 and EphB3 expression on both thymocytes and thymic epithelial cells (TECs). We used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and TEC development, and how they regulate interactions between both cell types. Objectives: In order to better define the importance in thymus of Eph-Ephrin B interactions we have analyzed the effects of the lack of Ephrin B1 and/or Ephrin B2, the ligands of EphB2 and EphB3 receptors. This approach is specially interesting taking into account that Eph-Ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. Methods: For this purpose we have used Cre-LoxP recombination systems for deleting EphrinB1 or EphrinB2 genes specifically on either thymocytes or TECs. Results: Animals with Ephrin B deficient thymocytes showed thymic hypocellularity and alterations on T-cell development whose severity depended on the background of the analyzed mice. In these mice only a few changes occurred in the cortical TEC network. On the contrary, mice with conditioned deletions in TEC, especially EphrinB1/B2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of CD45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. In addition, T-cell development was partially blocked at the DN cell stage. Conclusion: These data reveal an autonomous and non-autonomous role for EphrinB1 and EphrinB2 in the development of both T cells and TECs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. We discuss how Eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in T cell differentiation.

A. Rolink 1 , D. Vanhecke 1 1 University of Basel, Developmental and Molecular Immunology, Basel, Switzerland

The importance of normal T lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of T cells is defective. In order to identify molecules/genes and elucidate developmental processes that are essential for human T cell development we use a novel in vitro tool, the OP9-DL1 cell culture system (1) . Using this in vitro assay we obtain large numbers of human cyCD3 + and CD4 + CD8 + double positive thymocytes starting from Umbilical Cord Blood (UCB) derived CD34 + HSC. Signals and molecules that are involved in T cell development are being addressed by using blocking antibodies and/or chemical inhibitors. Similar as in mice we found an essential role for IL-7 and Notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. Among the molecules that are rapidly induced in CD34 + cells upon Notch signaling is CD7 followed by CD127. T cell specification is accompanied by the induction of CD1a and loss of CD34 on CD7 + CD127 + cells. These CD34 -CD1a + CD7 + cells become dependent on continuous IL7 and Notch signaling for sustained survival and further differentiation into CD4 + CD8ab + DP thymocytes. We found that Flt3L is not essential for the differentiation of CD4 + CD8 + human thymocytes but that addition of exogenous Flt3L in the co-cultures increases the number of CD34 + precursors and consequently result in higher yields of developing CD4 + CD8ab + DP thymocytes. Finally few mature TCRab + T lymphocytes develop from the cyCD3 + CD4 + CD8ab + DP subset in this in vitro assay suggesting that OP9 stromal cells lack the required selecting MHC-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. This in vitro assay will allow us now, by using RNA interference, to test additional genes for their role during human lymphoid development. From a clinical standpoint, a better understanding of the mechanisms controlling human T-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant T-cell disorders. Brain derived neurotrophic factor (BDNF) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. Emerging evidence also indicates an essential role for BDNF in the immune system, e.g. in the B and T cell lineages. We therefore investigated the impact of BDNF on thymocyte development using BDNF knockout (ko) mice and conditional ko mice lacking BDNF specifically in T cells. In both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. In contrast, the percentage of naturally occurring regulatory T cells and the expression of activation markers were unaltered. Moreover, the lack of BDNF did not result in enhanced thymocyte apoptosis. The increase in double negative thymocytes was due to a partial block in the transition from the DN3 to DN4 stage, where BDNF and its receptor p75 are expressed as revealed by real-time PCR. The observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral T cells, their homeostatic proliferation and without compromising peripheral immune responses in general. In summary, our findings point to a critical role of T cell lineage derived BDNF in thymocyte development acting in an autocrine and/or paracrine manner.

R. Berga 1 , C. López-Rodríguez 1 1 Pompeu Fabra University, Barcelona, Spain NFAT5 is a transcription factor that belongs to the Rel family (NF-kB and NFATc proteins). Its expression in primary cells and organs is restricted to certain proliferative tissues, like activated T lymphocytes and thymocytes, where levels of NFAT5 are relatively high and its subcellular distribution is predominantly nuclear. Recent mouse models suggest that NFAT5 participates in thymocyte development and also indicate its involvement in T cell proliferation and survival. NFAT5 deficient mice present a T cell immunodeficiency consistent on lymphopenia, which is more accused for CD8 + lymphocytes. These observations are of substantial relevance as we and others have described that, in vivo, NFAT5-null mice are unable to mount CD4+-and CD8 + -immune responses. Data from our laboratory indicate that NFAT5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. To selectively analyze the T-cell autonomous effects derived from the lack of NFAT5 during the development of T lymphocytes, we developed mouse models that delete NFAT5 at early (Lck-Cre + /NFAT5 flox/flox ) or late (CD4-Cre + /NFAT5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. Our work indicates that NFAT5 is expressed at all stages of T cell development. In addition, analysis of mouse models that lack NFAT5 at different points of T cell development indicate that it participates at early stages of the ontogeny of T cells. Objectives: Apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. We have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. Methods: We compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. Apoptosis was measured by staining with Annexin V in vitro and by detection of caspase activation in vitro and in vivo. We also compared their ability to undergo cell cycle arrest using BrdU staining. Tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. Results: Apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset B-cell lymphoma, but not the thymic T-cell tumors found in p53-/-mice. Interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated B-cells. Bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated B-cells in the bone marrow and spleen.

We propose that the apoptotic function of p53 has an important role in B-cell homeostasis, which, in turn, is important for prevention of B-cell lymphomas Moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic T-cell tumors.

S. Myrczek 1 , R. Pardi 2 , A. Gessner 1 1 Microbiological Institute-Institute for Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen, Erlangen, Germany, 2 Vita-Salute San Raffaele University School of Medicine, Milano, Italy JAB1 is the catalytic subunit of the highly conserved COP9 signalosome. This complex plays a central role in various cellular processes as proliferation and cell cycle control. JAB1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. Furthermore JAB1 regulates the activity of AP1 transciption factors. To date JAB1 is thought to be essential for every cell type as JAB1 knock out mice are embryonic lethal and T cell development is blocked by T cell selective absence of JAB1. To investigate the function of JAB1 in B cells we established a mouse strain deficient for JAB1 selectively in B cells. Mice with floxed alleles of JAB1 kindly provided by R. Pardi were crossed with a mouse strain expressing the Cre recombinase under control of the mb1-locus (M. Reth, Freiburg). Ablation of JAB1 expression resulted in an almost complete block of B cell development at the pro B cell stage. The absence of peripheral mature B1 and B2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with Borrelia burgdorferi. The observed block in B cell development is rescued by over expression of the anti apoptotic protein Bcl2 under the control of the m enhancer. FACS analyses revealed that all B cell subtypes analyzed in the JAB1-deficient, Bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. Serum immunoglobulin titers are detectable and after Borrelia infection specific antibodies are produced. We confirmed the absence of JAB1 in sorted spleen B cells by immunoblot analysis. In summary, we show for the first time that cells are viable and functional without JAB1 when apoptosis is prevented.

T. Nitta 1 , S. Murata 2 , K. Tanaka 3 , Y. Takahama 1 1 University of Tokushima, Tokushima, Japan, 2 University of Tokyo, Tokyo, Japan, 3 Rinshoken, Tokyo, Japan

How self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of T cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive T cells and thereby preventing autoimmunity is well established. A recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (cTEC) and is required for the optimum generation of CD8 T cells. Here we show that cTEC display a thymoproteasome-specific spectrum of class I MHC-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class I MHC-restricted T cells. Indeed, CD8 T cells generated in the absence of thymoproteasomes display a markedly altered TCR repertoire that is defective in both allogeneic and antiviral responses. These results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent CD8 T cells. Defects in helper T cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. Our understanding of the genetic control of helper T cell differentiation into specific functional subsets is still far from complete. Here we present the results to date from a genome-wide ENU mutation screen for mice with inherited deficits in specific helper cell subsets. These deficits were detected by multi-colour FACS analysis of peripheral blood samples, and by antibody production following immunization with heat-killed B.pertussis and CGG coupled with the hapten arsonate (ABA) in alum, which induce internally polarized Th1 and Th2 antibody responses, respectively. Using this screen, a number of new mutant strains have been isolated with complete or partial loss of CD4+ T cells or functional deficits that selectively interfere with Th1 or germinal centre responses. In this talk I will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. Systematic genetic analysis of helper T cell differentiation in the resulting strains will illuminate how T cell help is correctly polarized for immunity and to avoid immunopathology. Intrathymic T-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. In order to highlight the differences and similarities between fetal and adult T-cell development at the molecular level we performed a microarray study. Labelled RNA from FACS purified fetal and adult DN1 c-kit high (ETP), DN2 and DN3 thymocyte populations was hybridised to Affymetrix Mouse 430A-2.0 GeneChips. The resulting data were grouped into four distinct gene clusters: Cluster I contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. Conversely, cluster II consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as Acvr1, BMPR1, Fzd7, chemokine receptors CX3CR1, CXCR6 and integrins a2, a4, a9, aE and aV, pointing to a difference in microenvironments. Genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster III. Amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. On the other hand, cluster IV was limited to genes that were homogeneously up-regulated during development. These included GATA-3, TCF-1, Notch-1, Rag-1, Rag-2 and pre-Ta, which are indispensable for T cell development. Interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the ETP and DN2 stages, suggesting that the molecular program of T-cell development is more advanced in the early stages of fetal differentiation. Discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult DN3 population, therefore implying a more committed state of fetal progenitors. Finally, transcriptional signatures of each developmental stage were defined by "Recursive Feature Elimination" with support vector machines. This approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. TCRgd+ cells and TCRab+CD8aa+ intraepithelial lymphocytes (IELs) of the gut are unconventional T cells that reside in tissues and provide innate-like immune responses to "stressed-self". As these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional T cell developmental pathway based on TCR signal strength at the DN stage. The pre-T-alpha chain (pTa) that pairs with TCRb to generate the pre-TCR, has two isoforms; pTa a and pTa b . Both can form a functional pre-TCR with TCRb. Ligand-independent signalling by the pre-TCR is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pTa chain. pTa b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. We have hypothesised that pTa a and pTa b permit differential signal strength through the pre-TCR at the DN stage, facilitating the divergence of the conventional and unconventional lineages of TCRab+ T cells. Preliminary semi-quantitative PCR data suggest that pTa a and pTa b are differentially expressed in WT thymocytes at different stages of ontogeny. Retroviral transduction of pTa -/-E14 thymocytes with either pTa a or pTa b alone, followed by fetal thymic organ culture, confirmed the rescue of abT cell development by both isoforms. However the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pTa b expression generates a greater percentage of TCRab+ cells while pTa a expression results in the accumulation of ISPs. These results suggest different roles for the two isoforms of pTa in the thymic development of abT cells.

In order to determine the mechanism by which pTa a and pTa b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pTa a and pTa b that lack the "dimerisation residues" necessary for internalisation of the receptors. In addition, BAC transgenic mice that express singly either pTa a or pTa b under the pTa promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and T cells, and precursor of B cells can be detected and counted at day 18 of culture. If needed for further functional analyses, long-term proliferating lines and clones of progenitor T and B cells can be established at this point of "in vitro" development. Hence the "in vitro" differentiation of ES cells to different hematopoietic cell lineages and their progenitors can be quantified. It allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different ES or iPS cells. Aim: Increasing evidence includes Wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems.

Although their precise functions in bone marrow and thymus are still controversial, numerous studies show that Wnt signalling is able to control the proliferation of HSC and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. In the present work we analyse the effect of transient stimulation of canonical Wnt pathway in the differentiation potential of Lin -CD34 + CD1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into T cells, NK cells, monocytes, conventional dendritic cells (cDC) and plasmacytoid DCs (pDCs). Methods: Human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the Declaration of Helsinki. Transient b-catenin stabilization was triggered culturing purified thymic Lin -CD34 + CD1precursors with recombinant Wnt3a (100 ng/ml) or with LiCl (10 mM) for 12hr. Active b-catenin, was detected by flow cytometry using anti-human active b-catenin mAb (8E7) under conditions of phosphatase activity inhibition. Wnt3a or LiCl pre-treated precursor were assayed in chimeric human-mouse FTOC, in IL-15 and SCF-supported cultures for generation of NK cells and in co-cultures with murine bone marrow stromal ST2 cells suplemented with IL-7 and FLT3L. Phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. Expression profile of transcription factors was analysed by real-time quantitative RT-PCR .

Our results demonstrate that giving a boost to canonical Wnt signalling triggered by transient exposure of thymic progenitors to Wnt3a or LiCl, change their differentiation capacity enhancing NK cell production. On the contrary, Wnt3a or LiCl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cDC, as well as reduce their capacity to differentiate into pDC lineage. As a possible mechanism for this effect we show that Wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are IL-7 and FLT3L and modify the transcription factor profiles of CD34 + CD1thymocytes mainly increasing Hes-1 and Id3 expression levels. Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti-proliferative effect of TGF-beta than Th1 and Th2 clones or circulating Th1oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl-2 expression and reduced apoptosis in the presence of TGF-beta, in comparison with Th1 cells. Umbilical cord blood naï ve CD161(+)CD4(+) T cells, which contain the precursors of human Th17 cells, differentiated into IL-17A-producing cells only in response to IL-1beta plus IL-23, even in serum-free cultures. TGF-beta had no effect on constitutive RORgamma t expression by umbilical cord blood CD161(+) T cells but it increased the relative proportions of CD161(+) T cells differentiating into Th17 cells in response to IL-1beta plus IL-23, whereas under the same conditions it inhibited both T-bet expression and Th1 development. These data suggest that TGF-beta is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.

M. Irla 1 , W. Reith 1 1 University of Medecine, Pathology and Immunology, Geneva, Switzerland

Objectives: Medullary thymic epithelial cells (mTECs) are specialized for inducing central immunological tolerance to self-antigens. To accomplish this, mTECs must adopt a mature phenotype characterized by expression of the autoimmune regulator Aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. The mechanisms that control mature Aire(+) mTEC development in the postnatal thymus remain poorly understood. However, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and TEC control not only T cell development but also the differentiation and organization of TEC, a phenomenon designated as 'crosstalk'. The aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mTEC, key mediators of central tolerance.

We have demonstrated by generating different transgenic mouse models, that although either CD4(+) or CD8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mTEC population requires autoantigen-specific interactions between positively selected CD4(+) thymocytes bearing autoreactive T cell receptor (TCR) and mTECs displaying cognate self-peptide-MHC class II complexes. These interactions also involve the engagement of CD40 on mTECs by CD40L induced on the positively selected CD4(+) thymocytes. Conclusion: This antigen-specific TCR-MHC class II-mediated crosstalk between CD4(+) thymocytes and mTECs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mTEC population competent for ensuring central T cell tolerance.

Q. Qiu 1 , I. Ravens 1 , G. Bernhardt 1 1 Hannover Medical School, Institute of Immunology, Hannover, Germany CD155 is originally identified as human poliovirus receptor (PVR) and as rodent TAGE4, which is overexpressed in rodent colon carcinoma. CD155 is also known as NECL-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. CD155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. In addition, CD155 is overexpressed in transformed cells and promotes the cell cycle. Thus, CD155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. T-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (DN), double positive (DP) and single positive (SP) based on expression of the co-receptors CD4 and CD8; the DN subset is further subdivided into four stages (DN1-4) by differential expression of CD44 and CD25. Thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. During their final maturation in the medulla, semi-mature SP thymocytes down-regulate activation markers and subsequently exit into periphery. While semimature CD4+ SP are sensitive to negative selection, it remains elusive when negative selection occurs in the CD8 lineage. Here we show that the frequency of terminally matured CD8+ SP cells but not that of CD4+ SP present in thymus varies depending on age. In mice lacking expression of the adhesion receptor CD155, a selective deficiency of mature CD8+ SP thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. Evidence is provided that the mature cells emigrate prematurely when CD155 is absent thus cutting short their retention time in the medulla. Moreover, in unmanipulated wild type mice semi-mature CD8+ SP thymocytes are subjected to negative selection as reflected by the diverging T cell receptor repertoires present on semi-mature and mature CD8+ T cells. In CD155 deficient animals, a shift in the TCR repertoire displayed by the pool of CD8+ SP cells was found demonstrating that CD155 is involved in negative selection. In the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. Yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. The analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (Passegué et al. J. Exp. Med. 2005 , 202, 1599 . During development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. By using complementary, quantitative analyses (BrdU labelings, Hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from E9 (E, gestational day) on, in yolk sac, splanchnopleura/AGM, blood, liver, spleen and bone marrow. We have observed that around half of CD71 + Ter119 + erythroid and CD45 + CD11b + myeloid cells are simultaneously cycling (S/G2/M) in the post-gastrulation mouse embryo (E10-12). The peak of lymphohematopoietic cell proliferation occurs at E13 in a sort of wave-like pattern. These high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. Later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. Interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. We are also analysing the proliferation state of the first B and T lymphoid progenitors emerging at E11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. We attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. Objectives: The role of CD40-CD154 interactions in T cell activation of antigen presenting cells and B cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. Following an initial discovery that B lineage cells in the bone marrow (BM) as early as pro-B cells express CD40, we hypothesised a role for CD40-CD154 interactions in the control of B cell haematopoiesis. The objectives of this study were to investigate this hypothesis further. Methods: Flow cytometry was used to investigate CD40 expression by precursor B cells using B cell specific markers. Reverse transcription of BM stromal cell RNA and PCR were used to assess the presence of CD154 message and cell lineage specific mRNA. Irradiation and bone marrow transplantation (BMT) in both directions between CD154-/-and WT mice was used to assess potential functional contributions of stromal or haematopoietic CD154 on reconstitution of B cell numbers following depletion.

We show that CD40 is expressed by pro-B cells, and these cells proliferate in response to CD40 signalling in vitro. PCR identified a source of CD154, negative for CD3eta, in the BM of WT mice showing this CD154 is not provided by activated re-circulating T cells. We have shown that when CD154 -/-mice are recipients, but not donors of BMT, B cell recovery after irradiation is significantly delayed regardless of the donor cell source. In the in vitro experiments we found that the pTa gene is expressed from the DN1 (CD4 -/CD8 -/CD44 + /CD25 -) to the DP (CD4 + /CD8 + ) stage, whereas no YFP expression could be observed in the B lineage. The in vivo analysis of thymocytes confirms the appearance of YFP positive cells during T cell development from the DN1 stage on. In the bone marrow we found YFP + /B220 + and YFP + /B220populations. Thus these pTa expression analyses show closely similar pattern to those observed with huCD25 preTa-reporter transgenic mice (Gounari F. et al. 2002 , Martin et al. 2003 . The BAC pTa reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis.

Gounari F et al., Nat. Immunol. 3, 489-496 (2002 ) Martin C. H. et al., Nat. Immunol. 4, 866-873 (2003 . The individual functions and the reason for the tightly regulated expression of IgM and IgD during B cell development are poorly understood. Our data show, that IgD requires stronger stimuli than IgM to induce B cell activation and that this silences autoreactive VDJ recombination products when expressed as IgD. In agreement with this, mHC and dHC, the respective heavy chains of IgM and IgD, differ dramatically in pre-BCR signaling, which represents the prototype of an autoreactive receptor. Together with published data, our results reveal a novel role for IgD and suggest that the differential expression of IgM and IgD is important to raise the activation threshold of mature B cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens.

P. D. Rymkiewicz 1 , G. Klein 1 1 ZMF (Center for Medical Research), Section for Transplantation Immunology and Immunohematology, Tübingen, Germany

Thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. The core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type IV, nidogens and perlecan. A marker molecule for the conduits in the human thymus is the laminin isoform LM-332 which is synthesized by the medullary thymic epithelial cells (TECs) which tightly surround the conduits. Functionally the conduits are too small to transport cells but they are able to transport small molecules X 70kDa.MMP-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting LM-332. In the human thymus medullary, but not cortical thymic epithelial cells strongly express MMP-19. By Western blotting the zymogen and the activated form of MMP-19 can be detected in whole thymus lysates, whereas in lysates from isolated TECs mainly the activated form is present. An in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. Using confocal laser scanning microscopy double immunofluorescence staining showed that LM-332 and MMP-19 can be found in close neighbourhood, but they do not exactly co-localize. Why activated MMP-19 which can be secreted by medullary TECs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. Two natural inhibitors of MMP-19, TIMP-2 and TIMP-3, are found in the thymic medulla, but they are not expressed and secreted by TECs. Whether MMP-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. To study the process of T cells differentiation in more detail, we intend to establish an inducible gene expression system (Tet-On system) in primary T cells. The Tet-On System comprises two retroviral vectors. The response vector contains an inducible modified minimal CMV promoter which per se is unable to induce expression of the gene of interest (GOI). The second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. In this state, the transactivator enables the minimal CMV promoter to transcribe the gene of interest. Therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest.

To date we have tested two Tet-On Systems (RevTet System and Retro-X Tet-On Advanced Inducible Expression System) that differ in the sequence of their inducible promoters. To monitor successful transfection in retrovirus-generating Phoenix cells and transduction in T cells, respectively, we have cloned the reporter gene GFP under the control of a constitutive CMV promoter, into the response vector of the RevTet System. This allowed identification of transduced GFP-positive cells via FACS. However, when we used a red fluorescent protein, tomato, as a surrogate GOI, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. In contrast, we found comparably low leakiness when using the Retro-X Tet-On Advanced Inducible Expression System. Here, co-transfection of Phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. Therefore, the Retro-X Tet-On Advanced Inducible Expression System appears suitable for our studies. Future experiments will aim at establishing this system in primary T cells. Although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (GCs) on T-cell development, and thereby T-cell-mediated immune response, their relevance for these processes is still far from being understood. The study was undertaken to explore relevance of basal levels of GCs for T-cell differentiation/maturation. Eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by TCRab/CD4/CD8 expression were examined using flow cytometry analysis. It was found that adrenal GC deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of T-cell differentiation/maturation leading to an overrepresentation of the CD4+CD8+ double positive (DP) TCRab low cells entering selection, and their CD4+CD8+ DP TCRab-immediate precedents followed by underrepresentation of the selected CD4+CD8+ DP TCRab high and the most mature CD4-CD8+ and, particularly, CD4+CD8-single positive (SP) TCRab high cells.

The study suggests that withdrawal of adrenal GCs produces alteration in thymocyte selection processes that may affect diversity of functional T-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of CD4-CD8-double negative TCRab high cells. In addition, it indicates that GCs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature CD4+CD8-/CD4-CD8+ SP TCRab high cell ratio. In the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mTECs) -express a highly diverse set of tissue-restricted antigens (TRAs) representing essentially all tissues of the body, which is known as promiscuous gene expression (pGE). This allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing T cells.

The scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. Thus, even lack of a single TRA can result in spontaneous organ-specific autoimmunity. Promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for TRAs. For better understanding these features, we set out to precisely define the genomic organization of this gene pool. In particular, we probed to what extent and according to which rules predefined genomic clusters of TRAs are transcribed in mTECs. Our analysis proceeded from the bioinformatic definition of TRA clusters via gene expression analysis in mTECs using whole genome arrays to the in depth analysis of selected TRA clusters by RT-PCR at the population and single cell level. Patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. Conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pGE and adaptive immunity. Although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (AR) blockade failed to show any significant effects on thymic cellularity. Bearing that in mind, the present study was undertaken to explore: i) a 1 -AR expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -AR-mediated mechanisms in modulation of thymic cellularity and T-cell development.

For this purpose a 1 -AR expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (CD4/ CD8/TCRab), in adult Wistar rats subjected to 14-day-long treatment with a 1 -AR blocker urapidil (0.20mg/kg body weight/day s. c.). The a 1 -AR immunoreactivity was found in both thymocytes (mainly less mature CD3and CD3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ED1-postive cells, which comprise macrophages and dendritic cells). Chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. The thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. In addition, in these rats changes in distribution of major thymocyte subsets delineated by CD4/ CD8/TCRab expression were observed. These changes comprised of an increase in the percentage of CD4+8+ TCRabthymocytes, which was accompanied by the reduction in that of CD4+8+ TCRab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive TCRab high thymocytes. Compared with saline-administered controls, the percentage of CD4+8-TCRab high thymocytes in urapidil-administered rats was increased, while that of the CD4-8+ TCRab high was reduced. In addition, the percentage of CD4+ T regulatory and CD161+TCRab+ NKT cells was increased.

Collectively, this study clearly showed the expression of a 1 -AR on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -AR-mediated mechanisms may be implicated in modulation of multiple steps of T-cell development. 

We have recently shown that the thymus is a common target for mycobacterial infections. Of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with Mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. This observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. Since T cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. Methods: C57BL/6 mice infected with M. avium (10 6 CFUs, IV) were sacrificed at different time points after infection (5, 16 and 25 weeks). Non-infected animals were used as controls. Bacterial load was assessed in the spleen and thymus and cytokines (such as IFN-g, TNF, IL-10) were quantified by RT-PCR in tissues (normalized for HPRT expression) or by ELISA in the supernatants of cultured thymocytes and splenocytes. Statistical significances were determined by ANOVA.

We observe increased levels of IFN-g in infected thymi at 16 weeks post infection. This increased expression of IFN-g is concordant with the late bacterial growth arrest within the thymus. At 24 weeks post infection this difference is still present. Throughout the course of infection no significant differences are found in the expression of TNF and IL-10 in this organ. In the spleen, IFN-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased TNF expression. Conclusion: Our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. Since the cytokine milieu influences T cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. Such implications should next be investigated. Precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening DNA, but how trans-acting regulatory proteins work to establish and maintain DNA loops during gene activation remains largely unexplored. LPS-induced transcription of the mouse Igx gene in B lymphocytes utilizes three distal enhancers and requires the transcription factor NF-xB, whose family members include RelA and c-Rel. Using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that LPS-induced Igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and RNA polymerase II, the apparent molecular tie for the bases of these loops. RelA and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-Rel. We have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during Igx gene activation. This investigation was supported by Grants GM29935 and AI067906 from NIH and Grant I-823 from the Robert A. Welch Foundation to WTG, and by Grants HL067256 and HL61897 from NIH to LST. Allelic exclusion of immunoglobulin (Ig) genes supports Burnet's clonal selection theory. The recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the RAG complex. Attempts to prove the probabilistic or epigenetic nature of monoallelic V(D)J recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or X-chromosome inactive genes, Ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (Mostoslavsky, Singh et al. 2001) . We are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each B cell progenitor, which of the Ig alleles is going to be activated for rearrangement and expression. For this, we are generating B cell clones from B cell progenitors of (C57BL/6 X Balb/C)F1 mice, because the original strains have Ig heavy chain (Igh) alotypes distinguishable by monoclonal antibodies. We are analyzing if individual heterozygous clones show biased expression of a particular Igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. We have already shown, for the Igh gene, a clear segregation of monoallelic expressors among B cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell B cell clones. This result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. We are currently exploring this result by the analysis of the Igh silenced allele rearrangement status in sorted fractions of IgM+ B cell clones. We are also testing the same epigenetic hypothesis for the Ig kappa light chain gene (Igk), using F1 mice in which the Igk constant region from both alleles can be distinguished by antibodies.

A. Giniewski 1 , S. Lang 1 , M. Stein 1 , T. Winkler 1 1 Friedrich-Alexander University, Erlangen, Germany VDJ recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to RAG recombinase. Accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. H3Ac) or methylation of H3 on lysine 4 (H3K4Me2/3). Previous studies in our lab revealed three regions in the intergenic part of the distal V H cluster (IVARs), which are associated with high levels of active chromatin marks (H3Ac and H3K4Me2/3) only in pro B cells but not in pro T cells.

It is also known that VDJ H recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. Since one IVAR (IVAR #3) shows promoter activity in antisense orientation, it was analysed in more detail. We found three transcription start sites by 5'RLM RACE at the IVAR #3 element. Background: T-ALL is a malignancy of the lymphoblast committed to the T-cell lineage with translocations between TCR genes and oncogenes as a genetic hallmark. These translocations are thought to be driven by V(D)J-recombination mechanisms. We believe that these mechanisms only partly facilitate the occurrence of TCR translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. The LMO2 locus, is thought to be accessible only during the double-negative (DN) 1 and 2 thymocyte stages based on mRNA expression, implying that translocations between LMO2 and TCR genes can only occur within these stages. Gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as TLX1 (HOX11), which are not expressed in thymocytes, as being accessible for translocations to occur. Objectives: We aimed 1) to evaluate LMO2 and TLX1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for LMO2 or TLX1 translocation to occur based on this accessibility. Methods: DNA of Immunologically "healthy" sorted thymocytes was isolated using FAIRE (Formaldehyde-Assisted Isolation of Regulatory-Elements). This DNA was used to quantitatively assess LMO2 accessibility during thymocyte development at both the transcription start site (TSS) and negative regulatory element (NRE), and within different in T-ALL documented breakpoint-sites of both the LMO2 and TLX1 loci. Results: Quantitative analysis on the TSS showed a correlation with mRNA expression, with the DN1 and DN2 development stages showing the highest accessibility. The NRE, showed an inverse pattern of accessibility to the TSS region. Analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, DN1, DN2, DN3 and immature single positive (ISP) stages for both LMO2 and TLX1. Conclusion: Our findings show that both the LMO2 and TLX1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to DN1 and DN2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. We addressed this issue, by building a model able to account for of V-Ja gene rearrangements observed experimentally during thymus development of mice. We developed, based on experimental data, a numerical model on the whole TRA/TRD locus to estimate Va and Ja genes accessibility to rearrangements. The progressive opening of locus to V-J gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. Furthermore, the possibility of successive secondary V-J rearrangements was introduced in the modeling. The model points out some unbalanced V-J associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the J genes, depending on their location in the locus. The model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the V and the J genes of the locus. Finally, the model provides information on the kinetics of rearrangements and on the frequency of each V-J association. The model accounts for the essential features of the observed rearrangements on the TRA/TRD locus and may provide a reference for the repertoire of the V-J combinatorial diversity. The genetic programs of B-cell differentiation and the first DJ H gene rearrangements appear in the post-gastrulation mouse embryo (E10-12), shortly after the first multipotential hematopoietic progenitors do emerge. These DJ H joints represent the unselected baseline of the Ig repertoires. We have undergone a systematic sequencing of embryo DJ H joints obtained from normal BALB/c embryos and heterozygous embryos obtained from RAG2 -/mothers mated to BALB/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. The embryo DJ H s displayed unexpected mechanisms of diversity, including short stretches of non-templated N nucleotides in one-third of the studied sequences (in the absence of TdT expression) and frequent DJ H s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. Because the DNA polymerase m (Polm), a highly-homologous TdT member of the X DNA polymerase family, showed an increased expression in the embryo, we analysed the DJ H s of Polm -/mouse embryos. We observed that Polm was mainly responsible for introducing N nucleotides at the mouse embryo DJ H joints. Also, and based on its DNA-dependent polymerization ability, Polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. We think that, more than attempting to increase diversity, Polm acts as a "connector" in the embryo, subsequently participating in the repair of RAG-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. Along the end of gestation, further selective pressures acting over these first V-DJ H products will contribute to establish the differential neonatal Ig repertoires. Although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. Most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (RSV) infection, for which vaccines continue to be elusive. We now show that, compared to adults, the antibody response to RSV infection is very poor in neonatal mice and is unaffected by CD4 cell depletion. However, CD8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. To test the possibility that poor antibody boosting is caused by RSV-specific CD8 T cells killing RSV-infected B cells, we sorted cells from the lungs of infected neonates. Viral copy number was high in neonatal B cells, but viral load in surviving B cells was unaffected by CD8 cell depletion. In addition, Fas ligand (FasL) deficient gld mice responded to RSV infection in the same way as normal mice, indicating that FasL is not required for the inhibition of antibody responses. This new mechanism of regulation of B cell responses by CD8 T cells has important implications for vaccine development against neonatal infections. (2008) showed that IRF8 knockout mice had significantly reduced numbers of pre-pro-B cells in marrow and a phenotype similar to agammaglobulinemia. These results prompted us to consider ICSBP/IRF8 as a candidate gene in the pathogenesis of defective early B cell development. Therefore we decide to undertake direct sequencing of the gene encoding ICSBP/IRF8 in a small cohort of patients with autosomal recessive agammaglobulinemia. Methods: Eight patients affected by agammaglobulinemia were included in this study. All patients were under regular Ig replacement therapy. Informed consent was obtained from all patients. Genomic DNA was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. Direct gene sequencing of the eight exons of ICSBP/IRF8 were obtained using ABI PRISM sequencer. Results: Seven of the eight patients result wild type while only one patients present a synonymous SNP in exon V, yet documented as rs17444416.

Conclusions: Although recent findings indicated that IRF8 function is essential for early B cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in ICSBP/IRF8 that may be responsible for this disorder. The Hh/Ptch signaling system is known to control the development and neoplastic transformation of several cell types. However, the role of Hh/Ptch for the differentiation of B and T lymphocytes from hematopoietic stem cells (HSC) has not been assessed so far. To analyze the function of Hh/Ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the Ptch gene can be conditionally inactivated by virtue of the Cre/loxP recombination system. We show that targeted disruption of Ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated B and T lymphocytes from peripheral lymphoid organs. The developmental block occurs at the level of the common lymphoid progenitor cell (CLP), which defines an early branching point of HSC differentiation and lineage commitment. In contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (CMP) appears normal. Our data identify Hh/Ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. Hence, the progression of tumors, which are initiated upon oncogenic Hh/Ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system.

L. Calderon Dominguez 1 , T. Boehm 1 1 Max-Planck Institute for Immunbiology, Developmental Immunology, Freiburg, Germany

In many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. The ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. By analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. We demonstrate how an epithelial niche, rendered functionally inactive by disruption of the Foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the B and T cell lineages, respectively. The strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. Silencing of DC-SIGN using lentiviral RNA interference revealed its critical function for PD-L1 expression on DCs after M. tuberculosis infection. As a counterpart to expression of its ligand, we showed that CD4 and CD8 T cells from tuberculosis patients highly express PD-1 when compared to healthy uninfected individuals.

In addition, analysis of PD-1 expression in lung biopsies from tuberculosis patients revealed that PD-1 is expressed on CD4 and CD8 T cells confined to lung granulomatous lesions. Finally, blocking of the PD-1/PD-L1 axis using monoclonal antibodies abrogated the down-modulation of T cell proliferation and IFN-g production induced by ManLAM, a mycobacterial cell wall glycolipid and ligand for DC-SIGN. Taken together, our results suggest that the PD-1/PD-L1 pathway is involved in the exhaustion of T cell responses to M. tuberculosis. The inflammatory canonical NFkB pathway is critically involved in virtually all aspects of inflammation in general. Yet, the role of the alternative, non-canonical NFkB pathway in inflammation and adaptive immunity remains largely elusive. The alternative pathway is primarily mediated through the NFkappa-B inducing kinase (NIK) which in turn leads to the phosphorylation and the cleavage of p100 to p52. Among the receptors engaging NIK is the LTbR, which is also required to form the anlage for secondary lympoid tissues (SLTs). Due to a point mutation within NIK, alymphoplasia (aly) mice do not develop SLTs and are highly immunodeficient. However, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of NIK itself could potentially lesion the development of immune responses. To verify this notion, we generated a series of bone marrow chimeric mice (BMC) in which the absence of SLTs was disconnected from the hematopoietic loss of NIK function. We generated mice, which lack all SLTs, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal SLTs, but lacking NIK in all leukocytes (aly 1 wt). Surprisingly, we discovered that NIK is vital for the development of autoimmune disease, while SLTs (ie. LNs, spleen etc.) are essentially dispensable for cell-mediated immunity. We found that NIK is required for the polarization of effector T cells and that TH17 and TH1 cells cannot be generated in the absence of NIK. Preliminary data implicate the involvement of NIK in a discrete and novel pathway required for the formation of cell-mediated immune responses. The family of NFAT (Nuclear Factor of Activated T-cells) transcription factors is indispensable for T cells, for example playing an important role in cytokine gene regulation. In peripheral CD4 + T cells, NFATc1 and c2 are predominantly expressed. NFATc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. Here we address the functional difference of the short isoform NFATc1/A, which is highly induced upon T cell activation, and the long constitutively expressed isoform NFATc1/C.

As demonstrated by Y2H screen and Co-IPs, NFATc1/C-specific C-terminus can be highly SUMOylated. Confocal microscopy studies revealed that upon SUMOylation NFATc1/C -but not the unsumoylated NFATc1/A -translocates to Promyelocytic Leukemia-nuclear bodies (PML-nbs). This leads to interaction with HDACs followed by deacetylation of histones (Co-IPs), which in turn induces transcriptionally inactive chromatin (ChIP and confocal microscopy). As a consequence, multiple expression studies revealed SUMOylation dependent suppression of the NFATc1 target gene interleukin-2. Other lymphokines like IFNg and IL13 are reversely regulated. Interestingly, nTreg cells which do not express IL2 exerted only NFATc1/C, but no NFATc1/A expression (qRT-PCR). These findings demonstrate that the modification by SUMO converts NFATc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for NFATc1 function. Therefore, especially nTreg cells and anergized CD4 + T cells might be regulated by the long SUMOylatable isoform NFATc1/C. Lnk/SH2B3 and APS/SH2B2, two members of the Lnk/SH2B family of adaptor proteins, play an important role as negative regulators in B cell lymphopoiesis. They possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. Mice deficient for these proteins demonstrated that Lnk inhibits expansion of pro/pre-B cells while APS controls mature B-1 cell population, suggesting specific roles for these adaptors during B cell development. However, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. To address this question, we used primary and B cell lines at different stages of differentiation as our cellular system. Analysis of Lnk/APS expression pattern showed that Lnk is expressed at all developmental stages, while APS is only detected in immature and mature cells. We then first examined the role of Lnk in IL-7 signalling in pre-B cells Overexpression of Lnk dramatically inhibits IL-7-dependent growth demostrating that Lnk negatively regulates IL-7 pathways. Furthermore, we showed that IL-7 stimulation induces Lnk phosphorylation and its subsequent association with important signalling effectors, notably the E3 ubiquitin ligase Cbl. We next analyzed the role of APS In mature B cells by imaging and biochemical techniques. Our results showed that APS colocalizes with the BCR complex after BCR triggering. Interestingly, Lnk is not recruted to the BCR signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. Moreover, we showed, for the first time, that APS can associate, upon BCR stimulation, with the signalling molecules Cbl and Vav1. To address the functional implications of these interactions, we examined specific B cell responses, notably BCR trafficking and cytoskeleton remodelling. We demonstrated that overexpression of APS enhances ligand-induced endocytosis of BCR, possibly through interaction with Cbl and affects the kinetics of BCR-induced cell spreading. Our results therefore suggest a regulatory function of APS in BCR internalization and cytoskeleton dynamics. Altogether, our findings demonstrate that Lnk and APS display sequential specific regulatory roles during B cell development that are important for maintaining B cell homeostasis. Signaling through the T-cell receptor (TCR)-CD3 complex is a critical event in adaptive immunity. It is still not clear how ligand binding to the TCR is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the CD3 complex. It is widely accepted that dimerization or multimerization of TCR is required for TCR triggering. In our model T-cell activation is initiated by recognition of monomeric MHC/peptide complexes on the surface of antigen presenting cells (APC). Critical to TCR triggering is the movement of the T-cell across the APC. Engagement of a MHC/peptide complex on the surface of the APC will change the mobility of the TCR leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for T-cell activation. Furthermore, the change in mobility will lead to dislocation of the ITAMs from the plasma membrane so that they become accessible to tyrosine kinases.

To test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pMHC and a Fab that recognizes an epitope tag that we express on the T-cell surface. Binding of the Fab to the expressed epitope tag will constrain the lateral mobility of the TCR that is engaged by the pMHC arm of the same complex. The bifunctional complexes induced activation and proliferation as well as Ca influx and cytokine production in human CD4 + T-cell clones that displayed the epitope, but not in T-cells that did not display the epitope. Activation required interaction of the Fab with its epitope on the T-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the Fab binding site, was added. These results demonstrate that a monomeric copy of a pMHC is sufficient to trigger TCR and that formation of a TCR dimer is not an obligatory step in T-cell activation.

The bifunctional complex we generated may also have a great immunotherapeutic impact. Exchanging the Fab with a Fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of T-cell responses. Adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. Adaptor proteins containing characteristic SH2 or SH3 domains known to mediate protein-protein interactions are key players in these processes. SLy2 (SH3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a SH3 and a SAM domain as well as a bipartite NLS. SLy2 belongs to a family of three molecules: SLy1, SLy2 and Sash1.In humans, the SLy2 gene is located on chromosome 21, in mice on chromosome 16. SLy2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. Subcellular fractionation showed that the SLy2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.To elucidate the function of SLy2 we searched for possible interaction partners by yeast two hybrid screening with a mouse T cell lymphoma library. This approach identified Sin3-associated polypeptide p30 (SAP30) as a putative interaction partner of SLy2. SAP30 is a conserved member of the Sin3A-HDAC corepressor complex that contains histone deacetylase 1 (HDAC1) and histone deacetylase 2 (HDAC2) and acts as a transcriptional repressor for a variety of genes. We confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293T cells. In addition, we could show a direct interaction between SLy2 and HDAC1. To investigate the functional impact of this molecular interaction, we performed HDAC enzymatic activity assays. We were able to show that SLy2 increases the activity of HDAC1 in whole cell lysates and, more precisely, in nuclear extracts of 293T cells. The interaction of SLy2 with SAP30 and HDAC1 indicates a transcriptional function of this protein. Within the Sin3A-HDAC corepressor complex SLy2 might act as a switch for the activity of HDAC1.

CD46-Cyt1 and Cyt2 are co-expressed in human T cells and undistinguishable from the cell surface. In order to determine their specific role in T cell activation, we have expressed chimeric proteins consisting of the extracellular domains of CD19 (B cell marker) fused to the transmembrane and intracellular domain of CD46-Cyt1 or Cyt2 in primary T cells. We show that these two isoforms differently control human T cell function. Specific Cyt1 coengagement controlled IL-10 secretion, while Cyt2 coligation inhibited IFNg production. Moreover, our preliminary data suggest that CD46-Cyt2 inhibits the phosphorylation of several molecules known to be activated by CD46 stimulation. These data suggest that these two isoforms act as molecular switches for T cell activation, either promoting or turning off T cells. They demonstrate for the first time the distinct roles of CD46 cytoplasmic isoforms in primary human T cell activation. This also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. Nck is a ubiquitously expressed adapter protein that is almost exclusively built of one SH2 domain and three SH3 domains. Nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. In T cells, Nck participates in different and interdependent signalling pathways linking T cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. We previously showed that Nck directs the death factor FasL to the cytotoxic immunological synapse when T cells encounter putative target cells. We now performed a systematic screening for interaction partners of the four individual interaction modules of Nck in primary and leukemic T cells. We precipitated putative binding partners from untreated or pervanadate-treated PHA blasts, Jurkat and HUT78 cells with GST fusion proteins containing full length Nck, the three SH3 domains or the individual SH3 and SH2 domains. Binding proteins were excised from gels after staining with Coomassie, Silver or Flamingo Pink and processed by tryptic in gel digestion for mass spectrometrical analysis.

As expected, we observed major differences in Nck binding proteins precipitated from resting versus activated T cells. We not only verified established interactions (e. g. with the TCR signalling components Slp76 and CD3epsilon, the actin-regulatory proteins WASP and WIP and the nuclear protein Sam68) but also identified novel Nck-interacting proteins. The interaction with the actin-binding protein Hip55 once more underscores the fundamental role of Nck in TCR-mediated actinreorganization. The identification of the nuclear proteins SFPQ/NONO points to novel, yet unknown functions of Nck that might be associated with the recently reported nuclear translocation/localization of Nck. Accordingly, employing laser scanning microscopy, we clearly detected Nck within the nucleus also in human T cells.

The present data highlight that Nck serves versatile functions in T cells, which include the different interdependent pathways of TCR-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of Nck. Cytotoxic T lymphocytes (CTL) mediated killing is tightly regulated according to the strength of T cell receptor signal. Killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (cSMAC) within the immunological synapse. Although much has been learnt about the mechanisms controlling the strength of TcR signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the TcR is able to control the degree of CTL-mediated killing so finely. Here we examine how the strength of TcR signal controls polarization of the secretory apparatus leading to CTL-mediated killing using TcR transgenic OT-I CTL. Decreasing the TcR signal by reducing the concentration of OVA peptide or using the weak agonist peptide, G4, results in a slight reduction in the number of CTL target cell conjugates formed, and the number of conjugates in which a cSMAC (visualized by a patch of lck-staining at the immunological synapse) was formed. TcR signals result in reduced or absent (in the case of G4) staining with pSrc and pERK antibodies in the immunological synapse and reduced or absent (G4) degranulation as measured by CD107a assays. The centrosome docks at the cSMAC of the immunological synapse even with relatively weak TcR signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. Inhibitors support a role for PI3K in granule polarization. Together these data demonstrate that the strength of TcR signal controls the level of CTL mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the cSMAC and the accumulation of pSrc and pERK at the synapse. The centrosome polarizes to the cSMAC even with relatively weak TcR signals, but granule recruitment requires a higher threshold of signaling. These findings reveal how CTL can fine tune the degree of killing in response to TcR signals at the single cell level. Cytotoxic T cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. Cytolytic T-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. Perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. They are capable of undergoing regulated secretion in response to a T cell receptor engagement which involves binding to a cognate MHC class I-peptide complex. The intracellular transport of lysosomal proteins from the Golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the Vps10p-receptor Sortilin. Sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. In the secretory pathway, Sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. To explore whether Sortilin has a broader functional relevance, we asked if Sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic T cells. First, we demonstrate that Sortilin is expressed in T cells. To examine its function during an adaptive immune response, we analysed Sortilin-deficient cytotoxic T cells derived from a knockout mouse strain. In strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in Sortilin-deficient cytotoxic T cells. Whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of Interferon-g was found to be decreased. The enhanced release of cytotoxic molecules from Sortilin-deficient cytotoxic T cells translated into an increased cytotoxicity in vitro. Thus, the deletion of Sortilin imposed a specific phenotype in cytotoxic T cells which could not be compensated for by other sorting receptors. Our localisation studies of Sortilin in T cells were consistent with the results previously described in neuronal cells which indicated that Sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the Trans-Golgi-Network to endosomes and lysosomes. Taken together, we suggest that Sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. There is growing interest in the soluble splice variant of CTLA-4 (sCTLA-4) as an immune inhibitor secreted by T cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. However, little is known of the biology of sCTLA-4 in immune responses. Using a specific anti-human soluble CTLA-4 monoclonal antibody, JMW-3B3 that selectively binds the soluble isoform but not membrane bound CTLA-4, or cleaved fragments of it, we demonstrate that sCTLA-4 plays a vital role in regulating antigen-specific immune responses. We used antibody blockade to show that antigen-specific T cell responses are strongly enhanced upon blockade of sCTLA-4, secreting increased amounts of cytokines including interferon-g, IL-17 and TNF-a, but lower amounts of IL-10. Soluble CTLA-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. Addition of sCTLA-4 induced secretion of the immunoregulatory cytokine IL-10 by human PBMCs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. The immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sCTLA-4. It is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound CTLA-4 it is crucial to T cell inhibition. Membrane-bound CTLA-4 exists as a homo-dimer on T cells but sCTLA-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link B7 ligands on antigen presenting cells. A third important observation from this study is that sCTLA-4 exists both in serum and culture supernatants as a natural 46KDa homo-dimer, and not as a monomer. This goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. Together, these results lead us to reappraise sCTLA-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory T cell response to antigenic stimulus, rather than a product of resting T cells. This work also raises the possibility that where IL-10 dependent regulation is most critical, boosting sCTLA-4 secretion by regulatory T cells could be a novel therapy for immune mediated diseases. Recently, we identified a new adaptor protein, Swiprosin-1/EFhd-2, in lipid rafts of B cell lines that undergo apoptosis after B cell receptor (BCR) stimulation. Swiprosin-1/EFhd2 is expressed in immature B cells of the bone marrow, in resting and activated splenic B cells, in T cells, macrophages, mast cells and some nonlymphoid tissues. Ectopic expression of Swiprosin-1/EFhd-2 in the immature murine B cell line WEHI231 enhanced spontaneous and BCR-induced apoptosis. In contrast, shRNA-mediated down-regulation of Swiprosin-1/EFhd-2 impaired spontaneous and BCR-elicited apoptosis, but not BCR-induced G1 cell cycle arrest.

To understand how Swiprosin-1/EFhd2 enhances pro-apoptotic BCR signals, we analyzed whether Swiprosin-1/EFhd2 is involved in proximal BCR signalling. In fact, ectopic expression of Swiprosin-1/EFhd2 enhanced BCR-induced calcium flux in WEHI231 cells, whereas shRNA-mediated down-regulation of Swiprosin-1/ EFhd2 impaired BCR-elicited calcium signals. Concomitantly, GST-pulldown experiments revealed that Swiprosin-1/EFhd2 interacts with Phospholipase Cg2 (PLCg2) and with the tyrosine kinase Syk (splenic tyrosine kinase), both of which are important for BCR-induced calcium flux. The interaction of PLCg2 and Swiprosin-1/EFhd2 was further established by co-immunoprecipitation.

Reconstitution of BCR-elicited calcium signals through complementation of Swiprosin-1/EFhd2 silenced WEHI231 cells with Swiprosin-1/EFhd-2 was inhibited by the Syk inhibitor BAY 61-3606. In analogy, Swiprosin-1/EFhd2 regulated Syk activity positively. Moreover, Swiprosin-1/EFhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of PLCg2 and of Syk tyrosine residue 352, which is involved in Syk activation. Finally, reconstitution of Swiprosin-1/EFhd2 knock-down cells with Swiprosin-1/EFhd2 mutants revealed that the N-terminal putative SH3-binding site, the first EF-hand, and to a lesser extent, the second EF-hand and the C-terminal coiled-coil domain, are important for BCR-induced calcium flux in WEHI231 cells.

Interestingly, Swiprosin-1/EFhd2 re-expression in Swiprosin-1/EFhd2-silenced cells induced already in unstimulated cells raft partitioning of Syk, PLCg2 and the BCR, which was reversed after 2 min of BCR stimulation. In summary, Swiprosin-1/EFhd2 is an accelerator of proximal BCR signalling and acts through Syk and PLCg2 by assembling a Syk-dependent calcium initiation complex in lipid rafts. This might be relevant for memory B cell signalling or central B cell tolerance. To test the biological relevance of Cbl-b E3 ligase activity, these mice were analyzed for T cell proliferation, susceptibility to autoimmunity, in vivo T cell tolerance responses, and TC1 tumor rejection. Results: When stimulated, T cells from RF mutant mice hyperproliferate compared to wild type T cells, even in the absence of CD28 co-stimulation. Preliminary data also suggest that RF mutant mice are more susceptible to autoimmunity. In addition, RF/P14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in T cell tolerance induction. More importantly, Cbl-b E3 ligase dead mice can spontaneously reject TC1 tumors. Conclusion: Cbl-b E3 ligase dead mutant mice phenocopy total body Cbl-b knock out mice, thus indicating that Cbl-b E3 ligase activity is indispensable for its regulatory in vivo functions. Intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. To further elucidate the cellular mechanism of Cbl-b mediated tumor rejection we have now generated the conditional Cbl-b E3 ligase dead mutant mice to for the first time study the Cbl-b ubiquitination function in a tissue specific and temporal fashion. Our research is also currently focused on identifying the relevant in vivo Cbl-b ubiquitination substrates. Interferon alpha (IFN-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. For a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to IFN-a therapy. However, recent studies in mice have revealed that IFN-a/b also exerts effects on several host immune cells. IFN-a has been shown to enhance CD8 T cells (CTLs) responses against soluble antigens in mice. This immunostimulatory activity of IFN-a results at least partly from its direct ability to induce maturation of dendritic cells. Several studies have recently demonstrated that IFN-a/b also acts directly on murine CTLs, inducing clonal expansion and differentiation into effector and memory cells. To date, little is known about the effects of IFN-a on human CTLs. To approach this issue, magnetically sorted untouched human CD8 + CD45RO -T cells (mainly naï ve cells) were unstimulated or stimulated with human IFN-a and gene expression profiles were compared using an Affymetrix human array. Interestingly, IFN-a stimulation of highly purified human CTLs without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (TRAIL) and chemotaxis (IP10 and ITAC). In a second genome-wide array analysis, we analyzed the effects of IFN-a on human CTLs responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-CD3/CD28 antibodies. Gene expression patterns were compared for cells stimulated with anti-CD3/CD28 Beads alone or along with IFN-a. IFN-a regulates the expression of a number of genes that promote proliferation, activation and survival of CTLs, TCR stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in CTLs effector functions (Granzyme-B, IFN-g, TRAIL, FASL) and chemotaxis (IP10, ITAC). The enhanced expression of Granzyme-B, IFN-g, TRAIL and IP10 were further confirmed at the protein levels by flow cytometry analysis and/or ELISA. Enhancement of Granzyme-B-and TRAIL-mediated cytolitic functions was also found by functional assays using anti-CD3-coated p815 cells and TRAIL-sensitive CAKI-I cells as targets.

Our results show that IFN-a provides a strong signal-3 to human CTLs leading to their differentiation into effector CTLs. T cell activation is an important process of the adaptive immune system, which requires recognition of MHC-associated antigens by antigen presenting cells (APCs) via the T cell receptor (TCR). To induce a productive T cell response the interaction of T cells with APCs needs to be stabilized by adhesion molecules. Junction adhesion molecules (JAMs) are a recently discovered group of immunoglobulin (Ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. The third member of the JAM protein family, JAM-C, is highly expressed in platelets and endothelial cells, whereas expression in T cells is largely unknown. To investigate the regulation of JAM-C in T lymphocytes, we determined JAM-C gene expression in quiescent and activated human T cells. Treatment with the polyclonal T cell activator phytohemagglutinin (PHA) increased surface and total JAM-C expression in T cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. By contrast, no up-regulation of JAM-A in activated T cells was detectable. The highest level of JAM-C up-regulation by PHA was observed in CD3 + FoxP3 + and CD4 + CD25 high T cells. Moreover, T cell receptor activation with combined anti-CD3 and anti-CD28 stimulation induced JAM-C expression in T cells. JAM-C induction occurred at the mRNA level suggesting a transcriptional regulatory mechanism of JAM-C expression. Accordingly, we studied the regulation of the human JAM-C gene promoter in transiently transfected T cells. Luciferase activity of a JAM-C promoter gene construct with three potential consensus sites for the transcription factor NFAT was markedly induced in activated T cells. Finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin A and FK-506, but not with MAPK inhibitors, blocked JAM-C induction in activated T cells. In summary, the present data indicate that JAM-C is induced in activated human T lymphocytes via a transcriptional mechanism and suggests a major regulatory function of JAM-C for the T cell response. HIV-1 infection leads to immune dysfunction owing to a successive loss of the CD4 + T cell compartment. The molecular mechanisms underlying this depletion are not well-understood but may involve the viral Nef protein. Nef is a multifunctional accessory protein that is required for full HIV-1 virulence and the maintenance of high viral loads. Nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. CD4 and MHC class I, and modulating signal transduction pathways. The latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis.

In this study we identify a signaling complex assembling at the N-Terminus of Nef, which contains the kinases Lck and PKCV. Formation of this complex, termed NAKC for Nef-associated kinase complex, led to activation of Lck, as assessed by in-vitro kinase assay, and recruitment of PKCV to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. Recruitment of PKCV to membrane rafts is a hallmark of T cell activation and has been associated with activation of the NFxB transcription factor. However, contrary to our expectations, Nef-mediated NAKC formation did not activate NFxB. Instead, it led to a strong induction of ERK1/2. This correlated with a NAKC-mediated increase in HIV transcription that was demonstrated by luciferase reporter assays suggesting that ERK1/2 directly targets HIV transcription, possibly via induction of transcription factors. To our surprise, however, the effect of NAKC on HIV transcription was found to be independent of AP-1, NFAT and NFxB suggesting an alternative mechanism of NAKC-mediated enhancement of HIV transcription. On the basis of our previous results we propose that Nef enhances HIV transcription via removal of inhibitory factors and thus derepression of the HIV promoter. How ERK1/2 is involved in this mechanism and whether NAKC targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined.

P. Otahal 1 , T. Brdicka 1 , V. Horejsi 1 1 Institute of Molecular Genetics AS CR, Praha, Czech Republic

Aims: C-terminal src kinase (Csk) and CD45 are key regulators of src-family kinases in leukocytes. While CD45 is a transmembrane phosphatase, Csk is localized mostly in cytosol. However, a fraction of Csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. Currently, it is accepted that SH2 domain of Csk binds phosphotyrosine 317 of transmembrane adaptor protein PAG and via this interaction is recruited to the cell membrane and lipid rafts. However, PAG knock-out mice still have cell membrane-associated Csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane Csk. Thus, the mechanisms of membrane targeting of Csk remain unclear. To analyze the role of membrane and lipid raft targeting of Csk on lymphocyte signaling we targeted Csk to different membrane compartments by fusing Csk with transmembrane domains of LAT, LAX, CD25 and N-terminal part of Src kinase. Methods: Csk chimeras containing N-terminal membrane targeting motif and C-terminal orange fluorescent protein were cloned into retroviral vector pMXs. Jurkat T cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these Csk chimeras on T-cell receptor (TCR) signaling by measuring calcium flux and CD69 upregulation.

The efficiency of inhibition depended on the membrane targeting motif, while LAT-csk chimera completely inhibited TCR signaling and Src-csk chimera inhibited the signaling only partially; LAX-csk and CD25-csk chimeras showed almost no inhibition of TCR signaling despite efficient presence at the plasma membrane.

Conclusions: Our data demonstrate that the function of Csk strongly depends on its targeting to the specific areas of plasma membrane. It also strongly supports the idea that membrane compartmentalization is critical for regulation of T-cell signaling. Peripheral CD8 T cell tolerance can be generated outside lymphatic tissue in the liver. However, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating T cells is unclear. Here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (LSEC) and not by other antigen presenting cells in the liver or spleen. Uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve CD8 T cell-retention in the liver but again not in other organs. Using bone-marrow chimeras and Tie-2Kb mice, we could show that antigen cross-presentation by LSEC was both essential and sufficient to cause antigen-specific T cell-retention under non-inflammatory conditions, which was followed by CD8 T cell proliferation and expansion, but ultimately led to the development of T cell tolerance.

Our results show that CD8 T cell tolerance towards circulating systemic antigens is predominantly generated in the liver by LSEC, which preferentially take-up and cross-present circulating proteins to CD8 T cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. These insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of CD8 T cell responses. AlphaPIX is a Rho GTPase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. It has been shown that PIX proteins play roles in some immune cells, including neutrophils and T cells. In this study, we report the immune system phenotype of alphaPIX knockout mice. We extended alphaPIX expression experiments and found that whereas alphaPIX was specific to immune cells, its homolog betaPIX was expressed in a wider range of cells. Mice lacking alphaPIX had reduced numbers of mature lymphocytes and defective immune responses. Antigen receptor-directed proliferation of alphaPIX deficient T and B cells was also reduced, but basal migration was enhanced. Accompanying these defects, formation of T-cell-B-cell conjugates and recruitment of PAK and Lfa-1 integrin to the immune synapse were impaired in the absence of alphaPIX. Proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of PAK and expression of GIT2 in both T cells and B cells. These results reveal specific roles for alphaPIX in the immune system and suggest that redundancy with betaPIX precludes a more severe immune phenotype.

S. Merluzzi 1 , S. Parusso 1 , B. Frossi 1 , G. Gri 1 , C. Pucillo 1 1 University of Udine, DSTB, Udine, Italy

In this study, we investigated whether primary MCs could modulate the activation and proliferation of primary B cells. We performed co-culture assays using mouse splenic B cells and bone marrow-derived MCs. Naï ve and activated B cells proliferation could be induced by nonsensitized MCs while an increase in B cell proliferation was observed when MCs are activated. Moreover, B cell proliferation was partially abolished when MCs and B cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. Using both IL-6 -/-MCs and anti-IL-6 receptor antibody, we demonstrated that in co-culture of primary B cells and MCs, IL-6 derived from activated MCs is a key cytokine implicated in the B cell proliferation. Moreover, we showed that activated MCs can influence the surface expression of costimulatory molecules as CD40 on naï ve B cells and the interaction of CD40 on B cell surface and CD40L on MCs is important for the further differentiation of B cells to plasmacells. Indeed, we presented for the first time evidence that cytokines produced by activated MCs and interaction between CD40L e CD40 on MC and B cells respectively can contribute to differentiate mature B cells to IgA secreting cells.

In conclusion, in the present report, we showed a novel role of MCs as promoter of both the survival and activation of naive B cells and of the proliferation and further differentiation of activated B cells through soluble factors production and cell-cell contact, suggesting that MCs can contribute to the regulation of specific immune response.

E. Fourmentraux-Neves 1 , N. Bercovici 1 , A. Caignard 1 1 INSERM U567, Paris, France

Inhibitory killer Ig-like receptors (KIR2DL1-2/3) which bind to HLA-C molecules are expressed by human Natural Killer cells and effector memory CD8 + T cell subsets. These receptors suppress CD8+ T cell activation through recruitment of the Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1). To further analyse the yet largely unclear role of inhibitory KIR receptors on CD4+T cells, KIR2DL1 transfectants were obtained from a CD4 + T cell line and primary cells. The transfection of CD4 + T cells with KIR2DL1 dramatically increased the T cell receptor (TCR)-induced production of IL-2 independently of ligand binding, but inhibited TCR-induced activation after ligation. KIR-mediated TCR activation requires intact ITIM motifs, involves KIR2DL1-ITIM phosphorylation, SHP-2 recruitment, ZAP-70 and PKC-V phosphorylation. Synapses leading to activation were characterized by an increase in the recruitment of p-Tyr, SHP-2 and p-PKC-V but not of SHP-1. In contrast, the KIR2DL1/HLA-Cw4 interaction led to a strong synaptic accumulation of KIR2DL1 and the recruitment of SHP-1/2, inhibiting TCR-induced IL-2 production. KIR2DL1 may induce two opposite signaling outputs in CD4 + T cells, depending on whether the KIR receptor is bound to its ligand. These data highlight unexpected aspects of the regulation of T cells by KIR2DL1 receptors. B cell receptor (BCR) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific B cells to powerful co-stimulatory signals, such as T cell help or Toll-like receptor ligands. The role of BCR binding in modulating the access to these second signals is complex and varies between stimulatory conditions. By quantitative tracking of B cell responses in vitro we can measure which signals affect B cell proliferation or differentiation, or both, and thereby establish a novel understanding of how B cells respond appropriately to different combinations of stimuli. We utilised HEL-specific BCR transgenic SW HEL mice to assess the effect of a specific antigen signal on B cell responses to the T-independent mitogen lipopolysaccharide (LPS). The presence of antigen renders a greater proportion of cells responsive to LPS stimulation and profoundly influences effector cell differentiation. Antibody secreting cell formation is dramatically inhibited by HEL, but we found that isotype switching to IgG1 is strongly upregulated. Both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. When B cells are exposed to antigen for a limited period of time, switching to IgG1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of IgG1 antibody during these conditions. The observed IgG1 switching behaviour mimics that of B cells responding to LPS and IL-4, but is mediated by a different, STAT6-independent pathway. These data are indicative of the important role specific antigen signals play in regulating B cell responses in stimulatory environments.

A. Quintana 1 , C. Schwindling 1 , M. Pasche 2 , C. Junker 1 , C. Kummerow 1 , U. Becherer 2 , E.C. Schwarz 1 , J. Rettig 2 , M. Hoth 1 1 Saarland University, Biophysics, Homburg, Germany, 2 Saarland University, Physiology, Homburg, Germany

The adaptive immune response requires the interaction between antigen-presenting cells and T cells. This cell-cell interaction, called the immunological synapse (IS), facilitates the activation of T cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of CRAC/ORAI1 channels.

To allow sustained activity of CRAC/ORAI channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. Objectives: The purpose of the study was to analyze local and global calcium signals in T cells and to test the hypothesis that the IS controls these signals through mitochondrial positioning. Methods: We used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human T helper cells and cell lines. To test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. Results: Following IS formation, we found that mitochondria translocated to the IS in a calcium-dependent way. The distance between mitochondria and the plasma membrane at the IS was lower than 150 nm. Following accumulation at the IS, mitochondria limited calcium entry to the ORAI channels localized right at the IS by preventing their calcium-dependent inactivation. In contrast, no calcium influx was observed at sites where no mitochondria were accumulated as ORAI channels were inactivated at these sites. Mitochondrial positioning at the IS thus induced local calcium influx at the IS without the necessity to enrich ORAI channels at the IS. Mitochondria took up calcium at the IS distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the IS low enough to prevent calcium dependent ORAI inactivation and to prevent excessive calcium clearance by the calcium APTases in the plasma membrane, which could inhibit an efficient T cell activation. Conclusion: Mitochondria positioning at the IS controls local calcium entry through ORAI channels. Mitochondria prevent ORAI inactivation and excessive calcium clearance at the IS to facilitate calcium-dependent T lymphocyte calcium signalling. We aimed to determine the functional correlates of CD4 + T cell tolerance and immunity in vivo. Ovalbumin (OVA)-specific transgenic CD4 + T cells were adoptively transferred into syngeneic mice immunized with soluble OVA protein ± lipopolysaccharide (LPS) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. Under tolerogenic conditions (OVA alone), CD4 + T cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (ICOS, 4-1BB), from T cells activated by OVA+LPS. This difference in activation also translated into differing CD4 + T cell expansion and contraction kinetics in the early phase of the T cell response (days 1-6). In the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic CD4 + T cells in the spleen developed into mature effectors with a prominent capacity to secrete IL-2, IFN-g, and IL-17A, and only few OVA-specific FoxP3 + regulatory T cells ( X 10 %) were observed. Germinal centers were prominent and OVA-specific Ig of all isotypes were generated. In contrast, under tolerizing conditions, antigen-specific CD4 + T cells failed to migrate into the B cell follicles, but production of OVA-specific IgM was nevertheless observed. In these animals, the proportion of splenic OVA-specific regulatory T cells (30 %) was substantially increased. On day 14, both groups of mice were re-challenged via the airways with OVA+LPS to functionally assess their immune status. In tolerized animals, the transgenic T cell population in the lung infiltrate was composed of OVA-specific regulatory T cells (50 %) or T cells with a reduced capacity to secrete effector cytokines. In contrast, in immunized animals, this population almost exclusively consisted of CD4 + effector T cells with a pronounced inflammatory cytokine profile (IFN-g, IL-17A). With this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. We identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. Mast cells (MCs) are well-recognized as key effector cells in immunoglobulin E (IgE) -associated immune responses and as prototypic regulators of innate immunity. The characteristics, importance, and molecular requirements for interactions between mast cells (MC) and CD8 T cells (Tc) remain to be elucidated. Using myelin/oligodendrocyte glycoprotein (MOG), we demonstrated that MCs induce antigen-specific CD8 Tc activation and proliferation. The antigen crosspresentation by MCs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by CD8 Tc. In vivo evidence that MCs modulate T cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (EAE), the standard animal model for multiple sclerosis, in which both CD8 Tc and MCs are now recognized as key players. One of the main central nervous system (CNS) antigens recognized by autoreactive Tc in EAE is the myelin oligodendrocyte glycoprotein (MOG). To investigate the in vivo-relevance of the identified MC-CD8 Tc interactions, we have employed the EAE as a model of organ specific autoimmune disease in wild type mice and MC-deficient W/W sh mice. WT and W/W sh mice were immunized with the MOG 35-55 protein.

