key: cord-275104-imqmyqhz
authors: Fioravanti, Jessica; Di Lucia, Pietro; Magini, Diletta; Moalli, Federica; Boni, Carolina; Benechet, Alexandre Pierre; Fumagalli, Valeria; Inverso, Donato; Vecchi, Andrea; Fiocchi, Amleto; Wieland, Stefan; Purcell, Robert; Ferrari, Carlo; Chisari, Francis V.; Guidotti, Luca G.; Iannacone, Matteo
title: Effector CD8+ T cell-derived interleukin-10 enhances acute liver immunopathology
date: 2017-09-30
journal: Journal of Hepatology
DOI: 10.1016/j.jhep.2017.04.020
sha: 
doc_id: 275104
cord_uid: imqmyqhz

Background & Aims Besides secreting pro-inflammatory cytokines, chemokines and effector molecules, effector CD8+ T cells that arise upon acute infection with certain viruses have been shown to produce the regulatory cytokine interleukin (IL)-10 and, therefore, contain immunopathology. Whether the same occurs during acute hepatitis B virus (HBV) infection and role that IL-10 might play in liver disease is currently unknown. Methods Mouse models of acute HBV pathogenesis, as well as chimpanzees and patients acutely infected with HBV, were used to analyse the role of CD8+ T cell-derived IL-10 in liver immunopathology. Results Mouse HBV-specific effector CD8+ T cells produce significant amounts of IL-10 upon in vivo antigen encounter. This is corroborated by longitudinal data in a chimpanzee acutely infected with HBV, where serum IL-10 was readily detectable and correlated with intrahepatic CD8+ T cell infiltration and liver disease severity. Unexpectedly, mouse and human CD8+ T cell-derived IL-10 was found to act in an autocrine/paracrine fashion to enhance IL-2 responsiveness, thus preventing antigen-induced HBV-specific effector CD8+ T cell apoptosis. Accordingly, the use of mouse models of HBV pathogenesis revealed that the IL-10 produced by effector CD8+ T cells promoted their own intrahepatic survival and, thus supported, rather than suppressed liver immunopathology. Conclusion Effector CD8+ T cell-derived IL-10 enhances acute liver immunopathology. Altogether, these results extend our understanding of the cell- and tissue-specific role that IL-10 exerts in immune regulation. Lay summary: Interleukin-10 is mostly regarded as an immunosuppressive cytokine. We show here that HBV-specific CD8+ T cells produce IL-10 upon antigen recognition and that this cytokine enhances CD8+ T cell survival. As such, IL-10 paradoxically promotes rather than suppresses liver disease.

It is widely recognized that hepatitis B virus (HBV) replicates non-cytopathically in the hepatocyte. Most of the clinical complications related to this infection is reflected in the adaptive immune response, particularly the virus-specific effector CD8 + T cell response. [1] [2] [3] [4] [5] Indeed, by killing infected cells and secreting antiviral cytokines, effector CD8 + T cells (CD8 T E ) reaching the infection sites are major contributors to viral clearance as well as tissue immunopathology. [1] [2] [3] [4] [5] Paradoxically, during some acute viral infections (e.g. those caused by influenza virus, respiratory syncytial virus, coronavirus and vaccinia virus), CD8 T E have been shown to produce the regulatory cytokine interleukin (IL)-10, [6] [7] [8] [9] [10] [11] in addition to pro-inflammatory cytokines, chemokines and effector molecules. CD8 T E -derived IL-10 is generally thought to limit tissue damage, 6, 7, 11 however the net effect induced by this cytokine might depend on the pathophysiological context. 12 Whether HBV-specific CD8 T E produce IL-10 upon hepatocellular antigen (Ag) recognition and the role that this cytokine plays in liver immunopathology are currently unknown.

We show here that serum IL-10 is readily detectable in an acutely HBV-infected adult chimpanzee in a manner that coincides with intrahepatic CD8 + T cell infiltration. Using mouse models of acute HBV pathogenesis, we confirmed that IL-10 is indeed produced by HBV-specific CD8 T E upon hepatocellular Ag recognition in vivo. To our surprise, IL-10 blockade ameliorated rather than worsened liver disease. Ex vivo analyses of HBV-specific CD8 T E isolated from the livers of HBV replication-competent transgenic mice or from the blood of acutely infected patients revealed that IL-10 acts in an autocrine/paracrine fashion to increase IL-2 responsiveness and rescue CD8 T E from Ag-induced apoptosis.