Our results provide direct evidence that MC contribute to CD8-specific priming in EAE and show that the Tc proliferation failure is specific for CD8 Tc from MOG 35-55 -immunized W/W sh mice. The role of MC-CD8 Tc interaction in induction of autoimmunity will be further investigated in EAE.

In summary, we provide the first evidence that MCs regulate antigen-specific responses of primary CD8 Tc in vitro and in vivo. Our study further supports the emerging concept that MCs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding CD8 Tc responses. This newly uncovered MC function might be of great biological relevance in situations where effector CD8 Tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. Activation of resting T cells in vitro is triggered by combined T cell receptor (TCR) and CD28 engagement and can be modulated by simultaneous ligation of various other surface receptors. Although the FasL is best known for its capacity to initiate cell death in Fas-bearing cells, it has recently been implicated in the regulation of T cell activation. Thus, a crosstalk between the TCR and FasL is likely, but far from being biochemically elucidated. We report that FasL engagement by immobilized but not soluble FasFc fusion protein and anti-FasL antibodies blocks the activation of primary human peripheral T cells even in the presence of CD28 costimulation at the level of an early signal initiation. Inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in TCR signal transduction and also with a lowered calcium response. The data presented stress the importance of the Fas/FasL-system for signal initiation via the TCR/CD3complex and provide further arguments for a retrograde signaling capacity of FasL or a crucial role of Fas as a costimulatory molecule.

Golgi network (TGN). Moreover, TRIM specifically associates with the cytoplasmic tail of CTLA-4, but not via any conventional motifs in this region. Overexpression of TRIM augments CTLA-4 surface expression, whereas down-regulation of TRIM expression by shRNA results in disturbed CTLA-4 localisation, mainly restricted to the TGN. CTLA-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in CTLA-4 trafficking. Here, we identify additional transmembrane adapter protein (TRAP) family members as novel binding partners and regulators of CTLA-4 expression. Although there is some redundancy amongst TRAPs, our results highlight the importance of this family of proteins in CTLA-4 transport to the cell surface. It is imperative to reveal the mechanisms by which CTLA-4 is transported to the cell surface, given that minor changes in expression can have major effects on T-cell function and in the development of autoimmunity. Natural killer T (NKT) cells are found within the liver and are known to exhibit immune regulatory function. Upon recognition of glycolipids presented on CD1 molecules, NKT cells are activated and release cytokines, including IFN-g, IL-2 and IL-4. NKT cells are efficiently recruited to the liver via CXCR6-dependent chemotaxis toward CXCL16 and constitute a large proportion of the liver-resident lymphocytes. We have previously shown, that liver sinusoidal endothelial cells (LSEC) can scavenge circulating soluble antigens, and can cross-present these antigens to naive CD8 T cells. Cross-presentation leads to initial T cell activation and expansion, but ultimately these CD8 T cells are rendered tolerant. As both naive T cells and NKT cells come into close contact with LSEC in the hepatic sinusoids, we investigated whether NKT cells can modulate CD8 T cell tolerisation via interaction with LSEC. To this end we analysed CD1d expression on LSEC and their ability to activate NKT cells by presentation of the CD1d-binding glycolipid a-galactosylceramide (aGalCer). We found that LSEC express functional CD1d, as aGalCerpresenting-LSEC were capable to induce TNF-a, IL-2, IL-4 and IFN-g production in NKT cells in vitro. The interaction of aGalCer-presenting-LSEC with NKT cells led to the upregulation of CD54 and B7-H1 on LSEC. As naï ve CD8 T cell tolerisation by LSEC critically depends on B7-H1, we hypothesise that hepatic NKT cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of LSEC. Improved antibody responses by class-switched memory B cells require enhanced signaling from their antigen receptor (BCR). However all BCR classes on naïve and antigen-experienced B cells utilize the canonical Iga/Igb subunit for signaling. We identified the signal amplification mechanism of the IgG-and IgE-BCR. For these isotypes tyrosine-based signaling is not confined to Iga/Igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. The phosphorylated immunoglobulin tail tyrosine recruits the adaptor Grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. Hence membrane-bound IgG and IgE not only recognize antigen but also exert BCR-intrinsic costimulation to render memory B cells less dependent on T cell help for activation. Objectives: The majority of circulating human gd T cells harbor TCR containing Vg9, Jg1.2, and Vd2 gene products. They recognize nonpeptide antigens like (E)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (IPP) in malignant cells. Recently, we and others found out that gd T cells express a variety of costimulatory molecules including ICOS, and PD-1. One of the inhibitory receptors, PD-1, is a member of CD28/CTLA-4 family and contains a single Ig V-like domain in its extracellular region. PD-1 can bind to two B7 homologue molecules, PD-L1 and PD-L2. It has been reported that interaction of PD-1 with its ligands resulted in peripheral immune regulation and tolerance in ab T cells. In this study, we show that PD-1 is expressed on activated human gd T cells and regulates the effctor functions of gd T cells. Methods: Peripheral blood mononuclear cells were resuspended in Yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus IL-2 to obtain gd T cells. PD-L1+ and PD-L1-human tumor cell lines were established from cancer patients. In order to prepare anti-PD-L1 mAbs, the PD-L1 extracellular domain was expressed in E.coli as inclusion bodies and refolded in the standard arginine-based buffer. Mice were immunized with the refolded protein and mAbs were established. To determine the function of PD-1 in gd T cells, we determined cytokine production and cell mediated tumor lysis by activated gd T cells in the presence of inhibitors of PD-1/PD-L1 interaction. Results: gd T cells expressed PD-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed PD-L1. We first examined whether or not the engagement of PD-1 receptor could modulate the cytotoxic activity of gd T cells. PD-L1-expressing tumor cells tempered cytotoxic activity of PD-1+ gd T cells, and cytokine production such as TNF-a was down-regulated by PD-1 engagement. In addition, inclusion of anti-PD-L1 mAb reversed cytotoxic activity and cytokine production when PD-L1-expressing tumor cells were challenged by PD-1-expressing gd T cells. Conclusion: PD-1 delivers inhibitory signals in gd T cells upon engagement with PD-L1. Peripheral tolerance plays an important role in preventing T lymphocyte responses to self or harmless antigens. One of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and IL-2 production by T cells in response to antigenic challenge. The acquisition of the anergic phenotype is an active process, with negative regulators of T cell signalling being induced. Among these are the E3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. The E3 ubiquitin-protein ligases Cbl-b, Itch and Grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate TCR signalling elements. Our objectives were to determine the expression of Cbl-b, Itch and Grail in antigen-specific CD4 + T cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. In order to accomplish these objectives we induced priming or tolerance of ovalbumin (OVA 323-339 peptide)-specific T cells from DO11.10 TCR transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with E3 ubiquitin-protein ligases expression and the ubiquitination status of the TCR signalling machinery. Cbl-b, Itch, Grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and Western blotting studies. Moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using Laser Scanning Cytometry. Our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific T cells by mutant E3 ligase-, signal target-and ubiquitin-constructs. Collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific T cells.

S. J. Keppler 1 , P. Aichele 1 1 IMMH, University Freiburg, Immunology, Freiburg, Germany

Interleukin 12 (IL-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. It was postulated recently that IL-12 has a direct influence on CD 8 + T cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to TCR engagement (signal 1) and costimulation (signal 2). We analysed direct IL-12 signaling to CD8 T 

-12 signaling exhibited normal degranulation activity, cytolytic functions, IFN-g and TNF-a production. However, CD8 T cells lacking IL-12 signaling failed to up-regulate KLRG1 and to down-regulate CD127 in the context of Listeria but not viral infections. Thus direct IL-12 signaling to CD8 T cells determines the cell fate decision between short-lived effector cells (SLECs) and memory precursor effector cells (MPECs), dependent on the pathogen-determined local cytokine milieu. CD8 + T lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. We could now reveal that individual CD8 + T cells are tightly controlled in their effector functions by CD152 (CTLA-4). We demonstrate that signals induced by CD152 reduce the frequency of interferon-gamma (IFN-g) and GranzymeB expressing CD8 + T cells. For this novel function CD152 specifically represses the transcription factor Eomes, but not T-bet. A CD152 mediated induction of the inhibitory transcription factor cKrox has been ruled out. Ectopic expression of Eomes reversed CD152-mediated inhibition of effector molecule production. Additionally, enhanced cytotoxicity of individual CD8 + T cells differentiated in the absence of CD152 signaling could be demonstrated in vivo. The novel insights that CD152-mediated signal transduction in vivo indeed alters CD8 + T cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of CD8 + T cells. Objectives: ATP constitutes a damage associated molecular pattern (DAMP) and contributes together with pathogen associated molecular patterns (PAMP) to the efficient priming of the innate immune system. ATP is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed P2 receptors. P2x 1-7 receptors open to non-selective ion channels, whereas P2y1, 2, 4, 6, 11-14 are G-protein coupled receptors, which bind preferentially ADP, UDP, UTP or UDP-glucose. As the role of P2 receptors in the control of B cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular ATP production and release by human B cell subsets. Methods: Intracellular ATP measurement has been performed using a bioluminescence assay while extracellular ATP has been measured by HPLC.

Storage and release of ATP by B cells have been elucidated using confocal and TIRF microscopy, to study vesicles distribution and dynamics near the plasma membrane. Results: In both human naive and memory B cell we observed a prominent increase of ATP synthesis upon TLR9 but not BCR stimulation. Glycolytic pathway rather than oxidative phosphorilation was involved in ATP synthesis. P2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human B cells thus suggesting that ATP is released in the pericellular space. Labelling of resting and activated human memory B cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of ATP, confirmed with subcellular fractionation on sucrose equilibrium gradients. TIRF imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-Ig and this event was Ca(2+)-dependent. Conclusion: These data provide evidence that ATP is produced by B cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of ATP release in human B cells. ATP released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of B cells.

O. Ballek 1 , A. Brouckova 1 , D. Filipp 1 1 Institute of Molecular Genetics AS CR, Laboratory of Immunobiology, Prague, Czech Republic Two Src family tyrosine kinases Lck and Fyn are critical for the proximal T-cell signaling. We have previously demonstrated that induced Lck activation outside lipid rafts (LR) results in Lck translocation to LR. Central in this sequence of events is the rapid translocation of kinase active Lck to LR, yet the mechanism underpinning this process is unknown. The main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to LR and forming immunological synapse. We have recently characterized the C-terminal YQPQP sequence as a novel cis-acting component essential for partitioning of Lck to LR. Here we report that the expression of the C-terminal truncate of constitutively active Lck ( ¿ FQPQP) in NIH3T3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active Y505FLck. Comparative 2-D gel analyses followed by MS/MALDI identified RACK1 as a candidate protein for interaction with the C-terminal tail of Lck. Co-expression in NIH3T3 cells of HA-tagged RACK1 with either a wild type Lck or constitutively active Y505FLck revealed a significantly enhanced complex formation between Y505FLck and RACK1 compared to that of WTLck. Ectopic expression of Y505FLck with its domain-inactivating mutations showed that Lck-RACK1 interaction depends on functional SH2, SH3 and the C-terminal tail sequence of Lck. Lck-RACK1 interaction is readily detectable also in primary CD4 + lymph node T cells. Upon their activation, only the pool of Lck molecules associated with high molecular weight complexes can translocate to lipid rafts. Co-purification of RACK1 with these fractions further suggests that it plays a role in the translocation of Lck to LR. In addition, Lck and RACK1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. Moreover, the importance of interaction between activated Lck and RACK1 in the context of Lck translocation to LR is further strengthen by the observation that RACK1 is associated with elements of cytoskeleton. These results are the first to characterize RACK1 as a candidate molecule involved in the regulation of Lck translocation to LR through linking the C-terminal sequence of Lck to cytoskeletal network. Human B cells are currently not known to produce the pro-apoptotic protease granzyme B (GrB) in physiological settings. We have discovered that B cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (IL-21) can induce secretion of substantial amounts of GrB by human B cells. GrB response to viral antigens was significantly stronger in B cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. Both, naï ve and memory B cells differentiated into GrB-secreting cells, which featured a homogeneous CD19+CD20+CD27-CD38-IgD-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. B cellderived GrB was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic T cells. Our findings suggest GrB-secreting B cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum GrB levels found in various viral diseases. Further studies will elucidate whether B cell-derived GrB induces cytotoxicity towards virus-infected cells or exhibits other functions. Results: Transfer of OTII cells augments the wild type Th2 response to alumOVA inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to IgG1. The plasma cells up-regulate CXCR4, but not CXCR3, a chemokine receptor that attracts plasma cells to inflammatory sites. OTI cells respond to alumOVA by producing IFNg, a Th1-associated cytokine. When both OTI and OTII cells are transferred switching is diversified with plasma cells being IgM (˚5%), IgG2a (˚30 %), IgG2b (˚30 %) or IgG1 (˚30 %). In addition to CXCR4, some 70 % of these plasma cells strongly express CXCR3. The induction of CXCR3 in these plasma cells correlates with their increased expression of the transcription factor T-bet, which has been linked with IgG2a switching during Th1 responses. This is functionally significant for OTI-dependent CXCR3 expression, as well as induction of switching to IgG2a, are dependent on T-bet expression by the responding B cells, although T-bet-deficient B cells still switch to IgG2b. T-bet is known to be induced in B cells exposed to IFNg or TLR9 stimulation. These two hypothetical mechanisms are currently being tested in mice injected with blocking anti-IFNg antibodies or mice deficient in MyD88. Objective: A successful immune response against malaria has to be tightly controlled. The early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. Activation of T lymphocytes through TCR signalling takes place within the context of numerous other cell surface protein interactions. To prevent unnecessary activation of T cells the immune system has developed an intricate balance between positive and negative costimulatory signals. Costimulatory signals determine whether antigen recognition by T lymphocytes leads to full activation or to anergy. In contrast negative costimulators expressed by T cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. Recently, BTLA (B and T lymphocyte attenuator, CD272) was described as a novel negative costimulatory receptor. BTLA is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during experimental malaria infection.

To study the function of BTLA we employed a mouse model of blood-stage malaria using P. yoelii NL infection of BTLA-deficient and HVEM-deficient mice. P. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. Immunity in this model depends on CD4+ T cells and hence the role of negative costimulators that modulate T cell function can be studied using this model. Results: Peak parasitemia of P.yoelii-infected BTLA-deficient and HVEM-deficient mice was much lower compared to wild type mice. The increased immunity of BTLA-deficient mice depends largely on CD4+ T cells. We found that BTLA::HVEM interaction regulates the size and the cytokine-production of the responding T cell pool. However, in contrast to the CTLA-4 pathway, the manipulation of BTLA::HVEM interaction triggers no pathology during infection.

The HVEM::BTLA interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. 

. So far, the contribution of actin regulatory proteins to this process remained largely unknown. Here we demonstrate that the actin-bundling protein L-plastin is indispensable for segregation of LFA-1 and CD2 in the pSMAC of untransformed human T-cells. In marked contrast, TCR/CD3 accumulation in the cSMAC is not dependent on L-plastin. The relocalization of L-plastin in the immune synapse occurs within seconds of T-cell/APC contact formation and relies on actin polymerization. Importantly, binding of calmodulin to L-plastin is required for the maintenance of L-plastin in the immune synapse and inhibition of calmodulin prevents pSMAC formation. Thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein L-plastin and calmodulin. Protective T cell responses are based on expansion and persistence of clones with optimal affinity for antigen. Presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. Rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. Therefore we hypothesized that apoptosis might play an important role during this phase of T-cell biology. Here we show that the balance between the pro-apoptotic protein Noxa and its antagonist Mcl-1 regulates interclonal T cell competition during acute and chronic immune activation. We found p53-independent Noxa gene induction and Mcl-1 downregulation upon T cell activation. Concomitant we observed the release of death-inducing factor Bim from Mcl-1, which was delayed in Noxa -/cells. Using OT-1 cells and altered peptide ligands we observed that the level of Mcl-1 downregulation in activated T-cells depended on the antigen affinity of the T-cell receptor. Since Mcl-1 -/mice are embryonic lethal, Noxa -/mice were used to study the functional implications of this mechanism in vivo. At a young age Noxa -/mice have a normal lymphoid compartment, but accumulate effector T cells over time. Upon acute influenza infection, normal levels of effector cells were generated. However, the quality of the antiviral (NP366) response was impaired in these mice as many subdominant clones persisted in the effector T-cell population of Noxa -/mice at the peak of infection. This increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. In a model of chronic immune activation, effector T cells rapidly accumulated in the Noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. These results establish a novel role for the Noxa/Mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a Darwinian selection of T-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. Cytotoxic T lymphocytes (CTLs) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. Insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice.

Here, we refer to estrogen receptor-binding fragment-associated antigen 9 (EBAG9) as a negative regulator of secretory lysosome release. In gene deleted mice we show that loss of EBAG9 confers CTLs with enhanced cytolytic capacity, in vitro and in vivo. Here, we show that EBAG9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-Adaptin in T cells. Both interactions suggested an involvement of EBAG9 in endosome-lysosome related organelle biogenesis and membrane fusion. Efficiency of Granzyme B sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of Cathepsin D proteolytic processing. While the formation of the immunological synapse remained unaffected in EBAG9-/-CTL, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in EBAG9-deficient CTLs. These data imply a role for EBAG9 in regulating the formation of mature CTL granules and identify EBAG9 as a tunable inhibitor of CTL-mediated adaptive immune response functions. Finally, EBAG9 defines a novel negative regulator of secretory lysosome release in CTLs. Thus, the elucidation of the EBAG9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. The liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector T cells. Recently, we could show that liver sinusoidal endothelial cells (LSEC) inhibit activation of naive CD8 T cells by antigen-presenting DC. This regulatory effect of LSEC on DC function was MHC-independent and not limited to soluble mediators, but required physical contact. Interestingly, interaction with LSEC led to reduced DC expression levels of CD80/86 and IL-12.

In addition to indirect inhibition of T cell activation by de-licensing of DC, we now detected another influence of LSEC in form of direct inhibition of T cell priming.

In the presence of LSEC, stimulation with aCD3/aCD28 or PMA/Ionomycin could not significantly activate CD4 and CD8 T cells. Thus, LSEC did not only inhibit T cell priming triggered by TCR activation but also after elicitation of Ca 2+ influx into the cytoplasm. Furthermore, we found that IFN-g secreted by T cells in the early phase of activation is crucial for licensing the inhibitory function of LSEC. Taken together, these data indicate that the inhibitory effect of LSEC is mediated by a machinery induced at the early phase of T cell activation, however, interferes with late events in the T cell activation cascade. We propose a model of "inducible inhibition", where on the one hand naive T cell priming is directly inhibited by LSEC, and on the other hand tolerogenic priming by antigen-presenting LSEC is still allowed. Taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which LSEC not only tolerize T cells themselves, but also inhibit the responsiveness to local activation stimuli.

M. Almena 1 , S. Carrasco 1 , I. Merida 1 1 Centro Nacional de Biotecnología CSIC, Inmunología y Oncología, Madrid, Spain

Self-tolerance acquisition is essential for the immune system to control its own response. T cells achieve self-tolerance trough thymic selection and anergy, two processes where RasGRP1-Ras-Erk signal intensity is critical to determine the final cell outcome. RasGRP1 is a GEF for Ras that is activated in a diacylglycerol (DAG)dependent manner. DAG is generated by PLCg after TCR stimulation and is consumed by Diacylglycerol kinases (DGK). DAG generation, as a result of the concerted regulation of these two enzymes, activates Ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response.

1. Analyze the impact that DAG metabolism plays in T cell tolerance in vivo, using transgenic mice where DAG generation is impaired. 2. Develop a method to sense DAG production and localization, in both thymocytes and peripheral T cells, and its correlation with the strength of the stimulus used. Methods: We generated transgenic mice expressing a constitutively active DGK in T cell lineage. This protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after TCR stimulation. OT-I CD8 cells expressing GFP-C1 domains were used with peptide-pulsed APCs to study DAG generation and dynamics by confocal microscopy. Results: Transgene expression was obtained in thymic and peripheral T cells. No major defects were observed in T cell subsets but analysis of peripheral T cells demonstrated important defects in T cell activation. We are currently studying thymic selection breeding our transgenic with H-Y mice, in order to check if T cell populations are being properly selected. Using T cell-APC conjugates with peptides with different TCR binding affinities we found a clear correlation between the strength of the stimulus, DAG production and Ras-MAPK activation. Conclusion: Our data demonstrate that DAG generation not only activates C1 domain containing proteins but regulates a mechanism by which T cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in T tolerance. Future experiments will help to define the exact contribution of the lipid to TCR signaling pathways and to T cell homeostasis. The Inducible Costimulator (ICOS, H4, CD278), a CD28-like costimulatory molecule, has an important role in the development of efficient T cell responses. Early data showed that ICOS costimulation produced Th2 biased responses and high production of the anti-inflammatory cytokine IL-10, and was essential to the development of germinal centres. However, ICOS can also help in the IL-21-dependent differentiation of inflammatory Th17 cells. These different functions could be due to differences in the APCs bound by ICOS-expressing T cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of ICOS. ICOS shares with CD28 a YxxM cytoplasmic motif that can bind, upon Tyr phosphorylation, the regulatory p85 subunit of class IA PI-3 kinases. These can complex with one of the three 110 kDa catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate PIP 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. There is also evidence that the regulatory and catalytic class IA PI3K isoforms fulfill specific functions in macrophages and lymphocytes. We have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of ICOS, and particularly the presence of distinct PI3 kinase isoforms. Then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of ICOS activation.

Pull down of T cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of ICOS was carried out. Proteomic and immunoblot analysis of bound proteins showed that phosphorylated ICOS bound the different PI3-K regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) PI3-kinase subunits expressed by leukocytes. These data were confirmed in ICOS immunoprecipitates from pervanadate-activated cells. ICOS bound regulatory and catalytic subunits in the order p85a G p53a G G p85b and p110a G p110d G G p110b, in agreement with quantitative RT-PCR and immunochemical estimation of subunit abundance in the T cells and T cell lines used. The use of specific PI3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in ICOS function, including reorganization of actin cytoskeleton induced by ICOS ligands, or costimulation of TCR/CD3-induced secretion of IL-10 and IL-4. During the process of antigen recognition between T-cell and antigen-presenting cell (APC), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the Immune Synapse (IS) reorganize, displaying a segregated localization. In this context, the translocation of the microtubule-organizing center (MTOC) is an early event that occurs during the formation of the IS, bringing with it the Golgi apparatus, thus providing the basis for a polarized secretion. However, the molecular mechanisms involve in the localization of the MTOC at the contact area between the T cell and the APC are not completely understood yet. We have studied the possible role of scaffolding protein AKAP450, a member of the A-kinase anchoring protein (AKAP) family that localizes at the centrosome and interacts with PKA regulatory subunit and other signalling molecules, in MTOC polarization and immune synapse formation. Either the overexpression of GFPtagged C-terminal CG-NAP/AKAP450 construct that acts as a dominant negative, or siRNA knockdown of endogenous AKAP450 expression in T cells prevents the correct organization of CD3z and PKCV to the IS and MTOC reorientation towards T cell-APC contact area in antigen and superantigen-dependent human models, resulting in a disorganized IS; LFA-1 localization was also analyzed to assess p-SMAC architecture and, interestingly, confocal 3D reconstruction revealed that LFA-1 ring was not clear in the AKAP450-disrupted cells. Moreover, AKAP450 was required for TCR signalling since the knock down with specific siRNA and overexpression of C-terminal of AKAP450 decrease the phosphorylation of molecules such as LAT, PLCg1 and PKCV. These defective activation events as reflected in a reduction of IL-2 production. Together, our results underscore a key role for AKAP450 in the organization of the immune synapse and in the antigen-specific reorientation of the MTOC. The TCRbeta/pTalpha pre-TCR complex signals the expansion and differentiation of developing thymocytes. Functional properties of the pre-TCR rely on its unique pTalpha chain, which suggests the participation of specific intracellular adaptors. In fact, we have recently identified CMS, a member of the CIN85/CMS family of adaptors, as a pTalpha-binding protein that specifically interacted with the human pTalpha cytoplasmic domain via its SH3 domains, and to the actin cytoskeleton via its C-terminal region. We found that CMS co-localized with polymerized actin in pre-TCR clusters at the pre-TCR activation site, and also in the pTalpha endocytic compartment. Since actin polymerization plays a critical role in regulating signalling through the alpha/beta TCR in mature T cells, we decided to investigate the potential function of CMS as a regulator of actin polymerization and pre-TCR signalling in pre-T cells. Using pre-T cells expressing a mutant pre-TCR lacking the CMS-binding motif in the pTalpha tail and short hairpin iRNA-based gene silencing, we demonstrate that binding of CMS to pTalpha contributes to cytoskeleton dynamics and pre-TCR-mediated signalling in human pre-T cells. CMS-deficient cells specifically showed defects in pre-TCR-induced Ca2+ mobilization and cell activation involving the PI3K, NFAT, PLCg and ERK signalling pathways, together with defects in actin polymerization and cell motility. CMS therefore links cytoskeleton dynamics with the function of discrete pre-TCR signalling components, suggesting the functional implication of CMS in human T-cell development in vivo.

Abstract withdrawn by author

J Objectives: Most signaling pathways engaged after BCR activation have been described. However, several negative regulators of these pathways are unknown. The characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. Carabin (Tbc1d10c) has been recently described as a negative regulator of TCR signaling. It interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking NFAT nuclear transport. Moreover, Carabin maintains Ras protein under an inactive form, thus inhibiting Ras-MAPK cascade. Expression of Carabin is finely regulated following TCR signaling, and its knockdown (KD) enhances T cell activation. Considering the important molecular similarities of antigen receptor signaling pathways in T and B cells, we studied the role of Carabin in B cell. Could Carabin play a role of negative regulator of B cell function? Methods: We studied by quantitative RT-PCR 1) the expression of Carabin in different purified subsets of bone marrow and splenic mouse B cells, as well as 2) the kinetic of expression of Carabin in BCR stimulated murine splenic mature B cells.

3) We then studied the phenotype of Carabin KD (shRNA expressing) A20 B cells after BCR stimulation.

1) The expression of Carabin is significant in murine B cells, with an increase during B cell development, from bone marrow pro/preB to immature, to splenic T1 toT2 B cells and to follicular mature B cells.

2) The kinetic of expression of Carabin in BCR stimulated murine mature B cells suggests a fine regulation of Carabin expression.

3) BCR simulation, but not LPS stimulation, of Carabin KD A20 B cells shows an acceleration of Ras target Erk1/2 phosphorylation, without any for the phosphorylation of MAPK JNK, which is not targeted by Ras. Conclusion: Carabin is expressed in murine B cells in a developmental regulated manner, with the highest expression in mature compartment. BCR stimulation leads to a fine regulation of Carabin expression in wild-type mature B cells, and to a faster activation of Erk1/2 pathway in Carabin KD B cells. Altogether, these results strongly suggest a role of Carabin as a negative regulator of B cell function Toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. Toll like receptor 3 (TLR3) binds doublestranded RNA, a nucleic acid frequently associated with viral replication. We observed that freshly isolated human CD4 + T cells express TLR3 and respond to the well characterized synthetic TLR3 ligand polyinosinic-polycytidylic acid [poly(I:C)]. The expression of activation markers and cytokine production by CD4 + T cells upon T cell receptor (TCR) stimulation is enhanced in response to co-stimulation via TLR3. TLR3 stimulation on its own had no effect on expression of activation markers and cytokine production. To elicit the molecular basis of a potential cross-talk between TCR and poly(I:C) induced signaling, we used Jurkat cells to perform luciferase assays. We observed that costimulation with poly(I:C) in comparison to TCR stimulation alone enhanced NF-kB but not NFAT activation in Jurkat cells. Similarly to Jurkat cells, TCR stimulation activated NF-kB in primary CD4 + T cells. This effect was further enhanced by additional poly(I:C) stimulation as shown by real-time-PCR and Western blot analysis. On the other hand, we observed that poly(I:C) stimulation on its own activated the transcription factor interferon regulatory factor 3 (IRF3) as revealed by realtime-RCR analysis of IFN b and IRF7, whose transcription depends on the activity of IRF3. Combined TCR and poly(I:C) stimulation further enhanced the transcription of these two genes. These results indicate that TLR3 signaling modulates TCR-driven responses and vice versa both in Jurkat cells and in freshly isolated CD4 + T cells. This study was supported by DFG SPP 1110 "Innate Immunity" (Ka 502/8-3). The initiation of protective T cell responses requires the recognition of MHC-bound peptides from pathogen or tumor antigens by the T cell receptor (TCR). How this signal is transmitted across the T cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. In textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the CD3e cytoplasmic domain (CD3e CD ) binds to synthetic lipid vesicles that contain acidic phospholipids. This binding is predominantly due to electrostatic interactions between basic residues of CD3e CD and acidic phospholipids. In the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. Phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. To study the interaction of the CD3e CD with the membrane in live cells, we have developed a fluorescence resonance transfer (FRET) assay which measures the proximity between a fluorescent protein (TFP) attached to the C-terminus of CD3e CD and a fluorescent membrane dye (R18). With this assay, we show that the CD3e CD is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. Additionally, in vitro analysis confirm functional domains for CD3e CD lipid binding and conformational change. Finally, NMR spectroscopy analysis reveals key features in membrane binding dynamics of CD3e CD to lipid bicells. Membrane binding by the CD3e CD could thus be subject to dynamic regulation during the engagement of the TCR and further activation of the T cell.

M. Xydia 1 , Y. Ge 1 , U. Quitsch 1 , P. Beckhove 1 1 German Cancer Research Center (DKFZ), Heidelberg, Germany

in peripheral tissues and the factors affecting their proliferation. CD4+ T cell help is believed to contribute to optimal CD8+ memory expansion via CD40L on CD4+ T cells binding CD40 on dendritic cells. However, a few reports suggest that CD40L-CD40 engagement may mediate direct cell-cell contacts between CD4+ and CD8+ T cells. In this study, we investigated the importance of CD4-CD8 co-operation and CD40L-CD40 interactions for T EM proliferation. Methods: We isolated human CD4+ and CD8+ T EM cells from peripheral blood of healthy donors by FACS or MACS sorting. Separated or mixed CD4+ and CD8+ populations were activated in vitro using anti-CD3/CD28 beads. Proliferation was measured by [ 3 H]-Thymidine incorporation, in some experiments after irradiation of one T EM subset and/or incubation with blocking mAbs against CD40 or CD40L. Furthermore, FACS staining was used to assess cell-surface markers. Statistical comparison was performed by student's t test.

Results: Upon activation mixed T EM populations showed a highly better proliferative response than separated CD4+ or CD8+ T EM cells, demonstrating that optimal T EM expansion requires direct CD4-CD8 interactions. Surprisingly, not only CD8+ but also CD4+ T EM cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal CD4+ T EM proliferation depends on signals from CD8+ T EM cells. Activation induced the expression of CD40 on both populations and CD40L on subsets of CD4+ and CD8+ T cells. Blocking of CD40L on CD8+ T EM cells impaired significantly CD4+ T EM proliferation, which confirms that the improved expansive potential of CD4+ T EM cells in mixed populations depends on CD40L co-stimulation by the CD8 T EM subset. Conclusions: Our data demonstrate for the first time that activated CD8+ T EM cells deliver help to the CD4+ T EM subset via CD40L-CD40 signalling and may play an important role for CD4+ T EM expansion upon stimulation. The T cell surface glycoprotein CD5, a member of the Scavenger Receptor Cysteine-Rich (SRCR) family of proteins, targets to the immunological synapse upon T cell binding to antigen presenting cells (APC). However, it has not been established whether this translocation is due to the binding of a ligand expressed in the APC, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control CD5 localization upon T cell:APC conjugation.

We have questioned which domains of CD5 mediate the localization within the IS, and for this we have expressed CD5 mutants as GFP fusion proteins in human T lymphocytes. We have also used Jurkat cell lines expressing different CD5 mutants. T cells were incubated with superantigen-loaded Raji B cells, and following the establishment of stable interactions between the cells, we analyzed the localization of CD5 by immunofluorescence and confocal microscopy. Interestingly, our results show that the translocation of CD5 depends on sequences within the cytoplasmic domain, as a CD5 deletion mutant lacking most of the cytoplasmic tail, CD5.K384 stop , is randomly distributed through the whole cellular surface, even in sustained T-APC interactions. The cytoplasmic domain relevant to CD5 translocation was mapped within amino acids Glu 418 and His 449 since the CD5.H449 stop mutant, just short of 22 aa is still able to translocate to the IS, whereas CD5.E418 stop , that lacks 2 important tyrosine residues, is no longer transported to the IS upon T cell: APC interactions. Although these studies do not exclude a role for the extracellular domain binding to an elusive APC-expressed ligand, they suggest that a major mechanism of regulation of CD5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (Glu 418 -His 449 ) to intracellular signaling effectors. However, in Hodgkin's lymphoma (HL) EBNA-2 is missing, but LMP-1 is still expressed. Using a HL derived cell line, we have shown that the cytokine IL-4 can induce LMP-1 expression in vitro and can replace EBNA-2. We have investigated the molecular events for this mechanism. STAT proteins bind to the palindromic TTC(N) x GAA sequence, where × is 2, 3 or 4. A high affinity STAT6 binding site is spaced by 4 nucleotides. We found three potential STAT binding sites in the LMP-1 promoter, which we named LRS, TR and EDL1. They were spaced by 4, 3 and 2 nucleotides, respectively. Electrophoretic mobility shift (EMSA) experiments were performed with nuclear extracts prepared from IL-4-treated or non-treated KMH2-EBV cells. DNA binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (GL) epsilon promoter, which is known to contain a high affinity STAT6 binding site, or LRS-STAT6. A STAT6 complex binding to the GL-epsilon promoter and LRS-STAT6 was induced by IL-4. The specificity of the STAT6 complex was shown by supershift experiments with anti-STAT6, but not anti-STAT5 antibodies. When GL-epsilon or LRS-STAT6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the LRS-STAT6 contains a functional STAT6 binding site. Oligonucleotides, corresponding to LRS in which the STAT6 site had been mutated, could not compete for STAT6 binding. Interestingly, the unlabeled LRS-TR with 3 nucleotides as spacer could also function as competitor. However, when TTC/GAA palindrom was spaced by 2 nucleotides (LRS-EDL1), it could not compete. Thus, expression the transforming protein LMP-1 can be induced directly by the T cell derived cytokine IL-4 in a STAT6 dependent manner. It is likely that this mechanism operates in vivo as well and determines expression of the EBV encoded protein LMP-1 and thus the pathogenesis of EBV carrying HLs. established knockout/ knockin mice with a FasL deletion mutant that lacks the intracellular portion (FasL ¿ Intra). Co-culture experiments confirmed that the truncated FasL protein is still capable of inducing apoptosis in Fas-sensitive cells. Preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular FasL domain does not alter the intracellular FasL localization in activated T cells. We are currently investigating signalling and proliferative capacity of B-and T-cells derived from homozygous FasL ¿ Intra mice. Our data point to a rather inhibitory role of FasL reverse signaling during immune responses.

During an immune response numerous receptor-mediated signals delivered to T cells direct their proliferation, survival and differentiation. We are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms T cells use to process these multiple signals. Previous experiments with OT-I CD8 T cells revealed that TCR affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (JI 2007 (JI . 179:2250 . Furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. Here we examine the same question for CD4 T cells. Again we find that lower affinity peptides stimulate T cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. In contrast varying the number of dendritic cells used to stimulate CD4+ T cells primarily affect the proportion of CD4+ T cells going into division rather than affecting division time or cell death in subsequent divisions. Currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules CD40, CD80 and CD86 on parameters of CD4+ T cell proliferation to inform quantitative models of the immune response under different conditions. Our goal is to develop quantitative models of T cell behaviour that can accommodate information at the molecular, cellular and population level. Interaction between CD40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as B cells, and CD40L, a member of the tumor necrosis factor family transiently expressed on activated T cells, are essential for the development of humoral adaptative immune response.

Various studies have shown that dual stimulation of B cell through antigen binding on BCR and CD40 leads to an enhancement of Ig and cytokine production. The current dogma postulates that these 2 signals are necessary and sufficient to drive naive B cell proliferation and differentiation to Ig secreting plasma cells. However, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. Indeed, B cells can be activated through engagement of a variety of innate immune receptors, including Toll-Like Receptors (TLRs). Soluble CD40L is unable to induce murine B cell proliferation. However, we and others have shown that recombinant mouse CD40L (rmCD40L) can increase proliferation induced by TLR3 (Poly IC) and TLR4 (LPS) agonists. By contrast, we never observed any synergy between rmCD40L and TLR1/2 (Pam3CSK4) or TLR2/6 (Pam2CSK4) agonists. To go further in the study of CD40L/TLR agonist synergetic effect, we have developed trimeric synthetic molecule to mimick CD40L, named mini-CD40Ls, based on a C 3 -symmetry core holding CD40-binding motif Lys-Gly-Tyr-Tyr. In surface plasmon resonance experiments, mini-CD40Ls bind to immobilized human CD40 and compete with the binding of CD40L homotrimers and diplayed effector functions that matched those of the much larger recombinant CD40L homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of Trypanosoma cruzi infection. As soluble CD40L, mini-CD40Ls synergize TLR4 (LPS), TLR3 (Poly IC) and TLR7/8 (R848) agonist-induced murine B cell proliferation but no synergy was observed between mini-CD40Ls and TLR 1/2 (Pam3CSK4), TLR 2/6 (Pam2CSK4) and TLR9 (ODN 2395) agonists. Synergy between CD40L and TLR agonist provide the ground to use such a combination as adjuvant in vaccination strategy. However, to reach this goal, evaluation of CD40L/TLR combinations on murine and human B cell activation and differentiation in antibody producing cells are under investigation. Interaction of naïve CD8 + T cells with immature dendritic cells (iDC) expressing self-peptides can result in their abortive activation (AA), which leads to the induction of CD8 + T cell tolerance. We have defined a phenotypic profile for CD8 + T cells undergoing such AA. These cells undergo limited proliferation which is associated with lack of IFN-g production, low cell surface expression of CD25 and CD69, and high levels of expression of CD62L and Ly6C. Whereas, CD8 + T cells undergoing productive activation (PA), following encounter with mature DC, form effector CTL which is evidenced by extensive T cell proliferation, high levels of IFN-g, CD25 and CD69, and loss of CD62L and Ly6C expression. Ly6C is a GPI-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. In this study, we show that mAb-blocking of Ly6C in vivo and in vitro results in PA rather than AA. We hypothesize that the interaction of Ly6C, expressed on naïve CD8 + T cells, with its ligand on iDCs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive CD8 + T cells. Objectives: Organophosphorus compounds (OPCs) are commonly used in the manufacture of insecticides and pesticides. Exposure to OPCs is associated with neurological toxicity but the effect on the immune system remains ill-defined. In this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. Methods: BALB/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. During the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. At the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. Spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by ELISA. For in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of S. typhimurium. Animal survival was followed for up to 60 days after infection. Results: Daily injection of paraoxon induced G 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. Mice exposed to paraoxon exhibited G 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. At the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. To analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of S. typhimurium and followed for survival. Unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. Protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. Conclusion: Our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. This suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. T cell-dependent (TD) and T cell-independent (TI) IgG autoantibodies have been described in the context of the autoimmune disease Systemic lupus erythematosus (SLE). However, their different roles in autoimmunity are unknown. Here we show that TI antigens induce anti-inflammatory IgG antibodies and protect from antigen-specific immune pathology. Administration of antigen-specific anti-inflammatory IgG antibodies was sufficient to mediate this effect independent of the IgG inhibitory receptor FcgammaRIIB. TI but not TD IgG autoantibodies were further associated with inhibition of pro-inflammatory Th1 and Th17 cells and disease in mice deficient for FcgammaRIIB, a spontaneous model for SLE. The data suggest a novel immune regulatory function for TI immune responses through the generation of anti-inflammatory IgG antibodies. Objective: Class I phosphoinositide 3-kinases (PI3K) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase-associated receptors or G protein-coupled receptors (GPCR). The class I PI3K are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110g (p110gamma) isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a Tyr kinase-associated receptor, previous studies showed that p110g deletion affects TCR-induced T cell stimulation. Mice lacking p110g show a partial defect in T cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-TCR dependent differentiation and CD4+/CD8+ T cell lineage commitment. In the MRL/lpr mouse model of systemic lupus erythematosus, administration of a PI3Kg-specific inhibitor causes a reduction in the number of CD4+ memory T cells that mediate renal injury. Similarly, PI3Kg deletion in p65 PI3K transgenic mice also reduces the numbers of CD4+ memory T cells.

There is therefore evidence that PI3Kg has an important function in TCR-mediated T cell activation, although the mechanism by which PI3Kg regulates this process is not well understood. We studied the specific role of p110g in T cell activation. Methods: We studied whether the TCR activates p110g and the consequences of interfering with p110g expression or function on T cell activation. Results: We found that after TCR engagement, p110g interacts with and forms a complex with Ga q/11 , Lck and ZAP70. TCR stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110g controls Rac1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells (APC).

We show that p110g deletion affects the activation of many pathways downstream of TCR crosslinking, as well as the interaction between T cells and APC; these findings could explain the defective activation of p110g-/-T cells. Our observations clarify the activation mechanism and mode of action of p110g in the control of T cell activation, confirming a crucial role for p110g in TCR-induced T cell activation. We investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic T lymphocytes (CTL). We show that cytolytic granules in CTL can be delivered to the secretory domain via two different pathways -"short" and "long". The choice between these pathways is regulated by the kinetics of early TCR signaling which depends on the strength of TCR/pMHC/co-receptor interactions. Meanwhile, the molecular hardware used to deliver the granules remains the same. We conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (MTOC) and the MTOC polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of CTL cytolytic response. Our findings reveal a mechanism of well-documented flexibility in T cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. Objectives: In addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. Nicotine was shown to suppress both cellular and humoral immune responses. Previously we found that two nicotinic acetylcholine receptor (nAChR) subtypes, a4b2 and a7, expressed in mouse B lymphocytes regulate their development within the bone marrow. The aim of the present study was to evaluate the roles of these two nAChRs in B lymphocyte activation. Methods: B lymphocytes were magnetically separated from the spleens of C57Bl/6J mice. They were stained with fluorescently labeled IgM-, CD40-or CD23specific antibodies in the presence/absence of unlabeled nAChR subunit-specific antibodies to be examined by flow cytometry. B lymphocyte activation was studied by 3 H-thymidine incorporation upon stimulation with anti-CD40 and nAChR-specific agonists or antagonists. The antibody response of mice immunized with cytochrome c with or without a7 nAChR antagonist methyllicaconitine (MLA) was studied by ELISA. Results: Antibodies against a4 or b2 nAChR subunits inhibited binding of IgM-and CD23-specific antibodies but facilitated that of CD40-specific antibody. In contrast, antibody against a7 subunit prevented binding of anti-CD40 but not of anti-IgM or anti-CD23 suggesting that a7 nAChRs are located close to CD40, while a4b2 ones are close to BCR/CD23. Consequently, anti-CD40-induced B lymphocyte proliferation was increased by MLA much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. It was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. In contrast, proliferation of B lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. Mice co-injected with cytochrome c and MLA responded with IgM antibodies faster than those injected with cytochrome c alone, while the secondary / IgG responses were similar.

The CD40-mediated B lymphocyte proliferation, but not the IgM-IgG switch or memory B cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nAChRs. Therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.This work was supported by Philip Morris USA Inc. and Philip Morris International. Binding of CD4 + T H -lymphocytes to antigen presenting cells or of CD8 + cytotoxic T-lymphocytes (CTL) to their target cells lead to a tight contact between these two cells, called immunological synapse (IS). Formation of the IS induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the IS, all of which are crucial for T-cell functions such as cytokine release or target cell killing. Objectives: Using primary human T-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. For testing protein functions, down-regulation by RNAi technology is thus an important tool. We used short interfering RNAs (siRNAs) to analyze the role of proteins involved in calcium influx and proliferation (Stim1 and TRPC3), and to analyze SNARE proteins which were shown to accumulate at the IS and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. Methods: To validate down-regulation of different mRNAs quantitative RT-PCR was used. Down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). Results: Transfection efficiency of siRNAs in T-lymphocytes was about 96 %. Down-regulation of Stim1 was confirmed by qRT-PCR and by calcium imaging, but only for early time points following activation of CD4 + T H -lymphocytes, probably because of stability problems. To increase stability of siRNAs within T-lymphocytes we used modified siRNAs published by Mantei et al. (EJI, 2008) . We show that these siRNAs down-regulate various SNARE proteins in CTLs more efficiently than non-modified siRNAs. The optimal siRNA concentrations for transfection in primary human T-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. Conclusions: Following optimization, down-regulation of mRNAs by siRNA is a powerful tool to investigate the role of different proteins involved in the activation of T-lymphocytes in primary human cells. Chemical modifications increase the lifetime and efficiency of the siRNAs in primary human T-lymphocytes. Stress-inducible heat shock protein 70 (HSP70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific CD8 + cytotoxic T-lymphocyte (CTL) and CD4 + T-helper cell (Th1) responses. In this study, we investigated the behavior of T-cell subsets stimulated with endotoxin-free recombinant HSP70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic B-lymphoblastoid cell line (B-LCL) and K562 cells as well as targetindependent cytotoxicity. CD4 + cells exhibited a strong increase in proliferation after stimulation with HSP70, with rates of up to 29 %. In the presence of target cells, a 35-fold up-regulation of granzyme B mRNA was observed after stimulation of CD4 + T-helper cells with HSP70 in combination with IL-7, -12 and -15. The target cell-independent secretion of granzyme B by CD4 + cells was greatly augmented after stimulation with HSP70 plus IL-2 or IL-7, -12 and -15.

In this study, we have shown that HSP70 is capable of inducing a cytotoxic response of T-helper cells in the absence of LPS or any other PAMPs. The granzyme B secretion and the cytolytic activity of CD4 + T cells is induced in a target-independent way, whereas the cytotoxic activity of CD3 + and CD8 + T cells can be further enhanced in the presence of the target cells. Our data provide novel insights into the role of extracellular HSP70 on T-cell immune response concerning the induction of target-independent T-helper cell cytotoxicity. Jun N-terminal kinases (JNK) have been shown to play controversial role in regulation of cell fate. CD40, which is responsible for germinal centre formation in lymph nodes, trigger JNK activation. The role of other B cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. The aim of this study was to find out whether CD150 receptor contributes to JNK activation in mature human B cells. Protein expression and phosphorylation were studied by Western blot analysis. Protein associations were evaluated by immunoprecipitation and GST-pull down assays. HPK1 overexpression in a model system was achieved by transfection. pJNK1/2 expression in primary HRS cells was assessed by immunohistochemistry. Ligation of CD150 on resting (dense) and activated (buoyant) human tonsillar B cells lead to JNK2, but not JNK1 activation. CD40 ligation on primary tonsillar B cells also resulted in JNK2 activation. However, BCR crosslinking did not affect the level of JNK1/2 phosphorylation. CD150-mediated JNK2 activation was independent from SH2D1A/SAP adaptor protein expression, and was demonstrated for all studied B-lymphoblastoid, Burkitt's lymphoma and Hodgkin's lymphoma (HL) cell lines of B cell origin. We were searching for serine/threonine kinase that could coprecipitate with CD150 and link this receptor with JNK pathway. Using immunoprecipitation and GST-pull down assays we found that hematopoietic progenitor kinase 1 (HPK1) was associated with CD150 in primary B cells as well as in B cell lines. CD150-HPK1 association was independent from CD150 tyrosine phosphorylation and SH2D1A expression. Overexpression of HPK1 in a model system significantly enhanced CD150mediated JNK2 phosphorylation. It is known that TNF family receptors such as CD30, CD40, RANK trigger survival signals in HRS cells. We observed the expression of pJNK1/2 in HRS cells of primary classical HL. CD150 could be involved in sustained JNK2 activation in primary HRS cells, and this may reflect the role of CD150 receptor as well as other receptors in the regulation of HRS survival. Overall, it was shown that JNK2 is activated via CD150 in primary B cells and in all studied cell lines of B cell origin. Serine-threonine kinase HPK1 is involved in CD150-mediated JNK2 activation. Objectives: CD5 has been shown to act as a negative regulator of TCR signaling during thymocyte development. However, the molecular mechanisms involved in this process remain elusive. One potential key molecule involved in the downmodulation of TCR signaling is c-Cbl, a ubiquitin ligase that physically associates with CD5 upon TCR crosslinking in thymocytes. The objective of this study was to determine which sequences within the cytoplasmic tail of CD5 are involved in c-Cbl phosphorylation and association. Methods: EL4 thymoma cell line was stably transfected with wild-type human CD5 or hCD5 cytoplasmic tail mutants: CD5.K384STOP (maintaining only a pseudo ITIM); CD5.H449STOP (lacking the distal S and Y in the carboxy-terminal region); CD5. ¿ E418-L444STOP (lacking the pseudo-ITAM, putative site for c-Cbl association). Phosphorylaton of Y700 in c-Cbl was analyzed, which is required for Vav recruitment and C-Cbl dependent degradation by the proteasome. Stable clones were stimulated with anti-murine CD3 in combination or not with anti-human CD5 biotinylated antibodies and phosphorylation of c-Cbl was detected by flow cytometry after intracellular staining anti-phospho c-Cbl (pY700) antibody. Murine thymocytes were used as positive control. Data was analyzed using FlowJo software. Unpaired two-tailed Student T test was used to calculate statistical significance (p X 0.05).

In murine thymocytes, co-crosslinking of CD3 with CD5 induces an increase in c-Cbl phosphorylation compared to CD3 alone. Analysis of the EL-4 transfectants showed that mutants CD5.K384STOP and CD5.H449STOP lost the ability to costimulate CD3-mediated phosphorylation of c-Cbl. In contrast, CD5. ¿ E418-L444STOP mutant, was able to efficiently costimulate CD3-mediated c-Cbl phosphorylation, similarly to the hCD5wt.

Our results indicate that the absence of the pseudo ITAM in CD5 does not interfere with c-Cbl phosphorylation in response to CD3 plus CD5 crosslinkiing On the other hand, sequences present in the carboxy-terminal region of CD5 appear to be important for C-Cbl phosphorylation. Therefore, c-Cbl phosphorylation might not require physical association with the CD5 cytosplasmic tail, but rather, may indirectly associate with CD5 through the interaction with other SH2-SH3 domain-containing molecules, that may be recruited to CD5 through its carboxy-terminal region.

L. Kolly 1 , S. Narayan 1 , J. Tschopp 2 , A. So 1 , N. Busso 1 1 CHUV, Rheumatology, Lausanne, Switzerland, 2 UNIL, Biochemistry, Epalinges, Switzerland

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent IL-1b and IL-18 production. Despite recent evidence implicating ASC in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for ASC involvement in adaptive immune responses remains largely unexplored. To investigate the impact of ASC on T cell activation and subsequent effector function. ASC +/+ and ASC -/-T cells or purified CD4 + and CD8 + T cells were activated in vitro through anti-CD3 stimulation and their proliferative potential and cytokine profiles characterized. Proliferative responses by ASC -/-T cells were significantly inhibited two-fold following TCR-CD3 ligation when compared to ASC +/+ T cells. Furthermore, cytokine analysis revealed that anti-CD3 activated ASC -/-T cells predominantly displayed a more Th 2 phenotype, producing more IL-10 (199 vs. 692 pg/ml; ASC +/+ vs. ASC -/-T cells respectively; p=0.0074) and less IFN-g (15,831 vs. 6 ,921 pg/ml; ASC +/+ vs. ASC -/-T cells respectively; p = 0.0021). When ASC +/+ and ASC -/-T cells were purified into CD4 + and CD8 + T cell fractions and activated individually using anti-CD3, no inhibition in proliferation was observed amongst activated ASC -/-CD4 + and CD8 + T cells. Interestingly, the activated ASC -/-CD4 + T cell fraction produced significantly more IL-10 when compared to activated ASC -/-CD8 + T cells and ASC +/+ CD4 + and CD8 + T cells (ASC -/-CD4 + T cells = 380 pg/ml IL-10; ASC -/-CD8 + T cells = undetectable IL-10; ASC +/+ CD4 + T cells = 11 pg/ml IL-10; ASC +/+ CD8 + T cells = undetectable IL-10). CD4 + and CD8 + T cell mixing experiments revealed that ASC -/-CD4 + T cells are able to inhibit the proliferative ability of ASC -/-CD8 + T cells, ASC +/+ CD4 + and CD8 + T cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated ASC -/-CD4 + T cells. Collectively, these results demonstrate that the absence of ASC drives CD4 + T cells towards a suppressor cell phenotype, suggesting that ASC might play an important role in determining the fate of CD4 + T cells. Various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. In particular, E-series prostaglandins, PGE 1 and PGE 2 suppress some T-cell functions including proliferation, activation and cytokine production. PGE 2 signals through four types of GPCRs called the EP receptors. At low concentrations, PGE 2 is believed to be necessary for T cell function, whereas at higher concentrations, PGE 2 inhibits T cell proliferation. These effects are largely governed by various cell specific stimuli and tissue microenvironment. Objectives: To delineate, compare and contrast the effects of PGE 2 and EP receptor antagonists on T cell activation. Methods: Flow cytometry, proliferation assays, migration assays.

We have observed that PGE 2 diminishes expression of early, intermediate and late T cell activation markers. In contrast, pre-treatment of CD4 + T cells with EP receptor antagonists was found to impair cell surface expression of CD71, CD69, CD25 and OX40 but not CD44. Suppression of T cell proliferation by PGE 2 has already been widely studied. However, blocking EP receptors in CD4 + T cells by the use of EP antagonists prior to activation surprisingly caused a defect in T cell proliferation. Migration of CD4 + T cells to the chemokine SDF-1b was also found to be reduced due to pre-treatment with EP antagonists. In order to study the physiological relevance of these findings we studied the trafficking of basal and activated T cells to regional lymph nodes during inflammation in the presence and absence of EP receptor antagonists. This model revealed that the use of EP antagonists causes a reduction in the amount of CD44 + CD4 + adoptively transferred T cells in the regional lymph node following the induction of a local inflammatory response. Conclusions: In our study we show for the first time that EP receptors are required for expression of activation markers and activating proliferation in murine CD4 + T cells. Our results also suggest that considering PGE2-mediated cAMP signaling in CD4 + T cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in T cell activation.

Objective: Our objective was to investigate how ROS affect the different stages of T cell activation. Because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ROS affect calcium signalling.

The experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, T-cell activation assays and molecular biology. Results: We show by direct measurement of ROS that T-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. The effect of ROS on calcium signalling in Jurkat T-cells as well as in primary naï ve and effector CD4 + human T-cells was examined. Oxidation affects several Ca 2+ signalling pathways by altering the activity of IP 3 receptors, TRP channels and store operated Ca 2+ channels in a concentration dependent manner. Interestingly, calcium signalling is differentially affected in naï ve and effector T cells. Thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of Ca 2+ signalling in T-lymphocytes. Cysteins are the main carrier of thiol groups in proteins and we show that ORAI ion channels contain reactive cysteine groups that mediate ROS effects on the calcium influx pathway. Conclusion: ROS regulate the calcium dependent T-cell activation in a complex way, affecting all three major calcium signalling pathways. By mutational analyses of the ORAI proteins, we are able to pinpoint molecular targets of regulation. The activation of T cells during an immune response is a crucial but tightly regulated event. To make the grade, the T cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. This enables the T cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. In this study we present data of the signaling mechanisms underlying the potent T cell inhibitory receptor CD147 (EMMPRIN, basigin) , a member of the Ig-family. Previous studies reported that lymphocytes from CD147 knockout mouse possess enhanced mixed lymphocyte reactions and CD147 monoclonal antibodies can interfere with T cell activation. These observations already pointed to a negative crosstalk of CD147 signals with the T cell antigen receptor or co-stimulatory signals. Consistent with these studies, we found that RNA interference (RNAi) with CD147 in Jurkat T cells augments the secretion of the T cell growth-factor interleukin-2 (IL-2) upon T cell activation. This up-regulation is at least partially due to an increased activity of the nuclear factor of activated T cells (NFAT), which resulted in an enhanced IL-2 promoter activity. By reconstituting the RNAi-mediated knockdown with various truncated RNAi-resistant forms of CD147, we identified the immunomodulatory sub-domain of CD147. Supported by the GEN-AU Program of the Austrian Federal Ministry of Science and Research. miRNAs play a critical role in the control of hematopoiesis. The goal of this project is to determine whether miRNAs function also during the antigen-induced activation of mature B lymphocytes. Therefore, we determined miRNA profiles in primary splenic B cells before and after polyclonal activation with either LPS (simulates T cell-independent activation) or a combination of anti-IgM, anti-CD40 and IL4 (simulates T cell-dependent activation). Microarray assays identified about 104 miRNAs in unstimulated B cells. 35 of these were downregulated and one was upregulated upon stimulation. In silico analyses with various miRNA target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by miRNAs during the antigen-induced activation phase of mature B cells. Among these targets are BCR signalling molecules and transcription factors that control proliferation, IgH class switch as well as differentiation in antibody-secreting plasma cells. One of these transcripts codes for the interferon regulatory factor-4 (IRF-4). The graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. First results indicate that the expression kinetic of IRF-4 transcripts differs from that observed for IRF-4 protein abundance after B cell stimulation. Further analysis identified the IRF-4 transcript as a target whose expression is obviously fine-tuned by a miRNA upon antigen stimulation. We are in the process to biochemically verify potential targets for each of the differentially regulated miRNAs and determine the effect of ectopic and retrovirally mediated expression of miRNAs on B cell differentiation. The work was in part supported by the IZKF Erlangen, the DFG Graduiertenkolleg GK592 and the DFG Forschergruppe FOR832. Objective: Transforming growth factor-b (TGF-b) signals through type I (TGFbRI) and type II (TGFbRII) TGF-b receptors and receptor regulated Smad proteins. TGF-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. The mechanisms of resistance against TGF-b have not been fully elucidated. Our aim is to describe how B cell lymphoma cells respond to TGF-b compared to normal peripheral B cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to TGF-b. Methods: Proliferation assays were performed on 11 different B-cell lymphoma cell lines and normal peripheral B cells to screen for TGF-b-induced effects. Western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to TGF-b signaling pathways. FACS analysis was used to measure TGF-b receptor surface levels. Cells were treated with demethylating agents to examine changes in gene expression levels. 

S. Manthey 1 , F. Hauck 2 , I. Berberich 1 , F. Berberich-Siebelt 2 , GK 520 -Immunomodulation 1 Institute for Virology and Immunobiology, University of Wuerzburg, Würzburg, Germany, 2 University of Wuerzburg, Department of Molecular Pathology, Würzburg, Germany

The transcription factor CCAAT/enhancer-binding protein b (C/EBPb) can not act only as a transcriptional activator but also as a transcriptional repressor. In murine CD4+ T lymphocytes, the transcription factor is predominantly expressed in T helper 2 (Th2) compared to T helper 1 (Th1) cells. In contrast, by binding to the c-myc promoter(s), C/EBPb represses c-Myc expression thereby arresting T cells in the G1 phase of the cell cycle. Both, transactivation and repression depend on the N-terminal transactivation domain of C/EBPb. Blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of B cells to plasma cells. Furthermore, Blimp-1 is expressed in differentiated effector T cells where it is higher in Th2 than Th1 cells. The regulation of the Blimp1 expression is not fully understood. Interestingly, we found that C/EBPb can bind to the prdm1 promoter and activates Blimp-1 expression in T cells. As C/EBPb is also expressed in B cells, we hypothesize that this transcription factor might as well influence the expression of Blimp-1 in B cells. So far, we were able to show a similar expression profile of C/EBPb and Blimp-1 in B cells using Cre recombinase. Moreover we found a new putative Blimp-1 isoform lacking exon 2.

Currently, we analyze the expression of C/EBPb and Blimp-1 in primary B cells and B cell lines after various stimulations. To get more insights into the function of C/EBPb in B and T cells, we are generating mice carrying a B as well as a T cell-specific deletion of C/EBPb. Engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase C gamma (PLCy) and plays an important role in activation of cells. By screening 53 CVID patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in B and T cells isolated from the peripheral blood of patients in the group Ia of the Freiburg classification as compared to non-Ia patients and healthy donors (HD). Ia patients are characterized by the expansion of an unusual CD21low B cell population in which calcium mobilization is strikingly lower than in other B cell subsets. Common subpopulations like naï ve and MZ-like B cells as well as CD4 + T cells but not transitional B cells or CD8 + T cells also revealed significantly decreased calcium peaks. The cytometric data correspond to a semiquantitative RT-PCR assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (MIP-1a), and abrogated activation and proliferation, respectively. Preliminary data on B cell receptor (BCR) mediated phosphorylation of PLCy2 revealed constitutively high background levels in CD21low B cells of Ia patients. Since phosphorylation in the other B cell populations as well as calcium flux upon Ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of PLCy and upstream of internal store release, thus resulting in defective calcium signalling. The underlying mechanism yet remains to be elucidated and is part of our work in the future.

C. Balas 1 , V. Courtois 1 , K. De Luca 1 , R. Sodoyer 1 1 Sanofi Pasteur, Marcy l'Etoile, France

The presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. A potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. For instance monitoring of the expression level of cytokines, such as IL-2, IL-4 or IL-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. The analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. Such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. Methods: Genetic tools (primers and qPCR probes) capable of discriminating and quantifying alternatively spliced messenger RNAs from IL-2, IL-4 and IL-6. Furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated.

The genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract No A-136-0033-01835). Conclusion: Implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. In a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. Parasitic helminths affect about one third of the world population. Therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. From other parasitic infections it is known, that the regulatory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a crucial role during infections. Here, we use the Strongyloides ratti infection of mice as an experimental system to investigate the role of CTLA-4 during nematode infections. We employed a quantitative real-time PCR (qtPCR) analysis to quantify the migrating larvae (iL3) in the tissue and the released eggs and first stage larvae (L1) in the feaces. The cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mLN) upon stimulation with polyclonal a-CD3 and S. ratti antigen was determined. Additionally the humoral response was analysed in the primary and the secondary infection. To investigate the role of CTLA-4 during the infection, a neutralysing antibody (a-CTLA-4; 4F10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with S. ratti iL3. The in vivo neutralisation of CTLA-4-signalling by applying a-CTLA-4 during S. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. We detected an increase in Th2 cytokines, such as IL-4 and IL-5 and a reduction of the proinflammatory cytokines IFN-g and IL-17. The investigation of the humoral response showed a remarked increase of the IgG1-titer in the serum during secondary infection in mice that had been treated with a-CTLA-4 during primary infection. Furthermore, the blockade of CTLA-4 resulted in a diminished worm burden as indicated by reduced release of L1 in the faeces. These results suggest that the blockade of CTLA-4 during S. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. In particular the transition of the T cell cytokine profile towards a Th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. The strong increase of IgG1 during secondary infection also reflects the induction of a potent Th2 response. Objectives: CD36 is a class B scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. This clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (SLE). It was recently described that CD36 is highly expressed also on the Marginal Zone B cell subtype. We therefore set out to investigate the role of CD36 on the regulation of B cell in the setting of apoptotic cell clearance and autoimmune activation.

We used a mouse model for SLE where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. ELISA was used to measure antibody levels and flow cytometry to study cell activation as well as CD36 expression. CD36 knock-out (KO) and wild type mice were used. Results: Preliminary in vivo data show a tendency for a higher antibody response towards ds-DNA and the common self-antigen PC in CD36 KO compared to heterozygous mice. Since reduced levels of CD36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison.

In support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with LPS is gone in CD36 KO mice. After one injection of apoptotic cells, CD36 KO B cells are activated while wild type B cells are not. After four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that CD36 on B-cells are involved in setting this threshold. Conclusion: Our data suggest that CD36 is involved in the early regulation of B cell response towards apoptotic cells and production of autoreactive antibodies. It does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. Successful T cell immunity requires lymphocytes to be at the right time at the right place. The co-receptor CD152 acts as a major check-point of immune responses, but the mechanism by which CD152 controls peripheral T cell responses is unknown. The consequences of CD152 signaling on murine Th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. The genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19. Crosslinking of CD152 together with CD3 and CD28 stimulation on activated Th1 cells increased expression of the chemokine receptors CCR5 and CCR7, which in turn enhanced cell migration. Sensitive liposome technology reveals that mature dendritic cells but not activated B cells were potent at inducing surface CD152 expression and CD152-mediated migration-enhancing signals. Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased, as compared to CD152 negative counterparts, showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo. These data show that CD152 signaling does not just silence cells, but selects individual ones for migration. This novel activity of CD152 adds to the already significant role of CD152 in controlling peripheral immune responses by allowing T cells to localize correctly during infection. It also suggests that interference with CD152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. Here we analyzed the role of CD152 signaling on the longevity of CD28 null T cells. Using a sensitive staining method for CD152, we show that human CD4 + CD28 null and CD8 + CD28 null T cells rapidly express surface CD152. Serological inactivation of CD152 using specific Fab fragments or blockade of CD152 ligands using CTLA4Ig in CD4 + CD28 null and CD8 + CD28 null T cells reduces the number of non-apoptotic cells in a Fas/FasL-dependent manner. CD152 crosslinking on activated CD28 null cells prevents activation-induced cell death (AICD) as a result of reduced Caspase activity. Apoptosis protection conferred by CD152 is mediated by PI3'K dependent activation of the kinase Akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule Bad. We show that signals triggered by CD152 act directly on activated CD28 null T lymphocytes and, due to its exclusive expression as a receptor for CD80/CD86 on CD28 null T cells, prevention of CD152 mediated signaling is likely a major target mechanism taking place during therapy with CTLA4Ig. Objectives: CD45 is a transmembrane protein tyrosine phosphatase (PTP) expressed in all nucleated leukocytes. It activates Src family kinases (SFKs) by dephosphorylating inhibitory tyrosines in their C-terminal tails. In CD45 -/mouse T cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. At least in part, it is due to the activity of another transmembrane tyrosine phosphatase CD148 (PTPRJ, DEP-1) which acts as a positive regulator of SFK in CD45 -/-B cells and macrophages and can compensate for CD45 deficiency in these cells. Indeed, combined deficiency of CD45 and CD148 in mice results in defective macrophage and B cell signaling and development, a phenotype much more severe than the loss of either protein alone. Naïve murine T cells do not express CD148 and its expression is increased only after activation. Accordingly, no defects in T cell development and signaling in CD148 -/mice were reported so far. However, in human T cells the role of CD148 may be different since naive human T cells express CD148 at a level comparable to B cells. Using CD45 -/-/CD148 -/human T cell line (Jurkat-derived JS-7 cells) we tested the ability of CD148 to complement CD45 deficiency in T cells.

We used retroviral transduction to express human CD45 or CD148 in JS-7 cells and tested their ability to reconstitute major signaling pathways. We also employed substrate trapping mutant of CD148 to identify direct substrates of this phosphatase.

In agreement with previously published data, defective T cell receptor (TCR) signaling was observed in JS-7 cells. Expression of wild type CD45 or CD148 in JS-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased CD69 upregulation after TCR cross-linking. Moreover, the carboxy-terminal tyrosine of Lck, major T cell SFK, was hypophosphorylated in JS-7 cells expressing CD148 when compared to control cells. Finally, CD148 substrate trapping mutant expressed in JS-7 cells interacted with Lck in vivo suggesting that Lck is a direct substrate of CD148 in JS-7 cells.

The results suggest a level of redundancy between CD45 and CD148 in human T cells not appreciated so far. During the past decades, great efforts have been made to get insights into the complex process of antigen-induced T cell activation and the underlying signal transduction pathways. The T cell antigen receptor signaling cascade is initiated by phosphorylation of ITAM-tyrosine residues through the T-cell specific Src protein tyrosine kinase family member Lck. During T cell activation, Lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the ITAM-motifs. In order to resolve conformational changes of Lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive Förster resonance energy transfer (FRET) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. For the live-FRET imaging and biochemical assays we complemented Lck-deficient Jurkat T cells (JCaM1.6) with the biosensors. By introducing point mutations affecting the two major regulatory tyrosines Tyr 505 and Tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular FRET efficiency compared to the wild type biosensor. These results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. Thus, our biosensor is able to detect phosphorylation modifications of key residues. However, we could not detect any overall change in FRET and thus conformation of membrane-associated Lck molecules during T cell activation indicating that other mechanisms, presumably reorganization of localization, underlie Lck regulation. Furthermore, we observed a contribution of intermolecular FRET, which indicated homophilic interaction of Lck. Indeed, by performing single molecule analysis and native 2D immunoblotting we found Lck dimers and higher order oligomers. Together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the T cell antigen receptor. It has been reported that mitochondria accumulate under the immunological synapse (IS) in response to TCR (T cell receptor) stimulation. This process seems to be required to allow proper TCR-induced calcium influx in T cells in contact with antigen presenting cells (APCs), because mitochondria can sequester calcium and thus keep CRAC (Ca2+ release-activated Ca2+) channels open. However, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the IS. Thus, we speculated that other signals are involved in recruiting the organelles to the contact region between T cells and APCs. We found that the adhesion molecule LFA-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the IS. This process is antigenindependent and is enhanced by the presence of chemokines in the T cell environment. However, TCR triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. Our data suggest that, by recruiting mitochondria to the cell-cell contact region, LFA-1 prepares and facilitates TCR signaling. We are performing experiments to understand the signalling pathways involved in mitochondria translocation at the IS.

Burkitt lymphoma (BL) is a high grade B cell malignancy (Non-Hodgkin Lymphoma (NHL)) derived from germinal center B cells, that harbours a chromosomal translocation juxtaposing the protooncogene MYC next to the regulatory elements of one of the immunoglobulin loci. However, the precise contribution of Myc to the pathogenesis of this tumour is poorly understood. Based on the definition of a distinguishing gene expression signature for the molecular Burkitt Lymphoma (mBL) with Myc as one hallmarking signature gene (Hummel et al.2006) 

We describe a non-viral vector based approach (Vockerodt et al. 2008) to express Myc in primary human GCB cells from pediatric tonsils. Comparative whole genome gene expression profiling was performed in 9 independent preparations. Our data reveal a global change in gene expression in lymphoma precursor cells by Myc giving new insight into potential changes of the gene expression program of GCB cells on the accidental way to BL In addition as a first step the function of selected signature genes in BL is accomplished. In a representative cell line with a mBL signature and with a non-mBL signature RNAi directed inhibition of elements of the CD40 signaling cascade was conducted. After activating this particular signaling cascade (CD40) we analysed respective gene expression profiles of IKKs, TRAFs and MAPK deficient cells. Based on these different RNAi-mediated GE-profiles a comparison between both lymphoma types is performed. First attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (Markowetz et al. 2005 ). A rat thymic epithelial cell (TEC) line (R-TNC.1) was established from a long-term TEC culture. This line was characterized as a type of rat cortical TEC with nursing activity (TNC). Very little is known about molecular mechanism of the TNC/thymocyes interaction. In our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by R-TNC.1 cell line in vitro. It was found that a number of adhesion molecules, such as CD2, CD4, CD8, CD11a, CD18, CD54, CD90 was involved in these processes. Objectives: A main goal of this study was to define the adhesion molecules involved in the interaction between R-TNC.1 line and activated thymocytes. Methods: Experiments was performed on inbred AO rats. Monoclonal antibodies (mAbs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of ConA and IL-2 activated syngeneic thymocytes and unstimulated or IFN-g stimulated R-TNC.1 cells.

We found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mAb to CD2 and CD8 molecules (IFN-g unstimulated and IFN-g stimulated R-TNC.1 cells). Early adhesion was inhibited by mAb to CD90, abTCR, MHC class I molecule (IFN-g stimulated R-TNC.1 cells) and CD4 molecule (IFN-g unstimulated R-TNC.1 cells). After prolonged incubation, significant inhibition was obtained using anti-MHC class I mAb (IFN-g unstimulated R-TNC.1 cells). Almost all mAbs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mAb to CD2, CD4, CD8, CD90 molecule (IFN-g unstimulated and IFN-g stimulated R-TNC.1 cells) and MHC calss I and MHC class II molecule (IFN-g unstimulated R-TNC.1 cells). Our results also suggest the involvement of CD11a/CD18 dependent -CD54 independent pathway in adhesion and CD11a/CD18 dependent -CD54 dependent pathway in emperiopolesis.

The obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by R-TNC.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. The crucial roles of cytokines in shaping T cell responses have been documented in both healthy and disease conditions. Interleukin-27 (IL-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. IL-27 favours naï ve CD4 T cell differentiation into Th1 cells to the detriment of Th17 or Th2 differentiation. The IL-27 receptor (IL-27R) is a heterodimer composed of TCCR, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. IL-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse CD8 T cells, but the potential impact of IL-27 on human CD8 T cells has not been elucidated.

Our goal is to investigate the impact of IL-27 on human CD8 T cell functions. We used peripheral blood mononuclear cells (PBMC) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. We first assessed whether the IL-27R is detectable on ex-vivo T cells using flow cytometry. We observed a greater proportion of CD8 than CD4 T cells expressing the complete surface IL-27R (gp130+TCCR). However, we detected high amounts of intracellular TCCR in both, CD4 and CD8 T cells, but only polyclonal activation (anti-CD3) of CD8 T cells led to an actual increase of IL-27R surface expression. Purified CD8 T cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. IL-27 activated STAT1 and STAT3 signalling with rapid kinetics in both CD8 and CD4 T cells, indicating the capacity of IL-27 to signal through these molecules. Addition of IL-27 to anti-CD3 activated CD8 T cells led to a significant dose dependent increase of proliferation (as measured by CFSE-based assay) and IFN-gamma and Granzyme B production (determined by intracellular staining). These results demonstrate a pro-inflammatory impact of IL-27 on human CD8 T cells. Defects in immune regulation could result in the breakdown of immune tolerance leading to development of Multiple Sclerosis (MS). The PD-1/PD-L1 pathway is associated with production of the immunoregulatory cytokine IL-10, the suppression of T lymphocytes proliferation by inhibition of Akt phosphorylation (pAkt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in MS. We analysed by flow-cytometry the surface expression of PD-L1 and PD1, as well as myelin basic protein (MBP)-stimulated IL-10 production, pAkt inhibition, and apoptosis (Annexin V), in 50 MS patients with relapsing-remitting disease. Twenty-six patients were diagnosed as being affected by acute disease (AMS); 24 had a diagnosis of stable disease (SMS). Results showed that: 1) PD-L1 -expressing CD14+ and CD19+ cells are reduced in AMS compared to SMS individuals (p=0.04); and 2) PD1 expression is increased in CD4+ T cells of SMS individuals and is comparable on CD8+ T cells of AMS and SMS patients. This is associated with a significant decrease in IL-10 production by MBP-stimulated CD14+ and CD19+ cells of AMS patients (P=0.03). Additionally, CD8+ Anexin V+ (AV+) cells were diminished and CD8+ pAkt+ cells were higher in AMS compared to SMS patients, while similar percentages of CD4+AV+ and CD4+ pAkt+ were observed in both groups of individuals. Data herein show that the impairments of the PD-L1/PD-1 pathway seen in AMS patients result in a reduced MBP-specific IL-10 production by CD14+ and CD19+ cells as well as in a reduced apoptosis (Annexin V) and an augmented proliferation (pAkt) of MBP-specific CD8+ T. The PD1/PDL1 pathway plays an important role in the pathogenesis of Multiple Sclerosis. Monitoring of the expression of these proteins could be a novel diagnostic tool.

anti-4-1BB in CD8 cells. This difference could be due to down regulation of CD28 by activated lymphocytes and possible preferential response of CD8 cells to anti-4-1BB costimulation. Moreover, increase in IFN-g concentration in costimulated cultures also may enhance the suppressive function of MSCs which again could explain the inability of costimulation in proliferation recovery. Likewise, reducing TGF-ß by costimulation is not sufficient to abolish suppressive effect of MSCs. In overall, these results suggest that lack of costimulation expression by MSCs is not the mechanism of MSC suppression and other mechanisms are involved. Cytotoxic T lymphocytes (CTLs) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the CTL, the immunological synapse (IS). T cell receptor (TCR) enrichment at the IS is one of the early and key events of IS formation. Objectives: Soluble NSF attachment receptor (SNARE) proteins are required in almost all fusion events in cells.

In the present study we tested if the SNARE protein Syntaxin7 (Stx7) is part of the IS and whether it serves as a key player of IS formation and/or the fusion process itself. Methods: PCR-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (TIRF) were used on primary human CTLs to test the function of Stx7. RNA interference technique was also used to down regulate Stx7 expression in primary human CTLs. Results: We identified Stx7 in CTLs by PCR and immunocytochemistry. Stx7 accumulates at the IS after CTL/target cell contact. When Stx7 function was blocked by overexpression of a dominant negative Stx7 mutant (deletion of the transmembrane region), functional studies with TIRF showed a reduced accumulation and fusion of lytic granules at the IS. Furthermore, confocal studies showed a loss of TCR accumulation at the CTL/target contact side. Conclusion: These results imply that the SNARE protein Stx7 is present at the IS and moreover is required for IS formation in CTLs. The observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. Objectives: Despite the 20 years history of mouse T H 1 and T H 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. We have developed mouse T H hybridomas to study these differences between polarized T H cells. The in vitro established hybridomas were first characterized as T H 0, T H 1 or T H 2 phenotypes, based on their cytokine production (IL-2, IFNg or IL-4). A comparative analysis of T-bet, IFNg and IL-4 mRNA levels was also done on quiescent and activated T H hybridomas. In the present study, the Ca 2+response, membrane raft expression/organization, K + -and Ca 2+ -ion channel expression/function and sensitivity to apoptosis (AICD) were compared in these hybridomas.

Expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. Ion channnels were functionally analyzed by patch-clamp technique. Apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, DNA fragmentation) and flow cytometry. Results: Expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin B-staining) showed the following rank: T H 1 G T H 0 G T H 2, although the membrane cholesterol level (detected with anti-cholesterol Ab, AC8) was similar in the three cells. In connection, TCR displayed stronger colocalization with rafts and appeared more polarized in T H 1 cells upon activation than in T H 2 cells. T H 1 cells produced a more sustained calcium response with higher amplitude than T H 2 cells to the same TCR-mediated triggering signal. Interestingly, this does not coincide with the expression of Cav1.2 and Kv1.3 ion channels, major functional determinants of the sustained calcium influx. T H 2 cells expressed the highest levels of these two ion channels. There were also marked differences in their sensitivity to activation induced apoptosis (AICD) as assessed by three different markers of apoptosis.

The results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these T H hybridomas may be responsible for the observed differences in their functional characteristics. Objectives: Bone morphogenetic proteins (BMPs) belong to the TGF-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. Whereas TGF-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of BMPs in B lymphoid cells remains more elusive. In this study we investigated the effects of BMPs on mature human B-cells. Methods: B cells were isolated from peripheral blood of healthy donors using CD19-dynabeads. CD19 + isolated cells were FACS sorted into CD19 + CD27naïve B or CD19 + CD27 + memory B cells. DNA synthesis was measured by 3H-Thymidine incorporation, immunoglobulin (Ig) levels in cell supernatants were measured by ELISA and phospho-protein levels were measured by Western immunoblotting analysis. Results: All BMPs significantly suppressed anti-IgM-induced proliferation of CD19 + CD27naï ve B cells, of which BMP-6 and -7 were most efficient (40 % suppression). Similarly, all BMPs suppressed CpG-induced proliferation of CD19 + CD27 + memory B cells by 40 -50 %. To induce differentiation, both naï ve and memory B cells were stimulated with CD40L and IL-21. This increased the production of IgM, IgA and IgG 10 -100-fold compared to medium control, whereas addition of BMPs inhibited the production of all Ig classes. All BMPs highly induced phosphorylation of Smad1/5/8 in CD19 + B cells. The mechanisms for how BMPs mediate their inhibitory effects are currently being explored in more detail. Conclusion: BMPs have prominent inhibitory effects on anti-IgM-and CpG-induced proliferation of naive and memory human B cells, respectively. They also suppress CD40L/IL-21-induced production of Igs in mature human B cells.

S. Gutenberger 1 , K. Warnatz 1 1 University Medical Centre Freiburg, Freiburg, Germany

Background: Signals through the B cell receptor (BCR) and co-receptors are essential for the survival, differentiation and effector function of B cells. The stimulation of the BCR initiates several independent but interrelated signaling pathways. One important pathway leads to the activation of mitogen activated protein kinases (MAPK) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2). In a subgroup of patients with common variable immunodeficiency (CVID) we have previously demonstrated intrinsic defects in the activation of B cells revealed by the insufficient CD86 upregulation and proliferation after B cell receptor (BCR) stimulation. Therefore we assessed signaling pathways downstream of the BCR in order to identify defects in the activation of B cells. Methods: PBMC of 20 HD and 25 CVID patients were stimulated by anti-IgM. Different IgM expressing B cell subsets were analyzed separately for ERK1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to ERK1/2. To increase the signal intracellular phosphatases were inhibited by H2O2.

As markers of activation and initiation of proliferation, CD86 and Ki67 expression were measured after 2 days of in vitro stimulation. 

K. Theil 1 , P. Aichele 1 1 IMMH University Freiburg, Immunology, Freiburg, Germany

Type I interferons are homone-like molecules that are produced early after viral and bacterial infections. They signal via the type I interferon receptor (IFNAR) and have pleiotropic effects on different cells of the immune system. Their best known function is the antiviral activity.

To test the direct effect of type I interferons on CD8 T cells in vivo we adoptively transferred LCMV glycoprotein specific TCR transgenic P14 CD8 T cells that are deficient in type I interferon receptor (IFNAR-/-) into wild-type B6-recipient mice and compared their expansion with wild-type (wt) P14 T cells after viral infection. We could demonstrate a severe impairment in the capacity of P14 T cells lacking type I IFNR (IFNAR-/-) to expand after LCMV infection. Following infection of recipient mice with recombinant vaccinia virus, recombinant VSV (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing LCMV glycoprotein, P14 T cells expansion was considerably less dependent on type I IFNR expression. Therefore direct type I IFN signalling is essential for CD8 T cell expansion and survival only after LCMV infection. Our experiments showed that the LCMV generated cytokine milieu is responsible for the failure of expansion of IFNAR-/-T cells during LCMV infection. A suitable model for elucidating the impact of the LCMV generated cytokine milieu is the transfer of P14 T cells into H8 mice. H8 mice ubiquitously express the LCMV immunodominant glycoprotein-epitope gp33-41. Therefore the antigen-presentation can be uncoupled from the LCMV induced cytokine milieu when the H8 mice are infected with LCMV8.7. This is a LCMV variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the P14 T cells.

S. Frischbutter 1 , R. Baumgrass 1 1 Deutsches Rheuma-Forschungszentrum, Signal Transduction, Berlin, Germany

Antigen-specific stimulation of T helper cells induces activation of the main transcription factors NFAT, NF-KB and AP1 which are important for expression of cytokines such as IL-2, IFNg and IL17. It is known that the immunosuppressive drug Cyclosporin A (CsA) blocks the activity of the Ser/Thr phosphatase calcineurin and thereby the activation of the transcription factor NFAT. However, we and others observed that this drug also inhibits the activation of NF-KB.

To detect targets of calcineurin within the NF-KB pathway we analyzed phosphorylation and degradation levels of different NF-KB signaling proteins in the presence of CsA and other calcineurin inhibitors. We found that phosphorylation of the signaling protein Bcl-10 was prolonged in cells treated with inhibitors. Our data do not indicate an enhanced Bcl-10 phosphorylation but rather an inhibition of Bcl-10 dephosphorylation. Furthermore, calcineurin and Bcl-10 co-precipitated with each other. Interestingly, this interaction was observed only in T cell receptor-but not in TNFa-stimulated cells. In our proposed model, we hypothesize that calcineurin interacts with the CARMA/Bcl-10/MALT1 signaling complex and dephosphorylates Bcl-10 and, thus, promotes NF-KB activation. Therefore, calcineurin is not only a hub for NFAT but also for NF-KB activation.

A. 

T. Fulop 1 , J. Lamoureux 1 , C. Fortin 1 1 Université de Sherbrooke, Medicine, Sherbrooke, Canada

Objectives: Aging is accompanied by a decrease in immune functions, called immunosenescence. The exact cause is still not known. Changes in T cell subpopulations, thymic involution were invoked. We have demonstrated that the signal transduction is altered with aging. In the present work we studied the negative regulatory molecules in the T cell signaling to explain the altered activation of T cells with aging leading to decreased clonal expansion. Methods: 25 healthy young and elderly subjects were studied. Lymphocytes were separated by Fycoll-Hypaque. The molecules participating in the negative control loop of Lck were studied by Western blot and confocal microscopy. The surface expression of CTLA-4 has been studied by FACScan. The translocation of the molecules in the membrane lipid rafts (MLR) was also studied by Western blot. The activity of phosphatases was also determined. Results: We found that the phosphorylation of PAG was altered with aging explaining the decreased release of Csk from MLR and the decreased Lck activation. The activation of FynT was also altered. The phosphatase activity studies showed an increase in their activities with aging. The CTLA-4 expression was higher after stimulation in T cells of elderly. There was differences between CD4 and CD8 T cells with aging. Conclusion: These results suggest that the negative regulation is preponderant in T cells with aging on the positive activation and as such explaining the defect in T cell functions with aging. This opens new therapeutical avenues in the future. In contrast to other members of the tumour necrosis factor superfamily, Fas ligand (CD95L) contains a cytosolic proline-rich domain (PRD) that enables interactions with SH3 and WW domain proteins. Since FasL surface expression is regulated by ADAM10-mediated ectodomain shedding and FasL might be subsequently released into the cytosol by regulated intramembrane proteolysis (RIPing) through the secretase-like enzyme SPPL2a, we are interested in defining interactions involving the generated intracellular fragment of FasL. Employing a monoclonal antibody directed against the intracellular domain of FasL, we observed that previously described FasL-interacting proteins of the PCH family selectively bind to the full length molecule but not to N-terminal fragments (NTFs). In order to identify other SH3 domain proteins that potentially interact with the RIPed FasL PRD, we used a SH3 domain phage display library containing all 288 SH3 domains expressed in humans. The screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. Selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. It could be demonstrated that Tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the SNX9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known FasL-interacting proteins. Of note, in contrast to PCH proteins, the SNXs bound both NTFs and unprocessed FasL, indicating that individual interactors might influence different facets of FasL biology.

In conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of FasL to the full length protein or NTFs. This more detailed glance at the FasL interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conse- Optimal T cell activation requires the engagement of the T cell receptor (TCR) by the specific MHC/antigen complex and costimulatory signals as the interaction of B7 family members on antigen-presenting cells with CD28 on T cells. Remarkably, whereas classical glucocorticoids (GCs) effectively suppress solely TCRtriggered T cell activation in vitro, additional CD28 co-stimulation leads to GC-resistance.

In this study, we compared the non-steroidal selective glucocorticoid receptor agonist (SEGRA), Compound12, with classical GCs regarding their suppressive effect on CD28-costimulated T cells. Human primary T cell subpopulations and Jurkat cells were stimulated in vitro with plate-bound anti-CD3 and anti-CD28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. Remarkably, a clearly improved inhibition of IFN-gamma secretion was observed in CD28-costimulated human memory/effector CD4+ T cells by Compound12 than by classical GCs. Interestingly, apoptosis and activation antigen expression were similarly regulated. Improved inhibition of lymphokine secretion by Compound12 was also seen after PMA / Ionomycin stimulation of human primary T cells and Jurkat cells. When investigating the in vivo effects of Compound12 and prednisolone in acute and subacute DNFB-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of T cell-dependent skin inflammation when treating before hapten challenge. In contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for Compound12 than prednisolone. When evaluating possible mechanism for the increased activity of Compound12 in inhibition of T cell activation we got hints for a specific inhibition of the calcineurin pathway by Compound12 which was not prevented by the partial GC receptor antagonist, RU-486, in vitro. Moreover, in vivo we observed less induction of IL-1beta and TNF-alpha by pre-treatment with Compound12 than with prednisolone.

Our data indicate that the non-steroidal SEGRA, Compound12, may represent a promising drug candidate for the treatment of T cell-dependent inflammatory diseases where therapy with classical GCs is hampered by T cell resistance. Influenza A infection of B6 mice elicits robust CD8+ T cell responses, with virus-specific cells showing a distinct pattern of cytokine production: TNFa+ cells always express IFNg; and IL-2+ cells are contained entirely in the IFNg+TNFa+ subset. Interestingly, the co-expression of IFNg and TNFa varies for different epitope specificities. Almost all IFNg+ PA 224 -specific cells also express TNFa, but only about half of the IFNg+ NP 366 -specific cells co-express TNFa. This was originally linked to the avidity of the responding population for the specific peptide/MHC complex, with the IFNg+TNFa+ phenotype representing CD8+ T cells with higher avidity and a more differentiated phenotype. However, the same cytokine pattern is seen in adoptively transferred CD8+ T cells expressing a clonal TcR, implying avidity alone cannot control development of cytokine profiles. Co-expression of IFNg and TNFa by adoptively transferred CFSE-labelled OT-I cells following infection with influenza A virus expressing OVA 257-264 peptide shows a close correlation with division in vivo. Early after antigen encounter (0-2 divisions) the vast majority of cells express only TNFa. After 3-4 divisions cells begin to co-express IFNg and TNFa. The emergence of an IFNg+TNFa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both B cells and CD4+ T cells. Titration of adoptively transferred OT-I cells, which controls the level of expansion in vivo, reveals that more CD8+ T cells develop an IFNg+TNFa-phenotype with increased expansion. Thus we conclude that while TcR avidity and co-stimulation can impact the differentiation of CD8+ T cells, expansion plays a very important role in the regulation of CD8+ T cell effector function. In addition to its chemo-attractant function, SDF-1a (Stromal-cell Derived Factor-1a, CXCL12) has been described to costimulate CD4 + T cell during TCR triggering. Our objective is to clarify the mechanism regulating this costimulatory activity. TCR-driven proliferation of human CD4 + T cells was increased by immobilized SDF-1a to a level similar to that obtained with the costimulatory molecule CD28. As visualized by real time confocal microscopy, T cells entering in contact with SDF-1a formed a tether and displayed an active scanning activity. Since SDF-1a induced a similar activity in T cells stimulated with a sub-optimal dose of anti-CD3 mAbs, it is conceivable that the SDF-1a-driven scanning may favour productive TCR engagement. To test this hypothesis, we are studying the effect of SDF-1a on TCR internalization, calcium mobilization, MAPK activation and actin cytoskeleton reorganization. We are also studying the role of SDF-1a in the context of CD4 + T activation by antigen-presenting cells secreting SDF-1a. This study should help us to better define how SDF-1a modulates CD4 + T cell activation beyond its chemo-attractant function. Background: Propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. Caffeic acid phenethyl ester (CAPE), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. In this study, the effect of CAPE on the functions of T cells, which play the major role in chronic airway inflammation of asthma, was evaluated. Method: CD4 + T cells isolated from human peripheral mononuclear cells by autoMACS were stimulated with anti-CD3 and anti-CD28 antibodies and CAPE for 2 days. Cytokine levels were dertermined by ELISA and lymphoproliferation was analyzed by 3H-thymidine incorporation method. Signaling pathway of T cells was studied by Western blot. Result: It was found that CAPE significantly inhibited IFN-g and IL-5 production and lymphoproliferation in CD4 + T cells stimulated by anti-CD3/CD28. CAPE could inhibit nuclear factor-kB (NF-kB) activation, but not mitogen-activated protein kinase (MAPK) family phosphorylation in T cells. CAPE could also inhibit Akt phosphorylation. Conclusion: These results indicated that CAPE inhibits cytokine production and lymphoproliferation of T cells which might be related to the NF-kB and Akt signaling pathway. This study also provided a new insight into the mechanism of CAPE in immunology and the rationale for propolis in the treatment of allergic disorders. Objective: Upon activation, CD4 T cells express a variety of molecules on their surface, such as MHC-class II, CD80, CD86, CD70, whose ligands are constitutively expressed on resting T cells. Whereas these molecules are physiologically expressed on antigen presenting cells, their function on T cells is not understood. We tested the hypothesis that activated CD4 T cells might induce T cell proliferation and differentiation from CD4 resting T cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. Methods: CD4 T cells from the peripheral blood of healthy donors were co-cultured with fixed activated T cells from the same donor. After 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. Results: CD4 memory T cells but not naive T cells proliferated in response to contact with activated T cells. These cells showed a mild activated phenotype assessed by the expression of CD25, CD30, and CD69. Analysis of the cytokine profile of these cells revealed the differentiation of IL-10-and IFN-g-double-producing cells in response to contact with Th1 effector cells, and IL-4-producing cells in response to contact with Th2 effector cells. The levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-CD3/CD28 stimulation. Whereas neutralization of IFN-g or IL-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. Blocking particular receptor/ligand interactions by neutralizing antibodies against HLA-DR, CD70, CD80, and CD86 could not prevent cytokine production induced by T-T cell interaction. However, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. Conclusion: Interaction of CD4 memory T cells with activated T cells resulted in the production of the cytokines IL-4, IL-10, and IFN-g. Given the immunomodulatory capacity of IL-4 and IL-10, these findings might indicate a novel potential negative feedback mechanism to control T cell-driven immunity.

A. Nasir 1 , S. Thompson 1 , J.J. Murphy 1 1 King's College London, Division of Immunology Infection and Inflammatory Disease, London, United Kingdom

The murine BCL1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines IL-2 and IL-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted IgM. These cells were observed to express significant levels of the zinc fingercontaining protein ZFP36L1 by western blot analysis. This protein is reported to act in post-transcriptional regulation of gene expression by binding to AU rich elements (AREs) of mRNAs of certain genes and consequently promoting mRNA degradation. At a cell functional level, ZFP36L1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. Cytokine-induced BCL1 differentiation was observed to be associated with downregulation of ZFP36L1 protein.

In an attempt to determine whether ZFP36L1 downregulation was directly linked to BCL1 differentiation, a ZFP36L1 ShRNA expressing lentivirus (pSicoR) was employed to knockdown ZFP36L1 expression. This reagent downregulated ZFP36L1 expression very effectively . ShRNA infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. ZFP36L1 ShRNA infected cells also produced more secreted IgM per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. These results are consistent with a role for ZFP36L1 downregulation in promoting BCL1 plasmacytoid differentiation.

vidual lysates of peripheral blood lymphocytes (PBL) of 32 patients with IgG multiple myeloma and healthy controls were investigated for the expression of sialic acid (SA), galactose (Gal) and N-acetylglucosamine (GlcNAc), the sugars known to specify the glycoforms of human serum IgG. The degree of glycosylation and signaling status of all 32 isolated myeloma IgG BCRs were correlated and compared with the glycosylation of the IgG paraproteins isolated from sera of the same patients. It was shown that BCR IgG in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of IgG BCR is associated with higher levels of tyrosine phosphorylation (signaling activity) of the IgG BCR supramolecular complex and that BCR IgG and serum IgG paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. The results suggest that the development of the malignant clone in MM from postswitch B cells expressing IgG BCR at their surfaces to plasma cells secreting IgG paraprotein may be followed by permanent glycosylation changes in the IgG molecules.

caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to TPEN. Conclusion: The signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. In light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than TMS, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. Although best known for its pro-apoptotic function, it seems clear now that CD95 (Fas, APO-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. We investigated effects of Fas co-ligation during TCR/CD3/CD28-triggered activation of freshly isolated human T-lymphocytes. To this end, TCR-triggered cells were incubated in presence or absence of different ligand concentrations of anti-Apo1 mAb, FasLFc or FasLStrepFc fusion proteins, or leucin zipper (LZ-)CD95L. Interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and T cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked TCR-induced cell proliferation without inducing cell death.

The positive costimulatory effect of Fas at low concentrations is associated with elevated IL-2 and IFNg production, upregulation of activation markers, adhesion molecules and cell-cycle regulating CDKs and cyclins. In addition, we observed an increased activation of important signalling molecules including MAPK and caspases. Using pharmacological inhibitors, we demonstrate that Fas is internalized upon ligation. We also observed an increased TCR internalisation following Fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal T cell activation.

In stark contrast, most Fas ligands at high concentrations almost completely inhibited cell proliferation of TCR-triggered lymphocytes. In this context, crucial events associated with T cell activation, i. e. tyrosine and ERK1/2 phosphorylation, the expression of various activation markers, the IL-2 production and caspase activation were almost completely abrogated. These findings highlight that Fas-triggering accelerates or blocks T cell activation, depending on the strength of the stimulus. In addition, we provide further evidence for an anti-apoptotic function of FasL during signal initiation in human T lymphocytes. Sponsored by the DFG (SFB415) and the Medical Faculty Kiel (to OJ) It has been shown that glycosylation of cell surface proteins controls critical T cell processes, including homing, thymocytes maturation, activation, and cell death. Plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic T cell activation stimulus. The Galb1,3GalNAc-specific lectin from Amaranthus leucocarpus (ALL) shows a differential binding pattern to murine thymocytes and peripheral CD4+ and CD8+ T cells. In addition, mitogenic stimulus increase 3-fold the ALL binding to CD4+ T cells. Previous studies in human PBMC showed that ALL binds to human CD4+ T cells and ALL-binding increased after a mitogenic stimulus using total cell cultures as murine studies. These data suggest that ALL detects selectively activation-related changes in CD4+ T cell surface carbohydrate but none study has been performed to examine the ALL effect on human T cell activation. To examine the effect of ALL on human T cell activation, we analyzed the anti-CD3-dependent activation of purified CD4+ T cells from PBMC in presence or absent of ALL by measuring proliferation using CFDA-SE staining, expression of the surface activation marker CD25 and calcium influx by flow cytometry. Results showed that ALL did not induce significantly T cell proliferation or CD25 expression, but enhanced the anti-CD3-dependent proliferation and CD25 expression of purified CD4+ T cells. Analisis of calcium influx showed that ALL enhanced anti-CD3 dependent calcium influx. Our findings indicated that ALL alone does not affect T cell activation but suggested that ALL induces a costimulatory effect on human CD4+ T cells by up-regulating T cell activation mediated by anti-CD3 stimulus, as further studies have to be performed to elucidate ALL-induced costimulatory effect. Financed in part by PAPIIT-UNAM (IN214609)

A. The adaptor protein LAT (Linker for Activation of T cells) has a prominent role in the transduction of intracellular signals elicited by the TCR/CD3 complex. Upon TCR engagement, LAT becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. The balance between cell survival and cell death is critical for normal T cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95 receptor. It has been previously demonstrated that CD95 ligation in T cells induces the proteolytic cleavage of several adaptor proteins, including Gads, SLP-76, SLAP-130 and LAT. Given the dual role of LAT as a transducer of activation and negative signals in T cells, we have analyzed the role of the LAT cleavage in T cell functions and studied the proteases responsible for this cleavage. Objective: The study is designed to explore preliminarily the need of T cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of T cells, and by detecting the expressions of IL-Rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (HPBLs). The results may lay a theoretic and experimental basis for developing the condition media qualified especially to T cell culture. Methods: PBLs were isolated , and cultured in different media. Both immunocytochemistry staining and cell enzyme linked immunosorbent assay (CELISA) were used to detect the expressions of IL-1R, IL-2R, IL-3R, IL-7R, IL-12R, IL-18R, CD27, CD28, CDw137( 4-1BB), CD 95(Fas) and CD178 (FasL) on HPBLs in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. Using Typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. To evaluate the cellular activity, growth situation and cell cycle of T cells, both MTT and FCM analysis were also performed separately.

1. The expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) The expressions of membrane immune molecules before cultured.

2) Expressions of the membrane molecules on HPBLs during culturing. 5% FBS RPMI 1640 group (1640 group), IL-2 group, PHA group...

(1) MTT assay.

(2) Proliferative times and growth curves of HPBLs...

1. During cultured in vitro, there are expression changes of the IL-1Rs (IL-1Ra, IL-2Ra, IL-2Rg, IL-3R, IL-7R, IL-12R, IL-18R), co-stimulatory molecules (CD27, CD28, 4-1BB) and apoptosis associated molecules (Fas/FasL) on HPBLs in different time and cultured media. The expression patterns of the most molecules checked are similar in 1640 group, IL-2 group and PHA group, but the rests are different. 2. Our data also suggest that the HPBLs cultured in CD3McAb+CD28McAb+IL2+IL1a group has a great proliferative potential compared with the other groups.

Using this condition medium, may have a practical prospect to tumor therapy. 3. CELISA will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale.

F. Beceren-Braun 1 , R. Tauber 1 1 Zentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Berlin, Germany L-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. Apart from its role in adhesion, L-selectin functions as a signal transduction molecule. Crosslinking of L-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. In addition to process signals coming from outside of the cell, the intracellular part of L-selectin (LScyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the Ras/Rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-Jun N-terminal kinase (1), which leads to an enhanced binding of L-selectin to soluble ligands (3). In our previous work we described an association of LScyto with isozymes of the PKC family which phosphorylate the receptor on serine residues (4). Here we show that the Protein Phosphatase 2A inhibitor PhapII is a novel direct interacting partner of the LScyto. We propose a model in which the L-selectin mediated signalling is regulated by the interaction of PKC, PP2A and PhapII: PhapII binds to the unphosphorylated LScyto. Upon L-selectin crosslinking LScyto is phosphorylated, Pha-pII dissociates and inhibits the phosphatase PP2A. In addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of L-selectin. 

V. Heissmeyer 1 , E. Glasmacher 1 1 Helmholtz Center Munich, Molecular Immunology, Munich, Germany

During self-antigen recognition, Roquin dependent posttranscriptional downregulation of ICOS prevents T cell help to B cells and autoantibody production. The molecular mechanism by which Roquin interferes with ICOS translation remained unclear. We have identified two critical regions in Roquin. The amino-terminus is required for RNA binding and can be functionally replaced by conserved sequences from its paralog Mnab. The carboxy-terminus mediates P body localization and has specialized in Roquin for efficient repression of ICOS in T cells. Using knockout cells of dicer or ago1-4 genes, we prove that Roquin mediated repression of ICOS occurs in the absence of miRISC formation. Instead, Roquin function required intact P bodies, and was impaired after knockdown of Lsm1 and Rck or expression of dominant-negative GW182. Interestingly, Roquin activity is blocked through induced miRISC formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses.

S Objectives: Upon encountering their antigens, naï ve T cells are activated and driven to clonal expansion and differentiation into armed effector cells. According to the two-signal hypothesis, the induction of an optimal CD4 + T-cell immune response requires both antigen-specific and co-stimulatory signals. In contrast, stimulating naïve CD8 + T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. Thus, CD8 + T cells require additional signals for full activation and further differentiation into effector cells. Methods: In this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for CD8 + T-cell activation and differentiation. Naïve CD62L hi CD44 lo CD8 + T cells were sorted and stimulated by anti-CD3 and anti-CD28 antibodies.

Results: Firstly, we show that the activation and differentiation of CD8 + T cells require IL-2 provided by activated CD4 + T cells at the initial priming stage after stimulation. Secondly, this critical IL-2 signal is delivered through IL2Rbg of CD8 + cells and is independent of IL-2Ra. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNg and granzyme B produced by CD8 + T cells. Conclusion: Therefore, our studies demonstrate that a full CD8 + T-cell response is elicited by a critical temporal function of IL-2 released from CD4 + T cells, providing mechanistic insights into the regulation of CD8 + T cell activation and differentiation. Most antigenic peptides recognized by CD8 T lymphocytes are produced through degradation of intracellular proteins by the proteasome. However, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. MAGE-A3168-176 is a tumor antigenic peptide presented by HLA-A1 and widely used for vaccination of melanoma patients. We observed that proteasome and TPPII inhibitors failed to block presentation of the antigen by tumor cells. However, processing of this peptide occurred in the cytosol because TAP inhibition prevented its presentation. To characterize the cytosolic peptidase producing MAGE-A3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. We observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. This production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. This inhibitor also blocked the presentation of MAGE-A3168-176 by tumor cells. By electroporating HLA-A1 cells with a precursor peptide blocked at the C-and the N-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. One such enzyme is insulin-degrading enzyme (IDE). We observed that depletion of IDE abolished the capacity of a cytosolic fraction to produce the antigenic peptide. Furthermore, recombinant IDE was able to produce the peptide in vitro from the precursor peptide. Lastly, silencing of IDE with siRNA reduced presentation of the peptide by tumor cells. With TPPII, IDE is the second example of a proteasomealternative pathway in the production of class-I restricted peptides. Antigen-specific T cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. Immune escape, due to impairment of HLA dependent tumor epitope presentation is believed to be one major reason for this failure. To identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane MART-1/Melan-A tumor antigen were exposed to two or three rounds of brief co-culture with MART-1/Melan-A 26-35 specific cytotoxic T lymphocytes (CTLs). Immune selected melanoma cell clones, being resistant to lysis by MART-1/Melan-A 26-35 CTLs due to impaired epitope processing were further investigated. Our results show that in addition to previously described immune evasion mechanisms like down regulation of MHC class I and MART-1 expression, the IFN-gamma independent endoplasmic reticulum associated degradation (ERAD) pathway is crucial for MART-1/Melan-A 26-35 epitope generation. Moreover, deregulation of several ERAD components is essentially responsible for the observed immune escape of the immune selected melanoma cells. In support, re-expression of down-regulated ERAD components in CTL-resistant melanoma cells completely restored immune recognition by MART-1/Melan-A 26-35 CTLs. Thus, our studies demonstrate for the first time that ERAD not only plays a central role in the production of CD8 + T cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. Studies of T cell responses to hen egg lysozyme suggest that several conformers of peptide-MHC class II complexes can be generated for a single peptide epitope and that distinct CD4 T cell repertoires known as type A and type B recognise these different conformers (Lovitch and Unanue, Immunol Rev 207: 293-313, 2005) . Type A T cells recognise peptide-MHC complexes generated from intact proteins after intracellular antigen processing under H2-M (DM) control, where as type B T cells respond to synthetic peptides in the absence of DM editing, but fail to respond to processed intact protein. Type B T cells escape thymic deletion in mice (Petersen et al., Immunity 11: 453-462, 1999) , with implications for autoimmunity. So we studied whether type A and type B recognition patterns occur in T cell responses to autoantigens such as the Rheumatoid Arthritis (RA)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type B T cells are found in inflamed joints. Lymph node cells from aggrecan-immunised BALB/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. T cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type A and type B T cell recognition.

Responses to staggered and alanine-substituted peptide sets showed that type A and B T cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to MHC in distinct conformers. Type B T cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce RAassociated aggrecanases. Our data suggest that inflammation generates extracellular peptides that activate type B T cells. We are also characterising human type B T cell responses as well as searching for type B T cell ligands in synovial fluid from RA patients. We propose that extracellular cartilage degradation generates ligands that induce autoreactive type B T cell responses which participate in the pathogenesis of autoimmune arthritis.

A To cope with MHC I antigen presentation HCMV encodes for several post-translational strategies which have been extensively studied in transfected cells. In this study we analysed the PLC in naturally HCMV-infected cells and monitored the composition of the PLC throughout HCMV replication. Metabolic labeling experiments revealed the absence of tapasin incorporation into the PLC. In contrast, Western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. Tapasin mRNA levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. Taking advantage of a novel method, in which newly transcribed RNA is selectively labeled and analysed (Dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. Furthermore, also reduction of TAP1 and TAP2 transcription was observed contrasting to the elevated levels of ERp57 and MHC I transcripts. Importantly, ectopic expression of tapasin restored the incorporation of tapasin into the PLC in HCMV-infected cells. The data indicate that HCMV controls MHC I antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. Most peptides presented by MHC class I molecules are produced by the proteasome during degradation of intracellullar proteins. Two main proteasome types have been described, differing in their content of catalytic subunits. The standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their IFNg-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. The thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. The standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to IFNg and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. We produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. Using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. One comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). We quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. They are also present in dendritic cells, where they represent about 50% of the proteasome content. We characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor CTL. With respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. Importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( Tapasin is a multi-functional protein dedicated to MHC-I biosynthesis; it serves as a structural component in the so called MHC-I peptide loading complex (pLC), as a chaperone putatively acting as an active peptide editor and MHC-I quality control mechanism, as an ER retention signal for immature MHC-I, and as a chaperone stabilizing TAP expression and increasing TAP-performance. Furthermore, Tapasin has been found outside the ER, where it has been suggested to regulate retrograde transport of escaped immature MHC-I back to the ER from the trans-Golgi compartment. The role of Tapasin as an active peptide-editor has been debated and we here set out to study the effect of Tapasin on binding of peptides of both high-and low-affinity to a human MHC-I allele (HLA-A*0201) using protein interaction-and peptide-competition assays. Specifically we wanted to in detail compare the binding of two peptides of the same affinity. At high concentrations all of the tested HLA-A*0201 binding peptides (TAP-transported high-affinity peptide (TTP-HA), signal-peptide of high affinity (SP-HA), TAP-transported mediumaffinity peptide (TTP-MA)) induced dissociation of HLA-A*0201 from Tapasin, but only TTP-HA dissociated HLA-A*0201 from Tapasin at lower concentrations. Using peptide-competition assays against TTP-MA, a peptide of lower affinity, we could show that TTP-HA, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide SP-HA. However, analysis of MHC-I peptide loading in the Tapasin-negative cell line LCL-721.220-A2 showed no competitive advantage of TTP-HA compared to SP-HA supporting a role for Tapasin as a selective facilitator of MHC-I peptide binding. In conclusion, we here show that peptides of different affinities dissociate HLA-A*0201 from Tapasin in a dose-dependent manner, and that Tapasin facilitates TTP-HA, but not SP-HA replacement of a lower-affinity peptide (TTP-MA). Together these data strongly suggest a role for Tapasin as a selective facilitator of peptide binding to MHC-I. Importantly, this study implies that criteria in addition to peptide-affinity determines whether Tapasin will promote peptide binding to HLA-A*0201.

M. Basler 1,2 , C. Lauer 2 , M. Groettrup 1,2 1 Biotechnology Institute Thurgau, Kreuzlingen, Switzerland, 2 University of Constance, Division of Immunology, Department of Biology, Konstanz, Germany Two LMP7-dependent antigens have been described that relied on the 'structural presence' of LMP7 in the proteasome but not on the activity of LMP7. Here we have investigated processing of the H-2D b -restricted UTY 246-254 epitope of the male minor antigen UTY reported to be LMP7-dependent. Using splenocytes from LMP7 -/-, LMP2 -/and MECL-1 -/mice we found that the UTY 246-254 epitope requires LMP7 and LMP2 but not MECL-1. Curiously, a selective LMP2 inhibitor did not interfere with UTY 246-254 presentation. Objective: We investigated why the deletion but not the inhibition of LMP2 interferes with UTY 246-254 presentation. We hypothesized that the 'structural' requirement for LMP2 is based on replacement of the caspase-like activity of b1 in the proteasome. Methods: It was determined if T1A mutants of LMP2 and/or b1 can rescue the UTY 246-254 epitope. We used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. Finally we determined by mass spectrometry if the UTY 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by LMP2-deficient and proficient immunoproteasomes in vitro. Results: We found that T1A mutants of LMP2 and b1 rescue presentation of UTY [246] [247] [248] [249] [250] [251] [252] [253] [254] . Also inhibition of cells with a b1-selective inhibitor preserves UTY [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. An aspartate in position 7 of the UTY 246-254 sequence WMHHNMDLI is preferentially used as a cleavage site by LMP2-deficient but not half as frequently by LMP2-proficient immunoproteasomes.

The generation of the UTY 246-254 epitope relies on the replacement of the caspase-like activity of b1 by LMP2 because the b1 activity destroys the UTY 246-254 epitope. This is the first example for the 'structural' requirement of LMP2 for generation of an epitope. Eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. Thus we have discovered a so far unrecognized mechanism how LMP2 and perhaps also LMP7 and MECL-1 exert their function in antigen processing.

A. Linnemann 1 , A. Musiol 2 , R. Lindner 1 1 Hannover Medical School, Cell Biology, Hannover, Germany, 2 Hannover Veterinary School, Graduate School for Biomedical Sciences, Hannover, Germany

Objectives: MHC I molecules are constitutively endocytosed and recycled to the cell surface. This process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on MHC I. In fibroblasts, MHC I is known to internalize via a clathrin-independent, Arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. Although this observation suggests that membrane rafts are involved in the internalization of MHC I, no evidence for an association of MHC I with membrane rafts has been found in this cell type. Methods: A novel detergent extraction protocol was used to investigate the association of MHC I with membranes rafts. Endocytosis of MHC I was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. For characterization of MHC I internalization pathways, dominant negative mutants of GTPases (dynamin and Arf6) were overexpressed in 3T3 fibroblasts.

We show that antibody-mediated oligomerization of MHC I in 3T3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. This change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized MHC I internalized faster and more completely and arrived at different endocytic organelles. The two MHC I internalization pathways differed in their sensitivity towards dominant negative Arf6: endocytosis of oligomerized MHC I was not affected, whereas non-oligomerized MHC I endocytosed more slowly and changed its subcellular distribution. Unlike transferrin receptor internalization, none of the MHC I endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoA. Conclusion: We propose that MHC I switches from an Arf6-regulated to a novel, Arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of MHC I. Since MHC I is one of the cellular receptors for SV40 virus and since SV40 binding triggers MHC I oligomerization, this novel pathway may be involved in SV40 uptake. Antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. In this study, we tested the effect of siRNA-mediated knockdown of 57 Rab GTPases, the key regulators of membrane trafficking, on antigen cross-presentation. Twelve Rab GTPases were identified to be associated with antigen cross-presentation, and among which Rab3b, 3c were found to be colocalized with MHC class I molecules at perinuclear tubular structure. Tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the MHC class I molecules were internalized from plasma membrane to Rab3b and Rab3c postitive compartment. Moreover, the recycling ligand transferrin was enriched in the Rab3b or 3c positive vesicles. Furthermore, the Rab3b, 3c positive compartment were colocalizd with a fraction of Rab27a at a juxtaposition of phagosomes. Together these data demonstrate that Rab3b and Rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. Introduction: While the proteasome is thought to generate most of the HLA peptidome, other proteases were also proposed to be significant for this process. Both T cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model HLA peptides. The HLA-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the ER, binding stability at the cell surface and retrograde uptake of the HLA molecules back into the cytoplasm. Objectives: The role of the proteasome in HLA peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire HLA peptidome.

The peptidomes of both the authentic membranal and a recombinant soluble form of the HLA molecules were collected for analysis at different time points after the inhibition of the proteasomes. The turnover rates of the HLA-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. The HLA molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and Orbitrap tandem mass spectrometry. Both the endogenous membranal and soluble MHC molecules were studied in parallel from the same cells. Peptides were identified by their MS/MS fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide.

Results: A few thousands HLA-peptides were identified, and for a large portion of them, the turnover rates could be defined. Proteasome inhibition did not affect the complexity of the HLA peptidomes or reduced significantly the amounts of membranal HLA molecules. Many peptides were labeled relatively rapidly with heavy leucine, indicating that the HLA peptidome contains also the products of newly synthesized and rapidly degrading proteins. The source proteins of the HLA peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells.

The centrality of proteasomal degradation in HLA-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. Epstein-Barr virus (EBV) is a ubiquitous Y-herpesvirus, infecting over 90 % of adults worldwide. It can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for B-lymphocytes and epithelial cells. EBV persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. One such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. However, for EBV replication and dissemination to occur, it must enter the lytic phase. Here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic T -lymphocytes. EBV can circumvent possible eradication by CD8+ T lymphocytes during the lytic phase by interference with antigen processing and presentation through HLA class I in the infected cell. The viral proteins BNLF2a and BGLF5 have been shown to achieve this by impairing peptide-loading of HLA class I and inducing the degradation of mRNAs encoding HLA molecules, respectively. A third EBV lytic phase protein, the G-protein coupled receptor (GPCR) BILF1, has now been found to down-regulate cell surface HLA class I expression (Zuo et al, PLoS Pathogens 2009). This represents a novel function for a virally-encoded GPCR. BILF1 is expressed early in the EBV lytic cycle and is localized predominantly at the cell surface. There it can interact with HLA class I molecules, resulting in their internalization and lysosomal degradation. This has a profound effect on the ability of cytotoxic T-lymphocytes to recognize cells displaying antigens derived from EBV proteins. Interestingly, BILF1 displays a differential effect on distinct HLA class I haplotypes. Furthermore, we have shown that the intracellular C-terminal tail of BILF1 is required for its effect on HLA class I expression. However, the ability of the GPCR to activate intracellular signaling pathways is dispensable in this regard. Thus, by reducing the cell surface expression of HLA class I molecules, EBV BILF1 can hinder the recognition of virally-infected cells by cytotoxic CD8+ T lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. T lymphocytes mature in the thymus, generating a non-dangerous T cell repertoire. For the adquisition of tolerance, thymocytes suffer positive and negative selection processes. During T cell maturation, TCRs contact with different MHC-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. To obtain a non-self-reactive T cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of MHC-peptides complexes representative of the proteins that T cells will found in periphery, including tissue restricted antigens (TRAs)-derived peptides. In the last decade, transcription of TRAs in thymus has been well-reported. Furthermore, the expression of many genes codifying for TRAs are dependent.on the expression of the AutoImmune Regulator (AIRE). AIRE is mainly expressed in medullar thymic epithelial eells (mTECs), which are involved in negative selection. So far no systematic study have been made to describe the peptide repertoires associated to HLA molecules in the thymus. In addition, although many data of TRAs transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no HLA ligand arising from any TRA have been reported in thymus.

In this report we present the results of analyzing the HLA-DR-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. We describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. Using qPCR we demonstrate that SEMG1 is transcribed in thymus from both male and female individuals. Finally, we detected the SEMG1 mRNA expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. The proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for MHC class I antigen presentation. Vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). Deficiency in beta5i results in profound reduction of MHC class I expression, demonstrating the significance of this subunit for efficient antigen presentation. Currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. However, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. Here, we show that following infection with Listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. Our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. Further, MHC class I antigen presentation on beta5i-deficient cells could be restored by treatment with D3T, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. Consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for MHC class I cell surface expression. . We have previously shown that LC, in contrast to DDC, do not express cell surface TLR2, 4 and 5, which results in their inability to respond to both Gram-positive and Gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. Therefore, the question remained what the role is of LC in class II MHC-mediated activation of anti-bacterial T cells.

We determined the capacity of DDC and LC to internalize and process whole bacteria and present bacterial antigens to CD4 + T cells. In vitro generated LCs and DDCs were cocultured with GFP-expressing bacteria and subsequently analysed by CLSM and FACS for their uptake capacities. Furthermore we investigated their capacity to stimulate autologous bacteria-specific T cell lines as a measure for antigen presentation. Results: We found that LC are principally able to internalize bacteria, but far less efficient than DDC. Moreover, visualisation of bacterial uptake by EM revealed different uptake mechanisms by LC and DDC. Both in LC and DDC internalized bacteria were detected in the endosomal and lysosomal compartments of the MHCII processing route. Nevertheless, presentation of bacterial antigens by LC on MHCII was inefficient compared to that of DDC, as indicated by a low capacity to activate autologous bacteria-specific CD4 + T cells. The presence of exogenous TLR3 and TLR8 ligands did not overcome the differences between LC and DDC, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific T cells is not due to the lack of TLR signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of LC. Conclusion: We propose that the epidermis of the skin is an immune-privileged site where LC play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria.

E. James 1 , I. Bailey 1 , T. Elliott 1 1 University of Southampton, Cancer Sciences Division, Southampton, United Kingdom Regulatory T cells (Tregs) play a pivotal role in the suppression of tumour specific T cell responses. Depletion of Tregs in Balb/c mice results in a robust immunity to the normally poorly immunogenic CT26 colon carcinoma. This response is long lasting and mediated by both CD4 and CD8 T cells. Importantly, the Treg depleted CT26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (A20, C26, BCL1, RENCA) implying a broader repertoire of response.

We have characterised one of these cross-protective antigens, GSW11, which is H2-D d restricted. Analysis of the generation of GSW11 in CT26 revealed that the peptide is susceptible to over-processing by the ER-resident aminopeptidase ERAAP. Inhibition of ERAAP in CT26 cells substantially increased the amount of GSW11 present, observed by increased T cell responses to the tumour in vitro and HPLC analysis. This increase was in spite of an overall reduction of MHC class I molecules at the cell surface. To investigate whether the increase in immunogenicity following knockdown of ERAAP would protect mice, we generated stable ERAAP knockdown (KD) CT26 and immunised Balb/c mice. Greater than 80 % of mice injected with ERAAP KD CT26 were found to reject the tumour. Analysis of T cell responses revealed the presence of GSW11-specific T cells, however, these responses were small (0.5-1 %). This compared to a much larger response to CT26 (˚5 %). Preliminary results indicate that the majority of the T cell responses (non-GSW11-specific) in these mice are directed toward unstable peptide/MHC complexes, possibly indicating presentation of N-terminally extended peptide antigens. This highlights manipulation of the peptide repertoire as a potent tool for the generation of T cell responses in vivo. Minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of Graftversus-Host-Disease and in the Graft-versus-Tumor reactivity in HLA-identical stem cell transplantation [1] . The di-allelic HLA-A2 restricted minor histocompatibility antigen HA-1 locus codes for the highly immunogenic HA-1 His and the non-immunogenic HA-1 Arg nonapeptides, differing in one amino acid. The only difference that could explain the absence of the HA-1 Arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for HA-1 His and less than 5/ cell for HA-1 Arg [2] . As HA-1 His/Arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the HA-1 His allele is currently used for boosting the graft-versus-tumor responses after HLA matched HA-1 mismatched stem cell transplantation.

To elucidate the mechanisms underlying the differential cell surface presentation of the HA-1 allelic peptides, we investigated the impact of the HA-1 His/Arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of HLA class I-bound peptides. Therefore, proteasome-mediated digestion experiments, TAP translocation analyses, and HLA-dissociation assays with HA-1 His and HA-1 Arg peptides were performed. Moreover, the crystal structures of HLA-A2 in complex with either HA-1 His , HA-1 Arg or a HA-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the HLA-A2/HA-1 peptide complexes. Our results exclude a role for antigen processing in preventing HA-1 Arg to be presented at the cell surface and both the structural and HLA-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the HA-1 Arg allele in the HLA-A2 peptide binding groove [3] . They provide a rationale for the lack of HA-1 Arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. Proteasomes play an important role in MHC class I antigen processing. Exposure of cells to proinflammatory cytokines such as TNFa or IFNg leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. Immunoproteasomes generate many pathogen-derived CD8 T cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific CD8 T cell response. On the other hand, immunosubunit expression is not essential for development of CD8 T cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear.

We observed that mice that lack the immunosubunits LMP7 (ib5) and MECL-1 (ib2) develop a variety of autoimmune responses, including a latent form of T1D (or insulin dependent diabetes mellitus, IDDM), following irradiation and bone marrow reconstitution. IDDM development in these mice is characterized by inflammation of the islets of Langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of CD8 but not CD4 T cells. A CD8 T cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (IGRP) mRNA, was identified as an important target of the CD8 T cell response. This epitope, like many other known diabetes-associated epitopes, binds its presenting MHC class I molecule with low affinity. As T cells specific for low affinity binders most likely can escape central and peripheral tolerance while T cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind MHC class I molecules with low affinity, by a higher affine peptide species towards which T cell tolerance exists. Thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune CD8 T cells that enter inflamed tissues. Endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express HLA-II molecules with compact conformation and are therefore expected to stably bind autologous peptides. The role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response.

To study in situ T cell responses without characterizing self-reactive T cells, we have identified natural HLA-DR-associated peptides from autoimmune organs that will help finding peptide-specific T cells in situ. Here we report the first analysis of HLA-DR natural ligands from ex-vivo Graves' disease-affected thyroid tissue. Using mass spectrometry, 162 autologous peptides were identified from HLA-DR-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. Most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. Cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which HLA alleles were these peptides associated in vivo. All but two of the thyroglobulin peptides showed low binding with the corresponding alleles. The two peptides with relatively high binding affinity were presented in the context of DR3 and DR4. Analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a Lys and Arg was observed for all of them, independent of the restricting allele. Our data demonstrate that although the HLA-DR-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to HLA-DR in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. The T cell response generated following herpes simplex virus type 1 (HSV-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. Despite this, the relative contributions of CD4 + and CD8 + T cells in HSV-1 immunity have yet to be clearly elucidated. To better understand the role of HSV-1-specific CD4 + T cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein D of HSV-1. Following flank infection, gD-specific CD4 + T cells were first detected in the draining brachial and axillary lymph nodes (LN) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gD-specific CD4 + T cells were first recovered from the spleen, skin and dorsal root ganglia (DRG) at day 6 pi and peaked at day 9. While HSV-specific T cells were first observed in the draining LN at day 5 pi, hybridoma assays showed ex vivo presentation of the gD epitope by brachial LN cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. However presentation of the gD epitope was much more prolonged in vivo as proliferation of transgenic gDspecific CD4 + T cells was observed up to 23 days post-infection in the brachial LN. Ex vivo analyses suggest that only CD11c + cells were involved in gD antigen presentation at days 2, 5 and 15 post-infection. Subdivision of dendritic cells (DCs) populations indicated that both skin-derived DCs and CD8a + DCs can present the gD antigen to CD4 + T cells at day 2 pi, whereas by day 5 pi the skin-derived DCs were the predominant population presenting the gD epitope. Together these data show that following HSV-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. Furthermore, we present evidence that different DC populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection.

Complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. We also verified that the zippers did not influence the substrate "preferences" of the respective ERAP. Our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric ERAP1 and ERAP2 unable to form dimers. This observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. Conclusion: Allosteric interaction of ERAP1/ERAP2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human ER. During the biogenesis of class I molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (Cnx) and its associated thiol oxidoreductase ERp57. Upon assembly of the heavy chain with b 2 m, the class I molecule enters a peptide loading complex (PLC) that consists of the TAP transporter, tapasin, the calnexin homologue calreticulin (Crt) plus associated ERp57. Both Crt and ERp57 are required for efficient assembly of peptide-loaded class I molecules and their subsequent expression at the cell surface. We examined functional sites on Crt and ERp57 to gain insights into their mechanisms of action in class I biogenesis. For Crt, its lectin function is thought to be crucial for its association with class I molecules. However, when Crt mutants lacking lectin function were expressed in Crt-deficient cells, they completely complemented all class I biosynthetic defects. Thus polypeptide-based contacts either mediated through ERp57 or directly between Crt and the heavy chain are sufficient to effect the chaperone and quality control functions of Crt in class I biogenesis.

We also tested the notion that ERp57 must be recruited by Cnx or Crt to function on class I molecules. We found that the rates of heavy chain disulfide formation were normal in cells lacking Cnx, Crt or both chaperones. Furthermore, an ERp57 point mutant that fails to bind to Cnx or Crt was just as effective as wild type ERp57 in normalizing rates of disulfide formation. We conclude that ERp57 does not require recruitment by Cnx or Crt and likely acts directly on class I heavy chains to promote disulfide formation. Furthermore, in cells expressing the ERp57 point mutant, class I heavy chains, Crt and the tapasin-ERp57 disulfide conjugate were present at normal levels in the PLC, indicating that the interaction between ERp57 and Crt is not required for PLC assembly. Finally, we show that mutations that destroy the enzymatic function of ERp57 have no effect on PLC stability or class I surface expression, suggesting that ERp57 plays a structural as opposed to catalytic role in PLC function. Autoimmune pancreatitis (AIP) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. A strong association of AIP with the HLA-DRB1*0405/DQB1*0401 haplotype has been reported, but identification of the predisposing HLA gene(s) has been precluded by strong linkage disequilibrium. Here, we show that HLA-DR*0405 transgenic Ab0 NOD mice suffer from AIP and additional pneumonitis after sublethal irradiation and adoptive T cell transfer from syngenic donors, leading to complete pancreatic atrophy. Pancreas histology is characterized by destructive infiltration of the exocrine tissue with CD4+ and CD8+ T cells, B cells and macrophages. Mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. HLA-DR*0405 transgenic mice (CD4+ T cell competent) develop AIP even unprovoked, similar to Ab0 NOD mice (CD4+ T cell deficient), while HLA-DR*0401, HLA-DQ8 or HLA-DR*0405/DQ8 (double-) transgenic controls all remain normal after same treatment. We conclude that HLA-DR*0405 fails to protect from AIP, likely due to defects in the induction of CD4+ regulatory T cells. Our results identify HLA-DR*0405 as a prominent risk factor for AIP on the HLA-DRB1*0405/DQB1*0401 haplotype. This humanized mouse model should be useful to study mechanisms that underlie the HLA association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human AIP.

S. Khan 1 , C. Britten 1 , H. Overkleeft 2 , G. van der Marel 2 , K. Melief 1 , D. Filippov 2 , F. Ossendorp 1 1 Leiden University Medical Center, Section Tumorimmunology, Leiden, Netherlands, 2 Leiden University, Biosynthesis Group, Leiden, Netherlands

Objective: We have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic TLR ligands to study the impact on MHC class I and class II antigen presentation.

The potency of the vaccine was addressed by monitoring antigen presentation, priming of T-cells and tumor protection. Results: Our data show that this type of targeting of peptides greatly improves antigen presentation and T-cell priming compared to free peptide. Vaccination of mice with the TLR-ligand peptide conjugates induced high numbers of functional CD8 and CD4 T-cells that could protect mice for aggressive melanoma. This potency relies on TLR signaling since peptide coupled to a non-functional TLR ligand was unable to support induction of specific T-cells.

These data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal T-cell activation by dendritic cells, and show the potency of TLR-L peptide conjugates as a vaccine modality.

Y. Shi 1,2 , X. Hu 1,2 , A. Kawana- Tachikawa Objectives: Nef protein of human immunodeficiency virus (HIV) holds some important immunodominant CTL epitopes. Two overlapping 8-mer and 10-mer epitopes (RYPLTFGWCF (nef138-10) and RYPLTFGW (nef138-8)) were found to be presented by HLA-A*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. Y2F, Y2W, T5C, F6L, W8R, F10R, F10Y etc. or their combinations. It's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of HIV. Methods: Refolding method was used to attain the soluble protein pMHC. Crystals are grown using hanging drop vapor diffusion method and X-ray diffraction technology is used to determine the structure.

We have determined six peptide-MHC(pMHC) structures containing nef138-10 (Wild Type) and its four mostly common immune escape mutants (Y2F, T5C, Y2F&T5C, F6L), and also nef138-8 (Wild Type). We found that there was little difference between the nef138-10 (Wild Type) and nef138-10 (Y2F) when they were displayed in the peptide-binding groove of MHC molecule, except water molecule distribution near the anchor residue Y2 or F2. Interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (T5C) and nef138-10 (Y2F&T5C), which may affect the binding of peptide and the recognition of T cell receptor. For nef138-10 (F6L), the side chain of L6 was more flexible compared to the nef138-10 (Wild Type). Alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of F6 was not solvent-exposed due to shortening of the length of the peptide. Conclusion: As the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different TCR repertoire diversity in HIV-specific responses. Different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host CTLs, which indicates that the therapy strategy based on the cellular immune response should be diversity. For the in vivo or ex vivo activation of antigen-specific T cell responses long synthetic peptides are used to activate both CD8+ and CD4+ T cells. In this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in MHC class I. We observed a large variation in the effectiveness of activation of specific T cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. For the HLA-A2 restricted CMVpp65 derived NLV epitope specific T cells were most efficiently activated by N-terminally extended variants of the minimal epitope, while the use of C-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. This pattern was seen for 5/9 epitopes tested in different HLA restrictions. Furthermore, for all epitopes tested, extending both the C-terminus and N-terminus led to 2-5 log less efficient activation of the specific T cells, compared to the minimal peptide. Exchange of the C-terminal sequence of the C-terminal extended HLA-B7 restricted CMVpp65 RPH peptide with the C-terminal extended NLV peptide led to the enhancement of T cell activation by the exchanged NLV peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. TAP-deficient T2 cells loaded with extended NLV peptides efficiently activated NLV-specific T cells, indicating that the route of presentation was TAP-independent. Addition of lactacystin did not affect activation of specific T cells, illustrating that crosspre-sentation was proteasome-independent. Primaquine reduced the activation of specific T cells by extended NLV peptides, but not by the minimal NLV 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. These data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling MHC class I molecules. Not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. Therefore, a rational design of peptides is crucial for efficient activation of CD8+ T cells in approaches of vaccination, adoptive transfer and immune monitoring. Antigenic peptides presented by MHC class I molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. Recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. For two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. One of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. The other peptide is derived from protein SP110, and requires splicing in the reverse order of two segments initially separated by six amino acids. The first spliced antigenic peptide described was derived from Fibroblast Growth Factor-5 (FGF-5) and was recognized by human cytotoxic T lymphocytes directed against kidney cancer cells. It is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. The splicing mechanism of this peptide has not been worked out. The length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. We therefore evaluated the role of the proteasome in the splicing of this peptide. We observed that the spliced FGF-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. We evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. The results confirmed the transpeptidation model of splicing. We further compared the production of the FGF-5 spliced peptide by cells transfected with mutant constructs encoding FGF-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues.

We observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. Finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. The polymorphic residues within the peptide binding cleft of HLA class I molecules not only diversify the range of peptides presented to cytotoxic T lymphocytes but also influence the pathway of antigen presentation. In order to acquire high affinity peptides, some class I allotypes, such as HLA-B*4402, are heavily dependent upon tapasin and other molecules comprising the Peptide Loading Complex (PLC). Other class I molecules, like HLA-B*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. HLA-B*4402 and B*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. We have previously speculated that such tapasin-independent class I molecules may have been selected in response to viral inhibitors that target the PLC, such as the human cytomegalovirus US3 protein. To address this hypothesis, US3 was stably coexpressed in B lymphoblastoid cell lines expressing HLA-B*4402 or HLA-B*4405. In the presence of US3, the surface expression of HLA-B*4402 was substantially reduced whereas HLA-B*4405 expression was relatively unaffected. Although US3 was able to form complexes with both HLA class I allotypes, only HLA-B*4402 was retained intracellularly in an immature form whereas HLA-B*4405 was transported to the cell surface. Accordingly, in the presence of US3, HLA-B*4405, but not HLA-B*4402, constitutively presented a HLA-B44 restricted alloantigen to reporter T cells, suggesting that US3 binds HLA-B*4405 without interfering with peptide loading. US3 has been reported by others to bind the PLC but surprisingly we have not detected such US3-PLC complexes in our system. Rather, in the presence of US3 we identified a pool of class I molecules distinct from the PLC and only present in US3 expressing cells, implying that US3 may act independently of the PLC. These findings demonstrate how HLA class I polymorphism not only impacts upon the T cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation.