Chimpanzees Chimpanzee A0A006 has already been described. 13 The animal was handled according to humane use and care guidelines specified by Animal Research Committees at the National Institutes of Health, The Scripps Research Institute, and Bioqual Laboratories. The chimpanzee was individually housed at Bioqual Laboratories (Rockville, MD), an American Association for Accreditation of Laboratory Animal Care International-accredited institution under contract to the National Institute of Allergy and Infectious Diseases. Chimpanzee A0A006 was inoculated with 10 10 genome equivalents of HBV obtained from an HBV-positive serum of chimpanzee 5835 that was previously inoculated with a monoclonal HBV isolate (genotype D, ayw subtype; GenBank accession no. V01460) 25 contained in HBV transgenic mouse serum, 14 as described. Blood was obtained by venipuncture and analyzed for serum IL-10 (see below).

Mice C57BL/6, CD45.1 (inbred C57BL/6), and Balb/c mice were purchased from Charles River. IL-10 À/À mice (B6.129P2-Il10 tm1Cgn /J) were purchased from The Jackson Laboratory. HBV replication-competent transgenic mice (lineage 1.3.32, inbred C57BL/6, H-2 b ), that express all of the HBV antigens and replicate HBV in the liver at high levels without any evidence of cytopathology, were previously described. 13, 14 In indicated experiments, these mice were used as C57BL/6 x Balb/c H-2 bxd F1 hybrids. HBV nucleocapsid (Cor)-specific (referred to as Cor93 cells) T cell receptor (TCR) transgenic mice (lineage BC10.3, inbred CD45.1), in which >98% of the splenic CD8 + T cells recognize a K b -restricted epitope located between residues 93-100 in the HBV Core protein (MGLKFRQL), were previously described. 17 Env28 (envelope) TCR transgenic mice (lineage 6C2.36, inbred Balb/ c), in which $83% of the splenic CD8 + T cells recognize a L d -restricted epitope located between residues 28-39 of HBsAg (IPQSLDSWWTSL), were previously described. 17 In indicated experiments, lineage BC10.3 or lineage 1.3.32 were crossed with IL-10 À/À mice. Mice were housed under specific pathogen-free conditions and used at 8-10 weeks of age. In all experiments, mice were matched for age, sex and (for the 1.3.32 animals) serum HBeAg levels before experimental manipulations. All experimental animal procedures were approved by the Institutional Animal Committee of San Raffaele Scientific Institute.

Five patients with acute self-limited HBV infection were enrolled at the Unit of Infectious Diseases and Hepatology in Parma, Italy. Patients had clinical, biochemical, and virological evidence of acute HBV infection (aminotransferase levels at least 10 times the upper normal limit and detection of HBsAg and IgM anti-HBcAg Ab in the serum). Patients were negative for anti-HCV, anti-delta virus, anti-HIV-1 and anti-HIV-2 Ab and for other markers of viral or autoimmune hepatitis. T cell response was tested one month from the time of acute illness.

The study was approved by the Ethical Committee of the Azienda Ospedaliero-Universitaria of Parma, and all subjects gave written informed consent.

In vitro generation of CD8 T E was performed as described. 16, 26 Briefly, splenocytes from Cor93 or Env28 TCR transgenic mice were incubated with 10 lg/ml of Cor93-100 (K b ; MGLKFRQL) or Env28-39 (L d ; IPQSLDSWWTSL) peptides (Primm), respectively, at 37°C for 1 h, washed, and cultured in complete RPMI 1640 (10% FBS, 2 mM L-glutamine, 50 lM 2-mercaptoethanol, HEPES 10 mM, non essential amino acid 100 lM and penicillin plus streptomycin). Two days later, cells were cultured in fresh medium supplemented with 2.5% EL-4 supernatant. Media sup-plemented with cytokines were replaced every 2 days. After 8 or 9 days of culture, cells were tested for the expression of CD8, CD69, CD25, CD44, CD62L, CCR7, IFNc and granzyme B by FACS prior to subsequent use, as described. 16 10 7 cells of each cell type were injected intravenously into recipient animals.

Total RNA was isolated from cultured cells or frozen livers (left lobe) with Relia-Prep RNA Miniprep system (Promega) following the manufacturer's instructions.

For quantitative RT-PCR, 1 lg of total RNA was reverse transcribed prior to qPCR analysis for mouse il10 and ifng (TaqMan Mm01288386 and Mm01168134 probes, Applied Biosystems) in an ABI 7900HT fast real-time PCR system (Applied Biosystems). All experiments were performed in triplicate and normalized to the reference gene GAPDH.

Enzyme-linked immunosorbent assays IL-10 and IFN-c in mouse sera or cell supernatants were measured by ELISA (Biolegend and R&D, respectively), according to the manufacturer's instructions. HBeAg in mouse sera was measured by ELISA (Diapro) following the manufacturer's instructions. IL-10 in chimpanzee sera was measured using a human IL-10 ELISA (R&D Systems) according to the manufacturer's instructions.