C. Massa 1 , B. Seliger 1 1 Martin-Luther-University Halle-Wittenberg, Institute of Medical Immunology, Halle, Germany

In the attempt to optimize vaccine DC, modifications have been proposed both in the antigen loading and in the maturation protocols. For DC loading "whole antigens" are now preferred to peptides. Therefore, it is important to consider not only the costimulatory properties of the vaccine DC, but also their antigen processing abilities. This is even more important since there is the trend to stimulate DC with TLR ligands combined with IFN-Y in order to induce DC not only able to correctly migrate, but also secreting the bioactive IL12p70. Since IFN-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the DC in parallel to their costimulatory potential. For this purpose monocyte-derived DC were stimulated for 24h with the gold standard of maturation (TNFa, IL1b, IL6 and PGE2) or a combination of IFN-g and different TLR ligands. The DC obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic PBMC, but differ for the pattern of proteases expression as evaluated by real time PCR. With the exception of the downregulation of the tripeptidyl peptidase II (TPPII), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature DC. In response to the "IFN-gcontaining" cocktails there was a similar TPPII downregulation, but also the induction of many other enzymes. The cytosolic leucine aminopeptidase-3 (LAP3) had a more than 20-fold increase in transcription levels, whereas the mRNA expression of the aminopeptidases of the endoplasmic reticulum ERAP1 and ERAP2 and of the immunoproteasome subunits LMP2 and LMP10 was enhanced between 2 and 5-fold under these culture conditions. With regard to the different TLR ligands used in combination with IFN-g, there was a reproducible higher mRNA induction in the presence of the TLR4 ligand MPLA in comparison to the TLR-3 and 7/8 ligand polyI:C and R848. These data suggest that the maturation cocktail of DC may alter the peptide repertoire presented by HLA class I surface antigens. It has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for T cells. However, whether cognate interactions between mast cells and class II restricted CD4 + T cells actually occur, is still an open question. We addressed this question using peritoneal cell-derived mast cells (PCMC) as an antigen presenting cell model. Our results show that in vitro treatment of PCMC with IFN-g and IL-4 induced surface expression of mature MHC class II molecules and CD86. When IFN-g/IL-4 primed PCMC were used as antigen presenting cells for CD4 + T cells they induced activation of effector T cells but not of their naive counterparts as evidenced by CD69 up-regulation, induction of proliferation and cytokine production. Confocal laser scanning microscopy showed that helper OT-II T lymphocytes form with PCMC functional immunological synapses, characterized by PKCq enrichment and IFN-g polarized secretion towards the antigen-presenting mast cells. Finally, upon cognate interaction with OT-II T cells, mast cells lowered their threshold of activation via FceRI. Our results show that mast cells can establish cognate interactions with class II restricted helper T cells, implying that they can actually serve as resident APC in inflamed tissues.

H The vast majority of peptide ligands presented by MHC class I molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. Although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (ER) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of ER proteins remains little characterized. Studying processing and presentation of ER-targeted and cytosolic forms of proinsulin (PI), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that ER-targeting of this model antigen has profound effects not only on how PI is degraded, but also on regulation of its synthesis. As expected, proteasome inhibition inhibited degradation of cytosolic PI as well as presentation of the epitope insulin B15-23 to specific CD8+ T cells. In contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of ER-targeted PI (pre-PI) through induction of ER stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. Experiments using conditions permissive for pre-PI expression showed that ER-targeting modified proteolytic processing of PI for MHC class I presentation. These experiments suggested that two proteolytic pathways contribute to degradation of ER-targeted PI, with their relative contribution depending on the stability of the protein. While degradation of unmodified pre-PI was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-PI for presentation, while introduction of a site for N-glycosylation had the opposite effect. These findings imply that ER-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. CD8 + T cell immune response to exogenous antigens relies on cross presentation by dendritic cells (DCs) in secondary lymphoid organs. Recently, in several infectious murine models, it has been shown that in addition to DC located in tissues, de novo differentiating DC participate in the protective Th1 immune response. The role of de novo differentiating DC in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the APC subsets patrolling for exogenous Ag. A prerequisite for cross presentation is a moderate Ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to MHC molecules. This prerequisite is of special importance considering DC precursors (such as monocytes), which are not yet DCs and may take up antigen before differentiating into DCs. The objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to CD8+ T cells and whether they are able to sustain this cross presentation while differentiating into DCs. We have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate CD8+ tumor-specific T cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to CD8+ T cell clones, Faure, 2009, Eur J Immunol 39 (2): 380-90). In the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into DCs. We have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate CD8+ T cells after their in vitro differentiation into DC, 6 days following their antigenic pulse. The delineation of APC subsets able to sustain Ag cross-presentation and T cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. Viral genomes contain alternative reading frames (ARFs) encoding for MHC-I restricted epitopes (ARF-epitope). In the SIV/macaque model, CTL responses directed against ARF-epitopes participate in controlling viral replication. We previously described that HIV-1 genome contains ARFs within gag, pol and env genes encoding for a panel of HLA-B*07 restricted epitopes. QPRSDTHVF (Q9VF/5D) is one such epitope but its parental epitope QPRSNTHVF (Q9VF/5N) has a significant higher frequency among HIV-1 isolates. Strikingly, Q9VF/5D-or Q9VF/5N-specific CTLs recognize APCs infected with HIV strains encoding for Q9VF/5D (e. g. HIV LAI ). In contrast, HIV strains (e. g. HIV NL-AD8 ) encoding for Q9VF/5N do not activate CTL responses raising the possibility that Q9VF/5N epitope is not presented by infected cells. We asked whether introducing mutations within Q9VF might be a mean for the virus to escape CTL responses directed against this ARF-encoded epitopes.

We dissected the mechanism responsible for the lack of Q9VF/5N MHC-I presentation. We modified HIV LAI to introduce a D to N mutation in Q9VF. Introducing this single amino-acid mutation abrogated CTL recognition indicating that this asparagine (N) alters Q9VF MHC-I presentation. We performed in vitro proteasomal digestions of 28mer peptides encompassing Q9VF/5D or Q9VF/5N and cleaved polypeptides were analyzed by mass spectrometry. The asparagine (N) in Q9VF/5N is a preferential proteasomal cleavage site. Thus suggesting that proteasome cleavages within Q9VF/5N might be responsible for its lack of MHC-I presentation.

We then sought in HIV-infected patients for the presence of proviruses encoding for Q9VF/5D or Q9VF/5N, and CTLs responses directed against these epitopes. Far thus, two out of three donors tested recognized the Q9VF/5D peptide. We cloned and sequenced HIV-1 genomes from the three donors. Surprisingly, out of 20 HIV proviral genomes isolated from PBMCs of Q9VF/5D reactive donors, we could not find any virus bearing the Q9VF/5D sequence. The isolated HIV sequences either encoded for Q9VF/5N or had a stop codon within the epitope. In contrast, viruses encoding for Q9VF/5D were isolated from PBMCs of the Q9VF/ 5D nonreactive patient. Altogether, our data suggest that CTLs exert a selection pressure on viral ARFs. HIV-1 seems to escape immune surveillance by introducing mutations altering processing of ARF-derived epitopes.

I. E. Flesch 1 , Y. Wang 1 , D.C. Tscharke 1 1 The Australian National University, Biochemistry and Molecular Biology, Canberra, Australia

Vaccinia virus (VACV) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. CD8 + T cells recognizing viral peptides in association with MHC class I molecules on infected cells play a crucial role in the defence of viruses. Despite the large number of possible MHC class I-peptide combinations, CD8 + T cells only recognize a small number of epitopes, a phenomenon called immunodominance. Using recently defined CD8 + T cell epitopes for VACV in mice, we have investigated how heterozygosity of MHC class I molecules influences immunodominance patterns in H-2 bxd F 1 mice compared with their inbred parent strains. We find that the immunogenicity of VACV peptides defined using inbred mice is variable in F 1 progeny, with some peptides being almost equally immunogenic in F 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in F 1 mice. During acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in F 1 mice. In line with these findings, a multiepitope construct expressed by a recombinant VACV was less immunogenic in F 1 mice than would be predicted from its performance in parent strains. In terms of mechanism, we find evidence of altered TCR repertoires including in the case of one epitope, the loss of many diverse TCR Vb clones and outgrowth of CD8 + T cells with a restricted Vb usage in F 1 mice. These data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how MHC diversity can alter the range of epitopes that are immunogenic in outbred populations. Objective: TLR ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (DC). Therefore we aimed to study the efficacy of a TLR2 agonist, S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl polyethylene glycol (BPPcysMPEG), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide (MALP-2), as an adjuvant for cross-priming against cellular and soluble antigens. MALP-2 has been characterized as an effective mucosal adjuvant and synthesis of BPPcysMPEG further improved solubility and pharmacokinetic features of the adjuvant. Methods: DC isolation, in vitro and in vivo T cell stimulation, Intracellular cytokine staining, in vivo cytotoxicity assays.

Results: Systemic administration of BPPcysMPEG induced maturation of CD8 + and CD8 -DC in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. In addition, administration of BPPcysMPEG and cell-associated OVA resulted in generation of an effective CTL response against OVA in vivo in a T-helper cell-dependent manner, but independent of IFNa. Delivering antigenic peptides directly linked to BPPcysMPEG led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands such as CpG, systemic activation of DC with BPPcysMPEG did not result in shutdown of antigen presentation by splenic DC subsets, although cross-priming against subsequently encountered antigens was reduced.

We provide evidence that BPPcysMPEG stimulation of DC via TLR2/6 results in the generation of an effective CTL response and that delivering antigenic peptides linked to BPPcysMPEG is a promising strategy for vaccination. While BPPcysMPEG-matured DC retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after TLR-mediated DC maturation. Altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation.

tumor immunevasion. To verify whether the loss of ERAP1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of ERAAP (murine ERAP1) in a murine T lymphoma cell line, RMA. We used a method that allows an efficient and continuous expression of miRNAs that directly silence ERAAP and obtained several ERAAP-deficient RMA clones with different levels of ERAAP expression (up to 90 % of reduction at the protein level). Microsomal aminopeptidase activity and MHC class I surface expression were decreased in all clones proportionally to ERAAP expression. Moreover, low expression of ERAAP affected the stability of MHC class I molecules as evaluated after acid and brefeldin A treatment.

De-regulated ER peptide trimming also drastically affected the tumor formation of RMA cells and host survival. ERAAP-deficient RMA clones with different levels of ERAAP, 50 and 10 % as compared to control RMA cells, were injected s. c. in the flank of C57BL/6 syngenic mice, and analysed tumor growth. All mice injected with control RMA cells developed a tumor but survived up to 45 days after injection. All mice injected with RMA clone with a 50 % level of ERAAP expression developed a tumor and died within 23 days after injection. Surprisingly, any animal injected with RMA clone with a 10 % level of ERAAP died or showed a visible tumor. Thus, knockdown of ERAAP expression appears differently to affect the immunogenicity of RMA cells, depending on the ERAAP silencing level. Hemophilia A is an X-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor VIII (FVIII). Treatment consists of regular administration of FVIII, but is complicated by the formation of inhibiting antibodies against FVIII. Both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia A. The development of inhibitory antibodies in hemophilia A patients has been shown to be a CD4 + T-cell driven process. Therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of T cells against FVIII in hemophilia A patients using MHC class II tetramers.

CD4+ T-cell responses of two monozygotic twins with severe hemophilia A were analyzed. One of these subjects developed a high titer inhibitor (207 BU/ml) following intensive factor VIII (FVIII) treatment. High dose immune tolerance therapy together with anti-CD20 therapy resulted in eradication of the inhibitor. In contrast, his twin brother developed a low titer inhibitor (2.5 BU/ml) which declined rapidly after tolerance induction. Fundamental differences in the twins' antibody responses were further suggested by elevated and persistent IgG4 levels in the subject with the high titer inhibitor. In order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated DRB1*0701-restricted T-cell responses of the high titer inhibitor subject, using fluorescent MHC class II tetramers loaded with 20-mer synthetic FVIII peptides to stain epitope-specific CD4+ cells.CD4+ T-cells from the high-titre inhibitor subject recognized three peptides corresponding to the FVIII A2 domain: FVIII 405-424 , FVIII 421-440 and FVIII 653-672 , as well as the C1 domain peptide FVIII 2093-2112 , but not any C2 domain peptides. The C1 domain peptide contains a sequence that was reported as a promiscuous T-cell epitope (Jones TD et al., J Thromb Haemost.85:123-33, 2005 ). Analysis of T cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of T cells that may underlie the discordant immune responses of these twins to infused FVIII.

M. Forloni 1 , S. Albini 1 , M.Z. Limongi 1 , L. Cifaldi 1 , D. Fruci 1 1 Ospedale Pediatrico Bambin Gesù, Rome, Italy Neuroblastoma (NB) is a pediatric tumor that derives from neural crest. The most aggressive forms are characterized by amplification of the MYCN oncogene and severe reduction of HLA class I expression. MYCN has been claimed to hinder HLA class I expression through affecting the expression of the transcription factor p50 NF-kB subunit. Since in many human tumors the expression of HLA class I molecules is positively co-ordinated with that of ER aminopeptidases, ERAP1 and ERAP2, we wondered whether in NB cell lines MYCN may impair expression of these aminopeptidases. To explore this possibility, NB cell lines that differ in MYCN expression were quantified for expression of MYCN, ERAP1, ERAP2 and HLA class I heavy chains by Western blotting and for surface HLA class I expression by flow cytometry. We found that MYCN negatively correlates with expression of HLA class I, ERAP1 and ERAP2. This negative correlation was confirmed in a NB cell line expressing a tetracycline repressible MYCN transgene. Then, by the use of TNFa (a NF-kB nuclear translocation stimulator), sulfasazine and IKBa mutant (two NF-kB nuclear translocation inhibitors) and knockdown of p65 NF-kB subunit, we demonstrated that NF-kB is involved in ERAP1 and ERAP2 expression in NB cell lines and that MYCN does not affect NF-kB expression. Furthermore, we showed that MYCN and NF-kB are recruited to the promoter regions of ERAP1 and ERAP2 and that MYCN affects the recruitment of NF-kB binding to these promoter regions.

In conclusion, the present results indicate that an enhanced MYCN level, linked or not to MYCN amplification, represses ERAP1, ERAP2 and HLA class I expression in NB cell lines by affecting the recruitments of NF-kB binding to their promoters.

S. Brosch 1 , S. Tenzer 1 , H. Schild 1 , E. von Stebut-Borschitz 1 1 Uniklinik Mainz, Mainz, Germany

Infection of inbred mouse strains with the intracellular protozoan parasite Leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. C57BL/6 mice) or systemic disease (susceptible phenotype; BALB/c mice) depending on the genetic background of an individual. Healing of Leishmania infections is based on Th1 immunity, whereas IFNg secretion of both CD4 + Th1 and CD8 + Tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. Stimulation of antigen-specific effector T cells is driven by L. major-infected dendritic cells (DC) in an IL-12-dependent manner. Proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective CD8 + T-cell responses via the MHC class I. Here, we analysed the role of the IFNg inducible immunoproteasome for the priming of CD8 + T cells in L. major infections. Using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome LPM7 (LMP7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with L. major mimiking natural transmission of the parasite as compared to wild type C57BL/6 mice. In addition, ex vivo co-cultures with infected DC from either LMP7 -/or wild type mice together with antigen-specific T cells from infected wild types showed no differences in Tc1 cell IFNg secretion and the DC restimulatory capacity of CD8 + T cells. Furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble Leishmania antigen; SLA) CD8 + T cells, isolated from low dose infected C57BL/6 wildtype or LMP7 -/mice, were not detected. In summary, our data indicate that despite the fact that CD8 responses in L. major infections are important for disease outcome, processing of antigen and thus priming of CD8 + T cells against L. major is independent of the LMP7 subunit of the immunoproteasome. Studies have defined an essential requirement for autoantigen-specific B cells as antigen presenting cells in rheumatoid arthritis. However, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by B cells are unknown. In this study we have developed a system to investigate how antigen-specific B cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. We have utilised these cells to characterise the mechanisms by which aggrecan-specific B cells could induce autoimmunity. We have constructed plasmids encoding an aggrecan-specific B cell receptor and have transfected them into the B cell line A20, generating B cell lines that specifically recognise and target aggrecan for presentation to T cells. In addition, we have established conditions for a panel of aggrecan-specific T cell hybridomas to recognise aggrecan pulsed B cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. We used inhibitors of MHC class II transport, endosomal pH and enzymes involved in aggrecan degradation. We found that aggrecan-specific B cell lines presented the major arthritogenic CD4 + T cell epitope (84-103) from the G1 domain of aggrecan 10,000 times more efficiently than non-specific B cells and over 100 times more efficiently than the macrophage line J774. However, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by J774. Treatment of aggrecan-specific B cells with ammonium chloride to raise endosomal pH or brefeldin-A to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal pH and presentation by newly synthesised MHC class II. Interestingly, aggrecan presentation by antigen-specific B cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium Understanding the mechanisms of antigen processing and presentation by autoantigen-specific B cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. Tapasin is an MHC-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of MHC class I molecules within the peptide loading complex (PLC). Class I molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. Position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. The MHC class I antigen B27 is strongly associated to ankylosing spondylitis (AS) and other spondyloarthropathies. HLA-B27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the PLC. Tapasin also modulates B27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of B*2705, peptide loading is relatively independent of this chaperone. We analyzed the effect of B27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of B27 subtypes. The association of B27 heavy chain with tapasin was analyzed in C1R cells transfected with HLA-B27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody PaSta-1, which recognizes human tapasin. We also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the B27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the ME1 monoclonal antibody that recognizes B27properly folded B27/peptide complexes. The formation of fully assembled B27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody HC10, which recognizes MHC class I free heavy chains (HC), or with ME1. Maturation was analyzed by pulse-chase analysis, immunoprecipitation with ME1 and treatment with Endoglycosidase H (Endo H). HLA-B27 polymorphic positions other than 116, both at the A and C/F pockets modulate tapasin binding and the optimization of the peptide cargo. The stability of the peptide repertoires critically influences the folding efficiency of B27 subtypes. From as early as the initial phases of infection, HIV is coated with complement (C) fragments and following seroconversion, the circulating virus forms immunecomplexes with IgG and complement. Recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (iDCs) with differentially opsonized HIV.

The opsonization pattern of HIV may additionally have profound consequences for the outcomes of the antigen-presenting capacity of DCs and their ability to mount an adequate immune response. In this context, we compared the impact of differential HIV-opsonization on the antigen-presenting capacity of DCs and found that C-opsonized HIV triggered CTL responses, while IgG-coated virus did not. These in vitro generated CTLs showed an enhanced IFN-g secretion and recognized the help independent CTL epitope SLYNTVATL. C-generated CTLs also degranulated upon stimulation with specific HIV peptides and were able to elicit antiviral activity against HIV-infected CD4 + T cells.

Our results indicate that C-opsonization of HIV drives the virus towards the MHC class I pathway in DCs, thereby promoting a more efficient stimulation of naïve CD8 + T cells. This CTL-stimulating property of C could be exploited when searching for a novel approach against HIV.

IgG isolated from patients with high titers of anti-CCP antibodies showed a cross-reactivity with Hcit peptides. Vaccination experiments supported a triggering role of Hcit for the development of arthritis in mice model. Conclusions: Diamination process is significantly increased in patients with RA while carbamylation is suppressed. Production of specific antibodies against diaminated residues in RA patients may have a modulating role for the development of autoimmune arthritis. The classical pathway of MHC class I antigen presentation involves cytosolic degradation of viral proteins by the proteasome. Peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (TAP). Previous reports have shown that viral epitopes are presented to CTL independently of TAP in smaller viruses. We hypothesized that presentation of VACV by MHC class I might proceed by alternative pathways. The aim of this study was to characterize these alternative pathways in TAP-deficient mice. Our results show that CTL derived from C57BL/6 mice immunized with VACV, recognized TAPdeficient dendritic cells infected with the virus. Approximately 15 % of VACV global presentation in the context of H-2 b was independent of TAP. In addition, VACV infection induced a virus-specific CTL response in mice deficient in TAP. Dendritic cells (DC) initiate robust CTL immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class I molecules (pMHC). Two major DC subtypes have been described, CD8+ and CD8-DC, which differ in their MHCI antigen presentation capacities. CD8+ DC are the major DC subset responsible for cross presentation (presentation of exogenous antigen by MHCI), while CD8-DC display little cross presenting capacity. Here, we examined the MHCI antigen presentation pathway of CD8+ and CD8-DC in more detail. First, turnover (half-life) of total MHCI at the cell surface of CD8+ and CD8-DC was determined. Surprisingly, CD8+ DC exhibit rapid surface MHCI turnover compared to CD8-DC (following culture in the presence of brefeldin A). Following activation of DC with CpG, MHCI levels at the surface of both CD8+ and CD8-DC were stabilized and no longer underwent rapid turnover. This suggests that CD8+ and CD8-DC differ in their regulation of surface MHCI turnover and that this is subject to regulation by antigen-associated signals. Second, we examined the ability of CD8+ and CD8-DC to generate pMHCI complexes containing cross presented antigen. We utilized the model antigen ovalbumin (OVA) and an antibody that can detect H-2Kb loaded with the OVA-derived peptide, SIINFEKL. CD8+ and CD8-DC isolated from OVA-expressing mice (actin-OVA transgenics) displayed abundant Kb-SIINFEKL complexes at their cell surface. In contrast, in response to exogenous soluble OVA protein, only the CD8+ DC, but not the CD8-DC, displayed Kb-SIINFEKL complexes at the cell surface. Similarly, when DC were pulsed with OVA-coated splenocytes, Kb-SIINFEKL complexes were only detected on the surface of CD8+, and not CD8-DC. This data further validates the role for CD8+ DC as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other DC subsets from accessing the important cross presentation pathway. Objectives: The action radius of matrix metalloproteinases or MMPs is not restricted to massive extracellular matrix (ECM) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. Although many instances exist in which cells disintegrate, often in conjunction with induction of MMPs, the intracellular MMP substrate repertoire or degradome remains relatively unexplored. The aims of the present study were to identify novel intracellular MMP targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. Methods: Multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to THP-1 cytosol using gelatinase B/MMP-9 as a model enzyme. In the first dimension, ion exchange chromatography separated the THP-1 proteins by their net charge and/or isoelectric point (pI) followed by cleavage of the proteins by MMP-9. In the second dimension, potential substrates were separated by molecular weight on SDS-PAGE. To evaluate the effect of proteolysis by MMP-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with THP-1 cytosol in complete Freund's adjuvant. Lymph node T cells were isolated and stimulated with MMP-9-cleaved or intact THP-1 cytosol. Proliferation was assessed by measuring incorporated 3 Hthymidine.

Results: 100-200 MMP-9 candidate substrates were isolated, of which 69 were identified, revealing many novel MMP-9 (candidate) substrates from the intracellular matrix (ICM), such as actin, tubulin, stathmin,... About 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. Remarkably, a significantly lower T cell proliferation was observed in the presence of cleaved vs. intact cytosol. Conclusion: Multidimensional degradomics technology is a valuable tool for high-throughput identification of novel MMP substrates. Proteolysis by MMP-9 decreased the immunogenicity of the intracellular contents, suggesting that MMPs may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury.

a preference for HLA-A versus -B molecules; E3-19K did not detectably associate with HLA-C molecules under identical conditions. This locus specificity may provide a functional advantage to Ads by inactivating T-cell receptors, while avoiding activation of NK receptors. Finally, we showed that residue 56 in HLA-A11 and residue 93 in E3-19K are highly critical for association of both proteins. This defines a putative interaction surface between E3-19K and class I molecules.

Conclusions: Our studies provide novel insights into the functional relationship between E3-19K and the class I antigen presentation pathway. Moreover, because soluble E3-19K can differentiate between polymorphic gene products encoded in the MHC, our results may contribute to define paradigms for how class I substrate specificity is established for ER retention. Overall, our studies represent an important step towards a molecular understanding of the strategy evolved by Ads to establish life-long persistence in host cells. Objectives: The transporter associated with antigen processing (TAP) belongs to the ABC transporter superfamily and is a heterodimer consisting of the two subunits TAP1 and TAP2. TAP transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (ER) and is thus a key element of the MHC class I antigen processing machinery (APM). Methods: Target-specific TAP knock downs were generated by shRNA technology. The resulting transfectants were subsequently analysed regarding MHC class I surface expression using flow cytometry, whereas mRNA and protein expression levels of TAP1 and TAP2 were analysed by RT-PCR and Western blots, respectively. Furthermore, the protein stabilizing effect of TAP1 on TAP2 was investigated in the presence of two distinct proteasome inhibitors. Results: Previous findings obtained with rare TAP1 mutants suggested that the lack of TAP1 protein expression is associated with a strong reduction of TAP2 protein levels, which could be restored by TAP1 gene transfer, whereas no such regulation is found vice versa. To investigate this stabilizing effect of TAP1 on TAP2 different shRNA plasmids specifically targeting TAP1 or TAP2, respectively, were stably transfected into constitutively TAP1 and TAP2 expressing HaCaT keratinocytes and Colo794 melanoma cells. In both cell types the shRNA-mediated TAP1 and TAP2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. The knock down of TAP1 caused not only an almost complete loss of TAP1, but also a strong decrease of TAP2 protein expression. In contrast, the TAP2 knock down exhibited no influence on the TAP1 expression. Specific inhibition of the proteasome prevented the degradation of TAP2 in the TAP1 knock down variants.

The results of our study emphasize that an unidirectional stabilisation of TAP1 on TAP2 protein expression is not restricted to rare TAP1 mutants, but rather suggest a common regulatory mechanism for the TAP complex. UV injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. In this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following UV exposure. Therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of UV. Blister roofs were then collected before and 1, 4 and 10 days after UV-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. We demonstrated that, along with the rapid activation and migration of Langerhans cells (LC), UV skin radiation exposure promotes the infiltration into the epidermis of a monocytic CD14 + CD36 + and of a macrophagic CD14 -CD36 + cell subsets that emerge 1 and 4 days post exposition, respectively. More importantly whereas classical CD1a hi CD207 + LC are the unique dendritic cell (DC) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal DC namely CD1a low CD207and CD1a low CD207 + that emerged 1 and 4 days post irradiation, respectively. These two distinct populations of epidermal DC (EDC) differ from classical epidermal LC by their activation/maturation profile as assessed by the strong expression of CD86 and HLADR. Finally, 10 days post-exposure, we observed that LC represented almost the only haematopoietic cell population in the epidermis. These results suggesting that the UV-recruited EDC and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis.

I. Bailey 1 , E. Reeves 1 , T. Elliott 1 , E. James 1 1 University of Southampton, School of Medicine, Cancer Sciences Division, Southampton, United Kingdom

There is accumulating evidence that CD4+ CD25+ regulatory T cells (Treg) play an important role in anti-tumour immunity by preventing effective T cell responses to tumour antigens. Tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. The depletion of Tregs prior to challenge with the murine colorectal tumour, CT26, stimulates a robust, protective T cell response which is also protective to challenge with other tumours of different histological origins, such as B cell lymphomas and a renal cell carcinoma. This cross protection has not been seen with other tumour cell lines. We have identified a CT26-derived cross-protective antigen, GSW11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. This protein has previously been shown to encode CT26-specific CD8 and CD4 antigens, implicating it as a 'hot-spot' for CT26 tumour antigens. Interestingly, we have identified a truncated version of gp70 which may be responsible for generation of GSW11. Expression studies have revealed increased gp70 expression in CT26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (GSW11) generated. The current knowledge of HLA class II antigen presentation and peptide binding is mainly based on studies of HLA-DR molecules. They contain a large hydrophobic P1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while P4, P6 and P9 tune the peptide repertoire that can be presented by individual HLA-DR alleles. The same rules and requirements do not necessarily exists for peptide binding to HLA-DP molecules, however. The present study adresses this issue.

We have expressed and affinity purified soluble recombinant HLA-DP2 molecules from Drosophila melanogaster cells and studied its binding of a number of peptides known to bind HLA-DR, -DQ or DP molecules. Unexpectedly, the immunodominant epitope in multiple sclerosis (MS), the myelin basic protein derived peptide, MBP85-99, bound to HLA-DP2 with high affinity (10-30 nM). Binding studies of MBP85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (F91A, F92A and K93A) were affected, and only by a 20-50 fold reduction in affinity for HLA-DP2. The observation that none of the substitutions resulted in a complete loss of binding to HLA-DP2 indicates that 1) HLA-DP2 binding to peptides does not depend on a large hydrophobic residue accomodated in P1, or 2) MBP85-99 can bind in more than one register. We will present data addressing this issue. The HLA-DP peptide binding capacity was increased at neutral as compared to acidic pH, and by the presence of n-butanol, a small organic MHC loading enhancer (MLE). In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS, MBP85-99, possibly in more than one register. Additional studies are required to resolve the HLA-DP2 peptide binding properties, and to determine whether expression of HLA-DP2 affects the disease course in MS patients.

Results: Depletion of MNCs for CD14+ cells abrogated the TG-induced cytokine production and proliferation of CD4+ T cells, indicating a primary role for monocytes as APCs. However, the encounter of T cell with antigens presumably occurs in B cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. To mimic the conditions prevailing there, we depleted PBMCs for G 98 % of the monocytes (without significant loss of T-cells), and compared the TGelicited T-helper cell responses in the presence and absence of B cells. The TG-induced CD4+ T cell proliferation was significantly reduced in CD14/CD19-depleted MNC cultures, as compared to cultures depleted for CD14+ monocytes alone. The same applied to the production of IL-2, IL-6 and TNF-a. Production of IFN-g and IL-10 was generally not observed.

Our data indicate that normal B cells are capable of inducing a pro-inflammatory cytokine response in MNC-cultures, where monocytes and monocyte-derived cells are not preponderant. Studies addressing the relative contributions to this cytokine production, by B cells themselves and by T cells (following antigen-presentation by B cells), are in progress.

J. Kyosiimire-Lugemwa 1,2 , P. Pala 1 , G. Miiro 3 , J. Todd 3 , P. Kaleebu 1,2 , N. Imami 2 , F. Gotch 2,3 1 MRC Uganda, Basic Science, Entebbe, Uganda, 2 Imperial College, London, United Kingdom, 3 MRC Uganda, Entebbe, Uganda

Background: HIV-1-specific T-cell responses are preserved in HIV-1 infected individuals with non-progressing HIV-1 disease. "Long Term Non Progressors" (LTNPs) were defined as ART naï ve individuals infected with HIV-1 for G 8 years, maintaining CD4+ T-cell counts G 500, and with minimal CD4+ decline over time. We tested the hypothesis that Gag-specific T-cell responses are inversely correlated to disease progression whereas Nef-specific T-cell responses are not. Methods: 17 ART naï ve HIV-1 infected patients from the Entebbe cohort in Uganda were recruited and stratified by CD4+ T-cell count, CD4+ decline slopes, and time of enrolment, into 2 groups -10 LTNP and 7 Rapid progressors (RP). All patients were women reflecting the patient base at the Entebbe cohort. We measured plasma viral load, current CD4 T-cell count, and IFN-g, IL-2 and IL-4 ELISpot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). Peptides were based on consensus sequences of Gag and Nef from HIV-1 clades A1, A2 and D. Medians and inter-quartile ranges were calculated and comparisons between groups were performed using the Mann-Whitney U test. Correlations were presented using Spearmann's linear correlation coefficients. Results: Some Gag-specific IFN-g and IL-4 responses were significantly higher in the LTNP than in RP (p=0.02, IFN-g responses to GagA2 pool 1; p=0.04, IL-4 responses to GagA1 pool 2). IL-2 responses were low and not significantly different between LTNP and RP. There was a positive correlation between IL-4 responses to GagA1 pool 2 and CD4 T-cell counts (r 2 =0.502, P=0.04), but no correlation between either IL-4 or IFN-g responses and viral load. Cytokine responses to Nef peptides were not significantly different between the LTNP and RP. Conclusion: Overall, Gag HIV-1 specific responses were higher in LTNPs than in RPs confirming previous results. Non-specific IL-4 responses were high possibly reflecting baseline TH2 responses to helminths a common environmental exposure in the study population. Objectives: In human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the HLA class I antigen processing machinery (APM) have been described, which were associated with a reduced antigenicity of these cells. So far, the molecular mechanisms of such defects have not been elucidated in detail. To investigate whether impaired APM component expression was due to altered transcription and associated with cell growth properties murine Her2/neuand Her2/neu + fibroblasts were employed. Methods: Using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. Cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qRT-PCR and Western blot. Flow cytometry was employed for determination of the distinct phases using 7-AAD for DNA analysis and specific antibodies directed against the proliferation marker Ki-67, the M-phase specific pHistone H3 as well as for the H-2L d surface antigens. In addition, Chromatin Immunoprecipitation (ChIP) experiments with an antibody directed against RNA polymerase II were performed to investigate the transcriptional levels of tapasin in Her2/neuversus Her2/neu + cells. Results: Serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the G 0 /G 1 -, S-and G 2 /M-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. Tapasin mRNA level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the G 0 /G 1 -phase, which continuously raised and peaked within the S-phase. However, H-2L d surface antigen expression was not altered in Her2/neucells during cell cycle progression. In contrast to Her2/neufibroblasts the Her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered H-2L d surface expression. This was confirmed by a reduced promoter activity and decreased accessibility of the RNA polymerase II to the tapasin promoter. Conclusion: These findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. Recent studies suggest that dendritic cells (DCs) are key players in shaping the respiratory syncytial virus (RSV) specific immune response. Before, DCs within the airway epithelium were characterized as Langerhans cells. In this study, in vitro counterparts of Langerhans cells expressing Langerin (CD207) and CCR6 were cultured from CD34+ stem cells under the influence of TGF-b (TGF-b-DCs) and compared to CD34+ derived DCs, which passed through a monocytic stage . After infection with RSV, both types of DCs generated viral RNA and viral-proteins. Although TGF-b-DCs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by IL-4-DCs. The increased expression of viral proteins is most likely responsible for the pronounced inhibition of T-cell functions by TGF-b-DCs. Since there is evidence that Langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of RSV replication is characteristic of a more mature answer against RSV. The occurrence of inhibitory antibodies against exogenous factor VIII (FVIII) remains the major concern of FVIII replacement therapy in patients with hemophilia A. Initiation of the immune response implies the endocytosis of FVIII by professional antigen presenting cells (APCs): B lymphocytes, dendritic cells (DCs) and macrophages (MØ). The organ where the anti-FVIII immune response is initiated and the type of APCs involved in this process have not been investigated. We hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where APCs interact with FVIII to initiate the anti-FVIII immune response. We first administered radiolabeled FVIII at therapeutic doses to FVIII-deficient mice. FVIII was found to preferentially accumulate in the spleen and liver of the mice. Levels of FVIII in the spleen remained stable for up to 45 min following FVIII administration, while they rapidly decreased in the liver. Unlabelled FVIII was then administered to FVIII-deficient mice that had been splenectomized or sham operated and the anti-FVIII humoral responses were compared. Removal of the spleen resulted in significantly reduced levels of anti-FVIII IgG. Using flow cytometry, FVIII was found to preferentially accumulate with splenic MØ than DCs and B cells. Elimination of APCs by treatment of the mice with clodronate-containing liposomes prior to FVIII administration resulted in a drastic reduction of the anti-FVIII IgG response, as compared to control mice treated with PBS-containing liposomes. Taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-FVIII immune response and that splenic MØ have an important part in this process. The interactions between antigen presenting cells (APC) and T-lymphocytes are a relevant current issue. The area of contact between an antigen presenting cell and a T-lymphocyte is termed immunological synapse (Underhill et al, 1999) . The present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. In this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. Objectives: As central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be T cells.

Methods: Autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (Cabral and Novak, 1992, 1999) . Cytopreparations of each experiment were performed. Statistical analysis: Regression Model. Results: Experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p X 0,0001), ANOVA for Regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are T-cells, CD4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as Chloroquine or Brefeldin A, were added, rosettes were not formed, g) monoclonal antibodies anti-human MHC II precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. Conclusion: Taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. Objective: To investigate if patients with multiple sclerosis (MS), without the typical increase of antibodies in CSF, are less likely to develop neutralizing antibodies (NAbs) against IFNb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. Methods: Overall, 2252 patients were obtained from the Swedish Multiple Sclerosis Registry and the Swedish NAb Registry, and treatment information was available for 2207 of them. For 538 of these patients HLA-DRB1 data was available. Results: A significant correlation between lack of antibodies in CSF and NAb-negativity was found (p=0.02). Patients without CSF antibodies were to a lesser extent NAb-positive when treated with the IFNb-1a preparations, whereas no differences were shown for IFNb-1b. An association between HLA-DRB1*11 and NAb-negativity was detected (p=0.028). The known associations between HLA-DRB1*15 and CSF-positive MS and HLA-DRB1*04 and CSF-negative MS were confirmed. Conclusion: We show for the first time that patients without antibodies in CSF have a different propensity to induce NAbs compared to CSF-positive patients, indicating an extended immunological difference between the two MS sub-groups. HLA-DRB1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. In CSF-negative patients the reaction against IFNb-1a molecules, possibly through a T-cell dependent pathway, is lower than for CSF-positive patients. However, reaction against IFNb-1b, which might also be activated through a T-cell independent pathway, shows no difference in seroprevalence between the groups.

Abstract withdrawn by author

Tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using Tyrosinase-specific CTLs, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. There was no correlation between the level of antigen presentation and mRNA expression levels for the three antigens; however, our data suggest that Tyrosinase protein stability may play a major role in the high level presentation of this antigen. Measurement of the half lives of these proteins revealed a hierarchy in protein stability, with Mart-1 and gp100 more stable than tyrosinase. By the use of the cofactor DOPA, which stabilizes the tyrosinase protein, significant decrease of HLA-Tyr complexes presentation was achieved.

In addition to the study of antigen presentation, these TCR-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. By generating a whole IgG antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (ADCC). With the addition of point mutations in the Fc fragment, which increased the affinity of the Fc to the Fc receptor, enhanced tumor cell lysis was achieved.

G. Schiavoni 1 , S. Lorenzi 1 , F. Mattei 1 , F. Spadaro 1 , L. Gabriele 1 1 Istitituto Superiore di Sanità, Cell Biology and Neurosciences, Rome, Italy

Cross-presentation is a crucial mechanism for generating CD8 T cell responses against exogenous antigens (Ag), such as dead cell-derived Ag, and is mainly fulfilled by dendritic cells (DC), particularly CD8a + DC. However, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector CD8 T cell responses. Type I IFN are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. In particular, type I IFN have been shown to promote the cross-priming of CD8 T cells against soluble or viral antigens, partly through the stimulation of DC. In this study we evaluate the role of type I IFN to affect DC capacity to capture and cross-present apoptotic cell-derived Ag. By using UV-irradiated OVA-expressing EG7 thymoma line, we show that type I IFN promote the ability of CD8a + DC to capture apoptotic EG7 cells and to undergo phenotypic activation, both in vitro and in vivo. Remarkably, IFN-treatment prolongs the survival of Ag-bearing CD8a + DC and the persistence of apoptotic EG7-cell Ag within the phagosomal DC compartment, a process that is known to facilitate the recruitment of Ag into the MHC-I presentation pathway. Accordingly, type I IFN-treatment increases cross-presentation of apoptotic EG7-derived OVA Ag by DC, as revealed by higher expression levels of SIINFEKL peptide in association to MHC-I molecules on cell surface of phagocytic CD8a + DC. As a result, EG7-loaded DC become competent at inducing OT-I CD8 T cell proliferation and activation both in vitro and in vivo. Our data indicate that type I IFN promote the cross-presentation of apoptotic cell-derived Ag by CD8a + DC and suggest that these cytokines may act as a switch signal for cross-presenting DC, thus skewing the immune response from tolerogenic to immunogenic. (2).

We have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic DC subsets. Using biochemical methods, we show that only CD8+ DC efficiently transfer soluble antigen to their cytosol. The amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to TLR ligands CpG, Poly I:C or Pam3Csk4, and this correlated with enhanced cross-presentation. The increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. Measurement of the proteasome activity at different times after exposure to TLR ligands revealed that TLR signalling induced transient inhibition (maximum at two hours) of the proteasome in CD8+ DC but not CD8-DC, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting DC. This correlated with formation of aggresome-like structures only in cross-presenting DC exposed to TLR ligand. By limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. To our knowledge this is the first report of a direct, immediate effect of TLR activation on proteasome activity. Exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. They are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. Here we explore whether exosomes of different origin can selectively target different immune cells. We compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and B cells to peripheral blood mononuclear cells. Flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (ImageStream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an Epstein-Barr virus (EBV) transformed B cell line selectively target B cells. Our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. One of the hallmarks of cancer cells is the resistance to cell death. It has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. The proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. Here, we investigated that DM1, which is a saponin derivative isolated the stem bark of Dracaena mannii induced autophagocytosis on A549 human lung cancer cell line.

Methods: DM1 induced cell cytotoxicity was measured by MTT assay and Propidium iodide staining on A549 cells. We examined the morphological change using optical microscope. And GFP-LC3 punctation was measured using confocal. The protein expression was measured by western blot analysis and the mRNA expression was measured using reverse transcription poly chain reaction (RT-PCR). Results: DM1 was showed high cytotoxicity on various cancer cell line, especially A549 cells. DM1 treated A549 cells did not show regular DNA fragmentation and caspases activation. We also analyzed protein expression of apoptotic marker, but protein level didn't change. As a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. We checked LC3 and Beclin-1 protein and mRNA expression and inhibitory effect of autophagocytosis inhibitor. The expression level of LC3 was increased concentration and time-dependently. Beclin-1 also was increased. And then, we examined GFP-LC3 punctation on A549/ GFP-LC3 stable cells using confocal. A549 cells were formed GFP punctuation after DM 1 treatment. To confirm these data, we measured cell death ratio using 3-Methyladenine (3-MA), an autophagocytosis inhibitor. After 3-MA treatment, DM1 induced cell death was decreased comparing with DM1 treatment. Conclusion: DM1 did not induce apoptotic cell death. But DM1 showed several characteristics of autophagic cell death. Taken together, DM1 induced autophagocytosis on A549 cells. These finding indicated that therapeutic potential of DM1 by triggering autophagic cell death.

S. B. Rasmussen 1 , S. R. Paludan 1 1 University of Aarhus, Department of Medical Microbiology and Immunology, Aarhus, Denmark

During viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (PAMP)s. In the case of herpes simplex virus (HSV), detection is, among others, conducted by toll like receptor (TLR)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated CpG rich DNA of extracellular origin, e. g. viruses. Upon binding to DNA, TLR9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (IFN)a and IFNb being some of the most essential ones. The exact route of HSV to the endosomes and thus TLR9 recognition is not clear-cut. The endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in TLR mediated viral recognition has been drawn in to focus. We have found indications of an autophagy dependent IFN expression during HSV-1 infection. By use of an entry defect glycoprotein L and glycoprotein H deficient HSV-1, we found that TLR9 dependent IFN regulatory factor 3 activation was abrogated. In addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase Class III, which is crucial in autophagosome formation, abrogates IFNb induction. These findings points to a role for autophagy in TLR9 dependent recognition.

In the ongoing project we are examining how HSV-1 triggers formation of autophagosomes. Especially the role of endoplasmatic reticulum stress and doublestranded RNA-dependent protein kinase will receive attention. Also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated.

J. Zovko 1 , I. Berberich 1 , GK 520 -Immunomodulation 1 Universität Würzburg Institut für Virologie und Immunbiologie, Würzburg, Germany

Members of the Bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. Most Bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their C-terminal portion. Murine A1 and its human homologue Bfl-1 are anti-apoptotic members of the Bcl-2 family. A1 is expressed in small amounts in the bone marrow and immature B cells, but in high amounts in mature B cells. Thus the protein seems to be important for B cell maturation. We analyzed the function of the C-terminus of A1. Unless the C-terminal ends of other Bcl-2 proteins the tail of A1 does not function as a strong membrane anchor. Nevertheless, the last amino acids of A1 are important for the protein. In fact, the C-terminus of A1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. We show that A1 undergoes proteosomal degradation controlled by its C-terminus. Interestingly, binding to the proapoptotic Bcl-2 factor BimEL results in increased stability of A1. This is due to reduced ubiquitination of A1 after binding of BimEL. We conclude that the Cterminus of A1/Bfl-1 serves as a docking site for E3 ubiquitin ligase(s) that control the stability of A1 by targeting the protein to the proteasomal pathway. Very recently, we have identified a putative E3 ubiquitin ligase that interacted with A1 in a yeast two-hybrid screen. Currently, we are trying to confirm this interaction in mammalian cells. Suppressors of cytokine signaling (SOCS), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. Here we describe novel function of SOCS-1 in the suppression of ROSmediated apoptosis and associated mechanisms using TNF-alpha induced T cell apoptosis as a model system. Both in Jurkat T cells and primary splenocytes, SOCS-1 is induced under TNF alpha-induced apoptosis conditions. The TNF-induced apoptosis was mediated by generation of ROS, and the over-expression of SOCS-1 significantly suppressed TNF-induced ROS levels and the subsequent apoptosis. Such anti-apoptotic function of SOCS-1 was manifest not only by the suppression of Jaks acting upstream of p38 MAPK, but also protection of PTPs which down-regulate the TNF alpha -induced Jak 1/2 activities. We further show that TNF-alphainduced PTP inactivation can be prevented by SOCS1 which up-regulates thioredoxin levels. Finally we present data that there is a molecular interaction between thioredoxin and PTP which is formed in response to ROS-generating stimuli and sustained in SOCS-1 overexpressing cells. The results strongly suggest that SOCS-1 acts as an anti-oxidant and anti-apoptotic factor to sustain T cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. Aim: Inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. The effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the CNS by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. CXCL12/SDF-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. Moreover, it has been described that TNF-a mediates in CXCL12 effects on primary astrocytes, and it is clear that TNF-a participates in the pathogenesis of many neurological conditions. Methods: We used the astrocytoma cell line U-87, and SK-N-MC as neuroblastoma cells. Detection of TNF-a mRNA expression was carried out by RT-PCR. Transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. We performed cotransfection experiments of NFAT promoter construct with a dominant negative version of NFAT (dn-NFAT). Neuronal death was performed by MTT and TUNEL assays. NFAT translocation was confirmed by Western-Blot. P53 and Fas-L expression was carried out by Western-Blot. Results: CXCL12 induced mRNA-TNF-a and transcriptional activity of TNF-a promoter in human astrocytes. This cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on NB cultures, neuronal death increased in a direct and indirect way. Surprisingly, TNF-a did not induce NF-kB activation in NB cells but it induced NFAT activity. NFAT translocation was confirmed by Western-Blot. Neurotoxicity was absolutely reverted in the presence of ciclosporin. We discard p53 pathway as responsible for TNF-mediated toxicity since we did not find any alteration in p53-Ser46 levels in stimulated cells. In addition, we found increased FasL expression in neuroblastoma cells after 24h of TNF-a treatment.

Conclusions: CXCL12 induced-TNF-a promotes NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of FasL expression without alter p53function/levels.

S. Y. Demiroglu 1 , R. Dressel 1 1 Universitätsmedizin Göttingen, Zelluläre und Molekulare Immunologie, Göttingen, Germany

Objectives: Intracellular HSP70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. Extracellular HSP70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic T lymphocytes (CTL). Interestingly, HSP70 does not protect against cell death mediated by CTL. Acute overexpression of HSP70 can even increase the susceptibility of target cells to CTL, which use the granule-exocytosis pathway to induce apoptosis (Dressel et al. Cancer Res 63: 8212) . Granzyme B is one of the main effector proteases of cytotoxic granules. Therefore, we analyzed the effect of acute overexpression of HSP70 on granzyme B-induced apoptosis. Methods: HSP70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (Ge-tet). The effect of HSP70 induction on granzyme B-mediated apoptosis was now analyzed after delivery of granzyme B into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. Results: HSP70 did not protect the melanoma cells against granzyme B-mediated apoptosis when annexin V binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. Instead, we observed a moderate but significant pro-apoptotic effect of HSP70 in Ge-tet cells when late apoptosis was analyzed at the nuclear level by sub G1 peak measurements (p=0.01). In contrast, a partial protection of Ge-tet cells was observed after acute HSP70 overexpression when apoptosis was induced by staurosporine. Conclusion: Acute overexpression of HSP70 does not seem to protect tumor cells from granzyme B-induced apoptosis; it appears even to accelerate the progression of apoptosis to DNA fragmentation and nuclear condensation. It is conceivable that this HSP70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated DNase (Liu et al., Blood 102:1788) . Thus, granzyme B might be able to kill even those tumor cells that undergo an otherwise protective stress response. The work has been supported by the grants GRK1034 and DR394/2-3. The authors thank Prof. C. J. Froelich (Evanston, USA) for the granzyme B and Dr. T. Seidler (Göttingen) for the adenoviruses. Tumor necrosis factor (TNF) elicits its biological activities by stimulation of TNFR1 and TNFR2, both belonging to the TNF receptor superfamily. TNFR1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical NFkB pathway, cell death induction and MAP kinase signaling. In contrast TNFR2-associated signal transduction is poorly defined. Earlier findings demonstrated that only membrane TNF, but not soluble TNF, properly activates TNFR2, resulting in depletion of TRAF2 from the cytoplasm. Here we confirm that TNFR2 induced depletion of cytosolic TRAF2 by the use of TNFR1-and TNFR2-specific mutants of soluble and membrane TNF. Corresponding with the known inhibitory role of TRAF2 in the alternative NFkB pathway, we show that TNFR2 induced activation of the alternative NFkB pathway. Thus, we identified activation of the alternative NFkB pathway as a TNF signaling effect that can be specifically assigned to TNFR2 and membrane TNF.

J. C. Morales 1 , D. Ruiz-Magaña 1 , D. Carranza 1 , C. Ruiz-Ruiz 1 1 Universidad de Granada, Instituto de Biopatologí a y Medicina Regenerativa. Centro de Investigación Biomédica, Armilla, Spain Different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to TRAIL by regulating gene transcription of TRAIL receptors and other TRAIL pathway related-genes. In the present study we have analyzed the effect of several histone deacetylase inhibitors (HDACi), belonging to different structural families, on TRAIL-induced apoptosis in leukemic T cell lines. Moreover, we have analyzed the activity of HDACi in normal T lymphocytes. We have found that, to a greater or lesser extent, all HDACi potentiate the induction of apoptosis in leukemic T cells by enhancing the signals triggered upon TRAIL ligation, from the activation of the most apical caspase-8. In contrast, HDACi do not sensitize primary resting or activated T lymphocytes to TRAIL-mediated apoptosis. The analysis of the expression of several pro-and anti-apoptotic proteins involved in the TRAIL-signalling pathway indicates that most HDACi regulate the expression of TRAIL-R2 and c-FLIP in leukemic T cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. ZFP36L1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. It can bind to mRNAs containing adenine uridine rich (ARE) regions and subsequently mediate their degradation. We have previously reported a role for this protein in promotion of B cell apoptosis. One mechanism whereby ZFP36L1 may mediate cell apoptosis could be by degradation of cell survival gene mRNAs. The Bcl-2 protein is an important cell survival protein at different stages of B cell development. Bcl-2 mRNA also contains ARE regions that could possibly be targeted by ZFP36L1 protein.

In the present study, we have tested the ability of ZFP36L1 protein to bind to a Bcl-2 mRNA ARE probe and to degrade Bcl-2 mRNA. Recombinant bacterially expressed ZFP36L1 protein was shown to bind specifically to a Bcl-2 ARE probe by RNA electrophoretic shift assays (REMSAs). Furthermore, REMSAs using cell lysates of Ramos Burkitt lymphoma B cells stimulated to express high levels of endogenous ZFP36L1 also provided evidence that endogenous ZFP36L1 in B cells could bind to the Bcl-2 ARE. In order to examine whether ZFP36L1 binding to Bcl-2 ARE resulted in Bcl-2 mRNA degradation, actinomycin D RNA degradation assays were carried out on murine embryonic fibroblast (MEFs) cells from ZFP36L1 knockout mice and wild-type mice using quantitative real-time PCR analysis. Bcl-2 mRNA was expressed in both wild-type and knockout MEFs. The half-life of Bcl-2 mRNA was found to be extended in knockout MEFs compared to wild-type MEFs suggesting that ZFP36L1 does play a role in degradation of Bcl-2 mRNA. Overall, our data are consistent with a role for ZFP36L1 in degradation of Bcl-2 mRNA which could be a mechanism for the reported role of this protein in induction of B cell apoptosis. The Epstein-Barr virus (EBV) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. Its infection is associated with several human malignancies (Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein LMP1. Since LMP1 is able to transform numerous cell types, it is considered as the main oncogenic protein of EBV. The principal mechanism of LMP1 function is based on mimicry of activated member of the TNF receptor super family (TNFR), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like NFkB, c-Fos-JNK, PI3-kinase...involved in the regulation of cellular processes. We previously generated two unique model, a monocytic (TE1) and lymphocytic (NC5) immortalized by EBV and which expressing type II latency program. Here we developed original dominant negative (DN), by generating a fusion between GFP and TES1 or TES2 (Transforming Effectors Site) derived from the C-terminal intracellular part of LMP1, in inducible vectors. Then, we generated cell lines conditionally expressing these DNs. We showed these DNs not only inhibit survival processes resulting to the impairment of NFkB and akt pathway but increase apoptosis in this cell lines. We demonstrated that this pro-apoptotic effect is due to i) the depletion of LMP1's specific adapters and ii) the recruitment of theses adapters by DNs interestingly allowed generation of apoptotic complex involved TRADD, FADD and caspase-8.

Using this NC5 tumorigenic model in SCID mice, we showed that induction of the DN LMP1-TES2 prevent development of tumours and mouse death. These dominant negative derived from LMP1 could be used to develop therapeutic approaches in malignant diseases associated with Epstein-Barr virus, but also in inflammatory pathologies. Recent studies indicate that suppressors of cytokine signaling (SOCS) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. Since tumor cells often exhibit aberrant expression of SOCS genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of SOCS isoforms during DNA damageinduced apoptotic response and cell cycle changes in various tumor cell types. By using tumor cell lines transduced for over-expression or knock-down of distinct SOCS isoforms, it is found that SOCS1 and SOCS3 differentially affect apoptosis and cell cycle changes induced by DNA damaging agents in a cell type-specific manner. In T lymphocytic leukemia cell line Jurkat, SOCS1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38MAPK activities, while SOCS3 promoted apoptosis with an increase in p38 activities. In contrast, both SOCS1 and SOCS3 display proapoptotic effect in RKO colon cancer cell lines upon exposure to gamma radiation or ROS-generating agents. Notably, effects of SOCS proteins on cell cycle changes induced by DNA damaging agents were rather similar in that over-expression of either SOCS1 or SOCS3 induced a slight decrease in G1 or S phase cells and a prominent increase in G2/M cells, regardless of their distinct effects on apoptosis. The analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and CDK inhibitors affecting G1/S transition and G2/M arrest induced by SOCS1 or SOCS3. SOCS1 promoted DNA damage-induced p21 induction and G2/M cyclin B expression, while SOCS3 induced decrease in G1 cyclin E expression. The results suggest that SOCS isoforms potentially modulate growth of tumor cells exposed to DNA damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. The heat shock protein 90 (Hsp90) is a highly conserved a widely expressed molecular chaperone. It is known to regulate the activity of several protein kinases or the proper folding of client proteins. Hsp90 has also been identified as an important regulator of cellular survival. Besides these intracellular functions, extracellular Hsp90 can initiate cross presentation or immune responses. Apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. However the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. Hsp90 is released during necrotic cell death and proinflammatory effects of extracellular Hsp90 have been observed. Thus, we asked whether apoptozing cells cleave Hsp90 during apoptosis or how Hsp90 is disposed by these cells. We induced apoptosis either in activated or resting primary human cells and analyzed the Hsp90 protein content. We observed that Hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kDa. This fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. Analyzing the isoforms of Hsp90 (Hsp90 alpha and Hsp90 beta) we could show that the 55 kDA fragment is formed after degradation of the alpha isoform of Hsp90. Further, we were able to show, that Hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. Analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous Hsp90 and caspase inhibitors. We observed a rather proinflammatory cytokine profile, if cleavage of Hsp90 was inhibited or if exogenous Hsp90 was added. These results demonstrate that cleavage of Hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells.

activity. MIFC integrates the advantages of flow cytometry and fluorescence microscopy in one system, the ImageStream. Upon induction of autophagy, cytosolic LC3-I is processed to LC3-II, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. An increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. MCF-7 GFP-LC3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. Classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. We conclude that ImageStream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. Our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. JNK seems to mediate the Bcl-2/Beclin-1 control of autophagy. Recently, JNK was shown to be necessary for Beclin-1 upregulation, and JNK-mediated phosphorylation of Bcl-2 is associated with both, starvation-and ceramide-induced autophagy. The NFkappaB pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. We report here the novel findings that under conditions of starvation, pharmacological inhibition of NFkappaB decreased the autophagic flux in MCF-7 cells, while JNK inhibition shows an enhancing effect on autophagy induction. Ingenol 3-angelate (PEP005), a novel activator of protein kinase C (PKC), has been shown to induce apoptosis in acute myeloid leukemia cells. We show here, that in contrast to leukemic cells, PEP005 provides a strong survival signal to resting and activated T cells. This anti-apoptotic effect was dependent upon the activation of PKCV, a PKC isoform restricted to T cells and myocytes. Expression of PKCV in the acute myeloid leukemia cell line NB4 turned their response to PEP005 from an increased to decreased rate of apoptosis. Furthermore, our data show that PEP005 inhibited T cell apoptosis through the activation of NFxB downstream of PKCV, leading to increased expression of the anti-apoptotic proteins Mcl-1 and Bcl-xL. We conclude that PKCV expression determines whether PKC activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. This finding may be of considerable importance for the development of PKC -targeting antileukemic therapies. The neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. Several reports indicate that survival and activation of normal B lymphocytes are regulated by nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) autocrine circuits. However, the production and the role of neurotrophins were not evaluated in B lymphoma cells. Diffuse large B-cell lymphoma (DLBCL) is a common and often fatal malignancy. Despite major advance in the treatment (R-CHOP protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. We hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of DLBCL tumor cells. This hypothesis was investigated with DLBCL cell lines (SU-DHL). Thus, we evaluated the ability of SU-DHL cells to produce neurotrophins (NGF, BDNF) and to express their receptors (p75, TrkA and TrkB) in different cell culture conditions. Our preliminary data show for the first time the production of neurotrophins by DLBCL tumoral cells whose level decreased in apoptotic conditions, in association with Bad dephosphorylation suggesting its pro-apoptotic role. Furthermore our results suggest that up-regulation of autocrine circuits (expression of TrkA known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral B cells. Objectives: Ataxia telangiectasia (A-T) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. This gene encodes ATM, a protein kinase which has a major role in DNA double strand break response. A-T patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. The susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. Although ATM is known to have a central role in the induction of apoptosis in response to unrepaired DNA double strand breaks its role in extrinsic apoptotic pathways is unclear. This study aimed to investigate if ATM has a role in Fas induced apoptosis.

A bank of lymphoblastoid cell lines (LCLs) derived from A-T and normal individuals and tumour samples with wildtype or mutant ATM were used in the study. Apoptosis was induced by incubating cells with the Fas activating antibody CH11 and analysed by flow cytometry. Expression of the caspase 8 inhibitor cFLIP which inhibits Fas induced apoptosis was detected by western blot.

Results: There was no significant difference in the susceptibility to Fas induced apoptosis or cFLIP protein expression between ATM mutant and control groups. However cells expressing high levels of cFLIP protein do show greater resistance to Fas induced apoptosis than those with lower expression. Whilst the LCLs expressed both long and short forms of cFLIP, the tumour cells expressed only the long form. Conclusion: ATM mutations do not affect susceptibility to Fas induced apoptosis or alter cFLIP protein expression in LCLs or tumour cells. cFLIP protein levels and Fas susceptibility vary greatly between individuals but this is independent of ATM status. High expression of cFLIP protein correlates with reduced apoptosis in response to CH11 treatment but there is no clear difference in cFLIP expression between ATM wildtype and mutant cells. Labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. However, little is known about their possible role in the apoptotic cell death machinery. We report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. Activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. Diterpenoids also led to time-dependent cleavage of Bid. Inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. In addition, pretreating cells with neutralizing antibodies to Fas ligand, tumor necrosis factor receptor 1 (TNF-R1), and tumor necrosis factor (TNF)-a receptor 2 (TRAIL) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. Diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ROS). However, increased ROS production was not directly involved in diterpenoid-triggered apoptosis. These results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. 

Conclusion: Cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. Whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. Synthetic alkyl-lysophospholipids (ALPs) are a group of unnatural lipids with promising anticancer capability. A prototypic member is the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of Fas/CD95 independent of its ligand FasL/CD95L. Fas/CD95 is activated by edelfosine via its translocation in lipid rafts. In this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. Edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zVAD-fmk while FasL-stimulated cells stayed mostly alive. Furthermore cells could not be rescued by addition of zVAD-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. Fas resistant solid tumor cells overexpressing members of the anti-apoptotic Bcl2-family as well as cells overexpressing the cellular regulatory protein FLIP went in contrast to Fas stimulation to apoptosis after treatment with edelfosine. Therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. Apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present DNA mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. Thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. Many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. The present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. COLO 201, SW1116, LoVo, Caco-2, HT-29) . In addition, expression of antigens involved in tumour proliferation and apoptosis (Ki-67, PCNA, P53, Bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. Percentages of apoptotic cells were detected by using annexin V/FITC and propidium iodide double staining, while progression through cell cycle phases was evaluated by using PI staining. Correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from RT-PCR with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. Stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of DNA changed. Data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. The results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. The effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. Extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. The purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways.

The randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. Lymphocytes expressions of Bcl-2, Bax, caspase 3, 6, 9, NFkB, PARP1/89kDa, TNFR1/CD120a and CD95/Fas were assessed by Western-blot and flow cytometry.

Results: Before and after surgery the expression of Bcl-2, Bax, NFkB, PARP1 was significantly lower and expression of caspases, TNFR1 as well as percentage of CD95 cells significantly higher as compared with control group. Caspase 3 expression was significantly higher as compared with NFkB, PARP1 and TNFR1. In comparison to the standard nutrition preoperative immunonutrition increased Bcl-2 and NFkB expressions and decreased caspases and PARP1 expressions. In addition, we found a significant down-regulation of Bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. Conclusion: Preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. This modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. The capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. In this study the immunomodulatory effects of Salvia mirzayanii a native plant that is widely distributed to Iran was investigated. S. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. Study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p X 0.01). In flow cytometry analysis, accumulation of apoptotic cells in the sub-G1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. DNA fragmentation analysis of these cells showed a typical DNA laddering. Immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p X 0.007). These results indicated the presence of immunomodulatory compounds in the extract of S. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. A new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either La1 (10 9 CFU/day) or placebo, during 57 days prior to UV (2 × 1.5 MED). Blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after UV exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in UV-induced immune response, quantification of inflammatory cytokines, a DNA damage marker (p53). While a similar decrease of LC for both groups was observed on day 1 after UV exposure compared to placebo, La-1 group presented a faster increase of a new subset of epidermal dendritic cells (DC), namely early LC precursors (CD1a low CD207 -) associated with a minor recruitment of monocytes. Concomitantly, inhibition of IL-10 and a tendency to inhibit IL-6 was observed in La-1 group compared to placebo. On day 4, La-1 group presented a greater recruitment of early LC precursors and a trend to increase CD1a low CD207 + LC precursors compared to placebo. Additionally, a faster reduction of inflammatory and immunosuppressive cytokines (IL-6, TNFa, IL-8, and IL-10) was observed in La1 group compared to placebo. We show that La1 limits UV-induced immune-suppression and skin inflammation. This contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of La1 supplementation for photoprotection at a lower dose (1 log). The thymus is one of the primary lymphoid organs and plays a central role in the immune system. It provides the essential microenvironment for proper T cell development. In the thymus, the maturation of T cells depends on many interactions between T cells and different stromal cell types, mainly composed of epithelial cells (TECs). Foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. Foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around E11.5. Previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. Using the Cre-loxP system, we showed that a single epithelial cell, when reverted to express Foxn1 in a nude (Foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. Hence, we hypothesize that the epithelial progenitor cell expresses Foxn1. If true, it should be possible to target this cell type by use of Foxn1-promoter driven transgenes. Conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. Currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. Intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as Huntington's disease. Considering this, we are expressing transgenic EGFP variants containing either 19 or 82 glutamine residues under the control of Foxn1 promoter, leading to different degrees of TEC degeneration. Furthermore, also under the Foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (MTz) into a cytotoxic cross-linking agent for conditional cell ablation. Preliminary results describing the phenotypes of these mice will be presented.

T. Kamei 1,2 , Y. Toriumi 3 , K. Kimura 4 1 European University Viadrina, Frankfurt (Oder), Germany, 2 Shimane Institute of Health Science, Izumo, Japan, 3 Shimane University Faculty of Medicine, Department of Pediatrics, Izumo, Japan, 4 Japan Yoga Niketan, Yonago, Japan

As a method in relieving stress, yoga is popular today. Many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. We examined changes of electroencephalograph (EEG) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. The subjects consisted of eight yoga instructors who had been practicing yoga for several years. A 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. Throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (Fp2). Blood samples were drawn before and after each exercise. NK activity and percentages of T-cell and B-cell subsets were measured. During the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in NK activity (r=0.83, p X 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of T lymphocytes (r=0.77, p X 0.05) were observed. Furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of CD4 (r=0.96, p=0.0001). These findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of NK cells and T lymphocytes, mainly of CD4, within a short period of time. We conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. Yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. T regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. Several recent studies suggest also that diverse populations of regulatory T cell play an important role in regulating T-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. Here we demonstrate that CD4 + CD25 + T regulatory (T reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (MCs) without affecting cytokine release. This effect is shown to require a cell-cell contact and depends on interaction between OX40L expressed on MCs and the constitutive expression of OX40 (members of the tumor necrosis factor [TNF] and TNF receptor family, respectively) on T reg cells. This interaction does not alter the activation of PLC-g, Syk and LAT in IgE/Ag stimulated MCs upon co-incubation with T reg cells, whereas it induces a decrease in the phosphorylation levels of Akt. Moreover, we find that upon co-incubation with T reg cells, MCs show increased levels of cAMP, which is known to inhibit MCs function, as a result of OX40L signal. Antagonism of cAMP in MCs reverses the inhibitory effects of T reg cells restoring normal Ca 2+ responses and degranulation. The cross-talk between T reg cells and MCs through OX40-OX40L interaction defines a previously unrecognized mechanism controlling MCs degranulation. Loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. Active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. A healthy immune system responds to antigens with a combination of polarized Th1 or Th2 effector cells and the induction of antigen specific FOXP3+ regulatory T cells (Treg). It is believed that the dominant subset determines the quality of the eventual immune response. In allergic asthma there is a clear dominance of Th2 cell responses to non-pathogenic environmental antigens. Recently it was shown that the specific transcription factors that characterize the Th2 and Treg subset, GATA3 and FOXP3 respectively, counter regulate each other (Mantel Y et al., 2008) . We hypothesize that children with allergic asthma will respond to allergens with low induction of FOXP3+ Tregs and high GATA3+ Th2 cells.

In order to prove this hypothesis PBMC of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and LPS. Almost 100 million allergic patients are sensitized to the major birch pollen allergen Bet v 1, which cross-reacts with major allergens of Fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. The epitopes of Bet v 1 recognized by allergic patients' IgE antibodies belong to the conformational type and therefore have not been characterized in detail. Here we used antibodies raised against peptides spanning the Bet v 1 molecule in IgE competition experiments to search for sequences which are involved in IgE recognition. The strongest inhibition (i.e., G 90 %) of patients' IgE binding to Bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (P2) and aa 74-104 (P6) of Bet v 1. Cross-reactive IgE epitopes between Bet v 1 and related pollen allergens and plant food allergens involved primarily P2. P2 and P6 are not adjacent peptides in the Bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of Bet v 1. As determined by surface plasmon resonance, monoclonal antibody mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, i. e., dissociation constants, K(D) = 8.35e-11 M and K(D) = 1.05e-9 M, respectively. Interestingly, peptide-specific mAbs inhibited allergic patients' IgE antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. This finding indicates that the surface patch defined with anti-P2 and anti-P6 antibodies contains several IgE epitopes.

In summary, we have defined a surface-exposed patch on the Bet v 1 allergen which seems to harbor the majority of the IgE epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. Background: Antigen-specific Th1 cells as well as Tc1 cells, induced by biolistic gene transfer using plasmid DNA encoding the model allergen b-galactosidase (bGal) under control of the fascin promoter (pFascin-bGal), inhibited the elicitation of systemic Th2 immune responses and suppressed IgE production in an experimental mouse model. Moreover, protective biolistic DNA vaccination with pFascin-bGal prevented Th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bGal protein, but led to the recruitment of Th1/Tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (AHR). Objective: To analyze the modalities of AHR induction in mice biolistically vaccinated with pFascin-bGal. Methods: BALB/c mice were immunized with pFascin-bGal using the gene gun. Subsequently, mice were challenged by consecutive intranasal application of bGal protein. CD4 + and CD8 + T cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-CD4 (GK1.5) or anti-CD8 (53.6.72) monoclonal antibodies. Neutrophilic granulocytes were depleted by treatment of animals with either anti-Gr-1 monoclonal antibody RB6-8C5 or monoclonal antibody NIMP-R14. One day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (BAL) was performed and the frequency of IFN-g-producing CD8 + effector T cells in the lung was determined. Results: Whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of CD4 + T cells, AHR was not significantly affected, implicating that the elicitation of AHR by CD8 + T cells is dissociated from the activity of neutrophils. This notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered AHR. In contrast to CD8 + T cells, CD4 + T cells induced strong neutrophilic infiltration and AHR. Transfer experiments with CD4 + or CD8 + T cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of Th1 and Tc1 cells operative in the elicitation of airway inflammation. Conclusions: Robust type 1 immune responses, although highly effective in the counter-regulation of local Th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of AHR. Respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. Upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. House dust mite (HDM) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line NCI-H292. The proteolytic activity of these HDM allergens has been proposed to be involved in the activation process. The aim of this study was to compare the influence of HDM extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. In contrast to the HDM major allergen Der p 1, we were able to show that the major allergen of Phleum pratense, Phl p 1, although sharing molecular similarities with Der p 1, does not display any enzymatic activity under physiological conditions. Therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified Phl p 1. Chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mRNA was used for cDNA-microarray analysis. First data show that both, HDM extract and grass pollen allergens, induce the release of IL-6 and IL-8 from NCI-H292 cells. Furthermore, stimulation with HDM extract leads to the release of TNF-a, GM-CSF and IFN-g. Interestingly none of these mediators was induced after stimulation with grass pollen extract or purified Phl p 1. In contrast to HDM extract grass pollen allergens induce the release of MCP-1 from respiratory epithelial cells, as well as moderate levels of IL-12. Detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with HDM induced expression profiles will be helpful towards understanding the allergic response in general.

(Supported by DFG SFB TR22)

Results: Collectively, responses to bLG, but not to BSA, were observed in all groups analyzed, included healthy controls. Nevertheless, 3 distinct profiles of response were obtained: children with IgE mediated CMA had a significant increased level of proliferation (mean±SD of stimulation index(SI): 7.2±5.7) and of IL-13 (mean±SD: 1157±909 pg/ml), and reduced IL-10 (mean±SD of IL-10-spot forming units/2x10 5 cells (SFU): 912±510), compared to healthy subjects (3.4±2.7 SI, p X 0.05; 355±396 pg/ml, p X 0.05; 1272±623 SFU, for proliferation, IL-13 and IL-10, respectively); children with non-IgE mediated CMA had a significant reduction of IL-13 (192±362 pg/ml), compared to IgE patients (p X 0.0004) and an increased, although not statistically significant, production of IFN-g (33.7±54.6 SFU) compared to control (9.5±9.5 SFU) and to IgE-allergic patients (0.6±0.8 SFU). Finally, tolerant patients showed reduced IL-13 (636±1048 pg/ ml, p X 0.05) and proliferation (3.9±3.5 SI), compared to acute IgE-CMA children. Interestingly, the high level of IL-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to bLG; by contrast, IL-4 was undetectable in all patients even blocking the IL4-Receptor. Conclusion: bLG-specific, immune responses can be recalled in peripheral blood of CMA patients, as well as of normal and tolerant children. A Th2-like response with IL-13 and proliferation is dominant in IgE-mediated CMA patients; by contrast a Th1-skewed response with IFN-g is present in non-IgE-mediated allergic and in those children who outgrew IgE-allergy.

Y. F. Tang 1 , B. Chua 1 , F.C. Lew 1 , A. Ho 1 , K. Wong 1 , K.L. Wong 1 , D. M. Kemeny 1 1 National University of Singapore, Immunology Programme, Yong Loo Lin School of Medicine, Singapore, Singapore

Allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. Using a mouse model, this response can be divided into an induction phase, in which CD4 Th2 T cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. In the lung, recruited Th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (AHR). Previously we, and others, have shown that transfer of CD8 T cells inhibits the induction of the Th2 response. Here we have investigated the effect of CD8 T cells on the effector phase of the inflammatory lung response. In vitro activated OT-I CD8 T cells were transferred to ovalbumin (OVA)/ alum immunized mice one day before the first of 3 airway challenges with OVA. Eosinophil infiltration was inhibited by transfer of CD8 + T cells (36.7 %±4.1 % to 17.6 %±2.7 %). When IFN-gamma -/-OT-I CD8 T cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for CD8 T cell IFN-gamma. CD11c + CD103 + CD11blung DCs from CD8 transferred mice secreted higher levels (500pg/ml) of IL-12p70 following ex-vivo stimulation as compared with animals that were not given CD8 T cells. These data show that, in addition to regulating the induction of the allergic immune response, CD8 T cells can subsequently divert the local lung environment to one that favors Th1 immunity. The chain terminator drug Abacavir triggers a serious hypersensitivity reaction in 8 % of patients with HIV infection. This reaction is strongly associated with HLA-B*5701 and appears to be mediated by CD8+ T cells producing inflammatory cytokines. We show that CD8+T cell responses can be primed in vitro, in normal blood donors who are HLA-B*5701+, but not in non-B*5701+ donors. CD8 T cells, but not CD4 T cells, are expanded by Abacavir pulsed autologous APC over a13-day culture, producing IFN-gamma and TNF-alpha upon re-stimulation with APC expressing HLA-B*5701. Similar responses were detected in Abacavirhypersensitive HLAB*5701+ patients. Responses were not detected using mutant APC deficient in TAP or tapasin, or when normal APC were aldehyde fixed before loading with Abacavir, indicating a reliance on the conventional MHC-I Ag presentation. Responses were exquisitely restricted to HLA-B*5701 since they were undetectable using APC expressing closely related HLA-B57 or B58 allotypes. Responses to APC expressing mutants of HLA-B*5701 demonstrated a crucial role for residue 116. Isolation of peptide fractions from Abacavir-loaded cells has led to the identification of specific fractions recognised by an Abacavir-specific T cell line. Our data suggests that Abacavir forms a conjugate with an endogenous peptide that is presented by HLA-B*5701 triggering CD8 T cells. We speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic MHC-I, contributing to the responses observed in Abacavir naï ve individuals. The molecular mechanisms underlying altered HLA-B*5701 may be relevant to the role of other disease-associated class I allotypes such as HLA-B27 and B51.

A. Jenckel 1 , S. Bulfone-Paus 1 , N. Föger 1 1 Research Center Borstel, Immunobiology, Borstel, Germany

Mast cells play a crucial role in acute inflammatory and allergic reactions. Upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. Recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. The actin-associated protein Coro-nin1a (Coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. Recent data of our group also indicate a regulatory role of Coro1a in mast cell function. Coronin proteins have been described to be differentially phosphorylated in vivo. However the molecular mechanisms by which Coro1a is regulated in response to physiological stimuli are still poorly characterized. Here we investigated the modalities of Coro1a phosphorylation during the activation of mast cells. Immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of Coro1a on serine residues upon antigen-specific engagement of Fc-epsilon-receptors. As the phosphorylation status of Coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of Coro1a during mast cell activation. Cell fractionation experiments demonstrated that the association of Coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in Coro1a phosphorylation. A functional correlation between Coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of Coro1a. Thus, Coro1a is a downstream effector molecule of Fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. Allergen-specific T and B lymphocytes play an important role for the pathogenesis of asthma. T cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. Recently, we have shown in a mouse model for allergic airway inflammation that B cells directly interact with T cells in the inflamed tissue and locally produce IgE.

To analyse T/B-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific T cells and nitrophenolspecific B cells. Recipient mice are then challenged intranasally with an NP-OVA conjugate. This system allows to track single allergen-specific T and B cells in all stages of the immune reaction using flow cytometry and immunohistology. In addition, cells can be re-isolated by flow-sorting for in-depth analysis.

Using this system we could define several phases of the inflammatory reaction. T and B cells first become activated in the lung-associated lymph nodes. Granulocytes can be found very early in the lung tissue and also activated T cells very rapidly emigrate to the site of inflammation. However, clusters of allergen-specific T and B cells can only be found in later stages of the reaction. As another focus, we used our in vivo system to define the role of T cell costimulatory molecules for airway inflammation. Costimulatory receptors are key regulators of T cell activation and differentiation and therefore promising targets for therapeutic intervention. Of special interest is the T cell-specific ICOS molecule which is important for T/B cooperation as well as the regulation of chronic inflammatory reactions. Using ICOS knock-out mice we were able to delineate the specific role of ICOS for the different stages of airway inflammation. In particular, we analysed the impact on T cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. The integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. Coronins constitute a family of evolutionary highly conserved WDrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. In mammalians seven coronin family members have been described. The high degree of sequence conservation amongst coronin family proteins suggests common features and functions. However, individual family members may also have developed additional selective and specific functions. Our recent studies on Coronin1a (Coro1a) deficient mice have demonstrated that Coro1a exhibits an inhibitory function on the cellular steady-state F-actin content, is required for chemokine-mediated functions in T cells and is involved in the maintenance of T cell homeostasis. Coronin1b (Coro1b) is a closely related homolog of Coro1a and the two genes are co-expressed in hematopoietic cells. To address the question of functional redundancy in vivo, we have generated Coro1b deficient mice and crossed them with Coro1a deficient mice to obtain Coro1a/Coro1b double deficient mice. Analysis of T lymphocytes from Coro1a/Coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral T cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. An analysis of coronin deficient mast cells also revealed an involvement of Coro1a/Coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. However, in contrast to the inhibitory effects of Coro1a/1b deficiency on T cell function, mast cell degranulation and migration was enhanced in Coro1a/1b double deficient mast cells. Thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. Additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. Together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. Initially found by serial analysis of gene expression, murine Samsn1 (also known as Hacs1 or Sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (SAM), a Src homology 3 (SH3) domain and a predicted bipartite nuclear localization signal. The Samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. Initial overexpression studies suggest a contribution of Samsn1 in B cell activation and differentiation, however its physiological function is yet unknown.

To investigate Samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time PCR. Our data revealed an expression of Samsn1 in all tested hematopoietic cell types. The highest expression level of Samsn1 mRNA was seen in mast cells compared to lower levels in macrophages, dendritic cells, CD4+ and CD8+ T cells and B cells. The other two members of the Sly family of adaptor proteins -namely Sly1 (Hacs2) and Sly3 (Sash1) -were expressed only at a very low level in mast cells. The high level of Samsn1 mRNA expression in mast cells, together with minimal expression of other Sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. To address the potential role of Samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from Samsn1 deficient mice. Initial in vitro experiments indicate normal proliferation and differentiation of Samsn1 deficient mast cells. In additional studies we are now investigating the effects of Samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. Analyzing the role of Samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. Introduction: We showed previously that the ability of murine IgG1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain N-linked to the antibody Fc region (Silva et al; J.Immunol., 2008). Then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic IgG1 Abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in B cells. Objective and methods: To prove this hypothesis it was analyzed the expression of ST8SiaI-V; ST6GalNAc I-IV, ST3Gal II -V genes quantitatively by Real Time-PCR in the hybridomas producer of these two types of IgG1 Abs. Results: We observed that the expression of ST3Gal I, III and V coding genes was similar in both hybridomas, while the ST3Gal II and IV genes were less expressed in the hybridoma producer of non-anaphylactic IgG1. In addiction, the expression levels of ST8Sia and ST6GalNAc genes in the hybridoma producer of anaphylactic IgG1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic IgG1. Conclusion: These data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the IgG1 Abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. Financial support: CNPq, CAPES, FAPESP. Basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. Their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of Tolllike receptors (TLRs), which have been described in basophils, has a similar effect. We found that, in contrast to other TLR agonists tested, only the doublestranded RNA poly(A:U) induced the typical T H 2 cytokine and histamine production in vitro. This compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. We took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. Using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. This is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils.

ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. To investigate the effect of activation of NKT cells at different allergic asthma progression, we administered BALB/c mice with a-galactosylceramide (a-GalCer), a stimulator for NKT cell activation, before or after OVA immunization and measured the airway inflammation of that mice after 2 times of intranasal OVA challenge. In our results, the total numbers of bronchoalveolar lavage (BAL) cells were higher in mice administered with a-GalCer before OVA immunization compared to that of mice administered with a-GalCer after OVA immunization. Moreover, significant increased percentage and cell numbers of eosinophils in BAL of mice administered with a-GalCer before OVA immunization was noted. IL-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. IL-5 and eotaxin levels in the BAL fluid were higher in mice administered with a-GalCer before OVA immunization compared to that of mice administered with a-GalCer after OVA immunization. These data demonstrate that activation of NKT cells at different allergic asthma progression dictates the different outcome of asthma. In addition, the activation of NKT cells in naïve mice induces airway inflammatory responses. The potential risks of treatment with NKT cell activation on human diseases should be considered. Objective: Bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of IgE and inflammation. Histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. The transcription factor IRF1 mediates the differentiation into Th1 cells by activating multiple genes which are independently crucial for the development of naive T cells into Th1 cells. Because IRF1 is expressed in many different tissues, it can be considered as a master switch factor for Th1 cell differentiation.

Methods: Here, we tested mice deficient in IRF1 in the murine acute asthma model to evaluate its importance in this Th2 cell-mediated disease. The protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype CD8+ T cells prior to initial sensitization.

In our experiments, we could demonstrate that only after priming of IRF1 deficient mice with the help of adoptively transferred CD8+ T cells, asthma symptoms in these mice were more severe than in wildtype controls. As an example, eosinophil infiltration into the lung was increased by 3.5 fold. Likewise, ovaspecific antibodies and numbers of goblet cells (Fig. 1) were also significantly higher in IRF1 deficient mice.

Conclusion: Interferon regulatory factor 1 plays a role in the severity of the development of asthma. In its absence, proinflammatory parameters in the lung are increased significantly. This effect is only visible in the presence of wildtype CD8+ T cells. Mechanistically, a potential counterregulation of asthma by Th1 cells is not available in IRF1 knockout mice. Together with our previous report that IRF1 represents a susceptibility gene for allergy in the human, our data highlight IRF1 as key in regulating the severity of asthma. The sensory neuropeptide Substance P (SP) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (APC) activity. Here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. To address this question, atopic dermatitis-like allergic dermatitis (AD) was induced in C57BL/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. Animals were additionally exposed to noise stress for 24hrs prior to challenge. In this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. At the same time, an increased number of APC could be observed in these areas and contacts between nerve fibers and APC were found. Under the same conditions, we were able to show increased NK1-receptor and PPT1 mRNA levels. Accordingly, SP had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of CD11c expressing APC substets characterized by CD4 and CD8 expression in vitro. In vivo we found a stress dependent shift of cytokine mRNA levels towards a TH-1 cytokine profile and increased levels of IL-2 mRNA. Further the number of CD25 + T-regulatory cells was increased in vitro. Additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. Proton pump inhibitors (PPIs) that are the cornerstone of Gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (EE) in patients with associated symptoms. The most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in EE. Under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. Nonetheless, a number of evidences suggested the possibility that PPIs could have a direct effect in molecular pathways involved in asthma and EE: 1) the inhibitory mechanism of PPIs implies alkylation of cysteine residues in gastric ATPase3, 2) asthma and EE are prototypic Th2 diseases in which the cytokines IL-4 and IL-13 play a principal role through the activation of the transcription factor STAT6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate STAT6 by mechanisms involving cysteine alkylation.

On the theoretical basis that cysteine reactivity of PPIs may affect the regulation of STAT6, we analyzed its effect in the activation of STAT6 by IL-4 and IL-13. We found that treatment of cells with PPIs inhibited the ability of IL-4 and IL-13 to signal STAT6 activation in a dose-dependent manner in multiple cell types from different origin. Given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. In Argentina more than three million people suffer from asthma, and the number is rising. Asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. A Th2 mediated immune response prevails in these patients. In the asthmatic exacerbation period, Crisis (Cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. Our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (I).

We studied 12 asthmatic patients both at Cr and I. We evaluated Eosinophils, Basophils and IgE expressing B lymphocytes; as well as T regulatory cells (by expression of CD4 and CD25 high (Treg)), that might inhibit the development of a Th2 response, together with gdT cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. To evaluate the T cell response, mononuclear cells were cultured for 48 hours in the presence of M. tuberculosis (M) or S. pneumoniae (Spn), or absence of them (C). Then the percentage of activated cells was determined (Expression of CD69 or CD25 at 48 hours). All the parameters were evaluated in peripheral blood by Flow Cytometry. Discussion: Even though the pathophysiological characteristics of asthmatic patients in periods of Cr and I are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at Cr and I. We might be able to detect differences if we studied cells from the lungs, the target organ. We demonstrate that murine and human LC expressed the H4R. The level of intracellular CCL2 production in human LC was reduced after stimulation with H4R agonists and basal production could be restored when H4R was blocked with the specific antagonist JNJ7777120. Moreover histamine and a H4R specific agonist augmented the migration of LC from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice.

In conclusion, the H4R is expressed on murine and human LC and influences the immunomodulatory function and migration of these cells. These findings underline the relevance of the H4R in allergic skin diseases and encourage further exploration of the H4R as a therapeutic target in allergic skin diseases. Expression data of the non-coding RNA gene, PRINS (Psoriasis Susceptibility-Related RNA Gene Induced by Stress) identified and characterized by our workgroup, suggests a role for PRINS in psoriasis susceptibility and in cellular stress response. In order to asses the function of PRINS, we aimed to identify genes regulated by PRINS and intracellular molecules interacting with this stress-induced non-coding RNA. To identify PRINS regulated genes, we carried out a cDNA microarray chip experiment on HeLa cells where the expression of PRINS was silenced. This experiment identified G1P3, an interferon-inducible anti-apoptotic gene that was down-regulated by PRINS silencing. G1P3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. To detect PRINS interacting proteins we applied ribonucleoprotein (RNP) purification in HaCaT cells. With the help of Matrix-Assisted Laser-Desorption Ionization Time-of-Flight (MALDI-TOF) method we identified nucleophosmin, a protein that physically interacts with PRINS RNA. Nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. Immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. Our data indicate that the non-coding PRINS RNA forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. We suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic G1P3 which can contribute to the pathogenesis of psoriasis. Atopic Dermatitis (AD) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. Rituximab is a monoclonal anti-CD20 antibody therapy that targets pre-B cells and mature B cells, but not plasma cells. AD is generally considered as a biphasic, with switch to initial Th2 to chronic Th1-predominant disease, in which rituximab may have multiple effects. Objectives: To report three patients with severe AD refractory to conventional treatments and to anti-IgE Monoclonal treatment (Omalizumab). Materials and methods: Three patients with severe refractory AD with high levels of serum IgE that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. Subsets of lymphocytes were analyzed with multiparametric-flow cytometry (FACScalibur, BD) at baseline and at specific intervals after treatment. Serum immunoglobulins levels were quantified by nephelometry. Results: At baseline, all patients had highly elevated levels of total IgE ( G 5,000; G 6,000; G 19,000 mg/dl, respectively). All patients underwent prior treatment with omalizumab for 6 months, with only partial response. Then, we started rituximab therapy, resulting in a clear and complete improvement of AD eczema area and severity of skin lesions in all patients. Remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. Whereas allergen-specific IgE levels were not altered, we observed a large reduction in total serum IgE concentrations after initiation of therapy with rituximab. In the first treated patient (follow-up 1 year), IgE levels decreased from 5,512 to 1,500 mg/dl. The other two patients are in the 3 and 1-months of the follow-up period. Importantly, during follow-up no other therapies were required for AD control. Conclusions: Treatment with an anti-CD20 antibody led to a dramatical improvement in our series of patients with severe refractory AD. This study support further evidence on the efficacy and safety of rituximab in severe AD. We have previously demonstrated that chronic topical exposure of mice to the contact allergen DNCB or to the respiratory sensitiser trimellitic anhydride (TMA) preferentially activates T helper (h) 1 and Th2 cells, respectively. In addition, a single application results in divergent cutaneous cytokine production and the migration of Langerhans' cells (LC) with different tempos. To explore events occurring after allergen application, BALB/c strain mice were exposed to a single topical dose of either 1 %DNCB, 25 %TMA or to vehicle alone for 0.5-6h. Measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. Exposure to DNCB provoked rapid production of IL-2 (mean=131pg/ml, n=12, p X 0.001), IL-17 (mean=69pg/ml, n=12, p X 0.001) and IL-1a (683pg/ml, n=12, p X 0.001) in skin compared with TMA-or AOO-treated mice. In subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant IL-2 or of the known regulator of LC migration; IL-1b. Interleukin-1b induced a significant loss of epidermal LC numbers, measured as a function of reduced frequency of MHC class II positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p X 0.001). In contrast, IL-2 or control injections were without effect. However, IL-2 administration caused an increase in cutaneous IL-17 production (347pg/ml, n=12, p X 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). In addition, systemic treatment with anti-IL-2 antibody failed to impact on LC migration provoked by DNCB (33 % reduction; n=6, p X 0.001). In parallel experiments DNCB-induced LC migration was blocked by treatment with anti-tumour necrosis factor (TNF)-a antibody, another cytokine known to regulate LC migration. However, DNCB-induced cutaneous IL-1a (987pg/ml, n=10, p X 0.001) and IL-17 (248pg/ml, n=6, p X 0.01) expression was reduced to baseline levels by anti-IL-2 treatment. These data demonstrate that IL-2 is not involved in the regulation of LC migration, unlike IL-1b and TNF-a. However, IL-2 is involved in the regulation of the production of other cutaneous cytokines provoked by DNCB. Therefore it is hypothesised that IL-2 may influence LC and dermal DC maturation, via the expression of IL-1a and IL-17. Allergic contact dermatitis (ACD) caused by Nickel ions (Ni) represents the most common form of human contact hypersensitivity. Along with other allergies its incidence is increasing in the US and worldwide (NHANES III Survey 2005), but the majority of molecular events underlying this kind of T-cell mediated disease are still widely unknown.

To elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. By applying DIGE technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen Ni.

In the attempt to further characterize the role of a certain down regulated p38-MAPK-pathway related protein (p38PRP) in ACD, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. Our results strongly support an involvement of p38 MAPK pathway in allergenspecific signaling responses. It is expected that identification of differentially allergen-regulated proteins and detailed analysis of ACD-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (Work supported by EU-Project Novel Testing Strategies for In-Vitro-Assessment of Allergens, LSHB-CT-2005 -018681, www.sens-it-iv.eu.) Objectives: Schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. In the long term, 15 % of patients develop a lymphoproliferative disorder. Schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. The recent finding that treatment with IL-1 receptor antagonist (IL-1Ra; Kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine IL-1b and of IL-1-like cytokines in the pathogenesis of the disease. Methods: Two patients with recently diagnosed Schnitzler's syndrome were treated with Kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. Blood samples were collected before and after initiation of therapy. Serum cytokine levels were measured by ELISA, and expression of IL-1-related genes by real-time PCR on mRNA from blood CD14+ monocytes. Results: Compared to normal controls, Schniztler's monocytes had similar expression of IL-18, IL-18BP, and caspase-1, both before and after therapy with Kineret. IL-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. At the serum level, neither inflammatory (IL-1b, TNFa, IL-12) nor anti-inflammatory cytokines (IL-10, TGFb) were detected. As expected, IL-1Ra was only detectable after therapy. IL-18 was detectable in Schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pM) and decreased after therapy (13.2 pM). The circulating IL-18 inhibitor IL-18BP was lower than in controls and not affected by therapy. Thus, free IL-18 levels were increased in Schnitzler's patients as compared to controls (17.6 pM vs. 7.2 pM in controls) and decreased after therapy (9.9 pM). Conclusions: Schnitzler's syndrome is not associated to enhanced expression of IL-1-related cytokines (IL-1b, IL-18), nor of the IL-1/IL-18-converting enzyme caspase-1 in blood monocytes. However, the high circulating levels of IL-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. Experiments are in progress to test this possibility. 

Atopic dermatitis (AD) is a chronic relapsing allergic skin disease with a high and growing prevalence. Currently around 20 % of the children in industrialized countries are affected. In most cases patients exhibit increased systemic IgE-levels (so-called extrinsic form) accompanied by sensitization to allergens. While AD is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. Most AD-patients show topical colonization with Staphylococcus aureus indicating a defective innate immune response. As a class of pattern recognition receptors, Toll-like Receptors (TLRs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. All known TLRs except TLR3 signal via MyD88 to induce NFxB-dependent gene transcription. TLRs are also known to be involved in the pathogenesis of autoimmune diseases. As chronic AD also has an autoimmune component, the study of MyD88 signaling in AD might provide new insights into the function of TLRs.

To investigate the role of TLRs in the immunopathology of allergic reactions and skin infections, we induced AD-like symptoms in C57BL/6 MyD88-deficient mice by repeatedly sensitizing the mice to Ovalbumin (Ova) after mechanical disruption of the skin barrier by tape stripping.

First results show that MyD88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. Immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. Skin-draining lymph nodes are less enlarged in the Ova-treated knockout mice compared to wildtype and differ in cellular composition. Serum antibody levels determined by ELISA show reduced systemic total and Ova-specific IgG1-titers in Ova-treated MyD88-/-mice compared to wildtype mice, although the NaCl-treated MyD88-/-control group has higher total antibody titers than the wildtype NaCl control group. Total IgE levels are increased in the knockout mice compared to wildtype mice under both conditions. To further investigate the role of Staphylococcus aureus during AD development, we will include topical application of the superantigen Staphylococcal Enterotoxin B (SEB) or living bacteria into our analyses. Following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. Introdution: The skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. Among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. Little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. Bovines present variable and heritable levels of resistance to the tick Rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. In order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, Nelore (Bos taurus indicus) and Holstein (B. t. taurus). Methods: Skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype N = 4) or from non-infested contra-lateral sites. Expression of MIP-1a, IGF-1, MCP-1 and IP-10 genes was quantified with realtime RT-PCR. Sections of paraffin-embedded skin were stained with May Grünwald-Giemsa for differential cell counts. Lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. Results: As expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. However the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. Inflammation of Nelores contained significantly more basophils, eosinophils and mononuclear cells expressing CD3, CD4, CD8, CD21, MHC class II, and p46 than that of Holsteins. Lymphocytes expressing WC1 and CD21 antigens were significantly diminished in infested skin of Holsteins when compared with control skin (P X 0.05). Infested skin of Nelores contained significantly more message for MIP-1a, IGF-1, MCP-1 and IP-10 than that of Holsteins. Conclusions: Although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. Supported by CNPq, CAPES, FAPESP, and ICTTD. In the last decade it has become clear that keratinocytes play an important role in the skin immune system. Upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. In a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. It has been proposed (Goodman et al.) that keratinocytes rather support a Th2 response by interacting lymphocytes. We examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with CD3+ T cells from the same donor. During the coculture either SEB or antigen were added. In all experimental approaches the addition of keratinocytes resulted in higher production of IFNg by T cells. Furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. Again, the induction of IFNg by the presence of keratinocytes resulted in a marked and significant increase of IFNg production by T cells.

We were able to show that IL-1 plays a crucial role in the induction of IFNg in T cells keratinocyte interaction. In addition blocking of LFA-1 in the co-cultures resulted in significantly reduced IFNg production by T-cells underlining that ICAM-1/LFA-1 binding is also crucially important for IFNg induction. We conclude from our study that keratinocytes rather support a Th1 than a Th2 local response pattern by virtue of IL-1 secretion and ICAM-1/LFA-1 interaction. This property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a Th2 like micromilieu in acute towards a Th1 dominated milieu in chronic lesions.

The genotyping of CCL4L gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. Therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. This work was supported by project grants of Merck-Serono and Instituto de Salud Carlos III (PI07/0329). Psoriasis is an inflammatory dermatosis with 2 % prevalence among Caucasians. HLA-Cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to TNF loci with several SNP in promoter region. The most common polymorphisms are two G to A transitions in -308 and -238 positions. Strong association was found between polymorphisms in the -238 region with psoriasis. In several diseases, the association with HLA and clinical manifestation is different between genders, for example in spondyloarthropathy and HLA-B27, and this is a question of increasing interest. The objective of this study was to identify clinical and molecular differences between male and female in Brazilian psoriatic patients. Sixty-nine individuals assisted at the Dermatology Outpatient Clinic of the Teaching Hospital, University of Campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. HLA-A -B -C -DR -DQ alleles and TNF-238 and -308 SNP were differentiated by PCR/SSP. Analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. In the male group, the mean age at disease onset was 16,3 years. Severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). Seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. The main molecular risk factor for the disease, Cw*06 allele was positive in 10 cases (47,62 %), TNF 238 G/A genotype was found in 5 (23,8 %) and TNF 308 G/A in 4 (19,1 %). In the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. Twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. Cw*06 allele was positive in 26 cases (54,2 %), TNF 238 G/A genotype was presented in 16 (33,3%) and TNF 308 G/A in 8 (16,6 %). Severe disease was seen in male patients. There was no difference in frequency of Cw*06 allele between male and female groups, but there was a tendency of significant difference in TNF 238 G/A genotype. We found that C57Bl/6 mice were more susceptible than Balb/c and DBA/2 mice. Higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. Analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as IL-6 and MRP8/14, among different inbred strains of mice. Furthermore, we identified the cells which are involved in this cytokine production. As expected, histopathological analysis showed that S. aureus infection induces an influx of monocytes and granulocytes. Our study shows that not only bacteria-but also host-specific differences are associated with different courses of S. aureus skin infection. Aims: To investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. Methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. The statistical analysis of the data was held with the SPSS program.

The commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). The symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. Total IgE G 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. On the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. Finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered.

Conclusions: It has, therefore, been shown that 1)The anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used.

2) In particular, in many cases it is proved that there is a personal but also a family history of atopy.

3) Increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. Allergic contact dermatitis (ACD) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. As classical type IV delayed hypersensitivity reaction this response is mediated by T-cells. Since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. Up to now the identification of potential allergens completely relies on animal testing, like Buehler assay or guinea pig maximization test (GPMT). Due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., Cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. Since dendritic cells (DCs) play a pivotal role in the initiation of contact dermatitis we chose DCs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between DCs primed by allergens and T-cells in the test tube. The known allergens nickel, dinitrochlorobenzene (DNCB) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on DCs derived from monocytes that display a Langerhans cell (LC) type-similar phenotype. We used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. In addition to the upregulation of CD86 we could observe dose dependent upregulation of programmed death ligand 1 (PDL-1) and downregulation of the dendritic cell immunoreceptor (DCIR). Furthermore we observed enhanced T-cell proliferation in mixed leukocyte reactions (MLRs) applying LCs stimulated with allergens ex ante.

Since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour FACS and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. Our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. We want to present the case of a 60 year old Iraqui patient with Arabic ancestors who had been suffering from psoriatic arthritis since 35 years. In March 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. This led to the complete healing of the skin lesions. For this reason the dose could be reduced to one tablet Fumaric acid esters (120 mg) every second day. In April 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the Auricle. The histological examination showed an HHV-8 positive Kaposi sarcoma. The differential blood cell count demonstrated a lymphocytopenia. The HIV-serology was negative. The staging examinations (chest X-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. After the diagnosis the treatment with fumaric acid esters was discontinued. Over the course the livid papules showed a spontaneous complete regression. A spontaneous regression is known from the iatrogenic KS caused by immunosuppressive therapy when the immunosuppression is terminated. As our patient also showed a spontaneous regression of the Kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. Sarcoidosis is a multisystemic granulomatous disease with unknown etiology. Although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of Th1 type immune response. Recently, suppressor of cytokine signaling (SOCS) proteins have been identified as regulators of cytokine signaling pathways. In this study we aimed to evaluate the roles of SOCS1, SOCS3 and FoxP3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. Peripheral blood (PB) and broncholaveolar lavage (BAL) mononuclear (M) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage I/II sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for SOCS1, SOCS3 ve FOXP3 mRNA expressions by TaqMan PCR, and also flow cytometric analysis was performed for lymphocyte markers including CD3, CD4, CD25, FoxP3, CD4 + CD25 high , CD4 + Foxp3 + . Expression of SOCS3 and FoxP-3 mRNA in PBMCs and BALMCs from responders were found to be significantly higher in comparison to other two groups . SOCS1 was found significantly elevated in PBMCs of responders when compared with other two groups. It was also elevated in BALMCs of responders when compared with with those of untreated cases. The proportions of CD25, FoxP3, CD4+CD25 high , CD4 + Foxp3 + cells in PBMCs and BALMCs of responders were found to be increased in comparison to nonresponders and untreated cases. Our data demonstrates that SOCS1, SOCS3 and T regulatory cells may have potential roles in the control of sarcoidosis. We think that if the roles of SOCS1 and SOCS3 molecules and T regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. The inorganic dust, containing free crystalline silicon dioxide (FCS) is critical for the development of silicosis. Several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the FCS in affected individuals. The role of FCS in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.We studied the changes of neopterin, as a marker for IFN-g dependent macrophage activation and circulating immune complexes (CIC), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing FCS.We survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing FCS (Exposed), and 43 healthy workers without exposure to dust aerosol (Controls).The serum quantity of neopterin and CIC, containing IgA(IgACIC), IgG(IgGCIC) and IgM(IgMCIC) was detected by ELISA. Differences between investigated groups were detected by Student's t-test and a p-value less than 0.05 was considered significant.Neopterin level was significantly elevated in Exposed (3,2±0,8ng/ml) compared to Controls (1,8±1,3ng/ml; p=0,0001). Moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).The levels of IgGCIC was significantly elevated in the Exposed compared to controls (81,9±22,7AU vs 64,3±16,7AU p=0,0001) and to silicosis patients (69,6±20,0AU p=0,002). In contrast, IgMCIC was significantly elevated in silicosis than in Exposed (70,2±18,8AU vs 62,1±24,2AU; p=0,03).In comparison with Exposed, significantly higher IgMCIC was found only in mild, but no in moderate and severe silicosis. In contrast, the level of IgGCIC in mild and moderate silicosis was significantly lower compared to the Exposed (p=0,001 and 0,02 respectively).The obtained results showed that activation of alveolar macrophages mainly depends on the presence of FCS in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. The dynamics of CIC suggest the involvement of Fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing FCS. Described pathologic similarities between Sarcoidosis (SA) and Tuberculosis (TBC) suggest M. Tuberculosis antigens as caustaive agentes. It seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. So different HLA haplotypes have been described as a predisposing factor to develop Sarcoidosis (HLA A*01, B*08, DRB1*03 (these of good prognosis) *12/*13/*14/*15) or TBC (DRB1*02/*05/*14/*16 and associations with DQA1*02/*03/*05) We describe two cases of two female patients from the same geographic region with Mantoux and Zhiel-Neelsen negative tests and high levels of TNF diagnosed of TBC and SA respectively. Both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the TC study (pulmonary nodules and mesenteric adenopaties). We found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. After being treated with anti-TBC drugs, the first one presented a good clinical improvement, so she was diagnosed as TBC. The second one did not improved, so she was treated with corticosteroid, with good results. Therefore, she was diagnosed as Sarcoidosis.

After HLA analysis, we noticed that the TBC patient was HLA A*01, B*08 and DRB1*03 (Sarcoidosis good prognosis haplotype) and the patient diagnosed as Sarcoidosis was HLA A*01, DRB1*14.

As the results show, could there not be a direct relationship between the HLA system and the development of SA or TBC, or in contrast, was the first patient missdiagnosed of TBC being a good prognosis SA? Objectives: Experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. For example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. Experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. Ovalbumin (OVA), House-Dust-Mite (HDM) and Cockroach (CRA) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. The objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. Methods: For the induction of asthma, mice were initially sensitised by intraperitoneal injection of OVA, HDM, CRA or a combination of all three, followed by repeated challenge by intratracheal application of OVA, HDM or CRA. Inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (BAL) and by determination of chemokine and cytokine levels in BAL and lung tissue using ELISA and multiplex technology. Additionally, serum levels of IgE and IgG antibodies were measured. Airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. Results: Significant differences were measured in cell influx, chemokine/cytokine and total IgE levels. Compared to HDM and CRA, OVA induced an higher cell influx in the BAL, HDM showed an increase of chemokines in BAL and increased IgE levels in serum. Using a combination of all three proteins resulted in the most severe form of asthma. Conclusions: To our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. The most sustained and severe form of asthma was induced by the combination model. This model offers particular advantages for evaluating existing and novel therapeutic agents. Furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. The present study focused on peri-SMI connective tissue capsule formation, the most frequent post-operative local complication in patients receiving SMI. We investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. To this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (PBMCs) from the same patients were isolated and analyzed via FACS, concentrating on T-effector cells (Teff) and T-regulatory cells (Tregs: CD4 + , CD25 ++ , Foxp3 + ), cytokine profiles, T-cell receptor (TCR) repertoire and reactivity against human heat shock protein 60 (hHSP60). Intracapsular Tregs were visualized by immunohistochemistry and functionally tested in suppression assays. The cellular composition of intracapsular mononuclear cells showed a preponderance of CD4 + T-helper cells and a significant subset of TCRg/d + cells, exceeding that observed in peripheral blood. IL-17, IL-6, IL-8, TGF-b and IFN-g production prevailed, pointing to a TH17/TH1 weighted immune response. Furthermore, intracapsular T-cells displayed a restricted TCR a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hHSP60. Importantly, numbers of intracapsular Tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral Tregs. Our results suggest that silicone triggers a specific local immune response via activated TH17/TH1 cells, promoting fibrosis due to the production of profibrotic cytokines. Clonal restriction of the TCR repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. In this context, HSP60 might be a prominent candidate. Taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of SMI. The inverse correlation between the degree of capsular fibrosis and the number of intracapsular Tregs suggest that Tregs may initially be able to inhibit the progress of capsular fibrosis. However, as numbers of Tregs, as well as their suppressive capacity decreases over time, fibrosis develops. Supported by the Competence Center Medicine Tyrol (KMT) and the Lore-and-Udo-Saldow Donation. Objectives: Recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. The present project deals with the standardization and further development of a modified ELISA test system (SiLISA ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (SMIs) based on the protein signature adhering on the surfaces of such devices (1) .

The current SiLISA ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. The optimization of the SiLISA ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. The essay was further developed with antibodies against annotated proteins that were not yet tested in the past. All experiments were carried out in a 384 well plate format for high-throughput analysis. Statistical analysis has been performed using SPSS.

The extended essay has been successfully established in the system with antibodies against seven already tested proteins, including C-reactive protein, collagen-I, collagen-III, fibronectin, IgG, C3-complement, Myeloid Related Protein 14 and two new proteins, integrin-ß4 and fibrinogen. Data from more than 100 patients have been obtained and exploited so far. The intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. Patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. Conclusion: Applying the SiLISA ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. The distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. Muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. Necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. This work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. It was included at least 3 mice per group with distinct cytokine pattern: Th1 (C57BL/10, C57BL/6) and Th2 (BALB/c). Muscular injury was performed by injection of bupivacaine. Both Th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. Regional lymph nodes showed significant increase of cellularity and relative numbers of CD3 bupivacaine-inoculated BALB/c mice compared to non-inoculated matched mice at 4 dpi. BALB/c mice showed increased collagen expression and decrease of MMP-9 activity associated with more mRNA for TGF-b1. This study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (Th1 cytokines) or myonecrosis and collagen deposition (Th2 cytokines).

The severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (DCM) is imputed to the stimulatory autoantibodies against the second extracellular loop (EC II ) of the ß1-adrenergic receptor (anti-ß1EC II ). To analyze their pathogenic impact as a single causal factor we used a human-analogous Lewis rat model of DCM, where monthly subcutaneous immunization of the rats with the ß1EC II peptide as a Glutathione-S-transferase (GST) fusion protein induced production of anti-ß1EC II and eventually dilated cardiomyopathy. In this model we isolated a ß1EC II -specific rat monoclonal antibody (clone 13F6), and showed by ELISA that it binds to the linearized ß1EC II peptide. Additionally, we confirmed with flow cytometry that 13F6 also binds the ß1EC II in its native conformation, i. e. directly labeled circular ß1EC II (DyL649-ß1EC II ) peptide. Moreover, we demonstrated activation of the ß1-adrenoreceptor by 13F6 using a fluorescence resonance energy transfer (FRET) assay system in vitro. These data further corroborate the pathogenic role of anti-ß1EC II antibodies in mediating DCM in this animal model, thus rendering them a potential therapeutic target. Therefore, we investigated a novel anti-ß1EC II -specific peptide-based therapy, by intravenously applying a circular ß1EC II peptide in the DCM Lewis rat model to neutralize the anti-ß1EC II antibodies. While the peptide therapy strongly reduced the anti-ß1EC II titers in the serum by up to 80 % and consecutively lead to clinical remission, ELISPOT assays for the detection of ß1EC II -specific antibody-secreting cells (ASC) indicated no difference in the number of long-lived plasma cells in treated animals. In contrast, ELISPOT and flow cytometrical analyses revealed a decrease in the number of ß1EC II -specific memory B cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. Our newly developed anti-ß1EC II -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory B cells involved in the generation of these autoantibodies. These results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. Cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (DCM), are a frequent cause of mortality in humans with DCM being the most common reason for heart failure in young adults. Infections with Coxsackievirus B3 or Cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). Immunization with the well-characterized myhca 614-629 epitope elicits autoreactive CD4 + T cell responses that have been shown to be the major mediators of autoimmune myocarditis in BALB/c mice. It is known that professional antigen presenting cells (APCs) such as dendritic cells are crucial for initiating and maintaining T helper (Th) cell responses affecting the heart muscle. However, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. We therefore generated a TCR transgenic mouse harboring T cells that specifically recognize the myhca 614-629 peptide. In a first step, hybridoma cells were generated by fusing BW5147 TCRa -CD8lymphoma cells with myhca 614-629 -specific Th cells. TCR expression and antigen specificity was assessed by FACS analysis and ELISPOT assay. Following subcloning, the variable regions of the expressed TCR were characterized by PCR-sequencing. The rearranged V(D)J regions were subcloned into TCR cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. Using this novel TCR tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific T cells during the process of autoimmune myocarditis. Furthermore, this new tool will help to generate a high resolution analysis of the contribution of different APCs in the activation and differentiation of autoreactive Th cells during inflammatory heart disease.

M. Relle 1 , A. Schwarting 1 , P.R. Galle 1 1 University Medical Center of the Johannes Gutenberg University Mainz, Medical Clinic I, Mainz, Germany Several mouse or rat models have been established to explore the role of proteinase 3 (PR3), in ANCA-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that ANCA alone are not sufficient to induce these diseases directly. Therefore, we assessed the expression, mobilization and enzymatic activity of PR3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of PR3-positive neutrophils in the peripheral blood of frequently used mouse strains. Neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human IL-8. PR3-mRNA from the murine cancer cell line WEHI-274 was amplified by RACE-PCR and subsequently sequenced. Sequence comparisons were done with DNAsis software package and the blast tool of the NCBI. Promoter analyses were performed with the Genomatix software MatInspector. We could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of Furthermore, we identified an alternative PR3-promoter in the second intron of the mouse PR3 gene. This promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. Fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. Taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. The identification of an alternative PR3 transcript and its promoter indicates that the murine PR3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. Objective: Recent studies show that in vivo administration of OCH, a synthetic lipid that specifically activates natural killer T (NKT) cells, results in suppression of Th1 mediated immune responses in autoimmune diseases. NKT cell activation depends on lipid presentation via the MHC-I like molecule CD1d on antigen presenting cells such as mature dendritic cells (mDCs) and upon activation by OCH NKT cells rapidly produce large amounts of Th2 cytokines. The goal of this study was to investigate the effect of OCH and OCH-primed dendritic cells on atherogenesis.

Methods & results: LDL receptor deficient (LDLr -/-) mice were fed a Western type diet and atherosclerosis was induced via collar placement around both carotid arteries. Subsequently the mice were treated i. p. with OCH (N=13) or PBS (N=11) twice a week for seven weeks. The injections with OCH did not affect atherosclerotic lesion size. To improve the presentation of OCH to NKT cells in vivo, bone marrow-dendritic cells were maturated via TLR4 activation, in the presence/ absence of OCH. Subsequently we transferred 1.5x10 6 mDCs (N=11) or OCH-primed mDCs (N=11) (3 times) to LDLr -/mice. Afterwards the mice were put on a Western type diet to induce atherosclerosis. Vaccination with OCH-primed DCs resulted in a 70.6 % reduction in plaque size compared to mice treated with mDCs (p X 0.05). During the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p X 0.05) reduction in cholesterol levels in the mice treated with OCH-primed DCs. The number of NKT cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with OCH-primed DCs (p X 0.05). Additionally, the NKT cells in the liver of mice treated with OCH-primed DCs produced more IL-10. Discussion: We conclude that immunotherapy using OCH-primed dendritic cells efficiently activates NKT cells, resulting in a Th2 phenotype of the NKT cells and this leads to an efficient protection against atherosclerosis. These data indicate that immunotherapy based on ligand specific primed DCs may be a novel way to treat atherosclerosis. Systemic Lupus Erythematosus (SLE) is characterized by high serum titers of IgG anti-nuclear antibodies secreted by plasma cells. However, the characteristics of the IgG+ plasma cell antibody repertoire in SLE has never been determined on a single cell level and little is known about the role of germinal center (GC) reactions for the development of SLE autoantibodies. The IgG inhibitory FcgRIIB knock-out mouse on the C57BL/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsDNA and chromatin. To characterize the IgG+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 IgG antibodies from single isolated GC B cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type C57BL/6 and FcgRIIB deficient mice. IgH and IgL chain gene sequence analyses revealed no major differences in the Ig gene usage between wild-type and autoimmune mice, but GC B cells of FcgRIIB were enriched for antibodies with positively charged IgH CDR3 regions and anti-nuclear specificity. The overall frequency of autoantibodies was similiar between wild-type and FcgRIIB deficient mice. However, strongly autoreactive antibodies to dsDNA and murine IgG2c were isolated only from FcgRIIB deficient mice, but not from C57BL/6 control mice and somatic mutations contributed to their generation. In summary, our data suggest that the GC reaction plays an important role for the development of self-reactive antibodies in FcgRIIB deficient mice. The finding that the frequency of autoreactive antibodies is higher in GC B cells than in spleen or bone marrow plasma cells may indicate that autoreactive GC B cells are partly regulated even in the absence of FcgRIIB. Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes (NCs) represent a hallmark of systemic lupus erythematosus (SLE). However, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. High mobility group box 1 protein (HMGB1), a nuclear DNAbinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. It is not released, since apoptotic cells are immediately engulfed by phagocytes. Conversely, in conditions of clearance deficiency, which is observed in a subset of patients with SLE, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing NCs containing the "endogenous adjuvant" HMGB1. We investigated if HMGB1-containing NCs contribute to the breakdown of immunological tolerance against dsDNA and NCs. We found that HMGB1 remains associated with NCs released from late apoptotic cells in vitro. HMGB1-NCs complexes were detected also in the blood of patients with SLE. HMGB1 containing NCs from apoptotic cells induced secretion of IL-b, IL-6, IL-10, and TNFa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (DC), respectively. Cytokine release from murine macrophages was dependent on MyD88 and Toll-like receptor 2. Neither HMGB1-free NCs from living cells nor from apoptotic HMGB1-or HMGB1/2-deficient cells induced marked cytokine production or DC activation. Specific inhibition of HMGB1 activity by the antagonistic A box domain significantly reduced capacity of "apoptotic "NCs to induce TNFa and IL-10 release by macrophages. Immunizations with HMGB1-containing NCs from apoptotic cells induced anti-dsDNA and anti-histone IgG responses in non-autoimmune mice in TLR2-dependent manner.

In conclusion, HMGB1 in complex with NCs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against NCs/dsDNA and, hence, to the immunopathogenesis of SLE. Objective: Apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (SLE). In agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. Still, little is known about how apoptotic cell-derived self-antigens activate autoreactive B cells and where this takes place. Methods: Injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. Binding studies were performed using apoptotic cells and CHO cells transfected with Class A Scavenger Receptors (SR). Repeated injections of syngeneic apoptotic cells in SR deficient and wild type mice were conducted and antibody production by autoreactive B cells was measured. Autoreactivity against SR was followed in two SLE prone mice strains over the development of disease and in a cohort of SLE patients. An antibody against the SR was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. Results: In this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. Autoantibodies against SR are found before the onset of clinical symptoms in SLE-prone mice, and they are also found in diagnosed SLE patients. Furthermore, Injections of an antibody binding SR enhance the antibody production by B cells when co injected with either apoptotic cells or TNP-Ficoll. Conclusion: Our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. Because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of SLE.

E. Glasmacher 1 , K.P. Hoefig 1 , E. Kremmer 1 , V. Heissmeyer 1 stretches and helps in the selection of the correct splicing borders. A allele of (R61H) creates a strong binding site for a splicing enhancer protein SRp40 according to bioinformatics. Our findings indicate that, the putative branch point, R61H SNP and the T stretch located downstream of exon two, plays a role in the alternative splicing of BANK1. Finally, we believe that BANK1 delta 2 protein work as a dominant negative isoform in B cell activation and antobodies production, and may antagonize the effect of the full-length protein. These properties of the delta 2 protein may contribute to the observed reduction in SLE susceptibility.

S. Beermann 1 , R. Seifert 1 , D. Neumann 1 1 Hannover Medical School, Pharmacology, Hannover, Germany

The biological function of histamine is mediated by four different receptors, namely histamine H 1 receptor (H 1 R), H 2 R, H 3 R, and H 4 R. During an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and T cells. In these cells, histamine may modulate the synthesis of characteristic T cell cytokines such as IFNg, which plays a central role in a number of autoimmune diseases. The present study was initiated to analyze the involvement of histamine on the induced production of IFNg by immune cells. Mouse spleen cells were stimulated in vitro by either immobilized a-CD3 antibodies or CpG-oligonucleotides (CpG-ODN) in the presence or absence of histamine or 4-methylhistamine, a H 4 R-selective agonist. IFNg production was evaluated by analysis of cell culture supernatants by ELISA. Both, histamine and 4-methylhistamine concentration-dependently reduced IFNg production in splenocytes obtained from control C57Bl/6 mice induced by either a-CD3 antibodies or CpG-ODN. This histamine effect was completely inhibited by the H 2 R-specific antagonist famotidine, while H 4 R-, H 1 R-, and H 3 /H 4 R-selective antagonists had no or only moderate effects. Interestingly, the H 4 R-selective reagent JNJ7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-CD3 induced IFNg synthesis, but in contrast it slightly enhanced the histamine effect. Thus, at the murine H 4 R, JNJ7777120 may be a partial agonist. We conclude that histamine modulates the induced production of IFNg by T cells via mainly the H 2 R and, to a much lesser extend, the H 4 R. Using this assay system, cells obtained from control C57Bl/6 mice will be compared to those from SLE-prone MRL lpr/lpr mice and the respective wild type strain MRL +/+ .

I Objectives: Resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. In this study we examined their effects on cytokine release in healthy subjects and autoimmune patients.

To test the in vitro effects of 20 ng/ml resolvin E1(RvE1) on the release of TGFb, IL-6 and IL-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (PMA) (1nM), and the combination of PMA and ionomycine (3 mg/ml) for 72 hours. Methods: Mononuclear cells were prepared by Ficoll-Uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with SLE and Sjögren's syndrome (SS). Level of cytokines was measured by ELISA method. Results: In the patients with SLE (p = 0.010) and Sjögren's (p=0.017) mononuclear cell stimulation by PMA resulted in a reduced release of TGF b compared with controls. RvE1 significantly reduced TGFb release from control mononuclear cells stimulated by either PMA (p=0.041) or PMA+ionomycin (p=0.021), however RvE1 was ineffective at reducing TGFb release in the SLE and SS patients. RvE1 caused a non-significant decrease in IL-6 release from control mononuclear cells, but was again ineffective in SLE and SS patients. The production of IL-17 was not significantly modified by RvE1 in any of the groups tested.

The release of TGFb by 20 ng/ml of RvE1 can be significantly reduced in healthy control subjects but not in subjects with SLE or SS. At the single dose of RvE1 tested, IL-6 and IL-17 release were not significantly affected in healthy or autoimmune patients. Omega-3 fatty acid derived RvE1 may affect inflammation in healthy patients by reducing TGFb production but its effects on inflammation in SLE and SS patients may be expected to be smaller or non-existent. In addition, the TGFb release in the PMA activated mononuclear cells of SLE and Sjögren's patients is less than that of healthy subjects.

G. E. Fragoulis 1 , A.K. Tsirogianni 1 , M. Herrmann 2 , H. M. Moutsopoulos 1 , M.N. Manoussakis 1 1 University of Athens, Dpt Pathophysiology, Athens, Greece, 2 University of Erlangen-Numberg, Institute for Clinical Immunology, Erlangen-Numberg, Germany

Objectives: Altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (SLE) that is thought to lead to impaired clearance of apoptotic remnants. Herein, we assessed comparatively the phagocytic capacity in the peripheral blood of SS and SLE patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of SS patients. Methods: Patients studied included 29 with primary SS (American-European criteria 2002) and 14 with SLE (ACR criteria 1997). Age-and sex-matched healthy blood donors to the SS and SLE groups (13 donors each) were also studied in all assays. The phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (Gaipl et al, J Autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (MB-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal PBMC (SNEC-phagocytosis). Salivary gland biopsies of patients with SS with and without MALT lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (TUNEL assay) and the presence of macrophages (CD68-staining). Results: In agreement to previous studies, MB-phagocytosis was found significantly decreased in granulocytes and monocytes of SLE patients (both for P=0.0001).

In SS patients, defective MB-phagocytosis involved only monocytes (P X 0.0001) and significantly correlated with the presence of extraglandular manifestations (P=0.02). Compared to controls, SNEC-phagocytosis was significantly increased in the granulocytes of SLE (P X 0.0001) and of SS (P=0.001). In the salivary gland biopsies of SS patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. These lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. These phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ B-cell lymphoma. Conclusion: In a manner similar to SLE, SS patients appear to manifest altered phagocytic capacity. This may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. The autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (SLE) patients, but clinical value of such antibodies often is not understood. The purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -Adenozine Deaminase (ADA) in SLE and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. Methods: 30 healthy persons have been included in our study and 71 SLE patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (HNP) in anamnesis. Antibodies of IgG class to ADA (anti-ADA) determined by technique of indirect ELISA developed by us with the use of immobilized form of ADA as an antigenic matrix. b 2glicoprotein-I-dependent antiphospholipids (aPhL) of IgG classes were determined using commercial "Anti-Phospholipid Screen IgG/IgM" test set (Orgentec Diagnostica). Results: At admission an anti-ADA was revealed in 36,6 %, aPhL of IgG class -in 45,1 % SLE patients. It has been noted that IgG-aPhL were found out in anti-ADApositive patients more often and in higher antibody titer, than in anti-ADA-negative SLE patients (X 2 =6,4; p X 0,02). Development of cytopenic syndrome was noted reliable more often in SLE patients with associated presence of IgG-aPhL and an anti-ADA in comparison with patients who has not the combinations of these antibodies in blood (X 2 =3,9; p X 0,05). The increased levels of anti-ADA were revealed in 11 of 18 women with HNP, and the combination of anti-ADA and aPhL (9/18) was found out more often than isolated anti-ADA (2/18, X 2 =6,5; p X 0,02) or isolated aPhL (3/18, X 2 =4,5; p X 0,05). Conclusion: Taking into account the imbalance of immunoregulatory functions in SLE, the further studying of autoantibodies to ADA generation seems to be very promising. Presence of HNP in anamnesis is the evidence of necessity of careful biochemical monitoring of aPhL and anti-ADA in women for the prevention of abortus fetus and administration of adequate therapy. Objectives: Sjögren's syndrome (SS) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). Dendritic cells (DC) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. Two main subsets of DC have so far been identified in human peripheral blood: myeloid DC (mDC), which can be further divided into mDC1 and mDC2, and plasmacytoid DC (pDC), also known as IFN-a/b producing cells. The pivotal role of DC in inducing and maintaining tolerance could be critical in SS as alterations among DC populations might contribute to autoimmunity. Purpose of this study was to quantify mDC1, mDC2 and pDC in peripheral blood from primary SS patients by flow cytometry and compare the results with gender-and age-matched healthy controls.

Methods: Blood samples from 31 pSS patients fulfilling the American European Consensus group criteria (AECC) and 28 gender-and age-matched healthy controls were collected in heparin tubes. DC populations were stained with the Blood DC Enumeration kit, Miltenyi, according to the manufacturer's manual. Cells were analyzed on a FACS Canto II, BD, and data analysis was performed with FlowJo software, Tree Star. For the statistical analysis, a two-tailed Mann-Whitney U test was performed using Prism, GraphPad. Results: pSS patients have significantly reduced amounts of pDC (p=0,0002) and mDC2 (p X 0,0001) in peripheral blood. Conclusion: Alterations in DC populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (SLE) or diabetes. In SS patients, up-regulation of interferon-regulated genes has been shown previously. Therefore, decreased pDC numbers in peripheral blood from pSS patients might explain the fact that an increased IFN signature is found in salivary glands of pSS patients, but no elevated levels of IFN-a are measured in serum. Recently we reported that malignant CD5+ B cells from patients with B chronic lymphocytic leukemia (B-CLL) produce Granzyme B (GrB) and are rapidly undergoing apoptosis in a granzyme B-dependent manner following interleukin 21 (IL-21) stimulation. Several autoimmune diseases have been linked to both elevated frequencies of CD5+ B cells and increased IL-21 levels. We therefore hypothesized that IL-21 may have similar biological effects on CD5+ B cells in autoimmune diseases. Here we demonstrate that the amount of IL-21 in the serum of systemic lupus erythematosus (SLE) patients but not of healthy subjects highly correlated with serum levels of GrB. In contrast to B cells from healthy individuals, where no baseline GrB expression was found, we demonstrate that up to 14 % of CD5+ B cells in SLE individuals expressed GrB. In-vitro experiments revealed that IL-21 was able to induce expression of GrB in B cells from individuals with SLE and other autoimmune diseases including psoriasis and rheumatoid arthritis. This effect was direct and was strongly enhanced by engagement of the B cell receptor or toll-like receptor 9. Importantly, IL-21 significantly decreased the CD5+/CD5-B cell ratio in both SLE peripheral blood and healthy cord blood samples, suggesting a preferential induction of CD5+ B cell death. These results suggest that IL-21-induced GrB may play a regulatory role for CD5+ B cells similar to what we described earlier in B-CLL cells. This is the first report uncovering an interrelation between IL-21 and GrB levels in SLE and showing that IL-21 reduces the CD5+/ CD5-B cell ratio in B cells from SLE peripheral blood and healthy cord blood. Endogenous IL-21 may therefore play a disease-modifying role and may explain elevated GrB serum levels in autoimmune diseases. Further studies should evaluate the therapeutic potential of IL-21 in SLE and other autoimmune diseases.

R. De Palma 1 , E. D'Aiuto 1 , S. Vettori 1 , G. Abbate 1 , G. Valentini 1 1 Second University of Naples, Clinical & Experimental Medicine, Napoli, Italy

SSc is considered an autoimmune puzzling disease whose pathogenesis is unknown. In the last years, there have been increasing evidences that an interplay between activated T cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (SSc). We have previously shown that peripheral T cells from SSc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same T cell clonotypes found in the affected skin.

Here, using the same experimental approach, we found that the T cell clonotypes expanded in co-cultures are ab positive, HLA-DR positive, and promote apoptosis of autologous SSc fibroblasts. We also found that, in these co-cultures, SSc fibroblasts up-regulated Fas and underwent apoptosis that paired with the expression of Fas ligand (Fasl) on CD4+ T cells. Finally, when we added a blocking anti-Fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of Fas/Fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. It has to be reminded that the absence of Fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble Fas in sera of patients affected by SSc. Moreover, in the co-culture supernatants we found TGF-beta, IL-1beta, IL-6 and IL-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. Taken together, these data suggest that T cell response in SSc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. Indeed, fibroblasts of SSc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. However, the overall outcome of the T cell response triggered by fibroblasts in SSc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. These findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in B cell self-tolerance as reflected by elevated serum IgG levels of predominantly antinuclear antibodies (ANAs). Serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. However, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in SLE. To determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in SLE, we cloned and expressed 169 IgG antibodies from single FACS purified CD19+CD27+CD38+CD138+ bone marrow plasma cells of 3 patients with SLE and tested the recombinant monoclonal antibodies for self-reactivity. Our preliminary data on the Ig gene repertoire and reactivity profile of human IgG+ SLE bone marrow plasma cells in comparison to healthy controls will be discussed.

Z. Amirghofran 1 , E. Moazemi Godarzi 1 , E. Kamali Sarvestani 1 , E. Aflaki 1 1 Shiraz University of Medical Sciences, Shiraz, Iran, Islamic Republic of Interleukin 6 (IL-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (SLE). Two polymorphisms in the promoter region of IL-6 gene at positions -572 G/C and -174 G/C have been described that are key regulators of IL-6 gene. In the present study the relationship between these two polymorphic sites and disease susceptibility in a group of Iranian patients with SLE was investigated using polymerase chain reaction-restriction fragment length polymorphism method. The genotype distribution and allele frequencies of IL-6 gene polymorphism at -174 position showed no significant difference between SLE patients and controls. At this position the frequency of GG genotype as well as G allele was higher than C allele in both patients and control groups. In contrary, both allelic and genotypic frequencies at the -572 position significantly differed in SLE patients and controls. At this position GG genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p X 0.014). The frequency of -572 G allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). The haplotype study showed no significant difference between patients and healthy subjects. The relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p X 0.04). At -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. In conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to SLE and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. The eukaryotic constitutive proteasome is the main protease expressed in most tissues. Recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating NF-kB activity during the intestinal inflammation. In comparison with healthy controls and patients with ulcerative colitis (UC), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with Crohn's disease (CD) at both mRNA and protein levels. In our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between CD and UC patients.

Since IFN-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the IFN-g levels are highly elevated in inflamed intestine of CD patients, induction of immunoproteasomes in CD might be mediated by this cytokine. Our data with human leukemic T cell lines and primary macrophages show a significant increase in the NF-kB controlled production of proinflammatory cytokines after the IFN-g-mediated induction of immunoproteasomes in these cells. In the DSS-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. Therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing NF-kB activity in the epithelial and immune cells. However, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. Objectives: Inflammatory bowel diseases (IBD) including Crohn's disease (CD) and ulcerative colitis (UC) are characterized by unknown etiology and chronic intestinal inflammation. Noninvasive serological tests to differentiate CD from UC have been searched for a long time. Testing for pANCA together with ASCAs has good predictive values to identify patients with IBD.The aim of this study was to find evidences for diversity of ASCAs and anti-Mycobacterium paratuberculosis antibodies (anti-Mpt) by ELISA method. In addition, to examine whether combination of these ELISAs is useful for distinguishing CD from UC. Methods: The study population contained 161 patients with IBD (89 with CD, 41 with UC, 31 with gluten sensitive enteropathy, GSE) and 33 healthy control subjects. Serum ASCA IgG, ASCA IgA and anti-Mpt antibody levels were measured by solid phase enzyme immunoassay. Adsorption of 7 ASCA positive sera was performed by baker's yeast suspension. Results: Elevated level of ASCA IgG, IgA and anti-Mpt was shown in CD and GSE but not in UC compared to healthy controls. Serum levels of ASCA IgG, IgA showed a significant positive correlation with anti-Mpt antibody levels in CD. Repeated adsorptions with yeast removed ASCA IgG and IgA from sera of patients, but did not change levels of anti-Mpt.

These results indicate the diversity of ASCA IgG, IgA and anti-Mpt (accordingly their antigens) and suggest that combination of these ELISA can have a role in the differential diagnostics of IBD. It is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. We have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. We show that distinct populations of T cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative T cell population (ie TCRab+ve, CD4-ve CD8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating CD4(+) cells in inflammatory bowel disease (IBD). Having previously shown in mice that gut-resident T cells express high levels of the regulator of G protein signalling-1 (RGS-1) protein, we have now found substantial over-expression (10-100 fold) of RGS-1 in human gut-derived T cells, particularly in this unconventional T cell population. Furthermore, levels appear even higher in T cells derived from inflamed gut. Transfection of RGS-1 decreases primary T cell responses to CXCL12 and CCL19, strongly implying that it may regulate T cell localisation. Thus, RGS-1 may be a novel target for modulating T cells in IBD, consistent with which SNPs in RGS-1 have been associated with both coeliac disease and type 1 diabetes. Mechanisms involved in the induction of oral tolerance (OT) or systemic immunization through the oral rout are still poorly understood. In our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. Conversely, if they are immunized with peanut proteins prior to a challenge diet (CD) containing peanuts they develop chronic inflammation of the gut. Our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. Adult, female C57BL/6J mice were divided in control (C) and experimental (E) groups. C1-C3 received peanut protein immunization, animals of the Control groups C4 were sham immunized, and control group C5 received ovalbumin (OVA) immunization. The experimental group was immunized with peanut protein extract. Before initial exposure to a 30 day peanut containing CD, the experimental group was divided into 5 groups (E1-E5). OVA feeding began 7 days prior CD (E1) on day 0 (E2), 7 (E3), 14 (E4) and 21 (E5) during CD. Our results show that oral exposure to a novel protein (OVA) in the absence of gut inflammation (E1) leads to low levels of systemic antibody titers, comparable to tolerant animals. Conversely, as off initial induction of inflammation, groups submitted to OVA (OT) protocol develop increasingly higher systemic Antibody (Ab) titers similar to animals of the immune control group. In conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. Nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. Since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. Therefore, we assessed the effect of particles on dendritic cells (DC) as key players in the manifestation of intestinal immunity.In in vitro studies we could show that ultrafine TiO 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived DC. This effect appears to result from enhanced cell death in immature DC but also from direct stimulation of the cells.To analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive IL1-b.In our hands certain nanoparticles induced apoptosis of immature DC, as well as enhanced secretion of active IL1-b. We therefore hypothesize that particles can induce the inflammasome which leads to the activation of DC.To study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (DSS) in the drinking water for 8 days to wildtype mice. When ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. In future experiments we will also analyse the effect of different particles in the IL10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease.

M. Schmohl 1 , N. Schneiderhan-Marra 1 , M. Blum 2 , G. Stein 2 , M. Schmolz 2 , T. Joos 1 1 NMI-Natural and Medical Sciences Institute at the University of Tübingen, Biochemistry, Reutlingen, Germany, 2 EDI GmbH, Reutlingen, Germany The human immune system represents a highly complex system that protects the organism against diseases. There is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. Epithelial layers function as a barrier against pathogens. As the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. The ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. This ability appears to be breaking down with chronic inflammatory bowel diseases (IBD) like Crohn's disease and Ulcerative Colitis [1] . To date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. Therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. For this purpose a human organo-typic (HOT) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. The human CaCo-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. This co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. As a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular MAPK and RTK-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. This approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of Inflammatory Bowel Diseases. Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. Osteopontin (Opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of IBD patients. Opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental UC, indicating a controversy in this field, while its role in experimental CD remains unknown. In our study we investigated the role of Opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (TNBS) colitis, a T H 1-associated model that resembles CD, and dextran sulphate sodium (DSS) colitis, a T H 2-like-associated model for UC. Deficiency of Opn (either by antibody-mediated neutralization or use of Opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that Opn, and especially, the secreted isoform of Opn (Opn-s) is important for the initiation of acute intestinal inflammation. Importantly, we discovered that Opn drives IL-17 production and T H 17 polarization and decreases recruitment of CD4 + CD25 + Foxp3 + T regulatory (Treg) cells in mesenteric lymph nodes (MLNs) of mice with colitis. Also, there was an effect of Opn on recruitment of CD11c + DCs, which were significantly elevated in MLNs of Opn -/or anti-Opn-treated, as compared to Opn +/+ or Ig-treated control mice. This finding implies that Opn deficiency results in enhanced recruitment of regulatory CD11c + DCs which may mediate Treg induction and protect from colitis. Overall, our findings indicate that Opn is proinflammatory in both types of colitis, by promoting pathogenic T H 17 and attenuating Treg cell recruitment, implying also common mechanisms in the pathogenesis of CD and UC.

C. Shen 1,2 , G. Van Assche 3 , P. Rutgeerts 3 , A. Liston 1 , J. L. Ceuppens 2 1 K.U. Leuven, Autoimmune & Genetics Lab, VIB, Leuven, Belgium, 2 K. U. Leuven, Experimental Immunology Lab, Leuven, Belgium, 3 K. U. Leuven, Department of Pathophysiology, Gastroenterology Section, Leuven, Belgium

Background: Haptoglobin (Hp) is one of the acute phase proteins synthesized during inflammation. Hp-1 allele is associated with the disease behavior in Crohn's disease but not in ulcerative colitis. However its role in inflammatory bowel disease has not been defined. Aim: To determine whether Hp modulates the immune responses in experimental colitis. Methods: We induced 3 types of colitis DSS (Th1/Th17), TNBS (Th1) and oxazolone (Th2) in Hp KO mice. Neutralizing anti-IL-17 mAb was injected into DSS and TNBS Hp KO mice. Severity of colitis was evaluated by body weight, colon length and histology. Th17/Th1 cells were analyzed by flow cytometry. Cytokines were measured by ELISA or RT-PCR.

1) Compared to the WT mice, Hp KO mice developed much severer DSS and Oxa induced colitis. DSS induced lethal colitis in Hp KO but not in WT mice; 2) In DSS but not in Oxa colitis mice, IL-17, IFN-g, TGF-b and IL-6 were significantly increased (p X 0.01, DSS vs control) in lamina propria and mesenteric lymph nodes (MLN), and this is much evident in Hp KO mice compared to those in the WT (p X 0.05, KO vs WT). In TNBS colitis, we found elevated IL-12 and IFN-g (p X 0.01, TNBS vs control). Although not significant, IL-17 was also somewhat upregulated; 3) In DSS colitis we observed that IL-23 enhanced differentiated Th17 cells in vitro, this effect could be abrogated by coculture with serum from WT but not HpKO mice. Furthermore, in vitro in the presence of TGF-b, IL-6 and IL-21, more MLN-T cells from HpKO mice differentiated into Th17 cells; 4) Anti-IL-17 mAb improved DSS and TNBS colitis, and partially rescued Hp KO mice from lethal DSS colitis. In line with this, mice treated with anti-IL-17 showed reduced IL-6, IL-17 and IFN-g in both MLN and LP (p X 0.05, anti-IL-17 vs control).

Our results reveal that Hp has a protective role in the development of mucosal inflammation. In DSS and TNBS colitis Hp may exert its beneficial effect partially through inhibiting production of IL-17, supporting further pre-clinical and clinical application of Hp for treatment of Crohn's disease.

P. Engelmann 1 , G. Talabér 1 , G. Süt" o 2 , P. Németh 1 , T. Berki 1 1 University of Pécs, Clinical Center, Department of Immunology and Biotechnology, Pécs, Hungary, 2 University of Pécs, Clinical Center, Department of Immunology and Rheumatology, Pécs, Hungary

Objectives: Inflammatory bowel disease (IBD) resembles as an autoimmune-like disease. IBD is most common in developed countries: it is calculated that 2.2 million people in Europe suffer from IBD. Several hypotheses are raised in the pathogenesis of inflammatory bowel disease. One of the most favored is the dysregulation of the immune response due to failure of regulatory T cells. The most well known regulatory T cells are the CD4+CD25hi+ T (Treg) cells. Furthermore, other immune-regulatory cells are known such as invariant natural killer T (iNKT) cells producing both Th1 and Th2 cytokines rapidly upon antigen (lipid) stimulation. Methods: Based on this hypothesis we aimed to investigate the role of various immune-regulatory T cells in human IBD. We attempt to measure the proportions of iNKT cells, Treg cells in peripheral blood of patients with Crohn's disease (CD) and ulcerative colitis (UC) compared to normal controls. Blood samples were collected from normal controls and IBD patients; then lymphocytes were labeled for iNKT and Treg markers with specific monoclonal antibodies and measured with flow cytometry. Results: According to our results a decline in the total iNKT cells of IBD patients was observed, interestingly the proportions of CD4+ and double negative (DN) iNKT subgroups showed a characteristic shift among the study groups. Percentages of DN and CD4+ iNKT subpopulations were assessed after gating of total iNKT populations. In controls we observed high percentage of DN iNKT cells (74.5 ± 3.5 %, mean ± SEM), while CD4+ iNKT cells ratio was moderate (25.4 ± 3.5 %). In UC and CD patients we found a reduced proportion of DN iNKT cells (UC: 38.0 ± 7.1 %; CD: 34.0 ± 5.2 %, mean ± SEM), while the percentage of CD4+ iNKT cells was elevated (UC: 62.0 ± 7.1 %; CD: 65.9 ± 5.2 %, mean ± SEM) in both disease groups. Proportions of FoxP3+ Treg cells also showed a decline in IBD patients comparing to normal controls. Conclusion: This study can provide useful data about the pathogenesis of IBD and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. Objectives: The aim of this project is to explore whether exosomes from TGF-b1 gene modified bone marrow-derived immature dendritic cells (MD-imDC) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (IBD) in mice, the underlying mechanism was also investigated. Methods: Exosomes were isolated from supernatant of MD-imDC transfected with TGF-b1 adenovirus (TGF-b1-Exo). The T cell inhibitory function of TGF-b1-Exo was determined by mixed lymphocyte reaction (MLR) in vitro. To evaluate the protective effect of TGF-b1-Exo in the development of IBD, dextran sulfate sodium(DSS) induced murine IBD was established and mice were treated with TGF-b1-Exo. The main symptoms of IBD were observed. The inflammatory degree of colon was also evaluated by histological examination. The relative CD4 + Foxp3 + Treg cell numbers from spleens and mesentery lymph nodes (mLNs) were analyzed by FACS. Results: It was demonstrated that TGF-b1-Exo could inhibit the proliferation of T cells in MLR in vitro. In murine IBD model, after treated with TGF-b1-Exo, the main symptoms of IBD such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. Analysis of CD4 + Foxp3 + regulatory T cells (Treg) revealed that the relative numbers of CD4 + Foxp3 + Treg increased in lymphocytes from mesentery lymph nodes (mLNs) of inflammatory site but not from spleens. Conclusions: These results demonstrate that immunosuppressive exosomes obtained from TGF-b1 gene modified MD-imDC can delay the development of IBD. This protective effect is mediated by the induction of CD4 + Foxp3 + Treg. TGF-b1-Exo might provide a novel strategy for the therapy of IBD.

Results: HCV-specific cytokine expression by CD8+ T-cells was similar in the four vaccinees as observed by IFNg, IL-2 production-profiles. However, the killing capacity of expanded CD8+ T-cells was distinct as observed by the competence to kill NS3-peptide presenting transfectants in vitro. As depicted in figure 1 , CD8+ T-cells cells from both Vac1 (cleared ) and Vac2 (chronic) produced IL-2 and IFNg after stimulation with NS3-peptide59. However, specific killing of the peptide loaded transfectants was only observed in Vac 1, who was able to clear its HCV infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers.

[ Figure 1 ]

Killing of NS3 peptide presenting cells was restricted to the vaccinee that was able to clear HCV infection. These results suggest that controlling HCV replication as initiated by this DNA-prime MVA-boost vaccine-protocol was partly mediated by antigen specific CD8+ T-cells. Hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent CD8 T-cells. Objectives: Infection by hepatitis C virus (HCV) is characterized by its high tendency to chronicity, which is usually associated with a low or absent T-cell response against viral antigens. Immune response specific for non-structural protein NS3 from HCV was associated with viral clearance. We have demonstrated that fusion of an antigen to the extra domain A from fibronectin (EDA) targets the antigen to TLR4-expressing dendritic cells and improves its immunogenicity. Thus, we tested if covalent linkage between EDA and NS3 might constitute an alternative for vaccination against HCV infection. Methods: Recombinant plasmids expressing a secretable version of NS3 or EDA-NS3 under the control of CMV promoter were prepared. Recombinant NS3 and the fusion protein EDA-NS3 were produced in E. coli. The recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. HHD transgenic mice (expressing the human HLA-A2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(I:C) and anti-CD40 agonistic antibodies. ELISPOT and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. Intrahepatic expression of HCV-NS3 RNA was measured after a hydrodynamic injection with a plasmid encoding HCV NS3. Results: Immunization of mice with the plasmids expressing EDA-NS3, but not NS3 alone, induced strong T cell responses against the main HLA-A2 restricted cytotoxic T cell determinants from NS3. The recombinant EDA-NS3 fusion protein, but not NS3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of TNF-a by the THP-1 monocyte cell line. Immunization of HHD mice with EDA-NS3 fusion protein induced both CD4+ and CD8+ T cell responses against NS3 and, when immunized with poly(I:C) and anti-CD40 antibodies, was able to down-regulate the intrahepatic expression of HCV-NS3 RNA.

The recombinant EDA-NS3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against HCV infection. Vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of HIV infection even 25 years after virus detection. Therein we propose the intra-dermal inoculation of DNA vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus E2 protein encoding an artificial multi-component HIV antigen. This inoculation is followed by electroporation in order to increase DNA uptake. We used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. Among them, Langerhans cells (CD207+CD1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific CD8+ responses. The present work emphasizes molecular and cellular biodistribution of the DNA vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in HIV studies. Technical approach considers an intra-dermal injection of DNA followed by topical electroporation of the injection sites. Skin and draining LN biopsies were collected at different time points. These biopsies were used for IHC fluorescent staining in order to establish biodistribution DNA-encoded antigens and co-localisation with different cell types. Kinetic of antigen expression was studied by bioluminescence in vivo imaging. T cell responses were measured by IFN-g ELISPOT assays up to 3 years after DNA vaccination. We show that a DNA vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of CD1a+ cells in the draining LN in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. Our data suggest that electroporation after intradermal injection of DNA vaccine involves Langerhans cells from the epidermis that elucidate qualitative anti-HIV immune responses. This new approach that comprise new DNA vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. Murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human Melan-A, in combination with the immunostimulatory cytokine GM-CSF, selective targeted DCs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of DCs. In mice, a single application of only low doses elicited strong and long-lasting cytotoxic T-cell responses which provided protective antiviral and antitumor immunity. Furthermore, the efficient activation of human tumor-specific CD8+ T cells by mature DCs transduced with Melan-A-recombinant human coronavirus 229E indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. As the application of GM-CSF already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant Coronavirus-based vectors expressing different immunostimulatory cytokines. Thereby cytokines will be acting on T cell and DC level. To enhance T cell response Interleukin 2 (IL2) and Interleukin 15 (IL15) will be involved, and fms-like tyrosin kinase 3 ligand (Flt3L) will be expressed to modulate dendritic cells. IL2 is known to enhance early T cell expansion and limits T cell overshoot, whereaes IL15 guarantees survival of high affinity T cells during memory phase. On the other hand Flt3L enhances DC proliferation and accumulation. With these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific CTL response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches.

F. Demircik 1 , AG Waisman 1 Uniklinik Mainz, 1. Med, Mainz, Germany

In murine cytomegalovirus (MCMV) infection, cytotoxic CD8 T cells and NK cells play a critical role. Previously it was shown that mice deficient for B cells are more susceptible to MCMV-related disease, caused by virus reactivation. To better understand the role of B cells and antibodies in the response to MCMV, we made use of different mouse strains that lack B cells, secreted antibodies or IL-10 production by the B cells. We found that for the initial T cell response to the virus B cells are important, but antibodies do not play an important role. This implicates B cells as potential important antigen presenting cells (APCs) in the activation of the virus-specific T cells. The reduced T cell response to the virus was observed whether the mice were B cell deficient from birth or if they were depleted later in life. Six month after infection mice were tested for the memory CD8 T cell response. Interestingly, we found that in mice that lack antibodies (mice that lack B cells all together and mice that have B cells but no secreted antibodies) maintain a rather high T cell response to viral peptides, in a level similar to the acute response 7 days after infection. We conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of T cells. Finally, we tested the role of IL-10 produced by B cells by conditional deletion of the IL-10 gene in these cells. We found that B cell secreted IL-10 has a suppressive effect on the T cell response to MCMV, as this response is elevated in these mice. We conclude that B cells are important for an efficient acute response to MCMV and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for B cells in the efficient acute and memory response to MCMV. This work is supported by the Deutsche Forschungsgemeinschaft grant SFB490 to AW. Objectives: EBV infection leads to life-long viral persistence. Although EBV infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by T cells. EBV-specific IFNg-producing T cells could be demonstrated in acute and chronic infection by many researchers. Recent studies in HIV and Leishmania provide, however, evidence that assessing IFNg alone is insufficient to assess the quantity and quality of a memory T cell response and support the crucial role of multifunctional T cells in disease control. In this study we therefore analyzed EBV-specific T cell responses in peripheral blood (PB) and bone marrow (BM). Methods: Paired PB and BM samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. T cells were expanded for 10 days in the presence of IL-2 and IL-7 with exposure to overlapping peptide pools of latent EBNA-1 and lytic BZLF-1 antigens. EBV-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, CD3, CD4, CD8, CCR7, CD45RA, IL-2, TNFa, IFNg and CD107a.

The majority of ex vivo EBV-reactive CD4+ T cells as well as EBNA-1-reactive CD8+ T cells were IL-2 and TNFa-producing memory cells, the later being more frequent in bone marrow (CD4+, median, EBNA-1: BM 0.69 %;PB 0.12 %; BZLF-1: BM 0.37 %;PB 0.01 %, p=0.039). After in vitro expansion a major subset of EBV-specific CD4+ and CD8+T cells displayed a differentiated effector IFNg/TNFa phenotype. A comparable number of EBV-specific CD4+ and CD8+ T cells retained, however, a TNFa single, TNFa/IL-2 or triple producer phenotype resembling early differentiated or multifunctional memory T cells, respectively. Interestingly, both CD4+ and CD8+ T cells generated from BM revealed significantly higher cytotoxic potential. Sorting of CCR7/CD45RA differentiation subsets, revealed that EBV-specific T cells were predominantly expandable from the central memory compartment. Conclusion: Our data shows that multicolor assessment of IFNg, TNFa and IL-2 delineates various subsets of EBV-specific memory T cells, which reflect the profile of a protective immune response. Human adenovirus (HAdV) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (alloSCT).

Reconstitution of HAdV-specific T cells has been reported to be associated with sustained protection from HAdV disease, but epitope specificity of these responses has not been further characterized. Furthermore, the relative contribution of HAdV-specific CD4 + and CD8 + T cells in the protection from HAdV disease after alloSCT remains to be elucidated. In this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with CD154 or peptide-MHC tetramer staining, that clearance of HAdV was associated with a combined HAdV hexon specific CD4 + and CD8 + T cell response in both pediatric and adult alloSCT recipients. Based on this observation, we developed a clinical grade method for the rapid generation of T cell lines with high and defined specificity for HAdV hexon epitopes for adoptive immunotherapy. Activation of HAdV hexon-specific CD8 + and CD4 + T cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by IFNg-based isolation allowed rapid expansion of highly specific T cell lines from healthy adults, including donors without detectable frequencies of HAdV hexon-specific T cells. The frequency of HAdV-specific T cells was increased to 29-90 % in the T cell lines and the absolute numbers of both hexon-specific CD4+ and CD8+ T cells were 2 to 3 log increased compared to the starting material. Detailed analysis showed that HAdV-specific T cell lines recognized multiple MHC class I and II restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of HAdV infected target cells. This strategy may be used for adoptive transfer of donor-derived HAdV hexon-specific CD8 + and CD4 + T cells for treatment of disseminated HAdV infection after alloSCT. Several studies showed that HBV persistance correlates with a failure of an efficient virus-specific T-cell response. Induction of HBV-specific T cells by vaccination may be an innovative approach to overcome virus persistance. DNA prime-recombinant adenovirus serotype 5 (Ad5) boost strategy proved to be effective in stimulating T cell responses and control of viral infections. Woodchuck hepatitis virus (WHV) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. The efficacy of plasmid DNA and Ad5 vaccine vectors expressing WHV core protein was first examinated in C57BL6 mice. Groups of mice were immunized with a DNA prime-Ad5 boost regimen or with DNA and Ad5 alone. Ad5 was injected i. m. or s. c. T cell response was evaluated by intracellular IFNg staining of splenocytes stimulated in vitro with WHc-derived peptide pools. Anti-WHc antibodies were detected by ELISA.

We detected CD8+ T cell responses against peptide pools 1 and 3 in spleens of DNA and DNA-Ad5 immunized mice. However, in prime-boost group the percentage of of detected IFNg+ CD8+ T cells was lower in comparison to DNA group. In splenocytes of animals vaccinated with Ad5 very weak CD8+ T cell response was observed. In DNA vaccinated animals we determined high level of anti-WHc already after second immunization. After boosting with Ad5 level of antibodies did not change. Those antibodies were only IgG2a subclass what indicates Th1 T helper type of response. Ad5-immunized mice had over 3-fold lower level of anti-WHc: both IgG2a and IgG1 subtypes were detected. The weak response induced by Ad5 may be due to the low expression of WHcAg. In ongoing expreriments we improved the protein expression level by insertion of an intron. We currenly investigate the new construct in mice.

The new peptide construct containing four M2e-peptide sequences coupled to T helper epitopes from the Plasmodium falciparum CS protein and the hepatitis B virus antigen was administered together with adjuvants intranasally and subcutaneously as described (Mozdzanowska et al., Virology Journal 2007) into various mouse strains. In contrast to its predecessor peptide, we found that vaccination induced much higher anti-M2e serum Ab titers against peptide and native M2e. This correlated with a large number of M2-specific Ab-secreting cells in lungs and bone marrow. Moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype A/FM (H1N1), which contains a variant M2e-sequence different in 3 amino acid positions. Importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza A strains X31 (H3N2) and the highly pathogenic PR/8 (H1N1) with remarkably reduced viral titers in lungs and noses of mice.

In conclusion, our studies show promising results towards the further development of vaccination with M2e as a potential "universal" influenza vaccine. This research is supported by a NIH T32 fellowship CA09171-32, the NIH grant AI 46457 and a grant from the Commonwealth of PA.

L. Yu 1 1 Zhejiang University, Zhangzhou, China

Interleukin-18 (IL-18) is a cytokine produced by stimulated mononuclear macrophage system. In this report, 18-day-old chicken embryos were vaccined with the plasmid DNA (pCI-ChIL-18) encoding chicken interleukin-18 and the copy numbers of ChIL-18 in peripheral blood, spleen and bursa of Fabricius at different time points post-embryonic-vaccination were detected by Real-time fluorescent quantitative PCR. The polyprotein of infectious bursal disease virus (IBDV) was prepared into DNA vaccine, and the DNA vaccine was co-administrated with pCI-ChIL-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent IBDV four weeks later. The results indicated that allantoic cavity vaccinated with pCI-ChIL-18 could accelerate high concentrations of ChIL-18 in nonage peripheral blood, accelerate high expression of ChIL-18 in nonage spleen and bursa of Fabricius and promote the body early immune response capacity. Embryo vaccination with ChIL-18 could significantly enhance the nonage proliferation responses of T lymphocytes from spleen and B lymphocytes from bursa. Meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent IBDV induced by DNA vaccine. The results indicated that the nonage immune responsing to IBDV DNA vaccine was highly enhanced by embryonic coadministration with ChIL-18 (P X 0.05). Due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("Follicular Targeting"). Applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (APCs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles.

In in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. Transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal LCs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle.

The use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (DC) in CD8 cell cross-priming and suggests that vaccination via the transcutaneous (TC) route may be relevant in the induction of cellular immune responses. Advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. For this purpose, we developed a standard operating procedure (SOP) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. In a pilot study on 12 volunteers and a Phase I study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed SOP is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific CD8 cells. Induction of T cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. This study proposes new perspectives for the development of vaccination strategies that triggers T cell immune responses in humans. Objectives: All anti-HIV-1 neutralizing antibodies are directed toward the viral Envelope glycoproteins (gp) 120 and the transmembrane protein gp41. Two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (V3) is recognized by the human monoclonal antibody 447-52D and the epitopes in Membrane Proximal External Region (MPER) were recognized by the human monoclonal antibodies 4E10 and 2F5. In order to elicit anti-HIV-1 neutralizing antibodies we have designed virus like particles (VLPs) displaying either the gp120-V3 region or the gp41-MPER.

The VLPs are based on the acyltransferase component (E2 chain) of the pyruvate dehydrogenase complex of Geobacillus stearothermophilus. The E2 chain self-assembles into a 24 nm protein scaffold resembling a VLP and that contains 60 copies of E2. Efficient display and refolding of the V3 and MPER regions in E2 VLPs are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for E2. The priming and boosting with a combination of VLPs and specific HIV-1 envelope DNA were used to immunize mice and rabbits. Results: The V3-E2 and MPER-E2 VLPs were purified as stable 60mers from E. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. Binding of 447-52D, 4E10 and 2F5 antibodies to HIV-E2 monomers was confirmed by Western blot. We obtained high titers of HIV-1 gp140-specific antibodies in mice immunized with a combination of VLPs plus DNA (HIV-1 SF162 gp160). These antibodies generated a low (20 %-27 %) level of neutralization. Moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. Three doses of E2 VLPs plus DNA elicited a low titer of HIV-1 gp140 specific antibodies. Additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid DNA, to enahance the responses to Envelope and to induce neutralizing activity against these key epitopes.

Our results demonstrate that E2 VLPs are able to display antigenic determinants of HIV and to induce high titers of HIV-1-specific antibodies. The E2 VLPs represent a promising tool for a vaccine design. Now a day we paid for vaccination of previous generations. As a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. Every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. The most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. We offer a way of immunity according to the world scientific theory and practice. The method is based on biochemical, electrophysiology, and biology way of correction physical status. At first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. All of it attends indemnity necessary microelements that were extracted from wild officinal herbals. We don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. In our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. That's why when we secure new physical statement in the CNS program we forming new nearest and distant men health. We tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. It's completely different disturbances of physician state, which should have different therapeutic approach. The threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. We have previously shown that Baxter's Vero cell-derived H5N1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. More recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic H5N1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . The study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a H5N1 whole virus wild-type vaccine, could induce an immune response and protect mice against H5N1 influenza virus infection. A trivalent seasonal influenza vaccine was shown to elicit anti-H5N1 antibody and T cell responses and partially protected mice against a lethal challenge with wild-type H5N1 virus. The protective efficacy of the trivalent vaccine derived mainly from the H1N1 component. Moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with H5N1 virus, suggesting that antibodies are the main contributor to protection. H1N1 specific serum did not inhibit neuraminidase activity of H5N1 virus suggesting that protection was not mediated by neuraminidase N1-specific antibodies. Next, we investigated the combination of the trivalent seasonal influenza vaccine and the H5N1 whole virus wild-type vaccine. A prime with the seasonal influenza vaccine followed by immunisation with the H5N1 vaccine enhanced anti-H5N1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the H5N1 vaccine. Hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a H5N1 candidate vaccine.

[1] Gioia C et al. Aims: To register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. Methods: In the study we checked the vaccination history of 335 children in the first grade of primary school in the area of Central and West Macedonia. There were 234 greek and 101 foreign children. As fully vaccinated were considered those who had already undergone at least one dose of Hib, meningococcus and pneumococcus, two doses of HAV, as well as four doses of DTP-Sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. In all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. Results: The percentages of the compliance found, are presented in the following 

1) It should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%.

2) High percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media).

3) On the contrary, as far as the hepatitis A and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) Finally, a statistically significant difference was found relating the response to the vaccination coverage, between Greeks and foreigners, but also between the Greeks themselves, in relation to their educational and socioeconomic level. Objective: Over the past three decades, the incidence of type 1 diabetes has dramatically increased in Europe and North America, inversely correlated to the decrease of infections. According to the Hygiene hypothesis, pathogens may prevent the onset of the disease. OM-85, a bacterial extract of both Gram positive and Gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect Non Obese Diabetic (NOD) mice from diabetes development. We aimed here at understanding the mechanism underlying this protection. Methods: NOD mice and NOD-cd28 -/mice, which are devoid of natural regulatory T cells (Tregs), were treated with OM-85. Cytokine secretion, activation and proliferation of B cells and FoxP3+ Tregs were monitored. As toll-like receptors (TLR) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for TLR2, TLR4 or the MyD88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of OM-85. Two synthetic TLR4 agonists used as adjuvant in human (OM-174-DP and OM-197-MP-AC) were also tested for their capacity to protect NOD mice from diabetes.

The OM-85-induced protection of diabetes required natural Tregs, as NOD-cd28 -/mice were not protected. Remarkably, OM-85 activated B cells and not T cells, promoting their proliferation and IL-10 secretion, two phenomena that were TLR4-and MyD88-dependent. OM-174-DP and OM-197-MP-AC two synthetic murine TLR4 agonists effectively prevented diabetes onset in NOD mice, promoted the expansion of CD4 + CD25 + FoxP3 + T cells and the proliferation of IL-10 secreting B cells in a dose-dependent manner. Conclusion: Our results argue for the involvement of TLR4 signaling in the protective effect of OM-85 on development of diabetes and show that two other TLR4 agonists induce proliferation of B cells and their secretion of IL-10 as well as stimulation of regulatory CD4 + CD25 + FoxP3 + T cells. Activation of the innate immunity by TLR-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease.

D. De Wit 1 , A. Legat 1 , S. Thomas 1 , M. Van Mechelen 2 , P. Hermand 2 , M. Goldman 1 1 Institute for Medical Immunology/Université Libre de Bruxelles, Gosselies, Belgium, 2 GlaxoSmithKline Biologicals, Rixensart, Belgium

Aminoalkyl glucosaminide 4-phosphates (AGP) are lipid A mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting Toll-like receptor 4 (TLR4). Since natural lipid A from bacterial lipopolysaccharide (LPS) depends on membrane-bound or soluble CD14 (sCD14) for its TLR4 ligand activity, we investigated the involvement of both forms of CD14 in the responses elicited by CRX-527, a prototypical AGP. First, we found that CRX-527 efficiently induces NF-kB and IRF3 activation in HEK cells transfected with TLR4 and MD-2 genes, whereas the responses to LPS required co-transfection of the gene encoding membrane-bound CD14. Likewise, CRX-527 efficiently induces the synthesis of NF-kB and IRF-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound CD14 prevents LPS responses. We then observed that monocyte-derived dendritic cells (DC) which are devoid of membrane-bound CD14 respond to CRX-527 but not to LPS in serum-free medium. The addition of the soluble form of CD14 did not modify the levels of IL12 and TNF produced by CRX-527 stimulated DC but increased the levels of interferon-b (IFN-b). When sCD14 was added to HEK cells expressing TLR4/MD-2, NF-kB activity was not modified but IRF3 activity was increased in a dose-dependent manner in response to CRX-527. We will further compare the responses induced by CRX-527 in wild-type and CD14 deficient mice. We previously showed that the transcriptional transactivator (Tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. These observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. As we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (pTat) can raise a humoral immune response against two model peptides. These two peptides, which originate from diphtheria toxin (pDT) and from toxin alpha (pT), both contain an I-Ad restricted T-cell epitope but are nonetheless non-immunogenic in BALB/c mice of the H-2d haplotype when injected with Alum. The pTat, pDT, pT, pTatpT and pTatpDT constructs were prepared by chemical synthesis, purified by reverse phase HPLC and characterized by mass-spectrometry. pDT+pTat, pT+pTat, pTatpT and pTatpDT were respectively injected twice at two weeks interval in BALB/c mice and animals were bled 14 and 28 days after the second immunisation. The sera were subsequently incubated in microtiter ELISA plates previously coated with pT and pDT peptides respectively in order to assess the humoral immune response. We observed a lack of antibody response for the immunizations made with the mixture of peptides (pDT+pTat and pT+pTat) but an anti-pDT and anti-pT response for the immunizations made with the two hybrid constructs (pTatpT and pTatpDT). Our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of Tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect.

INTERLEUKIN-15 GENE POLYMORPH ISMS (C267T, G367A, C13687A AND A14035T) AND SUSCEPTIBILITY TO BRUCELLOSIS IN IRANIAN PATIENTS

Russian Federation Some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. Currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. As we previously reported the decreasing of intracellular IFN-g production by CBMC

Statistical analysis was performed using the Kruskal-Wallis and Mann-Whitney tests. Results: We revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype INFg+/CD69-, p=0.02) and activated (subtype INFg+/CD69+, p=0.028) CBMC, producing IFN-g, as compared with newborns from urban mothers (n=79)

Exposure to pets and the risk of allergic symptoms during the first 2 years of life

Intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study

PC09/16 TO WHAT EXTENT T-SPOT.TB COULD BE USED IN THE DIAGNOSIS OF TUBERCULOSIS IN CHILDREN EXPOSED TO TB INFECTION? S. A

TB) in children, especially in BCG-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. The purpose of the present study was to evaluate the diagnostic value of T-SPOT.TB (Oxford Immunotec, Oxford, UK) compared to tuberculin skin test (TST) in children exposed to TB contact in the family. Forty three children with a history for BCG vaccination/revaccination, treated in the University Clinic for Lung Diseases in Children Sofia, Bulgaria were enrolled in the study. The patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6)

TB has the highest diagnostic value in children n 4 years of age

In early childhood the diagnostic value of T-SPOT.TB and TST does not differ

CFP-10 antigen is more sensitive for detection of TB-specific T cells compared to ESAT-6 antigen. 4. In children with TST 5-14 mm T-SPOT.TB has a high diagnostic value

Objectives: The goal of this study is to determine the role of TLR2 and TLR4 in the development of spontaneous lupus disease by creating TLR2 or TLR4 deficient C57BL/6 lpr/lpr mice. Methods: TLR2 and TLR4 deficient lupus prone mice have been generated by crossing C57/BL6-TLR2 -/-or C57/BL6-TLR4 -/-mice with C57/BL6 lpr/lpr mice which develop a moderate type of lupus related to Fas deficiency. We analysed the phenotype of the disease, autoantibody production and renal injury. Statistical comparisons were performed using the Mann-Whitney U-test. Results: These mice developed a less severe disease and few immunological alterations. Indeed, in TLR2 or TLR4 deficient lpr mice, glomerular IgG deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsDNA and anti-cardiolipin autoantibody titers were significantly reduced. However, the response against nucleosome remained unaffected, indicating a role of TLR2 or TLR4 in the production of autoantibodies directed against certain SLErelated autoantigens. Analysis of B cell phenotype showed a significant reduction of MZ B cells, particularly in TLR4 deficient mice suggesting an important role of TLR4 in the sustained activation of these cells likely involved in autoantibody production. Interestingly, the lack of TLR4 also affected the production of cytokines involved in the development of lupus disease. Conclusion: Our data show that deficiency in TLR4

PC14/13 EXPRESSION OF FULL LENGTH MCL-1 AND ITS SPLICE VARIANT IN JUVENILE SYSTEMIC LUPUS ERYTHEMATOSUS (JSLE) NEUTROPHILS: DIFFERENTIAL MODULATION BY GM-CSF

Granulocytemacrophage colony-stimulating factor (GM-CSF) can prolong neutrophil survival by increasing Mcl-1, an anti-apoptotic protein. A splice variant of Mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. Here we investigate the expression of both the full length mcl-1 (mcl-1L) and its splice variant (mcl-1S) in JSLE neutrophils compared to controls and investigate whether the addition of GM-CSF changes the expression of both isoforms of mcl-1. Method: Neutrophils were isolated from children (diagnosed X 17 years) with JSLE (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, JSLE serum alone or with JSLE serum plus 20pg/ml GM-CSF. Quantitative real time PCR was used to assess mcl-1L and mcl-1S mRNA expression (Mean ± SEM) following incubation in the above conditions and immediately following neutrophil isolation

The ratio of mcl-1S to mcl-1L was also higher in JSLE patients compared to controls (p X 0.05). The addition of GM-CSF to JSLE serum was associated with an increase in mcl-1L (1.66 ± 0.31) and a decrease in mcl-1S (2.56 ± 1.1) mRNA expression

The addition of GM-CSF to JSLE serum can abrogate the increased neutrophil apoptosis. Alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. Our results suggest that JSLE neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with JSLE neutrophils having greater expression of the pro-apoptotic isoform mcl-1S, and less anti-apoptotic full length mcl-1

CYLD is a tumor suppressor gene known to play an important role in the NF-KB pathway. To analyze the function of CYLD in vivo we used the CYLD ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (sCYLD)

To further investigate the connection between sCYLD overexpression in T cells and colonic inflammation, we used an adoptive transfer model of colitis. Therefore naive CD4 + CYLD ex7/8 T cells were transferred into RAG1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. Here we could demonstrate that CYLD ex7/8 CD4 + T cells exhibit less capacity to induce colitis compared to control cells. Consequently we investigated if regulatory T cells (T regs ) of CYLD ex7/8 mice are capable to control inflammatory responses. For this purpose CD4 + CD25 + cells were co-transferred with naïve wt CD4 + T cells into Rag1 -/-recipients. Interestingly, RAG1 -/-recipients of CYLD ex7/8 T regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. Our findings demonstrate that overexpression of sCYLD leads to a hyperresponsive T cell phenotype and higher production of inflammatory cytokines by T cells

PC17/16 THE ROLE OF HLA COMPLEX IN INFLAMMATORY BOWEL DISEASE: CROHN'S DISEASE AND ULCERATIVE COLITIS

de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd). University Hospital Virgen Arrixaca

The allele frequencies of HLA class I in CD and UC patients were not different to those observed in controls, although we found an increased frequency of A*03 in CD vs UC. Haplotype frequencies of HLA class I and II in CD and UC were also not different to those observed in controls. However, we found increased frequencies of DRB1*13, *01 and *0103 alleles, and a decreased allele frequency of DRB1*15 in CD vs UC patients and controls. These data are in concordance with other previous studies suggesting that, in patients with isolated colonic CD, DRB1*0103 is associated with the development of severe disease and positive association of CD with DRB3*0301 and DRB1*13. Indeed, DRB1*15 was negatively associated with CD. This allele appears to confer protection against all subgroups of CD, in all ethnic groups including Japanese. However, HLA-DRB1*07 frequency allele, associated in unselected patients with CD in other studies, was not different in our CD and UC patients, and controls. Additionally, an increased frequency in HLA-DRB1*04 in CD was not found in our patients in a different manner to other reported studies. On the other hand, in our UC patients, allele frequencies of DRB1*15 were strongly increased with respect to CD and controls. However, the frequency of DRB1*04 was decreased in UC with respect to CD and controls. In this sense, our data are agreed with other reports showing that HLA-DRB1*15 is associated with UC in European, North American, Japanese and Korean populations

Methods: A total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for Epstein-Barr virus infection. The ELISA method was used to look for specific antibodies against the capsid of the virus VCAIgG and against the nuclear antigen EBV-IgM, while taking into consideration the possible increase of the VCAIgG title between two serum samples. Results: Totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 X 5 years of age, 12 5-10 years of age and 2 G 10 years of age) and 23 girls (10 X 5 years of age, 6 5-10 years of age and 7 G 10 years of age). Pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). The lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis G 20.000 in 9 cases (17,6 %). The most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). A past infection (negative EBV-IgM values and positive VCAIgG values) was

virus infection is common among children and teenagers

Serum negative are mainly the children of little age and 3) There is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks

General Hospital of Rethymno, Rethymno, Greece shows that the IL-13Ra2, previously believed to be a decoy for IL-13 only, is able to transmit a signal via IL-13. Our results support this and may suggest that IL-13/ IL-13Ra2 signalling causes disease in oxazolone-induced colitis. Currently we are dissecting the role of single cell populations expressing IL-4Ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. Together this data can define a role for IL-13 or IL-13Ra2 and identify specific cell populations

Methods: Splenic APCs exposed to enteroantigen (eAg) +/-probiotics were used to stimulate cultured CD4 + CD25 -T cells to which titrated numbers of Tregs were added. Neutralizing antibodies against IL-6 and IL-1b and ELISA-based cytokine analyses were used to monitor the effect of cytokines secreted in the T cell cultures. Results: Exposure of APCs to eAg and probiotics did not influence eAg-specific CD4 + CD25 -T cell proliferation. However, exposure to three of the six probiotics tested (B. bifidum BI-98, L. acidophilus NCFM TM and B. bifidum BI-504) consistently reduced regulatory activity of Tregs in a cell-dose dependent manner. The Tregreducing activity of probiotics was analyzed using fractionated components of the B. bifidum BI-98 strain. Data indicated that bacterial cell-wall components were responsible for reducing Treg activity and not components of nucleus or cytoplasm. The probiotic-induced down-regulation of Treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as IL-6 or IL-1b. Conclusion: We conclude that certain probiotic strains can modify APCs to cause reduced Treg activity in an eAg-specific T cell proliferation assay. This effect apparently depends on a direct APC-to-Treg cell contact and not secreted cytokines. The APC/probiotics-mediated inhibitory effect on Tregs may oppose antiinflammatory activities desired from probiotic therapy

Palmieri 1 1 'La Sapienza

Dysregulated innate and adaptive immune responses against commensal flora lead to Crohn disease (CD) and ulcerative colitis (UC), two different forms of Inflammatory Bowel Disease (IBD), a lifelong inflammatory condition of the gastrointestinal tract

Methods: We analyzed 21 pediatric CD patients (13 active, 8 remission), 24 pediatric UC patients (17 active, 7 remission), and 37 age-matched non-IBD controls. NKG2D/ligand expression was evaluated by immunostaining and multiparametric FACS analysis (on PBMC subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). Differences between groups were analyzed with non-parametric and parametric tests; a level of p X 0.05 was considered significant. Results: NKG2D expression is selectively upregulated on circulating "innate-like" T cell populations (g/d and CD3+CD56+ NKT cells), in active, but not in quiescent IBD patients; receptor upregulation correlates with disease type (observed in UC, but not in CD patients). In the same patient groups, the appearance of NKG2D ligands on circulating monocytes is also observed. The dramatic increase of NKG2D+ lymphocytes, and the strong upregulation of NKG2D ligands on both epithelial and immune components, are observed in active IBD lesions. Conclusions: Our observations document the dysregulated expression pattern of NKG2D/ligands on selected innate immunity populations in pediatric IBD patients, both at mucosal and systemic level

PC17/26 PERIPHERAL AND INTESTINAL REGULATORY T CELL DYNAMICS IN PEDIATRIC IBD PATIENTS

is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. Regulatory T cells (T reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. Aims: To evaluate the frequency of regulatory T cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric IBD and non

Mucosal regulatory T cells were identified by immunohistochemistry; circulating regulatory T cells were analysed by immunofluorescence and FACS analysis. Differences were analyzed with parametric and non-parametric testsconsidered significant. Results: FOXP3+ T reg were significantly increased in the intestinal lesions of active IBD patients (CD or UC), and returned to normal levels in post-therapy remission phase. At variance, circulating CD4+ T reg frequency was elevated in patients affected by both forms of IBD, independently of disease activity, as it persisted in the remission phase. A selective imbalance in the frequency of T and NK subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent IBD patients. Conclusions: Regulatory T cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; T reg dynamics in pediatric IBD patients only partially matches previous data obtained in the adults; quiescent IBD is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically

The increased expression of immunoproteasomes in the inflamed mucosa of IBD patients was shown to contribute to this pathology by enhancing NF-kB activation. Due to the relation between NF-kB and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in IBD. LMP7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. To test our hypothesis, we employed the DSS colitis model. In contrast to wild-type mice, colitis was attenuated in LMP7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. In addition, LMP7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by RT-PCR and ELISA. Especially NF-kB regulated genes show enhanced induction in wild-type mice unlike LMP7 knock-out mice

Synaptic Systems GmbH, Braunschweig, Germany Objectives: Although more than 200 million people worldwide are chronically infected with Hepatitis C virus (HCV) no prophylactic or therapeutic vaccines do exist to prevent or cure HCV infections. Our major objective is to develop a dendritic cell (DC)-based immunotherapy enhancing virus-specific cellular immune response for treatment of HCV infections

Based on this approach we aim at generating aDec-205 antibodies conjugated with immunodominant HCV proteins to induce HCV-specific protective immunity. Methods: Recombinant HCV proteins are expressed using "expression-ready-clones" containing N-terminally His-tagged HCV-Core (aa 1-191) or HCV-NS3 (aa 1027-1218) sequences. Protein purification is performed by metal-affinity chromatography on Ni-NTA-agarose

HCV-specific T cell responses are monitored at different time points after immunization by FACS and in vitro T cell proliferation assays. Results: To obtain high amounts of recombinant NS3 and Core we successfully optimized culture and protein purification conditions. Briefly, NS3 was purified natively using PBS-based buffers with pH-gradient. In contrast, purification of Core was performed under denaturing conditions in presence of GuHCl and urea and a pH-gradient elution. Moreover, optimized conditions allowing conjugation of aDec-205 to recombinant HCV proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. Efficient conjugation was verified by Western blot analysis. After successful generation of aDec-205/ HCV-protein conjugates we are currently establishing optimized vaccination conditions to induce HCV-specific immune responses

PD01/2 MVA-NEF VACCINATION INDUCES POLYFUNCTIONAL CD4 T-CELLS AND INCREASES THE PROLIFERATIVE CAPACITY OF CD8 T-CELLS IN HIV-1 INFECTED INDIVIDUALS UNDER HAART

Several vaccination trials have made use of the modified vaccinia virus Ankara (MVA) as delivery vector. In a therapeutic vaccination trial, we demonstrated that MVA expressing the HIV-1 protein Nef (MVA-nef) was safe in HIV-1 infected individuals under HAART and immunogenic in regard to the elicitation of IFN-g mediated CD4 T-cell responses. Recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of IFN-g provides limited information on the quality of antigen-specific T-cell responses. The evaluation of several functions is essential, as simultaneous production of multiple cytokines by T-cells is associated with superior control of viral replication. Methods: In a retrospective setting, we simultaneously assessed the production of IFN-g, IL-2 and MIP-1b, the expression of the activation marker CD154 and the differentiation marker CD45RA in Nef-specific CD4 and CD8 T-cell populations during the course of the vaccination trial. Furthermore we applied a multi-colour CFSE based proliferation assay investigating the proliferative capacity and the simultaneous expression of IFN-g, IL-2 and MIP-1b. Results: Following MVA-nef vaccination, we observed a significant increase of the total Nef-specific CD4 T-cell response and a significant increase of polyfunctional Nef-specific CD4 T-cells, simultaneously expressing IFN-g, IL-2 and CD154. Using the standard ICS no increase of Nef-specific CD8 T-cell responses was observed. However, by the CFSE based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of Nef-specific CD8 T-cells following MVA-nef vaccination. Notebly, we observed a correlation between the increase of IFN-g, IL-2 and CD154 expressing CD4 T-cells and the increase of proliferating CD8 T-cells suggesting the possibility of a causal link between the two functions. Conclusions: The MVA-nef vaccine is able to change the quality and quantity of the Nef-specific CD4 T-cell immune response and has the potential to increase the proliferative capacity of Nef-specific CD8 T-cells in HIV-1 infected subjects under HAART

This preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (SPR) tests. This Ab inhibited HIV-1 mediated membrane fusion and p24-detected replication. DB81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant ESN individuals. Further characterization of the antibody and of its binding epitope is ongoing

Following intradermal vaccination with 25mg DNA and electroporation of BALB/c mice, splenocytes have been incubated with peptides representing class I and II epitopes, and specific T cell-responses were examined by Elispot-assays. The specific antibody responses have been measured by sandwich ELI-SAs, and neutralizing antibodies have been investigated by HI-assays. Results: The vaccibody constructs have been found to be expressed and correctly folded in vitro. The in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific CD4 + and CD8 + T cells. Conclusion: Antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to APCs. Methods: The HCV recombinant proteins rNS4 (1677-1756 aa) and rNS5A (2061-2302 aa) were conjugated with Immunomax using the heterobifunctional reagent Sulfo-SMCC. BALB/c and DBA/2J mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with Immunomax, or with complete Freund's adjuvant (CFA)

the other combinations were not immunogenic at given doses. It should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of NS4 protein. Immunization with rNS5A-Immunomax conjugate and rNS5 in CFA (1.4 mg/mouse) induced a similar antibody activity, but a different T-cell responses. The conjugate induced splenic accumulation of T cells specifically reacting in vitro with NS5A recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. Immunization with rNS5A in CFA induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. Conclusion: Covalent conjugates of HCV nonstructural proteins with Immunomax effectively induce humoral and cell immune responses

PD01/21 DEGREE OF CROSS-GENOTYPE REACTIVITY OF HCV-SPECIFIC CD8 T CELLS DIRECTED AGAINST NS3

The existence of multiple HCV genotypes characterized by marked sequence differences is a challenge for immune control. The aim of this study was to compare the antiviral CD8 T cell response targeting HCV genotype 1 (GT1) and genotype 3 (GT3) as the most predominant genotypes in Germany and to determine the extent of cross-genotype reactivity of specific T cells. We analyzed a cohort of patients with past or ongoing intravenous drug use (IVDU) hypothesizing that multiple exposures to different genotypes may occur. Methods: 53 subjects (17 with GT1, 22 with GT3 and 14 anti-HCV-pos/HCV-RNA-neg) were analyzed. HCV-specific T cells were expanded from PBMC in the presence of peptide pools covering NS3 from GT1 or GT3. Individual reactive peptides and the degree of cross-reactivity between the GT1 and GT3 variants were determined by ICS. Complete NS3 is sequenced from all viremic patients

PD01/22 ANTI-RETROVIRAL EFFECTS OF TYPE I INTERFERON SUBTYPES IN VIVO

IFNa subtypes 1, 4, 6 or 9 suppressed FVreplication in vitro, but differed greatly in their antiviral efficacy in vivo. Treatment of FV-infected mice with the IFNa subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. Decreased splenic viral load after IFNa1 treatment correlated with an expansion of activated FV-specific CD8 + T cells and NK cells in the spleen, whereas in IFNa4-and IFNa9-treated mice it exclusively correlated with the activation of NK cells. Other IFNa subtypes like IFNa2, 5 and 11 are under investigation

PD01/23 ELIMINATION OF IMMUNODOMINANT EPITOPES FROM MULTISPECIFIC DNA-BASED VACCINES ALLOWS INDUCTION OF CD8 T CELLS THAT HAVE A STRIKING ANTI-VIRAL POTENTIAL

Immunodominance limits the TCR diversity of specific, anti-viral CD8 T cell responses elicited by vaccination or infection. To prime multispecific T cell responses, we constructed DNA vaccines that coexpress chimeric, multidomain antigens (with CD8 T cell-defined epitopes of the hepatitis B virus (HBV) surface (S), core (C) and polymerase (Pol) proteins, and/or the ovalbumin (OVA) antigen as stress protein-capturing fusion proteins. Priming of mono-or multispecific, HLA-A*0201-or K b -restricted CD8 T cell responses by these DNA vaccines differed. K b /OVA 257-264 -and K b /S 190-197 -specific CD8 T cell responses did

Although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening EBV-associated diseases, such as posttransplant lymphoproliferative disorders (PTLD). Thus, immunotherapeutic strategies using adoptively transferred EBV-specific T cells are promising. One option is the generation and expansion of CD4 + and CD8 + T lymphocytes by using EBV-specific synthetic peptides for the stimulation of pre-existing memory T cells. Aim of our study was to identify a set of MHC class-II peptides

For each antigen promiscuitive peptides with high SYFPEITHI scores were tested for immunogenicity using an IFN-g-ELISPOT. PBMCs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. Functional and phenotypic analysis of T cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as T-cell epitopes. Two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific T cell responses. So far, eight of the tested peptides could be identified as MHC class-II epitopes. Furthermore, a highly immunodominant class II peptide mix consisting of 5 peptides was selected. In conclusion, we could identify several new EBV-specific MHC class-II epitopes which can be used for

United Kingdom, 3 Hospital de Clínicas

During persistent HBV infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.In order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic HBV infections

A chimeric TCR against HBsAg, which aims to increase the percentage and quality of antigen-specific CD8 + T cells, was developed

Moreover, we pre-conditioned the liver microenvironment by injection of CpG oligodeoxynucleotides (ODN) to optimize the recruitment of transferred CD8 + T cells to the liver and to overcome the tolerogenic microenvironment of the liver. We found that the IL-12-exposed CD8 + T cells showed at least five-fold increase of survival rate in vivo than IL-2-exposed CD8 + T cells did

Treatment of the recipients with CpG-ODN could increase the percentage and also the total amount of transferred CD8 + T cells mainly in the liver. By in vivo BrdU incorporation, we demonstrated that the higher in vivo survival rate of IL-12-exposed CD8 + T cells and the effect of CpG-ODN were due to the up-regulation of the proliferation of those cells. To sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific CD8 + T cells to the liver to exhibit their effector functions. The detailed mechanisms responsible for the IL-12 and CpG-ODN effects on the regulation

Hyper IgM (HIM) and Wiskott-Aldrich syndrome (WAS) than those of corresponding controls (P X 0.01) . There was a significant elevation of t ADA and ADA1 activities in IgA deficient patients as compared to healthy individuals (P X 0.01) . Our results hypothesized that altered ADA activity may be associated with altered immunity. Therefore, serum ADA level could be used as an indicator along with other parameters

PD01/61 HIV-1 SEQUENCE EVOLUTION AFTER DENDRITIC CELL-BASED IMMUNE THERAPY IN A PHASE I/II CLINICAL TRIAL

HIV RNA was extracted from plasma samples collected before the startof HAART and early after vaccination when HAART was terminated. RNA was amplified by RT-PCR and sequenced using standard protocols. Sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. HIV sequences spanning known and predicted epitopes of the relevant HLA alleles from each participant were analyzed in detail. Results and conclusion: Immune therapy was well-tolerated and no severe adverse effects occurred. After HAART termination, plasma viral load became detectable in all patients after 2-6 weeks. Follwing the viral rebound a set point was reached, that was lower than the viral load before start of HAART. Using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract B. De Keersmaecker et al.). For studying sequence evolution, complete sets of both pre-HAART and post-vaccination sequences were obtained in 12 out of 17 patients. With one exception, variation in sequences of vaccine and control genes of pre-HAART samples compared to samples taken early after vaccination was limited. This indicates that there was no significant impact of the immune response on virus evolution at this stage. More focussed analysis on viral sequences spanning specific HLA

Islamic Republic of Newcastle disease (ND) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. The purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to ND live vaccine. One hundred and twenty, 10-day-old ROSS 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. The first experimental group vaccinated by a monovalent ND live vaccine with CL/79 strain, and the second experimental group vaccinated by a bivalent Newcastle disease and Infectious bronchitis live vaccine with CL/79 and H120 strains, via the drinking water at 10 days of age at the same time, and the control group received no ND vaccine. The antibody response to vaccination was assessed using the hemagglutination inhibition (HI) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. Results indicated that, although the strain of studied ND live vaccines were the same

United Kingdom T cell-based IFNg release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active TB from successfully treated disease or latent infection. T-cell cytokine profiles vary with in vivo antigen load in viral infections

CD4 T cells from 25 patients with active TB and 28 patients with successfully treated TB were analysed for simultaneous expression of IFNg and IL2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with PPD. Moreover, cells were stimulated with ESAT-6 and CFP-10

Receiver operator characteristics analysis revealed that a percentage of IFNg /IL2 dual positive cells X 56 % served as an accurate marker for active TB patients (specificity 100 %, sensitivity 65 %), while frequencies G 56 % were observed in treated as well as active TB patients. In conclusion, quantitation of antigen-specific T cells based on the analysis of IFNg only does not allow distinction of patients with active and successfully treated disease

PD03/7 NECESSITY OF POSTPONE BCG VACCINATION -LESSON FROM PRIMARY IMMUNODEFICIENCIES V. Thon

Methods and results: The Czech national database of primary immunodeficiencies (PID) was established in 1993 and is connected with the European database of primary immunodeficiencies (ESID). The prevalence of PID in the Czech Republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. Among these patients there are children diagnosed with severe combined immunodeficiency (SCID) and chronic granulomatous disease (CGD) too. According to the Czech national database of PID, 12 out of 14 children with later proved SCID were immunised with BCG vaccine in the first days of life. Nine of them developed disseminated and generalized BCG infections. Five children with SCID died. Moreover, reactivation of BCG was also seen in healthy children after admission of combined vaccines with hepatitis B given at the age of twelve weeks. On the other hand, this was not the case in thousands of children of HBsAg positive mothers who were vaccinated against hepatitis B after delivery in the first place and later immunized with BCG vaccine. Systematic vigilance against tuberculosis (TB) and vaccination significantly lower the prevalence and risks of TB. In the Czech Republic, the prevalence of TB is currently 6.12 to 100 000 inhabitants. Unfortunately, temporary interruption of BCG vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of TB and appearance of new cases of aviary mycobacteriosis. These complications were not observed in vaccinated children. Conclusion: We recommend a change of current practice of BCG vaccination considering new immunization schedule with hexavalent vaccine

PD03/8 NOVEL ANALOGUES OF THALIDOMIDE INHIBIT CD80 EXPRESSION AND PRODUCTION OF TNF-a, IL-12, IFN-g, CXCL-9

This work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. Our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating ENL and other diseases. Supported by: CNPq, FAPEMIG and CAPES, Brazil. of the B cell follicle. CTA1-DD augmented GC-formations, specific antibody responses and cell-mediated immunity to the T cell-dependent antigen NP-CGG, but failed to do so when used together with T cell independent antigens, such as NP-Ficoll or NP-Dextran. This effect required ADP-ribosyltransferase activity, as mutant CTA1R7K-DD failed to exert an adjuvant effect. The adjuvant function appeared to correlate with the FDC-localization and turned out to require complement and/or complement receptors (CR)

Chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by ELISA. We found that, in contrast to alum, PLG and PS particles, chitosan induced BMDC maturation on its own, as determined by the expression of CD80 and CD86. These effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. The effect of chitosan on cytokine production was investigated using a panel of different TLR agonists in combination with chitosan particles. Results show an increase in the secretion levels of IL-1a and IL-1b, while IL-6 levels were not affected. Finally we studied the role of inflammasome activation in the enhancement of IL-1b production. Using BMDC from nlrp3 -/-mice we examined IL-1b production in response to different TLR and chitosan combinations. Results show that the ability of chitosan to enhance IL-1b production is dependent on nlrp3. Collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan

Severe sepsis, induced in mice by cecal ligation and puncture (CLP), led to HO-1 expression in infiltrating peritoneal leukocytes, kidney and liver. Mortality rate of CLP increased from 20 % in wild type (Hmox1 +/+ ) mice to 87 % in HO 1 deficient (Hmox1 -/-) mice. Hmox1 -/-but not Hmox1 +/+ mice developed end-stage multiorgan failure. Mortality of Hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. CLP induced a significant increase in cell-free hemoglobin

Free heme was found to sensitize primary hepatocytes to TNF, anti-Fas antibody, H 2 O 2 or peroxynitrite mediated apoptosis. This cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine HMGB1. Similarly, circulating and cytoplasmic HMGB1 was increased in Hmox1 -/-relative to Hmox1 +/+ mice following CLP. In conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe

and b-1,2-linked mannose residues elicit inhibition effect. It was found that inhibition activity of oligosaccharides increases with chain length. Immunization with mannan-HSA conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. In the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. Acknowledgements: This work was supported by the Grant Agency of Slovak Academy of Sciences

All subjects received DTwP vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of Iran. Blood samples were collected before and 2-4 weeks after the vaccination. Immunogenicity of the vaccines was assessed by ELISA using commercial kits. Results: The geometric mean titers (GMT) of the antibodies induced against diphtheria and tetanus by DTwP-Local were 7.7 and 9.4 IU/ml and those of DTwP-Pasteur were 8.2 and 8.6 IU/ml, respectively. There was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. The GMTs of antibodies produced against pertussis were 30.2 EU/ml for DTwP-Local and 47.9 EU/ml for DTwP-Pasteur vaccines, respectively (P X 0.001). No significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. Conclusion: Immunogenicity against diphtheria and tetanus was similar for the two vaccines

PD05/18

United Kingdom Haemorrhagic Septicaemia (HS) is an acute disease of cattle and buffaloes in tropical countries, caused by Pasteurella multocida serotype B:2 , a Gram-negative coccobacillus. JRMT12, an aroA mutant of Pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. In this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 CFU of JRMT12. A possible contributory role of cellular immunity against HS was investigated in vaccinated and in control calves after challenge. A lymphocyte stimulation assay was used to assess the effects of a cell-free extract (CFE) of P. multocida on peripheral blood mononuclear cells (PBMCs) isolated from calves at different times after challenge. The results were indicative of a possible immunosuppressive effect of challenge with P. multocida B:2 on calf PBMCs. The suppressive effect was further investigated by in vitro experiments. Calf PBMCs obtained from normal calves were treated with CFE for 1 h before adding concanavalin-A (ConA)

PD06 -Vaccination and Immunotherapy Against Parasitic Diseases PD06/1 EVALUATION OF SIMIAN ADENOVIRAL VECTOR ADCH63 EXPRESSING MSP-1 AS A CANDIDATE BLOOD-STAGE MALARIA VACCINE

This successful regime incorporated a human adenovirus serotype 5 (AdHu5) prime, boosted eight weeks later with a modified vaccinia virus Ankara (MVA) vector. Adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. Their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. Recent clinical trials have suggested that the simian adenoviral vector AdCh63 is a promising clinical candidate. We have developed vectors (of human and simian origin) and MVA encoding a novel construct based on P. falciparum MSP-1 and have undertaken comparative immunogenicity studies in mice. The antigen, termed 'PfM128

While asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by Plasmointo carbon monoxide (CO), iron and biliverdin. When infected by Plasmodium Hb SAD mice are protected against experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. HO-1 expression and activity are strictly required to suppress ECM in Hb SAD mice, as demonstrated by functional deletion of the Hmox1 locus or pharmacologic inhibition of its enzymatic activity. The protective effect of HO-1 is mediated by CO, which inhibits the accumulation of protein-free heme in plasma following Plasmodium infection

Conclusion: Topical treatment of cutaneous leishmaniasis with GSNO accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben

GP63 expression was confirmed by SDS-PAGE and ELISA using monoclonal antibody against GP63. Discussion: Today researchers attempt to find a suitable vaccine for leishmaniasis. Although some researchers have reported proper vaccines

Of interest, A5-180recP is recognized only by sera collected from resistant bovines infested with all stages of R. microplus, but not by sera from similarly infested, susceptible hosts. Furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with A5-180recP. Our results show that Reverse Immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. Supported by CNPq and FAPESP. for the maintenance of Ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. Memory B-cell ELISPOT together with Ab titres might therefore prove useful as independent marker for duration of protection. Objective: This study focused on establishing experimental conditions and optimizing the performance of the memory B-cell ELISPOT assay by detection of specific memory B-cells against anti-tetanus vaccine and naturally acquired Toxoplasma gondii infection as a model. Methodology: Twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated from each donor using cell preparation tubes (CPT). Plasma was obtained after centrifugation of CPT and stored at -20°C until used for ELISA. Specific IgG-secreting B-cells were determined by ELISPOT assay, using tetanus toxoid (TT) and T. gondii surface antigen as model antigens. Results: To optimize our assay, conditions were changed and compared to the previously established protocol. We detected low frequencies of total IgG memory B-cells and TT-specific memory B-cells in all donors

Four seropositive and 2 seronegative donors had positive responses in ELISPOT. No correlations were found with serum antibody titers and frequencies of memory B cells (r=0.216, P=0.641) or with T. gondii-specific B-cells

Conclusions: Following optimization of several assay parameters, we demonstrated that the memory B cell ELISPOT could be reliably used to determine low numbers of antigen-specific memory B-cells in individuals naturally exposed to infection or following vaccination

Our previous work demonstrated that IL-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, BFU-E, but inhibiting the growth of late stage erythroid progenitors, CFU-E, from normal murine bone marrow. We also provided in vitro evidence that at least part of its effect on CFU-E is mediated by nitric oxide (NO) generation. In the present study we demonstrated that the in vivo reducing effect of IL-17 on bone marrow CFU-E was prevented by co-treatment with the NO synthase (NOS) inhibitor, L-NAME, implying that this effect is mediated through NOS activation. The data obtained in cultured bone marrow cells showed the ability of IL-17 to upregulate the expression of mRNA for both the inducible (i)NOS and the constitutive, endothelial (e)NOS isoform. Both the NOS-inducing effect of IL-17 and IL-17-related inhibition of CFU-E growth were dependent on p38 MAPK activity, since the p38 MAPK inhibitor, SB203580, markedly downregulated IL-17-induced activation of NOS and reversed the growth inhibitory effects of IL-17 on CFU-E. The in vivo stimulating effect of IL-17 on BFU-E colony growth in the bone marrow was not affected by co-treatment with the NOS inhibitor, pointing to different mechanisms for IL-17 effects on BFU-E and CFU-E. However, the in vivo exposure of the mice to L-NAME, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that NO itself is important regulator of hematopoietic progenitor cell activity. Overall, the data presented gave an insight into the mechanisms by which IL-17 acts on bone marrow cells and also revealed a link between the IL-17, NO and hematopoiesis. Further studies on IL-17-mediated induction of both iNOS and eNOS

Methods: A total of 1785 blood donor samples were tested for HBsAg and Anti-HBc with the immunoenzymic method ELISA, while simultaneously, molecular blood test (NAT) was applied. The positive samples for Anti-HBc were also tested for Anti-HBs and Anti-HBc IgM. Results: A total of 68 samples (3,8 %) were found Anti-HBc positive

Conclusions: It is proven, therefore, that in some cases the levels of HBsAg, following an infection from the Hepatitis B virus, are probable to remain low, so that it is not possible to detect them using ELISA method. In these cases Anti-HBc can be the only serological marker of the infection. Consequently, patients with positive Anti-HBc and levels of Anti-HBs X 100 IU/L are possibly not immune enough, so that they can become blood donors. That was the reason why some blood donation centers in our country, until recent years when there was no capability for NAT testing of blood donors, had 100 IU/L as a limit for Anti-HBs levels. However, in present days that NAT testing of blood donors is used in our country, it has offered great safety and it is possible that Anti-HBc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 IU/L Anti-HBs in all the blood units, which also goes for our study

PD14/20 NEONATAL ALLO IMMUNE THROMBOCYTOPENIA AND IGG GLYCOSYLATION PATTERNS

Michaelsen 1,2 1 National Institute of Public Health

In milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. The reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. Recently influence of glycosylation patterns of IgG on the biological activity has been realized. Variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with Fc-receptors, and hence a difference in thrombocyte elimination capacity of the IgG molecule. Patient sera from Norway and The Netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate IgG antibodies against the HPA-1A alloantigen and analyze the glycopeptides using mass spectrometry. The glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. So far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated N-glycans on their anti-HPA-1A IgGs

We monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. In 11 of them ALL was diagnosed for the first time. 1 subject had the second relapse of ALL. One patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. Methods: Leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. Hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. We valuated spontaneous and zymosan induced hemiluminescence. We used onsonised zymosan as the inductor of "respiratory paroxysm

Mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and RING finger containing protein Roquin, develop severe autoimmune disease. The observed lupus-like phenotype involves follicular helper T cells, which express higher levels of ICOS. These cells provide inappropriate T cell help to B cells, leading to the production of autoantibodies (Vinuesa et al. 2005, Nature 435, 452-8). It has been shown that the half-life of ICOS mRNA is shortened when Roquin is over-expressed. Such repression requires the 3'UTR of ICOS, in which a 47bp sequence, containing a possible miR-101 binding site, was sufficient (Yu et al. 2007, Nature, 450, 299-303). Mnab is the paralogue of Roquin, and has been shown to bind to nucleic acids (Siess et al. 2000, J Biol Chem 275, 33655-62). We demonstrate that in primary mouse T cells and embryonic fibroblasts Roquin, but not Mnab, inhibits translation of ICOS. We map critical domains in the Roquin protein to ICOS repression using deletion-and point-mutants of Roquin, as well as chimaeras that swap sequences from Roquin to Mnab and vice versa. Addressing the mechanism of Roquin mediated ICOS repression; we demonstrate binding of Roquin to ICOS mRNA in primary mouse T cells and in cotransfection experiments. Our current work dissects the requirement of cellular RNAi, the stress response pathway or P-body function by testing Roquin repression of ICOS mRNA in Dicer-, Tia-1-and Ago2-deficient MEF cells and in knockdown approaches. Acknowledments: J M M-V is a recipient of a Harvard Real Colegio Complutense (RCCH) grant. Work in Dr Tsokos' lab is supported by grant PHS NIH R01 AI 42269.

We have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-HMA) isolated from Brassica rappa inhibit nuclear factor-kappa B (NF-xB) activity in Raw 264.7 macrophages. In this report, we investigated the effect of 6-HMA on dextran sulfate sodium (DSS)-induced colitis model in mice. Methods: We induced colitis with DSS in mice and evaluated disease activity index (DAI), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (MPO) accumulation. Through H&E staining, histological change was observed. The expression of inducible nitric oxide synthase (iNOS), inhibitory kappa B-a (IxBa) and NF-xB were detected by western blot and immunohistochemical staining. In-vitro system, the expressions of interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) in HT-29 human colon epithelial cells were measured by RT-PCR. Results: In DSS colitis model, the DAI score and detection of MPO accumulation brevealed 6-HMA significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. Moreover, H&E staining also indicated 6-HMA suppressed the thickness of muscle layer, edema, mucosal damages by DSS. These results were related to the regulation of NF-xB activation. 6-HMA attenuated the DSS-induced phosphorylation and translocation of NF-xB subunit p65. In addition, this effect was accompanied with parallel blocking degradation of IxBa. Moreover, pretreatment of 6-HMA significantly reduced the mRNA levels of IL-8 and MCP-1 stimulated by tumor necrosis factor-a (TNF-a) in the HT-29 cells. Pretreatment of 6-HMA also significantly blocked the IxBa degradation and NF-xB p65 nuclear translocation stimulated by TNF-a in the HT-29 cells. These results were concurred with the effect on NF-xB activation in DSSinduced colitis model. Conclusions: These results for the first time demonstrated that alleviation of 6-HMA mediated by regulation of NF-xB activation and suppression of chemokines in vitro and in vivo. Therefore, 6-HMA could be new potential therapeutic agent for inflammatory bowel disease.CD4 serves as receptor of HIV and is a self-antigen. We have previously characterized the anti-CD4 IgG immune response in HIV-1-exposed, seronegative (ESN) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in HIV-infected patients (second CD4 domain preferred). To generate antibodies able to inhibit the infection of HIV virus trying to learn from what happen in nature in ESN we used a particular immunization procedure. We immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human CD4, in the absence of the target cells expressing the co-receptor CCR5. The latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the CD4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (CCR5 expressing) cells. Thus, in the absence of CCR5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during HIV-1 infections. A conventional protocol for the generation of monoclonal antibodies was used. DB-81 (IgG1, x), one of the anti-CD4 antibodies obtained, recognized preferentially CD4 complexed to gp120, as compared to CD4 alone, not competed for the gp120 binding site on CD4 and was specific for the second extracellular domain of CD4.

G. Röder 1 , L. Geironson 2 , A. Darabi 3 , M. Harndahl 1 , C. Schafer-Nielsen 4 , K. Skjödt 5 , S. Buus 1 , K. Paulsson 2 1 Copenhagen University, Institute of International Health, Immunology and Microbiology, Department of Experimental Immunology, Copenhagen, Denmark, 2 Lund University, Immunology BMC D14, Lund, Sweden, 3 Lund University, Rausing Laboratory, Division of Neurosurgery, Department of Clinical Sciences, Lund, Sweden, 4 Schafer-N, Copenhagen, Denmark, 5 Department of Immunology & Microbiology, University of Southern Denmark, Odense, DenmarkCytotoxic T-lymphocytes become activated by binding to MHC-I molecules presenting antigenic peptides. The loading of peptides onto MHC-I takes place in the ER and involves different chaperones and enzymes. Tapasin binds MHC-I molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported MHC-I molecules. How Tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. Here, we have generated the first 87 N-terminal amino acids of human Tapasin, Tpn , and shown that this fragment of Tapasin facilitates peptide dependent folding of HLA-A*0201. To further investigate the properties of Tpn and Tapasin, we generated multiple mouse monoclonal antibodies towards Tpn and wildtype human Tapasin. One clone, aTpn 1-87 /80 , was found to be specific for natural human Tapasin and stained cellular ER localized Tapasin. Using peptide chip technology, the epitope of aTpn /80 was demonstrated to be located on Tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the Tapasin structure. Together, these results demonstrate that, the first 87 N-terminal amino acids of Tapasin are able to facilitate peptide-binding to MHC-I, and as well, this fragment can be recombinantly expressed in E.coli and fold into a structure, which at least partially, resembles that of wild-type human Tapasin. We speculate that this region of Tapasin might support empty, open and receptive MHC-I peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this Tapasin region might be essential for enabling peptide editing.

A Objectives: Antigen processing and presentation through HLA class I molecules is critical for an effective destruction of infected or transformed cells by CD8+ T lymphocytes. Different intracellular routes governing the processing of endogenous and exogenous antigens have been described. We show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to CTLs. Methods: To produce genetic in-frame Tat fusion proteins, DNA sequence encoding for the amino acid region 301-498 of the influenza A virus nucleoprotein (NP) was inserted into the expression vector pTAT-HA. Starting from TATNpFlu recombinant protein we produce hybrid proteins, in which the HLA-B*2705-restricted NP-FLU epitope (aa 383-391) was replaced by HLA-B27 or HLA-A2-restricted epitopes of EBV and HCV, respectively. Cross-presentation was evaluated according to the standard 51 Cr release assay and through the IFN-g production. Results: Using HLA-B27 or HLA-A2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the HLA-B27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the HLA-A2 molecules present the epitopes in a classical proteasome-dependent pathway performed by DCs. Furthermore, different HLA-A2 restricted epitopes can be inserted in tandem and presented to the specific CTLs without interfering each other.

The data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. These findings also enlighten HLA-B27 as a remarkable HLA-class I molecule that, differently from HLA-A2, can present peptides through additional, unconventional antigen presenting routes. This could concur to an imbalance of the immunological properties of the HLA-B27 molecules leading to a more effective response towards viral as well as self -antigens. Objectives: Although cytotoxic T cells (CTL) in human immunodeficiency virus (HIV-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. CTL play a key role in limiting viral replication in infections caused by e. g. Epstein-Barr virus, Cytomegalovirus, Hepatitis C virus and HIV1. Consistent patterns of immunodominant and subdominant CTL-responses have been found between individuals with the same HLA-alleles in both acute and chronic infection. As the CTL-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. However, the factors determining these CTL-response hierarchies are largely unknown. While structural differences between peptide-HLA class I complexes may be important for TCR-repertoire selection and clonal expansion, it is less obvious how they impact CTL-response hierarchy formation and timing. Other factors may also contribute, e. g. epitope abundance at the cell surface. Methods: Antigen processing efficiency of CTL epitopes from the p17-Gag and p24 region was determined in vitro. 25mer peptides were digested with i20S and c20 proteasomes and the fragments identified by mass spectrometry. For epitope precursor peptides generated by the proteasome, we then determined TAP affinity, trimming by ERAAP and HLA-binding affinities and analyzed patient responses by EliSpot. Results: We show that CTL-immunodominance in regions of HIV-1 p17-and p24-Gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (TAP) affinity and endoplasmatic reticulum aminopeptidase (ERAAP)-mediated trimming, and moderately by HLA affinity. Proteasomal cleavage-preferences were affected by flanking and intra-epitope CTL-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting T cell response avidity and clonality. Conclusion: Our analyses reveal that antigen processing plays a pivotal role in determining CTL-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of CTL-epitope-based vaccines.

T. F. Gregers 1 , G. Koster 1 , O. Landsverk 1 , F. Skjeldal 1 , O. Bakke 1 1 University of Oslo, Molecular Biosciences, Oslo, Norway MHC II is synthesized and assembles in the ER together with Invariant chain (Ii). Ii facilitates MHC II assembly followed by transport to the MHC II loading compartment (MIIC) where peptide loading occurs. MIIC is multivesicular late endosomal compartments resembling conventional multivesicular bodies (MVBs) found in all cells. It is not known whether the biogenesis of MIICs is regulated by the same mechanisms as formation of MVBs. Expression of Ii induces the formation of enlarged endosomes and we have previously shown that Ii modulates antigen processing and presentation. We have suggested that Ii itself can act as a tethering factor involved in fusion of Ii containing endosomes, and our main question is whether Ii can regulate the formation of an endosomal pathway dedicated for antigen processing and MHC II loading.In order to investigate this we use cell lines expressing Ii controlled by an inducible promoter, thus being able to control the Ii expression level and thereby the endosomal size. Live imaging and high through put microscopy of Ii expressing cells treated with inhibitors and/or specific siRNAs have revealed that Ii induced endosomal fusion is independent on type III PI3 kinases and thus PtdIns(3)P. This is in contrast to conventional endosomal fusion and MVB formation. Thus other factors might be important for MIIC biogenesis. By using small RNAi libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in Ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell.

M. Bouvier 1 , L. Visvabharathy 1 , J. Fu 1 1 University of Illinois at Chicago, Microbiology and Immunology, Chicago, United StatesObjectives: Adenoviruses (Ads) cause persistent infections. The E3-19K protein from Ad targets class I MHC molecules for retention in the endoplasmic reticulum (ER), thereby preventing the cell-surface presentation of viral peptides. This escape from immune surveillance allows Ads to freely replicate in host cells. The molecular mechanism of E3-19K-mediated class I retention is mostly undefined. It is clear that further characterization of this mechanism is important to understand the susceptibility of the class I antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of Ad pathogenicity.

We used biophysical and cell-based approaches to examine interaction between Ad type 2 E3-19K and class I molecules.Results: We showed that E3-19K associates with immature (peptide-deficient) and mature (peptide-filled) HLA-A11 molecules, with the mature form being more tightly associated. We also provided evidence that E3-19K does not compete with the class I assembly proteins for binding onto class I molecules. Importantly, immature class I molecules sequestered by E3-19K can still bind peptides. Together, these results suggest that Ads have evolved to interfere with the early and late stages of the class I antigen presentation pathway. Evidence was also provided that E3-19K displays an allele-and locus-specificity towards class I molecules with High-density lipoprotein (HDL) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. Recent data indicate that qualitative changes of HDL including oxidative modifications and alterations of the protein cargo of HDL may alter its biological activity. Here we analyzed the anti-inflammatory potential of HDL and compared it with HDL obtained from patients with end-stage renal disease (ESRD), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. We demonstrate that freshly isolated, but not oxidized HDL from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (APC) such as monocytes and dendritic cells, which are regarded as the most potent APC. Production of typical proinflammatory cytokines (IL-12, IL-6, TNF-a) were significantly suppressed by HDL after stimulation of monocytes or dendritic cells with Toll-receptor ligands 2 and 4, but also with the T-celldependent stimulus CD40L (CD154) indicating an immunomodulatory effect independent of agonist neutralization by HDL. Moreover, surface expression of crucial activation and costimulatory molecules like CD40, CD83, and CD86 was inhibited by freshly isolated, but not oxidized HDL. The negative regulatory effect of HDL on cytokines and surface receptors occurred at the transcription level, while HDL did not modulate the activity of the major inflammatory transcription factor NF-kB or the MAP kinases p38 and Erk-1/2. Strikingly, HDL from ESRD patients not only failed to block, but rather promoted proinflammatory cytokine production and APC activation. These data identify HDL as a novel potent anti-inflammatory regulator of professional APC, which may help to dampen excessive inflammatory responses of the innate immune system. Conversely, qualitative changes of HDL leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in ESRD patients. Objectives: CD4+ T cell abnormalities may play a role in the autoimmune pathogenesis of Churg Strauss Syndrome (CSS). On one side, TH2 (IL-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of CSS. On the other, TH1 (IFN-g+) cells could participate in vessel wall damage and granuloma formation. In order to define this TH1 / TH2 balance and to identify potential T cell target antigens (Ags), we analyzed circulating CD4+ T cell responses to polyclonal stimuli and to myeloperoxidase (MPO) in CSS and healthy subjects. Methods: IFN-g and IL-4 expression in peripheral blood CD3+CD4+ lymphocytes were measured in 9 CCS patients and 7 healthy subjects (HS) upon polyclonal stimulation, both by intracellular staining and by ELISA. MPO-driven IL-4/IFN-g production was assessed by ELISPOT on T cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated MPO, negative control protein or hexavalent vaccine (positive control recall Ags). Results: Upon polyclonal stimulation, higher IL-4 and lower IFN-g intracellular expression were detected in CD4+ T cells from CSS patients, as compared to HS (IL-4: 1.3±0.3 % vs. 0.50±0.21 %, p X 0.05; IFN-g: 14.2±4.5 % vs. 27.0±4.8 %, p X 0.025). Similar results were obtained by ELISA (IL-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p X 0.05; IFN-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p X 0.05). ELISPOT counts of hexavalent vaccine-stimulated CD4+ cells were positive for IL-4 in 4/5 (80 %) CSS patients and in 5/5 (100 %) HS, and for IFN-g in 2/9 (22 %) CSS patients and 7/7 (100 %) HS. MPO stimulation determined significant IFN-g release in 5/8 (62 %) CSS patients, but not in HS (0/5) No IL-4 response to MPO in both groups was observed. Conclusion: Polyclonally or recall Ag-stimulated CD4+ cells from CSS patients show a TH2-polarized cytokine profile. MPO is here first identified as a CSS-related Ag targeted by CD4+ T cells, and responses towards it are instead TH1-polarized. These data unfold one molecular target and possible pathogenic mechanisms of CD4+ T cells in CSS.

A. Voigt 1 , E. Opitz 1 , K. Savvatis 2 , K. Klingel 3 , K. Stangl 1 , U. Kuckelkorn 1 , P.-M. Kloetzel 1 1 Charité -Universitätsmedizin Berlin Campus Mitte, Berlin, Germany, 2 Charite -Campus Benjamin Franklin, Berlin, Germany, 3 Universität Tübingen, Tübingen, GermanyMurine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. Immunoproteasomes (IP) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. Our previous work has established that IP expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.Here, we investigated the impact of IP function in enterovirus myocarditis. Mice, which are deficient in immunosubunit LMP2 of the stress-induced IP, were infected with 1x10e5 PFU CVB3 Nancy strain. In concurrence to wt littermates, we observed a pronounced up-regulation of cardiac IP subunit LMP7 as early as day 4 p. i. in LMP2-deficient mice. However, LMP2-deficiency was linked to less severe myocarditis at day 8 p. i. (HE stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. LMP2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p X 0.001). Whereas the cardiac output (CO) was reduced in wt littermates in enterovirusmyocarditis (p X 0.05), there was no difference in LMP2-deficient mice in comparison to sham-treated mice. Maximal left ventricular pressure and dPdt max were impaired in acute myocarditis in wt littermates. In contrast, systolic function was not affected by CVB3 infection in LMP2-deficient mice. Likewise, diastolic function was preserved in LMP2-deficient mice upon enterovirus infection. Our findings of less severe myocarditis in LMP2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.In conclusion, this study suggests an impact of LMP2-immunosubunit function in regulatory processes of viral replication. Absence of LMP2 confers host protection in enterovirus myocarditis.

H. W. Liao 1 , J. Xu 1 , J.Q. Huang 1 1 Sun Yat-Sen University, Guangzhou, ChinaThe characteristic of the Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. The pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. In this work, we showed that Dengue virus serotype 2 (DEN-2) strain induced apoptosis in human umbilical vein endothelial cells (HUVECs). Additionally, Fas expression was increased on infected HUVECs. TRAILR1 and TNFR1-2 were constantly very low whereas TRAILR2-4 decreased after DEN-2 infection. FasL was expressed at similar levels on HUVECs throughout DEN-2 infection. The apoptotic rates in HUVECs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by DEN-2 infection. There were no significant changes of NO in our study. We thus proposed that the Fas/FasL pathway might be involved in apoptosis induced by Dengue virus in vascular endothelial cells in vitro. Dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. It seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. Aim. To study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. Materials & Methods. Group I. S. epidermidis was injected daily for 7 days into WIS rat paw web tissue in saline containing 7.5x10 7 cells. Group II. S.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. Nodes were isolated on day 8.They were weighed, the cell number was counted and cells were stained with mAbs for immunohistochemical analysis. Immunohistochemical pictures were analyzed by Microimage program. Results. Group I. Skin contained some MHCII cells. The popliteal lymph nodes became enlarged on the bacteria injected side. There was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p X 0.05). Immunohistochemical pictures showed increase in percentage of OX62 (migrating dendritic cell), MHC II, His48 (granulocytes), OX7 (stem cells) and CD54 (ICAM I) subsets in the subcapsular , follicle, paracortex and medullary areas. Group II. After ligation of afferent lymphatics the weight of nodes was not significantly increased. Skin showed presence of multiple MHC II, ED1 (macrophages) and OX62 cells. Popliteal lymph nodes contained evidently less of OX62, His48 and MHCII cells than in group I (p X 0.05). Summary & conclusions. Afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. Local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. Interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. These observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. Omega-3 fatty acids, and in particular docosapentaenoic acid (DHA) and eicosapentaenoic acid (EPA) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. Our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. It is based on reviewing and processing data obtained by search of scientific and medical databases. Search terms used were: atheroma, atherosclerosis, cardiovascular disease (CAD) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, EPA, DHA. We also searched epidemiological research Web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption.

The results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. Omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. Combined therapy with omega-3 fatty acids and statins shows a synergistic effect. Methods: On ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. The bottom layer was shown to contain immune complexes (IC) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.Both these forms of Lp(a) were Western blotted and probed with jacalin-HRP, anti-Gal-HRP and anti apo(a)-HRP. Anti-Gal was prepared by affinity chromatography on guar galactomannan and complexed with Lp(a) in vitro. IC formation by Lp(a) was measured in terms of reduction in response in a new ELISA for Lp(a) involving addition of Lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-HRP detection. IC formation was also shown by migration of Lp(a) from free lipid layer to IC layer below in ultracentrifugation. Results: Anti-gal and Lp(a) could be liberated from precipitated IC using specific sugar. Immune complexed Lp(a) in serum was found to be more O-glycosylated, larger in size and binding more anti-Gal than Lp(a) in free form in Western blots. While IC formations within homologous free anti-Gal-free Lp(a) pairs were few, those within heterologous pairs were more rampant.

Conclusions: Lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. Anti-Gal comprising 1 % of serum IgG is rich in IgG capable of complement fixation and macrophage mobilisation. Present results offer a viable mechanism of Lp(a)-mediated immune injury to vessel walls leading to vascular damage. Even though no receptors have been detected for Lp(a), unlike for LDL, the present results may explain the internalization of Lp(a) in the form of Lp(a) immune complexes by macrophages since the latter can phagocytose IC. Extended specificity of the a-galactoside-specific anti-Gal for T-antigen in Lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. Objective: The aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the E2F2 mutation, and other affecting B cell apoptosis control, such as Bcl-2 over-expression, can induce the development of AIS. Methods: Mice: Mice with both genetic abnormalities were generated in a non-susceptible C57BL/6 (B6) genetic background. E2F2-/-Bcl-2Tg were obtained backcrossing E2F2-/-and E2F2+/+hBcl-2Tg mice. E2F2-/-Bcl-2Tg, E2F2-/-, E2F2+/+hBcl-2Tg and control mice (E2F2+/+) were followed up to 18mo-old. Serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for IgG and IgA ANA and anti-dsDNA by ELISA. Histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (H&E) and Masson's trichrome to identify histological changes. Immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse IgG, IgM and IgA. To evaluate B cell homeostasis, absolute number of B cell in blood, primary and secondary lymph organs were assessed by flow cytometry. In vitro proliferation was measured with [H3]-thymidine and BrdU was used to assess in vivo proliferation capacity of immature B cells. Results: Overexpression of hBcl-2Tg in B lymphocytes of E2F2-/-mice induced the production of high titres of IgG and IgA ANA and anti-dsDNA, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the IgA isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. E2F2-/-Bcl-2Tg mice showed an altered B cell homeostasis as demonstrated in proliferation and apoptosis studies. E2F2-/-mice showed neither autoantibodies nor nephropathy.

This study demonstrates that the isolated deficiency of E2F2 or the overexpression of a Bcl-2 Tg in the B6 genetic background do not induce an AIS. When combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild AIS with overproduction of IgA autoantibodies. An alteration in the B cell compartment, but not in the T cell compartment, seems to be underlying the syndrome described in the present work. In different mouse models of the autoimmune disease Systemic Lupus Erythematosus (SLE) loss of Toll-like receptor 9 (TLR9) abolishes the generation of antinucleosome IgG2a and IgG2b autoantibodies but exacerbates lupus disease. However, the TLR9-dependent tolerance mechanism is unknown. Here we show that loss of TLR9 in B cells of lupus prone mice prevents the generation of protective T cell-dependent self-reactive IgM and thereby enhances the development of Th1 and Th17 T cells. Transfer of a synthesized monoclonal polyreactive IgM to TLR9 deficient lupus prone mice inhibits T cell activation and abolishes development of lupus disease. Thus, these results document a protective TLR9-dependent tolerance mechanism in B cells that induces the generation of self-reactive IgM to prevent autoimmunity. Cloning and production of polyreactive or antigen-specific IgM might therefore be a powerful tool to treat autoimmunity. Objectives: To investigate cytokine and autoantibody levels in serum from patients with primary Sjögren's syndrome (pSS), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. Methods: Minor salivary gland tissue was obtained from a group of patients fulfilling the revised EU-US criteria for pSS (n=115) (Vitali et al. 2002) . Ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (FS) G 1 (FS+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (FS-). Germinal center (GC)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. Serum samples were used for cytokine and autoantibody evaluations.

The mean level of unstimulated whole saliva was significantly lower in the FS+ patients compared with the FS-patients, and in the GC+ patients compared with the GC-patients (p X 0.05). Interleukin (IL) 17, IL-1RA, IL-1beta, IL-12p40, IL-15, macrophage inflammatory protein (MIP) 1alpha, MIP-1beta, eotaxin, interferon (IFN) alpha, and IL-4 levels were significantly increased in the GC+ patients (n=27) compared with the GC-patients (n=70). In addition, minor differences in cytokine levels were found when comparing age groups. Degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pSS may represent "burned out" inflammation. No significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the FS-and FS+ subgroups.

The reduced salivary flow observed in GC+ patients may be influenced by the elevated levels of IL-4 found in these patients (Gao et al. 2006) . Increased titers of Th17-associated cytokines, IL-17, IL-1beta and the IL-23 subunit IL-12p40, may indicate a higher activity of these cells in GC+ patients (Nguyen et al. 2008) . Differences in cytokine levels may be utilized when sub-grouping the SS patients into disease phases and may consequently have implications for treatment. Objectives: C-reactive protein (CRP) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine IL-6. The rapid increase of CRP during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of CRP typically remain low in disease flares of systemic lupus erythematosus (SLE), a systemic autoimmune disease. Another feature of SLE is the so called 'Interferon (IFN) signature' which implies high levels of IFN-alpha and/or up-regulation of IFN-alpha related genes. IFN-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. Since high levels of IFN-alpha coexist with a muted CRP response in SLE disease flares and in viral infections, we hypothesized that IFNalpha inhibits CRP synthesis. Methods: CRP promoter activity was studied in a CRP-promoter and luciferase reporter transfected human hepatoma cell-line, HepG2. Production of the acute phase protein serum amyloid A (SAA) and the negative acute phase protein transferrin were analysed by ELISA as reference.

Results: The CRP-promoter activity was inhibited by all IFN-alpha subtypes. Mixes of type I IFNs that were induced by SLE-like immune complexes or virus also inhibited the CRP-promoter activity. Virus-induced purified leukocyte IFN-alpha had the most prominent inhibitory effect ( G 50 %) on CRP promoter activity. SAA synthesis was inhibited by IFN-alpha in a similar fashion as for CRP promoter activity, whereas transferrin was unaffected. Conclusion: Our data indicates that IFN-alpha is an inhibitor of CRP-promoter activity. We suggest that this could explain the muted CRP response seen in SLE disease exacerbations. Further, I may contribute to differences in CRP response between viral and bacterial infections. Background: B cell activating factor of the TNF family (BAFF) is an essential B cell survival and maturation cytokine. Mice overexpressing BAFF (BAFF Tg mice) develop lupuslike autoimmunity, B cell hyperplasia, and lymphomas. Autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be T cell-independent (TI) and MyD88-dependent. This suggested the involvement of transmembrane activator and CAML interactor (TACI), which is a receptor for BAFF that is essential for TI immune responses and is upregulated by MyD88-dependent TLR activation.

We assessed the role of TACI in BAFF-driven TI autoimmunity. Methods: We tested the importance of TACI in TI autoimmunity by generating BAFF Tg bone marrow (BM) chimeras reconstituted with TACI -/or TACI +/+ BM then comparing their disease severity by flow cytometry, autoantibody ELISA, immunofluorescence microscopy for Ig deposition. Results: As expected, BAFF Tg chimeras reconstituted with TACI +/+ BM produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (RhF), and Ig deposition in the kidneys and salivary glands was observed. By contrast, BAFF Tg chimeras reconstituted with TACI -/-BM had greatly ameliorated levels of circulating proinflammatory autoantibodies, RhF, and Ig deposition. B cell hyperplasia was greater in TACI -/-1 BAFF Tg chimeras. Defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (SLE). Autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. Physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize T-cells after phagocytosis of apoptotic material. These processes of silent apoptotic cell clearance seem to be disturbed in SLE patients. A characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). These microparticles have been recognized as mediators of intercellular communication. Therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human SLE. We observed an engulfment of microparticles by monocyte-derived dendritic cells. Further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. However, microparticles caused a remarkable downregulation of MHC class II molecules. Further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to LPS stimulated dendritic cells. Finally, these dendritic cells pulsed by membrane microparticles did not cause a significant T-cell expansion. Interestingly, dendritic cells obtained from SLE patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the MHC class II downregulation and a higher constitutive secretion of IL-8. Objectives: Increased levels of IL-18, an innate and inflammatory cytokine of the IL-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. Here, expression of IL-18 and other genes of the IL-1/IL-1R families was examined in human Systemic Lupus Erythematosus (SLE) and in MRL lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. Methods: Serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. Lymphoid (lymph-nodes, spleen, thymus, Peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from MRL +/+ and lpr/lpr mice of different ages. IL-18 and IL-18BP were measured by ELISA. Gene expression was assessed by real-time PCR and expressed relative to b-actin. Results: In SLE, serum and urine levels of total and free IL-18 are higher than in controls. Serum IL-18 correlates with disease activity and decreases upon remission. Monocyte expression of the receptor IL-18Rb is increased and correlates with disease severity, while expression of TIR8/SIGIRR (a down-regulatory receptor of the IL-1R/IL-18R family) is reduced. In MRL lpr/lpr mice, expression of IL-18, caspase-1 and IL-18Rb genes precedes disease onset in lymph-nodes. In other organs, changes in IL-1-related genes (IL-33 and TIGIRR-1 up-regulation, TIR8/SIGIRR down-regulation) occur after disease onset. Free IL-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. Conclusions: Free IL-18 levels correlate with autoimmune lupus both in mice and humans. Free IL-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in SLE. Both in human and mouse syndromes, upregulation of IL-18Rb is a marker of pathology, suggesting increased IL-18-dependent activation. Both in mouse organs and human monocytes, TIR8/SIGIRR expression decreases with disease, suggesting impaired control of IL-18R activation. Thus, IL-18 may be involved in autoimmune lupus pathology, and IL-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. In this study we compared the epitope specificity of anti-topo I autoantibodies present in sera of dcSSc, lcSSc and SLE patients. We have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with IgG purified from patients' sera. Regions of topo I selected from the library were expressed as recombinant fusion proteins and were tested with ELISA and western blot. We unexpectedly found that antibodies against a fragment of topo I (fragment F4 (amino acid (AA) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than SSc and SLE. Using sera of dcSSc, lcSSc and SLE patients we showed that the pattern of recognized epitopes is different between these patient groups. Fragment F4 was recognized by all patients. Fragment F1 (AA 5-30) was recognized by 9 of 34 dcSSc patients. Fragment F8 (AA 350-400) was recognized by 4 of 8 SLE patients. Analysis of clinical data revealed a significant difference between the F1 negative and F1 positive groups of SSc patients in age and in the duration of the disease. According to our results the newly identified fragments F1 and F8 could represent characteristic epitopes for dcSSc and SLE, respectively. Background: Previous studies demonstrated that depletion of regulatory T cells (Tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. Studies performed by us and others showed that in human connective-tissue diseases a reduced number of Tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. Objectives: Based on these observations and the fact that Rapamycin (Rapa) has the ability to expand Tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of CD4 + T cells isolated from Systemic Lupus Erythemaosus (SLE) patients by ex vivo expansion of Tregs. Methods: PBMCs or peripheral CD4 + T cells from SLE patients were cultured in the presence of specific stimulation with or without Rapa and rIL-2. By FACS the initial percent of Tregs and after expansion protocol were determined. In order to verify the suppressive capacity of expanded Tregs, CD4 + T cells enriched in Tregs were co-cultured with activated CD4 + effector T cells (Teff) stained with CFSE, after one week Teff cells proliferation was measured by FACS. Additionally, cytokine and IgG release in cell culture media were analyzed by multiplex and ELISA, respectively. Expanded CD4 + T cells anergy was also evaluated based on Cbl-b, Grail and Foxp3 mRNA by realtime RT-PCR. Results: In vitro expansion of Tregs was more efficient when the starting cells were CD4 + T cells. The presence of Rapa during expansion protocol significantly increased the number of Tregs. SLE Tregs cells expanded in vitro in the presence of Rapa had the capacity to suppress proliferation of both SLE and HD Teff cells. Rapa inhibits IgG secretion in the PBMCs culture, inhibition dependent on Tregs level. Rapa during Tregs expansion protocol stimulated some type of cytokines while suppressed others. Rapa had the capacity to re-establish SLE CD4 + T cells anergy by induction of anergy genes, GRAIL and cbl-b. Conclusions: Our data show that the above described protocol permits ex vivo Tregs expansion and that suppressive capacity of the expanded Tregs depends on the source of both Tregs and Teff cells. In this study, we look for a more specific approach to remove B-1 cells through targeting p110d by shRNAs strategy. Methods: We used the drugs, LY294002 and Wortmannin, pan-specific inhibitors against PI3Ks. Then we designed shRNAs carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. We then introduced either pan-specific inhibitors against all PI3Ks or p110d-targeting shRNAs into an SLE-prone animal model, NZB/W F1 mice, for therapeutic purposes.

The results suggested that PI3Ks are not only important for the development of B-1 cells but also remain essential to maintain their population after birth. shRNAs carried on lentiviral systems were designed to knock down the expression of p110d. Either pan-specific inhibitors against PI3Ks or p110d-targeting shRNAs were introduced into the SLE-prone animal model, NZB/W F1 mice. One inhibitor, LY294002, and shRNAs delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-DNA auto-antibodies and prolonged the life span. Conclusions: Our findings are promising for developing treatments for SLE. Moreover, knowing PI3Ks are critical for the maintenance of B-1 cell populations might shed light on future treating other diseases associated with B-1 cells, such as certain melanoma, lymphoma, or leukemia.

A. M. Zaghlool 1 , M. Alarcón-Riquelme 1 , S. Kozyrev 1 1 Institution of Genetic and Pathology, Uppsala University, Uppsala, SwedenRecently, we discovered that the BANK1 gene, which plays a role in B cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution G/A (rs10516487, R61H). We identified that BANK 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). The two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. To address the question of how BANK1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the BANK1 isoforms were tested in vitro. qPCR analysis revealed that, another T/C SNP (rs17266594), which is in complete LD with R61H SNP and located in the putative branch point, has a strong affect on the isoforms expression levels. Deletion of a polypyrimidine (PY) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein TIA1, which bind to T Objectives: Cerebral ischemia is the most common presentation of antiphospholipid syndrome (APS), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (aPL). We report the case of a subject with atypical movement disorder related to APS successfully treated with oral anticoagulation agents. Case report: A 57-year-old woman with a previous history of recurrent foetal losses was admitted to our Hospital due to cognitive dysfunction and headache. She presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. Mild executive dysfunction was observed. Her deep tendon reflexes were symmetric and normal. Pathological reflexes were absent. Biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. The immunoglobulin G (IgG) isotope of anticardiolipin antibody (aCL) was elevated, whereas IgM isotype and anti-2GPI antibodies were undetectable. The lupus anticoagulant (LA) was negative such as antinuclear antibodies (ANA). No evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. Brain magnetic resonance imaging (MRI) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. She was diagnosed as having an atypical neurologic manifestation probably linked to APS. She was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . The therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. Conclusions: Antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. Due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of APS (foetal losses, livedo reticularis, thrombosis). 

A.-S. Korganow 1 1 CNRS 9021, Strasbourg, France B lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. Several B cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. A few genes have been suggested to be implicated. One of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.We made the postulat that "intrinsic" abnormalities of B cells could be a common point in very quiescent patients. We choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. Known of them received immunosuppressive drugs since this period. The mean SLEDAI score was below 2. B cell surface markers expression was determined by flow cytometry. We analysed most of the already described and phenotypically distinctive B cell populations. We confirm the presence of activated B cells even in quiescent patients. We do not confirm the significant increase of a specific memory B cell compartment. Above all, we described a decreased expression of the CD19 surface protein for all patients. This CD19 lower expression is associated with CD45 lower levels. It is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. These findings suggest some mechanisms in lupus genesis. Objectives: TGF-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. Recent work from our group has shown that TGF-beta signalling in T-cells is protective in a mouse model of colitis associated cancer. Smad ubiquitin regulating factors (Smurf) are ubiquitin ligases that are involved in the regulation of TGF-beta signalling. The aim of this study was to determine the function of Smurf2 expression in T-cells on the pathogenesis of experimental colitis associated colon cancer. Methods: We could isolate a known splice variant of Smurf2 lacking an Exon in the C2-domain. To analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses Smurf2 in T-cells. Smurf2 Expression were analysed by qPCR. Wild type (WT) and Transgenic (TG) mice were treated once with the mutagenic agent Azoxymethan (AOM) followed by three cycles Dextran Sodiumsulfate (DSS). After each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. Results: Smurf2 Expression was upregulated by TGF-beta stimulation in T-cells and Smurf2 was markedly upregulated in tumor infiltrating CD4+ lymphocytes in AOM/DSS treated mice. Whereas WT mice suffered from severe colitis resulting in colon tumors beginning at day 42, Smurf2 transgenic mice had less colitis and were significantly protected from tumor development. Interestingly, T-lymphocytes overexpressing Smurf2 showed an upregulation of the TGFbRII and an activation of Smad2 and 3 as compared to wild-type T-lymphocytes, which were previously described as typical Smurf2 targets for degradation. In addition the transfection of Smurf2 and a CAGA-Luc Plasmid into Cos-cells for Smad3-Promotor studies yielded the same effect as shown by upregulation of the Smad3 activity. Conclusion: Although, WT-Smurf has been described as a negative regulator of the TGF-beta signalling pathway, our data show surprisingly that a Smurf2 splice variant upregulates the TGF-beta receptor expression and increases TGF-beta signalling effects. Due to immunosuppressive effects on T-cells Smurf2 has beneficial effects on mucosal inflammation and tumor development. Smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. The transcription factor STAT3 has important functions in cytokine signalling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IEC) both in humans and in mice. Studies in genetically engineered mice showed that epithelial STAT3 activation in DSS colitis is dependent on IL-22 rather than IL-6. IL-22 was secreted by colonic CD11c+ cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3 IEC-KO mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in IEC. Consistently, IL-22 and epithelial STAT3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing. STAT3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury.

MAP and MA infection induced an increase in both CD40 and TLR4 expression at day 3 and day 7 after infection. Mycobacterial infection did not result in differential TLR2 expression as compared to uninfected cells. CD40 is involved in stimulating Th1 pro-inflammatory responses, although MAP may interfere with CD40 signalling (1) . TLR4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .In conclusion, monocyte-derived macrophages from Crohn's disease patients show an increase in CD40 and TLR4 receptor expression in response to both MAP and MA infection. As MA is a known human pathogen of immunocompromised hosts, this findings further support a role for MAP in the immunopathology of Crohn's disease. Objectives: For our understanding of the pathogenesis of human IBD, animal models of intestinal inflammation are indispensable. Most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of IBD. The occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. To further dissect the pathophysiological mechanisms responsible for initiation and progression of IBD and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of CD8 T cells by exogenous antigen leads to colitis. Methods: Eight million naïve CD8 + OT-I cells, transgenic for a T-cell receptor specific for an OVA-derived peptide (SIINFEKL) in the context of H2-Kb, were transferred i. v. into B6 mice. At day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. Thirty minutes later, ovalbumin (OVA) or bovine serum albumine (BSA) were applicated i. r. Proliferation of CFSE-labelled cells was measured at day 2 after the injection of OT-I cells. The phenotype of effector cells was evaluated at day 5 by measuring IFNg production and by in vivo cytotoxicity assay. Based on histology and immunhistochemistry for CD8, the severity of colitis was scored.

Results: Local application of the exogenous antigen OVA but not of BSA led to antigen-specific activation and proliferation of adoptively transferred naïve OT-I CD8 + T cells. These cells differentiated into fully activated effector T cells with the capacity to secrete IFNg upon re-stimulation ex vivo and possessed in vivo cytotoxicity to SIINFEKL-loaded target spleen cells. Furthermore OVA treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of CD8 + T cells.

Our study demonstrates that the local activation of antigen-specific CD8 T cells by exogenous antigen in the colon leads to fully activated effector T cells with the capability to promote local intestinal inflammation in non-immune-compromised B6 mice.

Aims: To determine the immune system response of the Greek population against Helicobacter Pylori (HP), given the fact that HP infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc).

The results of express qualitative detection method for IgG and IgA antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. Patients who received antibiotics and excretory medicine in the last year were excluded. Anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using SPSS, x 2 and t-test programmes. Results: In 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. On the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. Out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). The statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, MCV and RDW). Conclusions: It is proven, therefore, that: 1) Helicobacter pylori infection is relatively common in the general population (30,5%).2) There is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) There seems to be no correlation with anemia. It is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of Helicobacter pylori infection in our country.

Abstract withdrawn by author

M. Durilova 1 , T. Ulmannova 1 , K. Stechova 1 , K. Tesarova-Flajsmanova 1 , V. Stavikova 1 , J. Nevoral 1 1 Charles University, Pediatrics, Prague, Czech RepublicObjective: Was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. Methods: Breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. Breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. Concentrations of the following cytokines were analyzed using ELISA method: IL-4, IL-6, IL-10, IL-17, IL-23, IFN-gamma, TGF-beta1, EGF and eotaxin. Man-Whitney U test was used for statistical analysis, p X 0.05 was considered statistically significant.Results: IL-10 as the only cytokine was not detected in any of the tested samples in both groups. Significant difference was seen in concentration of IFN-gamma, which was higher (p X 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). Higher concentrations of IL-4 and lower concentration of TGF-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. Conclusion: Immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. Although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. Inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. The results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. Supported by National Project No. 8310-5. Background: Ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. The chemokine IL-8 (murine homologs KC and MIP-2) and its receptor CXCR2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. This study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. Methods: Peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (FACS analysis) and functionally characterized in an in vitro chemotaxis assay. Four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (DSS) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. Anti-KC antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. Results: FACS analysis revealed 80 % neutrophil purity, 35 % of which were CXCR2 + . In vitro, the cells migrated towards KC and this was inhibited by anti-KC. In the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of DSS recipients at all time points. Neutrophil recruitment to the colon was detected only in DSS recipients and was inhibited by anti-KC, 4 h post cell transfer.

This study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. The human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. The barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (S)IgA and SIgM. SIgs are generated via epithelial polymeric Ig receptor (pIgR)-mediated transfer of IgA and IgM from the lamina propria to the intestinal lumen.

To assess the role of SIgs in colitis development, we constructed pIgR knockout (KO) mice and tested them in the dextran sodium sulfate (DSS) colitis model (1.5 % DSS in drinking water for 1 week, followed by pure drinking water for 1 week). pIgR KO mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of SIgs is to prevent pathology induced by commensal microbiota. In contrast, 2 % DSS was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. As previously documented by Medzhitov and co-workers (Rakoff-Nahoum et al, Cell 2004), treatment of commensal-depleted mice with the TLR4 ligand LPS in drinking water protected against the lethality of 2 % DSS. Thus, the commensal microbiota serve two distinct roles in the DSS colitis model. At DSS concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. At 2 % DSS commensals protect against the toxic effect of the chemical via their TLR ligands. In mice lacking SIgs, due to deleted pIgR, the severity of colitis induced by 1.5 % DSS was greatly enhanced suggesting that one role of SIgs is to prevent commensal microbiota from becoming pathogenic. Ulcerative Colitis (UC) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. Although UC is associated with a Type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. In a mouse model resembling UC, oxazolone induces IL-13 production which is an important pathological factor. Neutralizing IL-4 or IL-13 prevents or ameliorates disease significantly. As many aspects of the mechanisms involving these TH2 cytokines in colitis remain undefined, we used mice deficient in IL-4/IL-13 or the key receptor through which they signal, IL-4Ra, to further dissect their role in oxazolone-induced colitis. Disease was exacerbated in IL-4Ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. This was in contrast to IL-4/IL-13 double deficient mice which were protected from colitis. Removing IL-13 production from IL-4Ra -/mice, by using IL-4Ra/IL-13 double deficient mice, reversed the susceptible phenotype to protection. Together these data strongly suggest that IL-13 mediates susceptibility in an IL-4Ra independent manor. Recent evidence PC17/33 Introduction: The activation of CD4 + T-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (IBD). Whereas CD is associated with increased production of Th1-like cytokines, the cytokines profile in chronic UC is characterized by the increased production of several Th1 cytokines, such as IL-5,-6 and Il-13. However, the functional role of T cell transcription factors such as nuclear factor of activated T cells (NFAT) in IBD is poorly understood. The aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in UC.

Cryosesctions of UC and CD patients were analysed by immunohistochemically methods. A significantly higher expression of NFATc2 was found in UC and CD colonic tissue compared to control specimen. Transmitted to the Th2-mediated oxazolone-induced colitis model, NFATc2-production is significantly increased in both diseases, too. NFATc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. Interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in NFATc2 deficient mice compared to control mice, which can be observed by TUNEL assays, caspase3 and Annexin V staining, as well as in lamina propria T cells. Contrary, anti-apoptotic proteins, like bcl-2 and bcl-xL were downregulated for induction of apoptosis. This observation was associated with a reduced production of IL-6, IFN-gamma, IL-13 and IL-17 by mucosal T lymphocytes, tested by ELISA assays. Further studies with the oxazoloneinduced colitis model showed that NFATc2 regulates IL-23/IL-17 in an indirect way. Last, administration of IL-6 blocked the protective effects of the NFATc2 deficiency in experimental colitis, suggesting that NFATc through IL-6 signal transduction plays a direct pathogenic role in vivo. Conclusion: Our data define a unique regulatory role of NFATc2 in colitis by controlling mucosal T cell activation in an IL-6 dependent manner. The examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. The pivotal role of microRNAs in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (IBD) as well. The study of the expression of microRNAs in IBD will unravel their role in this disease. In addition, microRNAs by their mechanism of action, are promising new therapeutic agents or targets. A possible therapeutic application of microRNAs is the introduction of novel, artificial microRNAs or microRNA mimics to regulate specific genes. Because IBD is a heterogeneous disease in human we decided to define microRNA expression in a well defined model of experimental colitis. As a result of this study we found a number of microRNAs involved in different phases of experimental colitis.

To study the role of miRNAs in experimental colitis in mice we have used a well defined colitis model that resembles human IBD. This colitis is mediated by CD4CD45RB high T cells that are injected i. p. in SCID mice. In control mice in addition to the CD4CD45RB high T cells also regulatory CD4CD45RB low T cells are transferred and no colitis develops. To study miRNA expression we collected colonic tissue from the mice at 3 different time points during colitis progression. After 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. MicroRNA was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). From all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.The RNA isolation as well as the microarray analysis has been outsourced to Miltenyi Biotec GmbH, Bergisch Galdbach, Germany. We used the miRXplore TM Microarrays for microRNA expression profiling. From 11 microRNAs that demonstrated an induction during the development of disease we selected 4 microRNAs for in situ hybridization and for a proof of principle of the efficacy in the CD4CD45RB high transfer model. Objective: The purpose of this clinical trial (ID: NCT00287677 of www.ClinicalTrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (GH). Methods: Successfully Highly Active Antiretroviral Therapy (HAART) treated HIV infected patients that failed to elicit a humoral response to Tetanus Toxoid (TT), or to Hepatitis A (HVA) or to Hepatitis B (HVB) virus have been selected for the trial. Growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. Patients have been randomized in 3 groups: Group A (n=8) receiving HAART+ GH (for 6 months) + TT+HVA/B vaccines (at month 6 post GH adminsitration); Group B (n= 6) receiving HAART+GH but not vaccines; and Group C (HIV control group, n=7) with HAART+vaccines (at month 6) but without GH. All patients are followed up 6 months further. Results: Preliminary results show that an increase in thymic size was observed in GH recipients and not in controls. Furthernore after 24 weeks of administaring hormone the absolute numbers of CD4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (Mean and SEM; p X 0.0025). In contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the CD4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p X 0.02). Viral load remained undetectable in all patients. Despite the increase in CD4 counts the percentage of recent thymic emigrants (as assessed by the expression of CD31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. Finally, specific responses to Hepatitis A virus seem to be restored in a major proportion of patients treated with GH (group A) than in the other groups. Conclusions: Although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. A major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. In the course of the eradication of small-pox, freeze-dried Vaccinia Virus which proved to be extremely stable was used to overcome this limitation (Dryvax ® ). Before usage, Dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. An alarming report by the WHO has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. Each year, an overwhelming number of infections with HIV (80,000-160,000), hepatitis C virus (HCV; 2.3-4.7 million) and hepatitis B virus (HBV; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers.

In this report, we took advantage of the stability of freeze-dried vaccinia virus MVA and directly applied it into the nostrils of mice without prior reconstitution. This direct mucosal application induced systemic antibody and T cell responses comparable to those achieved by intramuscular administration. Importantly, mucosal application of lyophilized MVA conferred protective immunity against a lethal Vaccinia Virus challenge. Additionally, recombinant MVA expressing the model antigen ovalbumin induced long-term and protective immunity against Listeria monocytogenes-ova challenge. The data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized MVA. Methods and results: Seventeen HAART-treated asymptomatic HIV-1 infected patients with G 350 CD4 + T-cell counts and plasma HIV-RNA levels of X 1.7 log 10 copies/ml were treated with a DC-based vaccine. The vaccine consisted of autologous mature DC electroporated seperately with either sig-Tat-DC-Lamp, sig-Rev-DC-Lamp or sig-Nef-DC-Lamp mRNA and were each administered at a distinct anatomical site. After four monthly vaccinations HAART treatment was interrupted. PBMC from 4 timepoints, before during and after vaccination, were analysed for IFN-g production (Elispot), proliferation (CFSE assay) and lytic capacity (FATT-CTL) in response to the antigens used in the vaccine. PBMC were screened upon ex vivo stimulation with pools of overlapping Tat, Rev, Nef and Gag peptides and IFN-g secretion was analysed using Elispot ('peptide Elispot'). Elispot was also performed on re-stimulated T-cells with electroporated DC ('DC Elispot') in vitro. Responses were considered positive when the number of spot forming units per million T-cells was G 50 and when the responses were G 2 times the standard deviation above the mean of replicate negative controls (mock electroporated DC). 16/17 patients were screened with both peptide and DC Elispot. An increase of responses to the vaccine-antigens after vaccination was found in both assays. Based on the DC Elispot data, we observed immune reactivity against Tat in 4/16 patients before and 11/16 patients after vaccination. For Rev, 7/16 patients showed a pre-existing Rev specific response and 14/16 patients responded to Rev after vaccination. Nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. For our control antigen Gag, we observed an anti-Gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. The results of the other assays correspond to the DC Elispot results be it less pronounced. Conclusion: Therapeutic vaccination of HIV-infected patients with DC electroporated with Tat, Rev and Nef induces and/or enhances antigen-specific T-cell responses, especially when monitored with the DC Elispot. Background: Enterovirus 71 (EV71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. No effective vaccine for EV71 is available yet. Polysaccharide purified from Ganoderma lucidum (PS-G) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of PS-G as the possible adjuvant with EV71 inactivated virus. Methods: Mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: PBS, CFA/IFA, and PS-G. Each mouse received the same dose of boosters after 0, 2, and 4 weeks. Blood samples were collected at 0, 21, 35, and 45 days. The total serum anti-EV71 IgG level was determine by ELISA, and the neutralization assay was also done. To evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. Cytokines assay regarding IFN-g and IL-5 from splenocytes was also measured. Results: Immunization with EV71/PS-G showed that the anti-EV71 IgG levels were significantly increased compared with EV71 alone or EV71/CFA/IFA in BALB/c mice. Neutralization assays demonstrated an effective protection of EV71/PS-G group compared to EV71 alone. The splenocyte proliferation test showed that production of IFN-g significantly increased in EV71/PS-G-immunized mice compared to those of EV71 or EV71/CFA/IFA-immunized mice, indicating a Th1 cells response elicited by heat-inactivated EV71 vaccine with PS-G adjuvant. Conclusions: Vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. The results here suggested that PS-G can be used as a novel, safe and natural vaccine adjuvant. Objectives: The search for a vaccine against hepatitis C virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees.

Here we describe the differential modulation of CD8+ T-cell responses induced by a DNA prime MVA boost vaccine regimen in four individual chimpanzees and their association with viral clearance. Methods: An ex vivo expansion protocol was used to map peptide specific CD8+ T-cell responses against HCV-NS3, studying induction of IL-2, IFNg and TNFa cytokine production as well as killing capacity. To assess the killing capacity and MHC restriction of the peptides, a non-radioactive killing assay was designed. Peptides that induced both IL-2 and IFNg production by CD8+ T-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee MHC class I molecules. Introduction: High levels of HIV-1-recognizing CD8 + cytotoxic T lymphocytes (CTL) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of HIV-1 replication, and for the prolonged survival of infected individuals. A potential immunotherapeutic strategy would be the adoptive transfer of T cells, which are reprogrammed by introduction of an HIV-specific T cell receptor (TCR). Up to now, such CTL were generated by retroviral transfer of TCR-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). Methods: Therefore, we investigated the transfer of TCR-RNA into CD8 + T cells by electroporation, and chose TCRs which were able to recognize the HLA-A2 restricted HIVpol-peptide IV9, or the HIVgag-peptide SL9. Results: T cells, reprogrammed with these receptors, released the pro-inflammatory cytokines IL-2, TNF, and IFNg simultaneously, and showed up-regulation of the activation marker CD25, after stimulation with peptide-loaded target cells or target cells (i. e. EBV-transformed B cell and CD4 + T cells) presenting the naturally processed epitope. Furthermore, these cells maintained their ability to proliferate after stimulation. More importantly, killing assays demonstrated that the TCRreprogrammed CD8 + T cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. A comparison of our reprogrammed T cells with the parental CTL showed that the transfected T cells were only one order of magnitude lower in avidity than the parental CTL. Also, the parental clone's recognition pattern of mutant peptides was preserved in TCR-RNA-transfected T cells.

The transfection of TCR-encoding RNA into CD8 + T cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred TCRs is possible.

Background: Dendritic cells (DCs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. The aim of this study was to investigate the capacity of splenic DCs that ingest antigen coated magnetic beads to induce HCV cellular immune responses. Methods: Splenocytes of Flt3L pretreated Balb/c mice were incubated for 3 hrs with HCV NS5-coated magnetic beads. The cells were harvested and cells that contained beads were purified by passage over a magnetic column. The isolated population contained G 80 % DCs and was used for immunization. DC expression of the maturation markers CD40, CD80 and CD86 was determined before and after ingestion of NS5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. Cellular immunity was assessed using a conventional CTL assay, a CFSE-T-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably NS5 expressing SP2/0 cells). Results: In immunized animals, the CTL activity was increased 3-fold compared to mock immunized mice. Accordingly, tumor challenge with NS5 expressing tumor cells showed a significant reduction in tumor growth. The number of CD4 + IFN-g + cells was increased G 3-fold and the number of CD4 + IL-2 + increased G 5fold in the DC-NS5-bead immunization group. These results paralleled the proliferative response of splenocytes to NS5 protein obtained from immunized animals with the most significant response in the CD4+ population of DC-NS5-bead immunized animals.

The use of NS5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. The induction of a strong Th1 biased T cell response makes this approach a promising new tool in therapeutic immunization in chronically HCV infected patients. The success of anti-DEC-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. In their absence, anti-DEC-205 induces antigen-specific tolerance rather than immunity. We hypothesize that regulatory T-cell epitopes contained in anti-DEC-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. This hypothesis is based on our recent discovery of a set of natural regulatory T-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory T cells. We have verified experimentally that these epitopes generate an expansion of regulatory T cells and suppress inflammatory immune responses. Here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-DEC-205 antibody can be developed. We screened the anti-DEC-205 sequence computationally for putative HLA DR4-restricted, regulatory T-cell epitopes as targets for mutations that will reduce epitope binding affinity for HLA. Amino acid substitutions predicted to interfere with HLA binding were identified and are being incorporated into an array of anti-DEC-205 antibody variants recombinantly fused to a test antigen, HIV Gag. Variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced Gag immunogenicity, in terms of cellular and humoral responses. We predict that the modification of regulatory T-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-DEC-205 as a HIV antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. Supported by NIH 1R21AI078800 A live oral vaccine based on human adenovirus (Ad)4 has proved safe and effective in US military recruits for nearly 50 years. In these experiments, we have investigated whether replication-deficient Ad4 can be an efficient potential vaccine carrier for oral vaccination. Ad5 vectors were used throughout to provide a benchmark for efficacy. We generated novel Ad4 and Ad5 vector systems based on DNA recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://AdZ.cf.ac.uk). EGFP was inserted with a HCMV IE promoter as a model transgene. Preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with Ad4 than Ad5. Ad5 routinely infected and provided transgene expression in˚10 % of human CD4+ and CD8+ T cells. Stimulation of the HCMV IE promoter post infection increased detection of EGFP to 25 -30 % of CD8+ T cells present, showing that Ad5 infected a surprisingly large proportion of T cells. In comparison, Ad4 provided EGFP expression in X 2 % of either cell type, even after T cell activation. In contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with Ad4 and Ad5 vectors were comparable. Preferential infection of DCs is likely to be beneficial in the context of a vaccine. In vivo, we observed that following oral delivery both Ad4 and Ad5 induced restricted yet strong expression in the intestine. The vectors were rapidly taken up into the Peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. Interestingly, when delivered together Ad4 and Ad5 vectors targeted the same subset of cells. Together, these data show that Ad4 is a viable alternative to Ad5-based vaccines which may also avoid unwanted infection of activated T cells. Aims: Monoclonal antibodies (mAbs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; G 300 in clinical trials). To date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. However, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. As induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mAbs, in addition to blunt viral propagation, may also modulate the endogenous immune response. Methods: We have developed a preclinical mouse model of fatal leukemia induced by the FrCas E murine retrovirus. Mice were infected with FrCas E and treated, or not, with a neutralizing mAb (the 667 mAb). Viral propagation, survival and development of immune responses were followed up for several months. Results: Using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. These results show that neutralizing mAbs act as immunomodulatory agents capable of inducing long-term protective immunity ( G 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (Gros et al, 2005; Gros et al, 2008; Michaud et al. submitted) . Although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral T-cell responses. Conclusions: Our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral T-cell responses. Our observation might have important consequences on the design of antiviral mAb-based immunotherapies. Objectives: We have analyzed the potential of virus-like particles (VLPs) from rabbit hemorrhagic disease virus (RHDV) as a delivery system for foreign T-cell epitopes.

To accomplish this goal, we generated chimeric RHDV VLPs incorporating a CD8 + T-cell epitope (SIINFEKL) derived from chicken ovalbumin (OVA). The OVA epitope was inserted in the capsid protein (VP60) of RHDV at two different locations: 1) the N-terminus, predicted to be facing to the inner core of the VLPs (RHDV-VLP-2), and 2) a novel insertion site predicted to be located within an exposed loop (RHDV-VLP-306). Both constructions correctly assembled into VLPs and we analyzed the immunogenic potential of both chimeric RHDV VLPs in vitro and in vivo.

In vitro, dendritic cells (DCs) were able to process and present SIINFEKL peptide in the context of MHC class I from chimeric RHDV VLPs for CD8 + specific recognition in a dose-and insert position-dependent manner. Moreover, chimeric RHDV VLPs activated DCs for TNF-alpha secretion.In vivo, mice immunized with the chimeric RHDV VLPs without adjuvant were able to induce specific cellular responses mediated by cytotoxic (CTL) and memory T cells. Although both chimeric RHDV VLPs were able to induce specific IFN-g-producing cell priming, insertion of the SIINFEKL peptide at the amino-terminal position (RHDV-VLP-2) was more immunogenic than insertion at position 306 for induction of CTLs and anti-viral immunity.More importantly, immunization of mice with chimeric RHDV VLPs at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing OVA in target organs. In addition, immunization with chimeric RHDV-VLP-2 at the highest dose tested was able to resolve VV-OVA infection. Conclusion: Our data demonstrated that immunization with chimeric RHDV VLPs was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. Objectives: A major issue pertaining to use of DNA vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. In this study, we describe the targeting of antigen-encoded DNA directly to dendritic cells (DCs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-S-glyceryl cysteine, a known TLR-2 ligand. Methods: Agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to DNA encoding for green fluorescent protein (GFP), influenza hemagglutinin (HA) or nucleoprotein (NP). Transfection efficiencies of DCs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. Stimulation of T cells by NP-transfected DCs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific CD8+ T cells. Subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. Results: Electrostatic binding of the lipopeptide to DNA plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the DNA backbone and retard its ability to migrate towards the anode. High levels of GFP, HA or NP were detected in murine spleen-derived cultured DCs following transfection with these complexes concomitant with the upregulation of surface MHC Class II molecules compared to when DNA alone was used. The ability of transfected DCs to stimulate CD8+ T cells from influenza virus-infected mice was also investigated. Subsequently, vaccination by lipopeptide-DNA complexes resulted in the induction of higher numbers of IFN-g producing NP 147-155 specific CD8+ T cells in the spleen and lymph nodes of mice compared to those that received DNA alone. Conclusion: Altogether these results demonstrate that the use of a TLR-2 targeting lipopeptide-based system that can facilitate the delivery of DNA by directly targeting and concurrently activating antigen-presenting cells, such as the DC, could prove to be advantageous in enhancing cellular responses induced by DNA vaccination. The level of interferon in blood serum of non-infected mice was determined by ELISA Kit and by the cytopathic test in the L929cell culture after aplication of Ridostine and Ridostine ointment. The effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. The statistical processing of the data was carried out by the Student t-criterion. Ridostine was administered to patients once or twice in the case of high temperature. The clinical signs were recorded (temperature, rhinitis, headache etc). For prophylactic and treatment purposes the Ridostine ointment was intranasally applied twice per day during 7 days. The effectiveness of the preparation was evaluated by clinical sings and the level of CD2+, CD4+, CD8+, CD16+ T -lymphocytes. Results: Ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. Ridostine and Ridostine ointment stimulated synthesis of interferon and phagocytic activity. Ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. Prophylactic intranasal application of Ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of T-cell immunity. Application of ointment at an early stage of disease markedly activated T-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. Conclusion: Interferon inducer based on natural dsRNA stimulates some reactions of innate immunity and resistance to influenza virus. The drugs based on dsRNA show promise for treatment of influenza. Objectives: The creation of gene engineering vaccines against hepatitis C virus (HCV) based on recombinant proteins is one of relevant approach. Since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. The aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural NS4 and NS5A HCV proteins and Immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. Objectives: IFN-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. A/H5N1 influenza virus inhibits the IFN-g synthesis (Mibayashi et al., 2007) and causes a decrease in CD4 + and CD8 + T-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (Tumpley T. M. et al., 2000) . Thus, IFN-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. An essential defect of this cytokine is its fast degradation in blood. The goal of this work was to study immunomodulating properties of an IFN-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded IFN inducers. Methods: A recombinant human IFN-g analog Deltaferon is distinguished by a 10 amino acid deletion at the C-end of the molecule and substitutions of amino acids in positions 129-131 (Tat'kov C. I. et al., 2000) . Deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 IU once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast RNA preparation (5 mg/kg). The content of IFN-a and IFN-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by MTT-test (Mosmann T., 1983) . Results: When Deltaferon was administered in doses of 2-20*10 4 IU alone it did not influence the content IFN-a in blood, but caused a transient increase in the level of IFN-g. The injection of the preparation in a dose of 2*10 4 IU led to a an increase in both spontaneous and conconavalin A-induced proliferation of splenocytes. The two-fold administration of Deltaferon in the maximal dose increased a level of IFN-g and inhibited cell proliferative activity. The combined administration of Deltaferon (2*10 4 IU) and dsRNA markedly increased the level of IFN-a and enhanced splenocyte proliferation.

The recombinant human IFN-g analog is able to enhance IFN-g synthesis, splenocyte proliferation and to modulate the effect of IFN inducer. These data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. By using a combined approach of routes of immunization and vaccine delivery systems such as Poly-Lactic Acid (PLA) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. We showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (ID), transcutaneous (TC), subcutaneous (SC)). While ID and TC route remain mostly efficient for the induction of antigen-specific CD8 responses (tetramer+ HIV-1 gag p24 cells), the quality of humoral responses (IgG, IgA) remained distinct between the two routes. In addition, SC route is less efficient than ID and TC routes for the induction of CD8 responses after PLA-p24 immunization. We have also shown that a lower antigenic charge of PLA particles was needed when PLA-p24 were injected using ID and TC routes of immunization. Currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. This unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system.

B. Dí az-Freitas 1 , C. Prego 2 , S. Vicente 2 , M.J. Alonso 2 , A. González-Fernández 1 1 University of Vigo. Area of Immunology, Department of Biochemistry, Genetics and Immunology, Vigo, Spain, 2 University of Santiago de Compostela, Nanobiofar Group, Department of Pharmacy and Pharmaceutical Technology, Santiago de Compostela, SpainObjectives: The design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. In this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis B surface antigen (rHBsAg) as a model. Our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. Materials and methods: Loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rHBsAg. The immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to BALB/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. Blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). Specific IgG antibodies levels directed against rHBsAg in serum were measured by indirect ELISA in mIU/ml. Results: Chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rHBsAg loading were prepared. The results of the specific IgG levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. The response elicited was delayed respect to the alum based vaccine, and very interestingly, IgG concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. After intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. The IgG titers elicited were enough to induce full seroprotection against the disease (100 mIU/ml).

Polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. More studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. The success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (DCs) in an environment that provides optimal immunostimulatory signals. In our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigen- Background: Infection with human immunodeficiency virus type 1 (HIV-1) is characterized by dysfunction of HIV-1-specific T cells. To control the virus, antigenloaded dendritic cells (DCs) might be useful to boost and broaden HIV-specific T-cell responses. Poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. We investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from HIV-1 and the TLR3 ligand poly I:C as a maturation factor. Methods: Monocyte-derived DC (MDDC) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly I:C. Potential maturation of DC was evaluated by flow cytometry. MDDC from HIV-infected patients under highly active anti-retroviral therapy (HAART) were similarly pre-incubated with p24 microcapsules containing p24 and poly I:C. These antigen loaded MDDC were used to directly stimulate autologous peripheral blood lymphocytes (PBL) in ELIS-POT. They were also co-cultivated for 10 days with autologous PBL to evaluate the immunogenic capacity. Potential expansion of specific T cells was measured by comparing ELISPOT responses of PBL before and after coculture, using a pool of overlapping p24 peptides. Intracellular staining of interferon-gamma (IFN-g), interleukin-2 (IL-2) and CD107a after p24 stimulation was also performed. Results: MDDC from HIV(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of DC, but when poly I:C was present the DC did mature. MDDC from HAART treated HIV-infected individuals, cultivated with p24 containing microcapsules with poly I:C, were able to efficiently expand and broaden autologous effector memory T cells which contain and secrete IFN-g and IL-2, upon p24 peptide restimulation. Objectives: We aimed at investigating whether and how the distance of a cytokine from the VLP surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. Methods: We inserted one, two or four Ig-like domains of hCD16b between our model cytokine IL-2 and the minimal GPI-anchor acceptor sequence. Subsequently, we compared particle production by Western blotting for p30Gag, targeting of cytokines to lipid rafts and and VLP upon isopycnic membrane separation and biological activity in IL-2 dependent proliferation assays of IL-2 variants. Results: Murine IL-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the IL-2 dependent cell line HT-2. We found that the membrane anchors comprising one and four Ig-like domains (IL-2::1IgGPI and IL-2::4IgGPI) resulted in severely reduced VLP production by 293 producer cells and despite of an increased targeting of IL-2::4IgGPI to VLP, a reduced stimulatory capacity of producer cell crude supernatant, when compared to IL-2 fused to the minimal GPI-anchor acceptor sequence of hCD16b (IL-2::GPI). IL-2::2IgGPI, however, showed comparable particle production and biological activity in vitro when compared to IL-2::GPI. Furthermore, IL-2 fused to 2Ig::GPI showed an increased capacity to co-stimulate primary P14 TCR transgenic T-cells specific for LCMV-GP 33-41 in the context of H2-D b .

Conclustions: Besides the minimal GPI-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to VLP. 2Ig::GPI has a biological activity in vitro, which is comparable to the minimal GPI anchor. Moreover, IL-2::GPI displays increased co-stimulatory potential in the context of specific MHCp complexes. This work was supported by grants SFB F1816-B13 of the Austrian Science Foundation, the Austrian Research Promotion Agency (Forschungsförderungsgesellschaft) Bridge grant 812079 & Biomay AG, and the Christian Doppler Laboratory for Immunomodulation.

A. Roemhild 1 , Interdisciplinary transplant laboratory 1 Charite Berlin, Insitute of Nephrology and Medical Immunology, Berlin, Germany Immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. Missing viral immunity, particularly against EBV, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (PTLD). PTLD affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. Healthy individuals control EBV infection by EBV-specific cytotoxic T lymphocytes (CTLs), but some patients under immunosuppression are unable to do so. In these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific T cells into the patient. Currently these T cells are generated by repetitive stimulations with EBV-infected autologous lymphoblastoid cells (LCLs). Due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. Therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. T cell lines were generated with two different protocols. In the first protocol T cells are generated by repetitive stimulation with EBV-infected autologous LCLs. The second protocol is based on stimulation with 6 different overlapping EBV peptide-pools and immunomagnetic cell isolation. Expanded T cells were analysed using multicolour flow cytometry. Cells were stained for diverse surface markers and intracellular cytokine production. Cytotoxic capacity and specificity was determined by a Calcein release assay. Our group developed a new protocol for the production of EBV specific T cells, thereby shortening the generation time from 2,5 month to 16 days. T cell lines are composed of CD8 and CD4 cells with a mainly effector memory like phenotyp. After restimulation the cells produce more TNFa than IFNg. Depending on the generation protocol T cells specifically recognized and lysed autologous LCLs alone or loaded with EBV-peptides. The detailed characterization of EBV-specific T cell lines should help to further improve the adoptive immunotherapy and its outcome. The novel, short time generation protocol did not affect phenotyp and cytokine production of the T cells. Nevertheless their therapeutic potential in vivo has to be tested in further experiments.

S. S. Schmucker 1 , M. Assenmacher 1 , A. Richter 1 1 Miltenyi Biotec GmbH, R&D Cell Biology, Bergisch Gladbach, GermanyAdoptive transfer of virus-specific T cells provides a promising treatment of infection in immunocompromized patients. As expansion of virus-specific T cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific T cells from naive hosts exist. In this study we established a cell culture system for priming of highly rare naive CMV pp65-specific CD4 + and CD8 + T cells from CMV-seronegative donors in vitro.Magnetically isolated naïve (CD45RO -CD25 -) CD4 + and CD8 + T cells from PBMC of CMV-seronegative donors were co-cultured with autologous mature monocytederived DC loaded with CMV pp65 peptide pool and CD3-depleted autologous PBMC as feeder cells in the presence of IL-2, IL-7, and IL-15. Already 9-13 days after primary activation pp65 495-503 /A2-tetramer + CD8 + T cells were detectable for 3 HLA-A2 + blood donors. To analyze CD8 + T cells having other specificities than for the peptide pp65 495-503 as well as probably primed CD4 + T cells, we looked for the production of cytokines after a second stimulation. We found IFN-g secretion in up to 3.9% of the CD8 + T cells and up to 3.8% of the CD4 + T cells after restimulation with pp65 peptide pool, but not with either irrelevant IE-1 peptide pool or without antigen, in each of eight donors tested. For generation of T cell lines, we magnetically enriched the primed T cells according to their IFN-g secretion. Subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific T cells, defined by their sustained capability to produce IFN-g. Evaluation of the antigen-specificity of the expanded T cells also showed upregulation of the activation markers CD154 and CD137 only if restimulated with the pp65 peptide pool. Further cytokine analysis of the cells revealed co-production of IFN-g, TNF-a, and IL-2, indicating the functionality of the in vitro primed and expanded T cells.In conclusion, we established a cell culture system, which enables the in vitro priming and expansion of CMV-specific CD4 + and CD8 + T cells derived from the naive compartment. This should extend the application of adoptive T cell therapy to patients for whom immune donors are not available.

A. I. Wolf 1 , K. Mozdzanowska 1 , L. Otvos 2 , J. Erikson 1 1 The Wistar Institute, Philadelphia, United States, 2 Temple University, Philadelphia, United StatesThe influenza virus A matrix protein 2 ectodomain (M2e) sequence has remained highly conserved among various human influenza A strains and is therefore a promising target for a protective vaccine. Based on previous work using a synthetic M2e-based multi-antigenic peptide vaccine (Mozdzanowska at al., Vaccine 2003; Virology Journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-M2e antibody (Ab) response and its ability to confer protection against viral challenge.Objectives: Cytomegalovirus (CMV) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (HSCT). Due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived CMV specific CD8 + T cells, have been considered. Clinical data confirm a crucial role for antiviral CD8 + T cells inversely correlating with the incidence of CMV reactivation and disease. CMV specific cells have to reach protective levels in order to be effective. Levels of such cells correlating with protection against CMV infection and disease have only been reported in patients expressing HLA-A*0201 and HLA-B*0702 previously. Considering other frequent HLA alleles CMV specific CD8 + T cells were monitored longitudinally in 30 HSCT patients in this study to establish the cell number thresholds at which patients are protected from CMV reactivation. Methods: We have correlated the pattern of different ex vivo CMV peptide specific CD8 + T cell responses (frequency analysis using tetramer staining and interferon gamma ELISPOT analysis) with the CMV viral load (DNAemia) and clinical status in patients. Different response groups were compared using the Mann-Whitney-U test.Results: Our results demonstrate that the presence of different CMV specific CD8 + T cells inversely correlates with the ability to detect of CMV reactivation in patients at different cell number thresholds. We show that the cell number thresholds for HLA-A*2402/pp65 (341-349) (7.6x10 6 cells/l) and HLA-B*3501/pp65 (123-131) (4.4x10 6 cells/l) specific CD8+ T cells are significantly lower than those for HLA-A*0101/pp50 (245-253) (17.2x10 6 cells/l) and HLA-A*0201/pp65 (495-503) (13.2x10 6 cells/l) specific CD8 + T cells in HSCT recipients post transplant. This difference is also evident in healthy CMV seropositive volunteers. Conclusion: These findings suggest a differing efficiency of the responses restricted by the two sets of alleles. The data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of Ag specific T cells to be monitored after therapeutic strategies for CMV in HSCT patients. (2,5 -50 mcg) . No adverse effects were indicated during trials (up to 25 month of observation).HIV-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of VICHREPOL.No differences were detected in CD4+ and CD8+ T cell counts and CD4+/CD8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to HIV infection. The results of phase I clinical trials of VICHREPOL vaccine were approved by WHO authorized Russian National Control Institution and transition to phase II immunogenicity trials was recommended. Objectives: To improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pIX. The adenoviral nanoparticle vaccine was evaluated in the Friend virus (FV) mouse model and compared to conventional adenoviral vectors. Methods: Adenoviral nanoparticle vectors were constructed by deletion of pIX from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. For vaccination against FV, that is a retrovirus complex of Friend murine leukemia virus (F-MuLV) and spleen focus forming virus, we constructed fusion proteins of pIX and the F-MuLV surface Env protein gp70 or Gag. To elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid DNA encoding either pIXgp70 or gp70 alone. Conventional adenoviral vectors were used that express full-length F-MuLV Env and Gag. The vaccines were tested in CB6F1 hybrid mice that are highly susceptible to FV infection and develop viremia and splenomegaly after FV infection. Results: Vaccination of CB6F1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after FV challenge that was superior to vaccination with conventional vectors. Immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of F-MuLV Env-specific CD4 + T cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. We could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. Conclusion: Adenoviral nanoparticles are a useful tool for the induction of antibody and CD4 + T cell responses that are superior to conventional adenoviral vectors. This new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . Antibody levels were monitored by ELISA and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative RT-PCR, and cellular production of IFN-gamma was measured via flow cytometry. Results: We found that animals vaccinated with CAF01 exhibited higher levels of serum IgG and mucosal IgA than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. Animals that received only Vaxigrip were producing IFN-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also CAF01 did not show any increase in their levels of IFN-gamma. Conclusion: CAF01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. Evaluation of the T-cell specific immune response is very important for global eradication of measles and rubella. Peripheral blood lymphocytes (PBL) from 13 children aged 1-2 years old (6 boys and 7 girls) -Group 1, and 11 children (6 boys and 5 girls) 6-7 years old -Group 2 were isolated on a gradient of density before vaccination ( or revaccination) with Priorix, 1 week, and 2 months after and incubated with CFSE. Then 2 million/ ml PBL were incubated in RPMI-1640 supplemented with 10 % FCS (the negative control), at presence of 5 mcg/ml PHA (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % CÎ 2 at 37°C within 7 day. Intensity of a fluorescence estimated on FL1 by flow cytometr FACSCalibur (BD Biosciences, USA). Cytokines production was measured in the same cultures by BioPlex technology (BioRad, USA). In the negative control 90 % PBL in both groups did not enter mitosis. In the positive control 90 % of cells have passed one and more mitoses. In Group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. In Group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. In a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. Production of the interleukin (IL) 6, IFN-g, TNF-a, IL-4, IL-1 was more informative than IL-5, IL-7, IL-8, IL-12. Measles and rubella antigens induced cytokines production in PBL of immune children and did not influence on PBL of intact children. Thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. Objectives: Broadly neutralizing human monoclonal antibodies (mAb) and patients' sera recognizing functionally conserved epitopes on HIV envelope (Env), such as the gp120 CD4-binding site (CD4bs), appear to be uncommon. Therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. Here we describe the generation of two anti-idiotype (AI) murine antibodies recognizing human anti-HIV-1 neutralizing polyclonal IgGs directed against the CD4bs. The mAbs were shown to react with an anti-CD4bs human neutralizing mAb (b12), to elicit antibodies that recognize the gp120 molecule and an anti-HIV-1 neutralizing response in rabbits, confirming them as CD4bs mimotopes. These mAbs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of HIV-infected individuals. Methods: Broadly neutralizing sera were collected from long-term non-progressor patients. Anti-CD4bs IgGs were purified and used to immunize mice for hybridoma generation. mAbs reacting in ELISA with the anti-CD4bs IgG fraction were used to immunize rabbits. Rabbit sera were then tested for anti-gp120 titer and HIV neutralizing activity by pseudovirus-based neutralization assay. Sera from HIV-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the AIs. Serial dilution of b12 in sera from healthy HIV-negative donors were used to determine ELISA sensitivity. Results: Two clones (P1 and P2) reacted in ELISA only with the CD4bs-directed IgG fraction. The clones were also recognized in ELISA by b12. P1 and P2-immunized rabbit sera showed a strong anti-gp120 titer. In the pseudovirus assay the AIs-immunized rabbits showed a neutralization activity against virions bearing HXB2 strain glycoproteins. In particular, 3/5 rabbits in the P1 group and 1/5 in the P2 group showed an 80 % HIV neutralization at dilutions ranging from 1:20 to 1:150. The immunoenzymatic assay used, allowed to detect a P1 and P2 reactivity in HIV-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. Conclusions: These data demonstrate that immunogens designed on the idiotype of broadly neutralizing Abs are feasible and could help in the design of effective anti-HIV vaccines or diagnostic assays. Yellow fever vaccines (17D and 17DD) are well tolerated, with a very low rate of severe adverse events (YF-SAE), such as serious allergic reactions, neurotropic (YF-AND) and viscerotropic (YF-AVD) diseases. Viral and host factors have been postulated to explain the basis of YF-SAE, especially those able to modify the host immune response to the YF vaccine. However, the mechanisms underlying the occurrence of YF-SAE still remain unknown. In the present investigation, we present a detailed immunological analysis of a 23-year-old US citizen female patient, who developed YF-AND characterized by encephalitis associated with pancreatitis and myositis following 17D-204 vaccination. Our findings highlighted that YF-AND exhibited decreased expression of Fc-g-R in monocytes (CD16, CD32 and CD64) along with increased levels of NKT-cells (CD3 + CD16 +/-CD56 +/-/CD3 + ratio) and activated T-cells (CD4 + and CD8 + ) and B-lymphocytes. Enhanced levels of plasmatic cytokines (IL-6, IL-17, IL-4, IL-5 and IL-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within NK-cells (INF-g + , TNF-a + and IL-4 + ), CD8 + T-cells (IL-4 + and IL-5 + ) and B-lymphocytes (TNF-a + , IL-4 + and IL-10 + ). The analysis of CD4 + T-cells revealed a complex profile with increased frequency of IL-12 + and IFN-g + and decreased percentage of TNF-a + , IL-4 + and IL-5 + cells. Depressed cytokine synthesis was observed in monocytes (TNF-a + ) following in vitro antigenic stimuli. These results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of YF-SAE. This is the first case report of YF-SAE investigated by members of the International Laboratory Network for Yellow Fever Vaccine Associated Adverse Events.

G. Mester 1 , H.-G. Rammensee 1 , S. Stevanović 1 1 Eberhard-Karls-Universität Tübingen, Department of Immunology, Tübingen, Germany Adenovirus (ADV) is a widespread pathogen in humans and can persist in its hosts after infection. Persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. BMT recipients, with high rates of mortality. However, the cellular immune response against ADV is poorly characterised, and very few T cell epitopes have been published up to now. Thus, our aim was to detect dominantly immunogenic adenoviral CD8 T cell epitopes by analysing the responses of healthy blood donors who have overcome infection. We have predicted possible CD8 T cell epitopes for the frequent MHC class I alleles A*01, A*02, and A*24 from the proteins pII (hexon), pVIII, and E1A of ADV strains Ad2 and Ad5 by using the Syfpeithi software developed by our group (www.syfpeithi.de). Subsequently we performed a 12-day recall stimulation of PBMCs from at least 16 healthy donors with synthetic peptide followed by IFN-g ELISPOT screenings to identify naturally occurring T cell responses and assess their frequency in the population. Tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific CD8 T cells. We could identify 27 new peptides eliciting IFN-g responses, several of which were confirmed as novel CD8 T cell epitopes. Amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined HLA restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. These findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro T cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. 

Methods: SLE (5 ARA criteria) was diagnosed in a 40-year-old African female patient with HIV-1 (clade C) infection. Good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of CD4 T-cell count X 200 cells/mm 3 . Initiation of 500 mg MMF bid was associated with biological and clinical remission of SLE and CD4 T-cell increase. No opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to ART. HIV-1-specific CD4 and CD8 T-cell responses were analyzed after 18 months of MMF by IFN-g ELISpot assay and polychromatic flow cytometry assessing IFN-g, TNF-a and IL-2 production following stimulation with a panel of 192 HIV-1-derived optimal epitopes (9/10-mers) covering various HIV regions and a pool of 105 HIV-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. All peptides are derived from HIV-1 consensus strain IIIB. Results: Highly polyfunctional HIV-1 specific CD4 and CD8 T-cell responses against gag were detected. 11 epitope-specific CD8 T-cell responses were identified: except for one response restricted by HLA A*23 and another one by HLA Cw*07, all the others were restricted by HLA-B alleles and mostly by B*58 (n = 5). Seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. IFN-g+, TNF-a+ and IL-2+) regardless of the viral region and HLA restriction. Conclusion: Strong, broad and polyfunctional HIV-1 specific CD4 and CD8 T-cell responses known to be associated with nonprogressive infection were detected during MMF treatment.We therefore suggest that MMF use in the context of SLE-HIV is not detrimental to the establishment or preservation of protective HIV-1 T-cell immunity. 

The rabies virus was propagated in the Vero cell line. Virus was titrated by focus fluorescent units. Virus preparations having a titer of 10 6 DL50/mL were inactivated with b-propiolactone. Aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. Male mice of the strain CF-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group A was immunized with virussqualene, group B with virus-aluminium hydroxide, group C with the antigen alone, group D with saline buffer-squalene, group E with saline buffer-aluminium hydroxide and group E was inoculated with mock-infected cell culture supernatant. Mice were boosted at the 7 th day. All mice were properly bled to prepare preimmune sera and hyper-immune sera. At the end of the immunization protocol the IgG raised against the rabies virus was tested by an indirect ELISA. Results: The highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. There was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected Vero cell supernatant. Conclusions: The use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. Aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. Supported by: CONCyT-46767, COFAA and CGPI-20090305. . State of vaccine-induced measles immunity was determined by means of ELISA in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (LMV) before and after tuberculosis chemoprophylaxis. Statistic data were processed with t-, W-and U-criteria. Results: During the first three years since LMV revaccination IgG level was middle (children with negative and long-term positive MT) and high (children with conversion and hyperergic MT). In 4-6 years since LMV revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with MT conversion. In 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected IgG level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. Comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles IgG level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with MT conversion in 1-3 years since LMV revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic MT IgG level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values.

-At the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-In remote periods since LMV revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-In countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. Objectives: To evaluate the balance of IFN-gamma and IL-17 producing cells in lungs during the immunotherapy of tuberculosis with the DNA vaccine encoding the heat-shock protein 65 (DNAhsp65). Methods: Balb/c female mice were infected by intra-tracheal route with 10 5 H37Rv Mycobacterium tuberculosis. Immunotherapy with endotoxin free DNAhsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. Each dose consisted of 100 micrograms of DNA vaccine in the quadriceps. Intracellular cytokine staining of CD4+, CD8+ and gamma-delta T cells from lungs were determined 10 and 50 days after the end of the therapy. Bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. Differences of P X 0.05 were considered significant (T test). Results: At day 10 after the end of the immunotherapy, DNAhsp65 treated mice exhibit increased numbers of absolute CD8+ and gamma-delta T cells when compared to non-treated animals. The percentage of IFN-gamma and IL-17 producing gamma-delta T cells were the same between treated and non-treated animals.

In contrast, DNAhsp65 treated mice showed more IFN-gamma producing cells in both CD8+ and CD4+ cell populations. At day 50 after the end of the therapy, the main observation in mice which received DNAhsp65 treatment was the augment of all three populations producing IFN-gamma. Although non-treated animals also increased the frequency of CD4+ and gamma-delta T cells positive for IFN-gamma, they did not increase the numbers of IFN-gamma CD8+ cells, together with a more frequency of gamma-delta T cells producing IL-17. Finally, the immunotherapeutic effects of DNAhsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis.

The results presented here indicates that CD8+ cells producing IFN-gamma and the reduction of the frequency of gamma-delta T cells secreting IL-17, are the main effects of DNAhsp65 immunotherapy of murine tuberculosis. Furthermore, these results have important implications since they indicate the importance of an appropriate balance of IL-17 and IFN-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. The detection of quantitative changes in mRNA expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time PCR) screening methods. Because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a Reverse-Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA) method. RT-MLPA is a reliable, robust, low cost and user friendly technique permitting rapid mRNA expression profiling of as many as 60 loci in a single reaction. Genes of interest can be selected on a tailor-made basis. The assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a PCR amplification step within the RT-MLPA ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. Since this assay is relatively high throughput (96-well format), requires only 100 ng RNA per sample, and allows mRNA profiling in direct ex vivo whole blood samples (from e. g. Pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. To illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-Saharan Africa. Thus far we have applied RT-MLPA to characterize the human immune response to Mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. A particularly useful application of the RT-MLPA is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (TB) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active TB patients. Initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with TB disease. These biomarkers may provide new and relevant information that can be applied in future TB studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. Preclinical M. leprae infection is a major source for leprosy transmission. Therefore, early detection of individuals infected with M. leprae is crucial. However, to date there are no diagnostic tests available that can identify preclinical leprosy. Such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.Newly developed HLA based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of M. leprae infection.Using this post genomic approach, we were able to identify candidate proteins and peptides unique to M. leprae containing predicted T cell epitopes restricted via several major HLA-class I and II alleles. Since the selected genes were of unknown function, their expression in M. leprae bacilli was assessed.Evaluation of the immunogenicity of these M. leprae proteins in PBMC from a Brazilian population showed that 5 candidate antigens induced significant IFN-g levels in M. leprae infected individuals but not in healthy controls from an endemic area.Importantly, among exposed healthy controls 71 % had no detectable IgM antibodies to the M. leprae specific PGL-I, but instead responded to one or more M. leprae antigen(s). To further improve the diagnostic potential of these M. leprae sequences, synthetic peptides spanning all 5 M. leprae proteins were analyzed similarly. Determination of cumulative T cell responses towards 4 of these peptides that activated PBMC of leprosy patients increased the sensitivity compared to single peptides to 100 % in PB, 75 % in Rx and 93 % in HHC, without compromising specificity.Since diagnostic tools should be applicable in several populations regardless of the genetic background, these M. leprae antigens are also tested in populations on the African (Ethiopia) and Asian (Nepal) continent.In addition, we have applied these antigens in a new user-friendly UCP-LF assay to detect different cytokines. This assay proved to be more sensitive than ELISA for detection of IFN-g and can be easily applied in field sites. Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis (Mtb), affects millions of people. M. bovis BCG is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. Paratuberculosis, an enzootic bacterial disease in ruminants, due to Mycobacterium avium subsp. paratuberculosis (Map), has a significant economic impact on livestock production, and moreover, Map infection may be one of the microbial triggers of Crohn's disease in humans. Map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. CD40L, a co-stimulatory molecule preferentially expressed on activated CD4+ T cells, is the ligand of CD40. CD40-CD40L interaction induces the production of IL-12 and the initiation of a Th1-type immune response. Several studies show that CD40L is required for the activation of macrophages and the maturation of DCs. Moreover, CD40L enhances the capacity of CD8+ T cells to produce IFN-g and to lyse Mtb-infected monocytes. In this study we attempt to improve existing TB and Map vaccines with a recombinant BCG expressing CD40L. We have constructed the recombinant BCG strain expressing CD40L (rBCG2) by electroporation of BCG with pGFM11/SignalSequenceAg85B-CD40lec and an another recombinant strain with empty vector pGFM11 (rBCG1) as a control. The expression of CD40L has been evaluated by Western blotting. BALB/c mice were vaccinated with the recombinant BCG vaccines. BCG growth kinetics were compared by counting viable bacteria (CFU) in spleen and lungs. The immune response was evaluated by measurement of Th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. Two months post vaccination, mice were challenged with Mtb and Map and protection was evaluated. Preliminary results show normal persistence of the two recombinant BCGs. Analysis of the immune response shows an effect of CD40L 2 weeks after vaccination but not at 4 and 8 weeks. rBCG2 seems to be more protective against paratuberculosis than rBCG1, but not against tuberculosis. Another vaccination experiment is required to confirm these results. The effects of BCG-CD40L on cultured DCs in vitro will further be explored. Objectives: Tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. Iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. The regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. Methods: Using the guinea pig model of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (PPD) of Mycobacterium tuberculosis or infected with BCG or M. tuberculosis. The expression of ferritin in co-ordination with key immuno-regulatory proteins, TNFa, IFNg and IL-1a, was examined using real-time PCR. To determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the PPD re-stimulation studies by the addition of guinea pig specific TNFa and IFNg antibodies. Results: A typical pro-inflammatory immune response was observed with significant up-regulation (p X 0.05) of TNFa, IFNg and IL-1a after re-stimulation with PPD and mycobacteria. Of interest was a trend in ferritin down-regulation after re-stimulation with PPD and BCG and this was significant (p X 0.05) after restimulation with M. tuberculosis. The down-regulation of ferritin was also affected by the addition of TNFa antibody in the PPD re-stimulation study. Conclusions: Ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. The data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve TNFa. These results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. This work is funded by Health Protection Agency PhD studentship award.

Methods: Anti-CD40 mAb and Ag85A were chemically treated with SATA and Sulfo-SMCC respectively, in order to produce a stable crosslinker between both proteins. Crosslinking was confirmed by western blotting and CD40 binding on CD40 transfected L929 fibroblasts. The conjugates were tested in vivo in wild type and CD4 + cell-depleted mice for the induction of specific anti-Ag85A serum antibodies. Splenocytes were challenged ex vivo with Ag85A and were examined for their ability to produce Th1-related cytokines. ELISPOT assays were performed to determine IFNg production and flow cytometry was used to analyse intracellular cytokine staining for TNFa, IFNg and IL-2.

We developed a method to successfully crosslink anti-CD40 mAb to Ag85A. Serum antibodies against Ag85A were detected after immunisation with this conjugate vaccine in both wild type and CD4 + cell-depleted mice. T cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in IFNg production (ELISPOT), when compared to mice vaccinated with Ag85A alone. Production of two other Th1-related cytokines, TNFa and IL2, was also increased in these T cells as shown by intracellular cytokine staining. Conclusion: Our results suggest that anti-CD40 conjugate vaccines could provide a new way to increase vaccine efficacy. This new conjugate vaccine may be able to by-pass the need for CD4 + T cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients.

H. Schäfer 1 , R. Burger 2 1 Robert Koch-Institute, Cellular Immunology, Berlin, Germany, 2 Robert Koch-Institute, Infectious Diseases, Berlin, GermanyObjective: Immunity against mycobacterial infections is mediated by both CD4-positive and CD8-positive T-lymphocytes. CD8-positve cells respond to peptides derived from cytosolic proteins and presented on MHC class I molecules of antigen presenting cells (APC) via the endogenous pathway. Some APC however, are able to take up extracellular antigens and present peptides thereof on MHC class I molecules. This process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. To define the contribution of cross-presentation to activation of CD8+ T cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. Methods: Purified T lymphocytes from guinea pigs immunized with BCG or complete Feund's adjuvant were labeled with the intracellular fluorescent dye CFSE and incubated with PPD and/or APC for 5 days. Surface phenotype and proliferation of T-lymphocytes were analyzed by flow cytometry.Results: Up to 30 % of lymph node T lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with PPD-pulsed macrophages. No difference was observed between BCG-(living mycobacteria) and Freund's adjuvant (heat killed mycobacteria)-immunized animals. The responses of both T cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime CD4+ T-lymphocytes in vivo. Similarly, the CD4-positive subpopulation should primarily respond to soluble antigens presented on MHC class II molecules. Proliferation of both the CD4+ and the CD8+ subpopulation depended on the presence of APC. Stimulation oft CD8+ cells as a consequence of direct loading of peptides onto MHC class I molecules was ruled out by using MHC-class I-positive fibroblast cells instead of professional APC, which did not lead to proliferation of primed T-lymphocytes. Conclusion: Cross-presentation of soluble antigens to CD8-positive T cells is a highly effective means to stimulate the response of cytotoxic T cells against mycobacterial antigens even without direct contact to infected cells. Therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. Objectives: Due to broad antigenic cross reactivity of purified protein derivatives (PPD) with BCG vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (TST) is not reliable to evaluate the specific anti-tuberculosis immune response. Therefore, new tools are required to improve Mycobacterim tuberculosis (TB) diagnosis and treatment, including enhanced ability to compare new treatment strategies. Among different antigens early secretory antigenic target 6 (ESAT-6) protein is highly specific for TB complex and elicit strong T-cell response in human.

In order to monitor the immune response against the pathogen, 83 Iranian and Afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of ESAT-6 and PPD specific T-cells in their peripheral blood by home made ELISPOT IFN-gamma assay. Results: Considering cut off of 4 spot forming unit ( G 20 spots per million), we found detectable response to ESAT-6 in almost 80 % of patients with active disease. This frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable ESAT-6 specific response even after six months completing treatment. Neither of healthy individuals showed such response. T cell response against PPD was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. Conclusion: ELISPOT IFN-gamma assay showed a sharp induction of Th1 immune response, against ESAT-6, in TB patients which persists after successful treatment and full recovery. These results may show potential application of tuberculosis-specific ELISPOT testing as a proxy measure of TB diagnosis and treatment. BCG is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. Both environmental and genetic host factors as well BCG strain variability and virulence of the intruding M. tuberculosis strain have been suggested to affect the efficacy of BCG vaccination. In mouse and bovine models it has been shown that pre-exposure to Mycobacterium spp. negatively affected protective efficacy of BCG. We use non-human primates (NHP) for the evaluation of new TB vaccine candidates and possible identification of immune mechanisms of protection. However, in naive rhesus macaques a variable efficacy of BCG reminiscent of the clinical situation was revealed. By meta-analysis we compared immune response parameters measured after vaccination using BCG strain Danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant M. tuberculsosis strain Erdman. Although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of BCG. Most remarkably, while BCG induced levels of IFNg secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. Our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of TB diagnostics for NHP, negatively affects the protective efficacy of BCG. Together, these results are relevant for evaluation and interpretation of TB vaccine tests in NHP and support further research into the identification of (mechanisms of) protective immunity in the primate host.

P. S. Nagpal 1 , P.K. Upadhyay 1 1 National Institute of Immunology, PDC-1, Delhi, IndiaObjective: Study was aimed for the preparation of dry powder formulation containing live Mycobacterium indicus pranii for pulmonary immunization against tuberculosis. Pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. Secondly, pulmonary delivery is a Needle-free delivery system, long desired for vaccine delivery. Dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. Method: Sodium Alginate solution with suspended Mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. Aerosols so generated were entrapped in CaCl 2 solution with Poly-Vinyl Alcohol (PVA) as surfactant. Particle so formed collected by centrifugation and lyophilized for dry powder formulation. PVA and alginate concentration varies the size, surface and shape of the particles. Formulation so prepared was delivered to C57BL/6 mice directly into lung by endotracheal intubations of mice. Proliferation index (PI) of spleenocytes of immunized mice was measured after in-vitro stimulation with Mycobacterium tuberculosis antigen. Result: 2.4 % alginate, 0.012 % PVA concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. Viability of the Mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % PVP (poly vinyl pyrollidone). There was 6 fold increase in Proliferation Index of spleenocytes and releases 600 pico-gram of Interferon gamma after 4 week of immunization. Formulation also induces the Activation of Dendritic cells after their in-vitro incubation as shown by 10 % increase in CD86 and 10.5 % in CCR7 expression as compared to blank alginate formulation. Bacterial exopolysaccharides (EPSs) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. EPSs are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. Some EPSs contain sulphate groups as inorganic substituents. The presence of these groups contributes to the biological activity of EPSs, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. B100 is a constitutively sulphated EPS produced by Halomonas maura, a recently discovered halophilic bacterium. In preliminary experiments we found that modification of its EPS by adding sulphate groups to the native polymer (thus obtaining B100S) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. At the same time we found that other EPSs produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. It was therefore of interest to determine whether the antiproliferative activity of B100S was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by B100S. By cell cycle analysis we determined that B100S is able to induce apoptosis in up to 80 % of Jurkat and MOLT-4 T cell leukemias. The examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by B100S is restricted to cells of the haematopoietic lineage and that leukemic T cells are particularly sensitive to death induced by B100S, but that untransformed cells are not. A time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that B100S triggers apoptosis through the mitochondrial pathway. It is noteworthy that B100S also induces vigorous apoptosis in primary leukemic T cells obtained from the peripheral blood of patients. Therefore, B100S may well provide a satisfactory therapeutic alternative to patients with acute T cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. Particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting DCs. Polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. This layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. By using the biopolyelectrolytes dextran-sulphate and poly-L-arginine, biodegradable microcapsules can be obtained. In this study, we have chosen the lungs as a non-invasive route for vaccine delivery. As demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-L-arginine microcapsules were readily taken up by local pulmonary APCs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards APCs. Microcapsule instillation also affected the pulmonary APC activation status, indicated by the emergence of an APC population expressing increased levels of MHCII, the co-stimulatory ligands CD40, CD80 and CD86, and of the inflammatory cytokines IL-6, IL-23 and MCP-1.

Using ovalbumin (OVA) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. Analysis of the alveolar infiltrate, CD4 T cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard Th2 and Th1/Th17 skewing adjuvants Alum and complete Freund's adjuvant (CFA). In response to OVA aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while Alum and CFA respectively induced typical eosinophilic and neutrophilic inflammations. Striking differences were also observed on the level of CD4 T cell responses. Microcapsule based vaccination resulted in a marked induction of IL-17 secreting Th17 cells, without inducing strong Th1 (CFA) or Th2 (Alum) responses. These differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (IgG1, IgG2c and IgE).In conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung APCs, and possess immune stimulating activities distinct from Alum and CFA. Due to their capacity to generate Th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. Pneumolysin is an important virulence factor produced by virtually all clinical isolates of Streptococcus pneumoniae. The protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. Published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of CD4 + T cells and increased production of pro-inflammatory cytokines in immune cells. In this study we investigated the interaction of pneumolysin with murine dendritic cells (DC). We found that pneumolysin induced the activation of DC, reflected in the enhanced expression of the costimulatory molecules CD80, CD86 and CD40 and MHC class II molecules. The toxin alone was found to be a poor inducer of cytokine production by DC but it did enhance the secretion of TLR agonist-induced cytokines such as IL-6 and TNF-a. Previous published findings have shown that pneumolysin activates peritoneal macrophages in a TLR4-dependent manner. However, we found that pneumolysin was capable of activating DC from both wildtype and TLR4-defective C3H/HeJ mice, by inducing cell maturation and synergising with TLR agonists to enhance cytokine secretion. Importantly, we also found that pneumolysin is a strong inducer of IL-1b secretion by DC, through its effects on caspase-1 processing, which is also TLR4-independent. The results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require TLR4 signalling. Objectives: Transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. The objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. Methods: Pregnant BALB/c mice were supplemented with placebo (maltodextrin) or probiotics (Lactobacillus paracasei NCC2461 (ST11) or Lactobacillus rhamnosus NCC4007 (LPR), each at 5x10 8 CFU/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. At weaning, pups were immunized with live attenuated measles vaccine (MV-S, Aventis-Pateur). Weight evolution of pups was followed from week 1 to week 7 of life. Fresh feces were collected at 3, 5 and 7 weeks of life for determination of IgA levels (assessed by ELISA). Pups were bled 2 and 4 weeks after immunization for determination of measles-specific IgG1 and IgG2a antibodies. Analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. Results: All newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. Fecal IgA production increased progressively in all pups from weaning, reflecting a normal development status. Nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning S-IgA production in pups. LPR supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. Interestingly, no significant effect was observed in the ST11-fed group. Finally, a modification of the microbiota composition was observed in pups of supplemented mothers. Particularly, there was a significant increase in Lactobacilli in pups from the LPR group as compared to controls. Conclusion: This study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. Moreover, these first results seem indicate that the effects are strain specific. Chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. Chitosan (Chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. Here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. Nanoparticles (Chi-NP) were prepared from 200 kDa chitosan by ionotropic gel formation. Bovine serum albumin (BSA) and myoglobin, human immunoglobulin G and superoxidedismutase (SOD), and chicken lysozyme were FITC labeled. Chi-NP were preincubated with proteins at 3:1 ration and washed 3 times. After washing Chi-NP containing bound proteins were run by denaturating gel electrophoresis. All proteins were able to form complexes. Most effective binding was shown for BSA, SOD, and lysozyme. The stability of Chi-NP complexes with proteins was studied in vitro on macrophage cell line RAW264.7 by confocal microscopy. For this Chi was labeled with rhodamine and nanoparticles were coincubated with FITC labeled proteins before addition to the cells. We showed co-localization of Chi and FITC for all proteins studied. These results demonstrate that Chi-NP form stable complexes which are internalized by macrophages. The family Euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. In the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 Euphorbiaceaes on peripheral blood mononuclear cells (PBMC) from healthy individuals were evaluated. The PBMC were exposed to the extracts w/o phytohaemagglutinin A (PHA), Cycloheximide (CHX) or lipopolysaccharide (LPS) as agents that induce proliferation, apoptosis and cytokine production in PBMC. The lymphoproliferative activity of PBMC was evaluated by thymidine incorporation and CFSE dilution assay using flow cytometry. The mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using DiOC6/propidium Iodide staining by flow cytometry and TNF-a secretion in the culture supernatans by ELISA. We found that 15 up to 24 Euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. However, only the bark extract of Croton spp. insoluble in Hexane:Diclorometane:Methanol (HDM) and the latex extracts of Euphorbia cotinifolia and Euphorbia tirucalli induced strong proliferation, apoptosis and also TNF-a production in PBMC. These extracts were subfractioned by Sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. Additionally, we started with the characterization of the specific immune effects of these subfractions on PBMC. All three subfractions induced proliferation predominantly on CD3+ cells. These effect was also observed in isolated T cells indicating that accessory cells are not necessary for the subfractions'activity. The lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates D-(+) Galactose or a-methyl-mannopyranoside. These results demonstrate the presence of immunomodulatory compounds in plants from the Euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. 

We found significant increase on lymphocytes and eosinophils populations obtained from LPS + P. acnes-treated group in relation to control group.On 35 th day, we detected a significant negative correlation between eosinophils absolute number and FEC. Both IL-5 and IgE serum levels were increased on animals from group I when compared to control.The enhancement on Th2 immune response pattern induced by LPS and P. acnes treatment diminished drastically parasitic load. Conclusion: Our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. Vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. Indeed, current influenza A vaccines do not generate significant immunity against serological influenza A virus subtypes and would thus be ineffective in the face of a pandemic novel variant. Hence adjuvant usually needs to be added to those types of vaccines. Here we show that Wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. Experiments performed in mice showed induction of specific CD8+ CTL cells against conserved proteins that were accompanied by the induction of IFN-g producing CD4+T cells, following single immunisation. In addition increased HI titres and higher levels of specific IgG2a and IgG2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. Consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. This promising finding on the improvement of seasonal influenza vaccines by Wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase I clinical trials in humans.

S. Lindgren 1,2 , N. Almqvist 2 , A. Lönnqvist 1 , S. Östman 1 , C. Rask 1 , E. Telemo 2 , A.E. Wold 1 1 University of Göteborg, Department of Clinical Bacteriology, Göteborg, Sweden, 2 University of Göteborg, Department of Rheumatology and Inflammation Research, Göteborg, SwedenObjective: Dietary antigens normally evoke immunological tolerance. A prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. The gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in Western societies in parallel with reduced complexity of the infantile commensal microflora. We have observed that children neonatally colonized with S. aureus in the gut seem protected against development of food allergy. Here we examine whether a S. aureus toxin affects tolerogenic processing by the intestinal epithelium. Methods: Mice were given S. aureus enterotoxin A (SEA; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. One hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). Results: Recipients of serum from SEA pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with SEA. This was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. Examination of gut sections from SEA treated donor mice revealed increased density of CD8a + intraepithelial lymphocytes.

Our results show that SEA promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes.

Abstract withdrawn by author To develop an efficient vaccine against Cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like OmpA, OmcB, and Pmp21, proteins of the inclusion membrane like IncC, secreted proteins like CpaF, and the heat shock protein GroEL. CpG-DNA 1826, a highly stimulatory oligonucleotide for APCs, was used as adjuvans. Subcutaneous co-injection of OmpA, OmcB, Pmp21, or GroEL together with CpG-DNA 1826 reduced the chlamdial burden in nasally infected mice. However, symptoms like substantial loss of body weight were not influenced. In contrast, a low dose infection with Cp. pneumoniae almost completely prevented the loss of body weight upon challenge. To improve the efficacy of the vaccine we used Poly-DL-lactide-co-glycolide microspheres loaded with the protein OmpA or Pmp21 together with CpG-DNA 1826. The microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same APC. Intranasal but not subcutaneous vaccination of mice with OmpA or Pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. Pmp21 microspheres induced protective IFNg-secreting CD4 + T-cells and raised pulmonary Pmp21-specific IgA levels in vivo. Also, Pmp21 microspheres caused lower IL-6 serum levels upon administration than the injection of Pmp21 together with CpG-DNA 1826, indicating fewer side effects. Objectives: Staphylococcus (S.) aureus superantigens are highly potent T cell mitogens and the causative agents of toxic shock syndrome (TSS) and food poisoning. Most S. aureus have superantigens and patterns are highly variable. To date, the role of superantigens in bacteraemia is not well defined.To analyse whether superantigens play a role in bacteraemia, we investigated S. aureus strains and anti-superantigen antibody responses in 44 cases of S. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. A rise in neutralising antibody titers indicates that superantigens are produced during infection.

The study comprised 44 iv drug users with positive S. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original FINTROVA and FINLEVO trials (Ruotsalainen 2006).All S. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-PCR was applied to determine the superantigen gene pattern. Sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). Neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.Results: Genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (Holtfreter 2007) . There were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. Interestingly, the staphylococcal lineage ST59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).Most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. We frequently observed a rise in antibody titers during infection. Surprisingly patients with ST59 strains did not show any elevations in neutralising antibody levels. Conclusion: S. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. This indicates that superantigens are produced during infection. However, the action of superantigens is frequently modulated by specific neutralising antibodies. This and the special behaviour of S. aureus ST59 strains need further investigation. Objectives: Down Syndrome (DS) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. To test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (TT), which is part of the Dutch immunization program. Methods: After booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) TT responses were investigated in 15 and 7 DS children, respectively. Samples were taken before and 3-4 weeks after vaccination. TT-specific IgG and IgG-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (ELISA), avidity of IgG 1 -anti-TT by an avidity ELISA. The results were compared with reference values from the laboratory. Results: At 4 years, post-vaccination anti-TT-titers were decreased (geometric mean total IgG, IgG 1 , IgG 2 and IgG 4 ). At 9 years, DS children had lower postvaccination geometric mean IgG 4 anti-TT-titers only. Post-vaccination IgG 1 -anti-TT avidity levels were decreased in 8/15 and 4/7 DS children at four years and nine years of age, respectively.

The quantitative and qualitative anti-TT-responses in both DS groups are shifted downwards compared to the reference values. Although the anti-TTresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that DS children have profound and lasting difficulties with specific anti-TT antibody formation. 2 kDa; 30, 6 kDa; 23, 9 kDa; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kDa and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kDa were expressed by a majority of examined strains independently of the associated diseases. We assume that these OMPs could be conservative proteins of H. pylori. Conclusion: Considering OMPs as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of H. pylori OMPs as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. The increasing cases of severe diarrhoea and invasive lethal infections in children caused by Salmonella Typhimurium are a major public health problem in Mexico. The rapid dissemination of multidrug-resistant S. Typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. The objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant Salmonella infections was associated with a reduced in anti-Salmonella immunity. A cohort of 100 families, from a Mexican agricultural community with a high incidence of endemic Salmonella infections, was followed prospectively for an 8-month period. Sera were obtained from healthy subjects from the same community (2 months to 88 years of age). The highest incidence of Salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. The lowest incidence, 10/1000, was observed in the population aged 10 to 59. Whereas serum from individuals ranging 15-70 years of age showed maximum IgG1, IgG3 and IgA anti-S. Typhimurium titres, children less than 5 years-old did not show detectable IgG1 and IgG3 titres and had weak IgG2, IgA and IgM antibody levels; only their IgG4 levels were comparable to those detected in adults. Moreover, the levels of IgG2 and IgG3 antibodies were lower in adults with a diarrheal-associated episode. Interestingly, S. Typhimurium YUHS 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to Fc-mediated phagocytosis by human monocytes. These data contributes to define the protective immune response involved in anti-S. Typhimurium immunity. Diseases caused by the yeast of Candida genus are a serious clinical and social problem. Despite this fact, there is no effective prevention against these opportunistic pathogens yet. Although C. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. C. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates.

The surface mannan of Candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. Because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). In our study we prepared oligosaccharides from acid-stable part of mannan C. dubliniensis by conventional acetolysis. This procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. Obtained oligosaccharides were used in inhibition ELISA and SPR (Surface Plasmon Resonance) measurements. The reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-HSA conjugate injections in week intervals as well with inactivated C. dubliniensis whole

Z. Neščáková 1 , S. Bystrický 1 1 Institute of Chemistry, Slovak Academy of Sciences, Bratislava, SlovakiaOur newest approach to sub-cellular vaccine against Gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified LPS) as the target antigen. This is achieved by conjugation of carbohydrate to a protein carrier which secures the T cell dependent immune response. The goal of the immunization with this conjugate is to generate the effective production of memory B cells. Here we prepared subcellular conjugate with the detoxified LPS from Vibrio cholerae strain O135 using polymer carrier and a protein. The cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. Activation and differentiation of B-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. A single-platform approach based on flow cytometry and defined number of fluorospheres was used to count B cells. However in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. The protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. Like pan-B cell marker CD19, expressed almost on all blood and tissue B cells, was used. Here we investigate the characteristics and development of antibody (iso)types after secondary immunization with MenCC or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. Methods: Volunteers, age 18-55 years, were immunized with MenCC or received a secondary immunization with MenCC or plain MenC PS. Blood samples were obtained before and seven time-points after immunization. IgG, IgA, IgM, IgG1, IgG2 and avidity were assessed by a multiplex immunoassay. Functional antibodies were determined by a serum bactericidal assay. Results: High levels of antibodies were still present 5 years after primary MenCC immunization. Secondary immunization resulted in increased IgG and SBA titers after 5 to 7 days. In primed individuals, IgM was still present, and this only increased following a secondary immunization with plain PS. In addition, immunization with PS induced a higher IgG2 response compared to MenCC immunization. Discussion: Secondary immune responses are quiet slow. The composition of the Ig (iso)type distribution is different between MenCC and plain PS and might be of influence on functional titers. Although this study indicates that immunological memory was previously induced by a single MenCC vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as N. meningitidis. The immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of IgG, IgA and IgM isotypes and by phagocytic and respiratory metabolic activities of granulocytes. Results: Prime-boost immunization strategy resulted in enhanced production especially dLPS-specific IgG and IgM isotype antibodies in both experimental groups (peak titers 1:3200). IgM-IgG isotype switch was more pronounced with O-SP. Peak values of dLPS specific IgA isotype were signicantly lower than IgG and IgM ones (1:800 vs. 1:3200). Flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with O-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). Subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. The second de-OAc conjugate exerted different pattern of stimulation, sc intervention was more effective.

Our results are indicative for immunological effectiveness of novel dLPS derived glycoconjugates; thus promising further application in cholera subcellular vaccine. This work was supported by APVV 0032-06 and VEGA 2/7029/27 Grants of the Slovak Grant Agencies. Background: The yeast Candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. A long-acting, effective and safe vaccine that protects against medically important Candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. Mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked D-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. Investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. Objective: In this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, BSA) was used.

Methods: In order to study the immunogenicity of heptamannoside -BSA conjugate as inducer of hummoral and cell-mediated responses, BALB/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. Cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using ELISA. Results: Prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. Intraperitoneal boost elicited significantly higher IgG and IgM levels than subcutaneous boost. Immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. Introduction: Bulgarian immunomodulator Respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. It is composed of killed bacterial bodies and lysates of six microbial species (Streptococcus pneumoniae, Branhamella catarrhalis, Streptococcus pyogenes, Haemophilus influenzae, Staphylococcus aureus, Klebsiella pneumoniae). The immune response in respiratory tract includes not only systemic immunity in lungs, but also BALT as a part of common mucosal immune system. Most animals develop BALT after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. Therefore, immunostimulation of BALT may contribute to more efficient mucosal immunity in respiratory tract. Aim: To study BALT development in different terms after oral application of Respivax in guinea pigs. Methods: Male guinea pigs (250g-350g) were treated orally with 50 mg Respivax five consecutive days. After the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. Morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. The populations of CD8, CD4 and B cells were identified on cryosections by using indirect peroxidase immunostaining. ZIO technique was used to detect intraepithelial dendritic cells (DC). Results: BALT was not identified in control animals. In the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. The following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. They were composed mainly of CD8 positive cells, which number reached maximum at the end of the second week. On day 14 B cell lymphoid follicles with different size were found. Lamina propria was presented by abundant lymphocyte infiltrates, composed of CD4 and CD8 positive cells. On days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. Numerous intraepithelial DC's were detected in treated animals, comparing to controls in which only a few were identified. Conclusion: Oral administration of Respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of DC and BALT development.

G. Gupta 1 , S. Majumdar 1 1 Bose Institute, Molecular Medicine, Kolkata, IndiaVisceral leishmaniasis (VL) caused by the protozoan parasite, Leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. Chemokines, particularly CC chemokines, have been shown to render protection against Leishmania infection. There is no clear understanding about the immunoprotective role of CXC-chemokines in VL.In the present study, the comparative potential of CXC chemokines, Interferon Gamma inducible protein-10 (IP-10) and interleukin-8 (IL-8) in restricting Leishmania donovani infection via the release of nitric oxide (NO) and proinflammatory cytokines was studied in an in vitro model. NO, a crucial mediator for IP-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (iNOS2) expression and was linked to the MAPK signaling pathway via antagonistic regulation of p38MAPK and ERK1/2. Further, IP-10 was also able to abrogate the survival of Leishmania in an in vivo model of VL by restoration of Th1 cytokines and NO. Thus this study strongly demonstrates that IP-10, like CC chemokines, is involved in rendering a protective response in VL via upregulation of proinflammatory mediators. African Trypanosomiaisis (AT), known as Sleeping Sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. AT is caused by the protozoan Trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. The DNA vaccines could be the answer for this disease by being able to induce production of IgG antibodies and induce of Th1/Th2 cytokines mediated by CD8+ T cells and activating CD4+ T helper cells.

In this study, we shows that Balb-C mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from Trypanosoma brucei, named Invariant Surface Glycoprotein (ISG), trans-sialidase (TSA), and fosfolipase C (PLC) are able to produce IgG antibodies anti-trypanosoma. This immunization process was able to control the mortality level when mice were submitted to challenger assay with Trypanosoma brucei brucei parasites. In mice co-infected with S. ratti and L. major (NL) neither clearance of L. major nor Strongyloides infection was changed. Mice co-infected with S. ratti and P. yoelii (NL) showed the same course of parasitaemia as single infected mice. These results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, S. ratti and L. major or P. yoelii. If this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. In the present study ,we evaluated TSA -encoded DNA vaccine against L.major in BALB/C mice. IgG and IFN-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p X 0.05) following immunization and after challenge with Leishmania major. IL-4 values were increased in all groups, but there was no statistical difference between the groups(p G 0.05) following immunization and after challenge with Leishmania major. The immunized mice with the DNA vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p X 0.05) in this regard . The survival time of the immunized mice with the vaccine was significantly higher than the control groups (p X 0.05) after the challenge with Leishmania major. The immunized mice had significantly lower parasite load comparing to the control mice(p X 0.05). The findings of this study indicated that the TSA -encoded DNA vaccine increased the cellular response and induced protection against infection with Leishmania in the mice. The TSA -encoded DNA vaccine may be an excellent candidate for future vaccine developments against Leishmania. There is a lot of evidence showing that BCG vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. In this study the protective effect of autoclaved Leishmania major (ALM) vaccine in combination of either rectal or subcutaneous BCG on susceptible BALB/c mice was evaluated.One month after BCG vaccination, BALB/c mice were immunized subcutaneously twice with ALM+alum at 3 week intervals. Three weeks after booster injection, 5×10 5 stationary phase L. major promastigotes were inoculated subcutaneously in one footpad. Immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. High level of interferon gamma was induced in the spleen and significant increase in the serum ratio of IgG2a/IgG1was observed only in rectal immunized group. Rectal immunized mice showed comparable nitric oxide production and iNOS induction in peritoneal macrophages .The obtained results in rectal BCG vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal BCG immunization against leishmaniasis might be due to the long-lasting induction of Type 1 immunity. Methods: Two groups of BALB/c mice were infected by L. tropica. One group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. Mice were challenged by L. major in the right footpad after establishment of L. tropica infection. The immune response was evaluated at two intervals: one week and one month after challenge. Single cell suspensions were prepared from draining lymph nodes of mice. Cells were stimulated by phorbol myristate acetate (PMA). Cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. The following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (DTH) to L. major challenge, production of gamma interferon (IFN-g) and interleukin 10 (IL-10), and cellular expression of CD4 and CD25. Results: Infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of DTH and IFN-g, lower levels of CD4+ lymphocytes, and higher protection against L. major challenges. Conclusion: Intradermal infection of L. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in BALB/c mice. Therefore, we propose the route of infection as an important variable in this experimental model. This factor should be considered for development of an appropriate experimental model for human L. tropica infections. Objectives: Many mammals exhibit a periparturient relaxation of immunity (PPRI) to gastrointestinal nematode parasites culminating in increased worm burdens. It has been suggested that the extent of PPRI may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. Subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. However, this effect is often confounded with increased food intake and thus increased energy levels. Here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host.

The Nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian PPRI. Lactating rats, re-infected with 1,600 infective N. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (ME). Parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. Histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mRNA levels in the mesenteric lymph nodes were carried out to assess the host immune status. Results: Increasing CP supply, but not increased ME supply, reduced worm burdens. Whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased CP supply increased mucosal mast cell numbers and levels of N. brasiliensis specific antibody (total IgG, IgE, IgG1 and IgG2a). This was independent of level of ME supply. Feeding regime did not affect levels of the type-2 cytokines IL-4 and IL-13. Conclusion: This study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. This anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. This data emphasises the role of immunonutrition in combating infectious disease. Protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. Background: Gp63 is the major surface glycoprotein of Leishmania that exhibits protease activity and has an important role in the biology of the parasite. The aim of this study was cloning and expression of Gp63 of L.major strain MRHO/IR/75/ER. Methods: L.major promastigotes were grown in RPMI1640 supplemented with 10 % FCS. L.major RNA extraction and cDNA synthesis were carried out. Gp63 gene segment was amplified by specific primers and cloned into pTZ57R to construct pTZ57R/gp63. The presence of gp63 into pTZ57R was confirmed by PCR. Then, pTZ57R/gp63 was sent to determine the sequence of its nucleotides. After that the GP63 gene segment was sub-cloned into pET32 a (+) expression vector and transformed into E.coli BL21 (DE3) pLysS and GP63 protein was expressed in presence of 1mM IPTG. Objectives: The development of a vaccine against malaria caused by Plasmodium falciparum is an urgent public health priority. Influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. At appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (Genton et al., Plos One, 2007) . The aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. Objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. Particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated.

The study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. Subjects received virosomal formulations containing 50 mg of AMA 49-C1 (PEV301T), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (PE)-peptide conjugate and 10 ug of UK39 (PEV302T), a circumsporozoite protein derived synthetic PE-peptide conjugate. Comparator groups received the influenza vaccine Inflexal V. Volunteers received two injections at study days 0, and 90. Results: Safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. This vaccine showed a very good safety and tolerability profile in all study participants. Curcumin dissolved in DMSO when administered orally to P.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. Under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. We therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. Under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to Plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. Curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. Like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. We believe that it is one of the ways by which curcumin may be killing the parasite. Among immune cells, NK and gamma-delta T cells are suspected to play a critical role in the early control of Plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. Gamma-delta Vgamma9Vdelta2 T cells, a non-conventional T cell subset specific of primates, are activated and expanded during primary P.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of IFN gamma. Furthermore these cells inhibit in vitro growth of P.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an NK and T cell specific cytotoxic molecule has been incriminated. So far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear.

To further investigate the anti-parasitic activity of gamma-delta T cells an RNAi strategy based on a lentiviral vector approach was undertaken. We demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta T cells. Concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta T cells. In addition, we also provide evidence that such a mechanism may occur in infected patients. Altogether these data highlight a new mechanism by which gamma-delta T cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta T cells to prevent or cure malaria. The immune system has a number of mechanisms to prevent self-destructive responses. Amongst these, regulatory T cells (Treg) have the ability to actively suppress effector responses. Many questions surround the issue of antigen specificity of Treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. The purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. Such a model is provided by murine autoimmune hemolytic anemia (AIHA) induced by immunisation with cross-reactive rat red blood cells (RBC). Mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. The re-establishment of tolerance is associated with the loss of T-cell proliferative responses, and emergence of IL-10 responses, to epitopes on the dominant RBC autoantigen, anion exchanger-1 (AE-1, or Band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. Here we show that transfer of tolerance to naï ve recipients is dependent on IDO mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of IDO, were refractory to tolerance and developed hemolytic disease. Induction of IDO is therefore an important process in antigen-specific tolerance, and initiators of IDO activity, including CTLA-4 + regulatory T cells or soluble forms of CTLA-4, may also be crucial components of this regulatory pathway. Consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. Objectives: It was shown alpha-fetoprotein (AFP) induced immunosuppression of cell-mediated immunity in vivo. Our previous work discovered AFP-activated mice bone marrow hematopoietic stem cells (HSCs) suppressed effector reactions of cell-mediated immunity in vitro. We investigated relationship existed between AFP-induced HSCs suppressor activity and immunosuppression of cell-mediated immunity during AFP-produced teratocarcinoma development. Methods: Animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and RNA interference (RNAi) were used in this work. Results: As a result, there was a negative correlation (r medium =-0.81) between dynamics of HSCs suppressor activity elevation in spleen and inhibition of NK cells, NKT cells and CD8 + T cells cytotoxic activities ex vivo during tumor growth. Besides, the inhibition of spontaneous and induced cytokines productions such as IFNg, TNF-a and TNF-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as TGF-b 1 (r medium =-0.74) and IL-10 (r medium =-0.65) in isolated splenic HSCs ex vivo. Analysis of effector CD4 + T cells in spleen showed decrease of T H 1 cells quantity and simultaneous T H 2 cells number increase during teratocarcinoma development. Moreover, it were found elevated numbers of CD4 + CD25 + CTLA-4 + -and CD4 + CD25 -IL-10 + IL-4regulatory T cells in spleen as well as increasing suppressor activity of isolated regulatory T cells ex vivo. Number boost kinetics of T H 1, T H 2 and regulatory T cells were correlated (r Th1 =-0.74, r Th2 =0.86 and r Th3 =0.80 and r Tr1 =0.68) with kinetics of HSCs suppressor activity level. In addition, dynamics of regulatory T cells activity were linear (r Th3 =0.84 and r Tr1 =0.76) to HSCs suppressor activity level in spleen during tumor growth. Quantities of TGF-b1-and IL-10-produced HSCs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. However, inhibition of AFP expression by RNAi caused to inhibition as immunosuppression activity of HSCs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. Conclusion: Thus, HSCs suppression activity is correlated with changes in cell-mediated immunity during endogenous AFP productions by teratocarcinoma cells and may play a role in AFP-mediated dysfunction of normal immunoregulation during AFP-produced tumor development. Syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. Although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. Our studies showed that naturally aquired S.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected CBA mice. While the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. Beside cellular changes, we also evaluated the impact of the S.obvelata on mitogen-activated protein kinases (MAPK) signaling in bone marrow cells and found that infection upregulated all three MAPK families, p38, JNK and ERK. Additionally, S.obvelata enhanced the expression of mRNA for the inducible nitric oxide synthase (iNOS). To evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, T cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. Bone marrow myeloid and erythroid progenitors from S.obvelata-infected mice displayed altered sensitivity to IL-17, as compared to non-infected controls. The infection also altered the effect of IL-17 on MAPK activation by preventing its stimulating effect on p38 MAPK. Moreover, in S.obvelata-infected animals IL-17 markedly down-regulated the expression of both iNOS and constitutive, endothelial (e)NOS, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. IL-17 induced NO production through the activation of both iNOS and eNOS. Besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-Validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified MC were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-Implementation as a standard method for analysis of the MC in patients with paraproteinemia.

The method showed good linearity: R 2 G 0.98. The analytical range was from 1 g/L to 70 g/L. The coefficient of variation (CV) was X 10 % for [MC] n 1 g/dL, and X 20 % for [MC] X 1 g/dL. This procedure was successfully implemented to quantify the MC in 1100 serum samples between March 2008 and February, 2009. Among these samples, we have quantified light chains, heavy chains, IgD and biclonal paraproteinemias. Conclusions: 1. We have developed a simple, reproducible and low-cost method to quantify the MC using standard analyses of Serum Protein Electrophoresis (SPE), serum Albumin, and densitometric quantification of MC and Albumin regions. 2. The procedure allows monitorization of the MC in patients at diagnosis, after therapy, and evaluation of complete remission. Objectives: Direct influence of alpha-fetoprotein (AFP) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (HSCs) was detected in our previous work in vitro. We investigated possible role of endogenous-produced AFP in induction HSCs immunosuppressor activity at tumor-bearing mice. Methods: Animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric ELISA, methods of molecular biology and RNA interference (RNAi) were used in this work. Results: Our results demonstrated AFP endogenous synthesis adduced to elevation of immunosuppression activity of HSCs in bone marrow. This AFP effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. Moreover, CD34 + CD38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. However, immunosuppression activities of purified HSCs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. Besides, immunosuppression activity of HSCs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. Isolated HSCs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as TGF-b 1 , IL-10, PGE 2 and NO. Inhibition of such suppressor factors lead to levelling of HSCs immunosuppression activity ex vivo. Kinetic of HSCs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with AFP level dynamics in blood serum. In addition, inhibition of AFP expression by RNAi caused to diminishing of HSCs immunosuppression activity as well as HSCs appearance in spleen and lymph nodes. Conclusion: Therefore, AFP plays role not only specific inducer of HSCs immunosuppression activity but also as a factor of activated HSCs penetration into spleen and lymph nodes during AFP-produced tumor development. CD40-Ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated CD40L is cleaved to soluble (s)CD40L. We sought to examine the levels of sCD40L in platelet concentrates (PCs) having led to an acute transfusion reactions (ATR), and to test for its biological effect on B-lymphocytes. We recorded 5 ATR episodes that could lead to investigation of residual platelets in container. Two fractions of aliquots from each PC (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; sCD40L -along with other products -were assayed by quantitative ELISA. Levels of IL8, CD62p and PDGF-AB in PC supernatants and lysates from PCs associated with ATR were similar to that in controls. Supernatants of PCs associated with an ATR contained higher sCD40L levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of sCD40L . To examine if sCD40L was biologically active, we stimulated purified B cells recovered from healthy blood donors and exposed those normal B cells to supernatants and cell lysates of PCs implicated in ATR, or control material, and we measured IL-6 secretion. The IL-6 concentration was consistently below 5-10 pg/mL in PC supernatants and lysates, and unstimulated B cells did not secrete detectable levels of IL-6. The addition of supernatant from ATR-associated PC samples to purified B cells consistently resulted in sustained IL-6 production over control (P X 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of sCD40L (P X 0.05). Pre-incubation of B cells with CD40-blocking antibodies substantially abrogated IL-6 secretion, unlike isotype-matched control. The partial blocking of CD40 binding on CD40 + B cells strongly suggests a potentially synergistic role in B cells for cytokines other than sCD40L (under investigation) and indicates a sustained role for PC-derived sCD40L. These data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of sCD40L by collected/stored CPA. Objectives: There is accumulating evidence for a role of natural killer (NK) cells in the antitumor response against hematological malignances. NK cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. Analysis of activating receptor pathways in NK cells has revealed a dominant role for natural cytotoxic receptors (NCRs) and DNAX-accessory molecule-1 (DNAM-1) in the lysis of acute myeloid leukemia (AML) blasts. Here, we investigate the expression of these activating receptors on NK cells from AML patients stratified by age. Methods: We analyses by flow cytometry peripheral blood mononuclear cells (PBMCs) from 30 AML patients before specific anti-leukemia therapy and 47 healthy donors. All results were analyzed statistically using SPSS version 15. Results: AML patients under 65 years showed a significant reduction in the expression of DNAM-1, NKp30 and NKp46 compared with age-matched controls. Both healthy individuals and AML patients older than 65 years showed a reduction of these receptors compared with young donors. In contrast, we have found that NKp44 expression was increased in some patients of AML.

On the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or FAB subtype. In addition, an inverse correlation in the expression of DNAM-1 on NK cells and its ligand CD112 on AML blasts has been found in AML patients under 65 years. To analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy NK cells.

The initial recognition of AML cells by NK cells may represent a crucial process to prevent tumor development. Here we described for the first time, a decrease of DNAM-1 expression on AML patients and confirm previous reports showing a significant decrease of NKp30 and NKp46 on AML-NK cells. Altogether, these alterations of the major receptors involved in NK cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival.

A. Stelmaszczyk-Emmel 1 , E. Gorska 1 , U. Demkow 1 , M. Wasik 1 1 Medical University of Warsaw, Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Warsaw, PolandGlucocorticosteroids are often used in leukemia treatment. Their therapeutic use is limited due to several side effects. One of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. Dexamethasone is used in schedule of children's ALL-B treatment and the response on glucocorticosteroids therapy is very important. The aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in ALL-B and their tendency to begin apoptosis. The study involved 10 children with ALL-B. Bone marrow cells isolated by centrifugation on Histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 M. Analysis of: P-gp surface expression, P-gp function (rhodamine 123 test), pHi (Carboxy-SNARF) and apoptosis test (annexin-V and PI test) were performed with the use of flow cytometer Coulter EPICS XL. For statistical analysis nonparametric Wilcoxon test was used.The results showed that P-gp expression on lymphoblasts was 14,52 %±11,32. After 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. Average percentage of lymphoblasts with rhodamine 123 efflux, which characterized P-gp activity, was 2,62 %±3,15. After 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). Average pHi in lymphoblasts was 7,78±0,19. Acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . Rest of lymphoblasts showed alkalization (pHi -8,00).The percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-V test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). We concluded that dexamethasone does not influence surface P-gp expression on lymphoblasts of patients with ALL B but significantly increases activation of this protein. Functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of P-gp is not identical with its activity. Moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. Those effects may contribute to the treatment outcome. . However, small numbers of clonal PC can also be detected in the peripheral blood (PB) of the majority of patients and, in a minority of cases, MM is transformed into PC leukaemia (PCL). Here, we describe that tumoral PC can express CD49d/CD29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the BM, respectively. Objectives: To evaluate the activation state of CD29 on malignant PC from BM and PB, as well as its regulation. Methods: PC and active integrin expression on these cells were detected with anti-CD38 and anti-CD29 (clon HUTS21) MoAb, respectively, by flow cytometry. To study the integrin activation with divalent cations and PC index proliferation (BrdU+ cells), we used short-term PC cultures (18 hours). Results: CD29 active form was expressed in the majority of normal and tumoral BM PC from healthy subjects (67.3±6.6, n=9) and MM patients in the early stages of the disease (32.6±7.5, n=17). In these cells, HUTS21 epitope was clearly upregulated by Mn 2+ . In contrast, circulating PC were almost all HUTS21 negative, and levels did not significantly augment when these cells were exposed to Mn 2+ . Moreover, not only PB but also BM malignant cells from PCL patients were also HUTS21 negative and divalent cation refractory. It was also observed that PC from PCL patients showed an increased proliferative index (2.35±0.9 BrdU+ cells, n=3) in comparison to PC from MM patients in the first stages of disease (1.3±0.2 BrdU+ cells, n=5).

These results suggest that the active form of CD29 must be expressed on PC to retain these cells within the BM environment. Moreover, its downregulation is associated with increased numbers of circulating PC and disease progression. Multipotent mesenchymal stem cells derived from (hUCB) represent promising candidates for the development of future cellular therapy strategies. They are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues.

In the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hMSC) starting from diagnostic samples of Bone Marrow aspirates and the Cord Blood (CB). The protocols differed in the presence of either 10 % fetal bovine serum (FBS) with and without FGF2,or 10 % human platelet lysate (HPL), 5 %HPL, (10 % FBS + 10 % HPL), (10 % FBS + 5 % HPL). We obtained a significantly better expansion With HPL, compared to cells with a selected batch of FBS and in fewer days.In the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. We compared the quality and the quantity of protein expression before and after differentiation (day 18). Two BM and two CB differentially expressed spots were observed between the two groups (before and after differentiation).We noted the low pourcentage of hMSC in CB samples: in ten samples, only two made MSC colonies as in BM samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red S staining, for adipogenic differentiation, the cells were stained with Oil Red O to visualize lipids droplets. Background: Inherited bone marrow failure syndromes (IBMFS) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. Activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. We studied the presence of p53 DNA (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (FA) and those with acquired aplastic anemia (AAA). Subjects and methods: This is a cross sectional study that involved: 1) ten cases with FA diagnosed on the basis of DNA breakage analysis, 2) ten cases with AAA, and 3) ten normal control cases. The presence of p53 DNA was measured in both bone marrow and peripheral blood samples using a real-time quantitative PCR by taqman assay.

Results: p53 DNA was demonstrated in bone marrow of 90 % of children with FA, compared to 10 % in children with AAA (p X 0.001), while, no p53 DNA was seen in normal control. A positive correlation between DNA breakage and presence of p53 DNA was seen in bone marrow from FA (p X 0.02, r0.81).

The presence of p53 tumor suppressor gene by real time PCR in bone marrow of FA may represent an early indicator of significant DNA genetic alteration in those patients.