The extent of hepatocellular injury was monitored by measuring serum alanine aminotransferase (ALT) activity at multiple time points after treatment, as previously described. 16 In vivo IL-10R-specific antibody treatment

Primary hepatocytes were isolated from wild-type or IL-10 À/À HBV replicationcompetent transgenic mice (inbred C57BL/6) exactly as described. 16 Hepatocyte purity (assessed by flow cytometry-based parameters of size) and viability (assessed by light microscopy-based morphology and Trypan blue dye exclusion) were routinely greater than 70% and 80%, respectively. Hepatocytes (10 6 cells/ml) were incubated at a 1:2 ratio with Cor93 CD8 T E for 4 h in the presence of 10 lg/ml brefeldin A (BFA, Sigma) prior to intracellular IFN-c staining.

Single-cell suspensions of livers were generated as described. 18 For analysis of ex vivo intracellular cytokine production, cell suspensions of livers were obtained as described above except that 1 lg/ml of BFA (Sigma) was included in the digestion buffer. All flow cytometry stainings of surface-expressed and intracellular molecules were performed as described. 27 Antibodies (Abs) used included PB-and PE-conjugated anti-CD8a (53-6.7), Alexa Fluor 488-, PerCP-, and APC-Cy7-conjugated anti-CD45.1 (A20), Alexa Fluor 488-, and Alexa Fluor 647-conjugated anti-IFN-c (XMG1.2), PE-and PB-conjugated anti-CD25 (PC61), APC-conjugated Annexin V (EOS9.1), 7AAD (BD Pharmingen). All Abs were purchased from BioLegend, unless otherwise indicated. For phosphorylated STAT5 analysis, cells were fixed with 4% paraformaldehyde, permeabilized with absolute methanol and stained with Pe-Cy7-conjugated anti-phosphoSTAT5 (47/Stat5pY694, eBioscience). All flow cytometry analyses were performed in FACS buffer containing PBS with 2 mM EDTA and 2% FBS on a FACS CANTO (BD Pharmingen) and analyzed with FlowJo software (Treestar).

For Haemotoxylin and Eosin staining, livers were perfused with PBS, harvested in Zn-formalin and transferred into 70% ethanol 24 h later. Tissue was then processed, embedded in paraffin and stained as previously described. 16 Bright-field images were acquired through an Aperio Scanscope System CS2 microscope and an ImageScope program (Leica Biosystem) following the manufacturer's instructions. The extent of hepatocellular injury was monitored by histopathological and quantitative morphometric analyses as described. 18 The number of injured hepatocytes (identified as either apoptotic or necrotic based on standard cytopathological criteria) and intrahepatic inflammatory cells (mononuclear and polymorphonuclear) were counted in at least 50 high power fields of liver tissue (corresponding to about 2 mm 2 ). Results are expressed as number of cells per mm 2 .

In vitro cell culture assays with murine CD8 T E To test the expression and production of IL-10 and IFN-c by Cor93 T E , cells were incubated in complete RPMI 1640 media with 2 lg/ml of rhIL-2 (Roche) at 5Â10 6 cells/ml for 4 h at 37°C in the presence or absence of the Cor93 peptide. To assess the effect of IL-10 on Ag-induced apoptosis, Cor93 CD8 T E (10 7 cells/ ml) were incubated for 1 h at 37°C with 18 lg/ml of anti-IL-10R Ab (BioXCell), 400 ng/ml of recombinant mIL-10 (Biolegend) or left untreated prior to the addition of Cor93 peptide (1 mg/ml) and human IL-2 (1 mg/ml). For the assessment of cell viability, cells were harvested 8 h later and stained with Annexin V and 7AAD, as described above. For the assessment of CD25 expression, cells were harvested 24 h after peptide stimulation. For STAT5 phosphorylation, Cor93 CD8 T E were cultured overnight in serum-free RPMI complete medium (Gibco, Life Technologies), prior to treating them as described above. Cells were harvested 15 min after peptide stimulation.

To measure the effect of IL-10 on Ag-induced apoptosis, peripheral blood mononuclear cells (PBMC) from five HLA-A201 + patients with acute self-limited hepatitis B (concentration of 2Â10 6 /ml) were incubated for 1 h at 37°C with 20 lg/ml of anti-IL-10R Ab (BD Pharmingen), 200 ng/ml of recombinant human IL-10 (BioLegend) or left untreated prior to the addition of Core 18-27 peptide (1 lg/ml) and human IL-2 (100 IU/ml). After a 5 h incubation, cells were extensively washed, stained with Core 18-27 dextramer, anti-CD8 and anti-CD3 mouse Abs for 15 min in the dark, then stained with Annexin V and 7AAD (BD Pharmingen), according to the Annexin V staining protocol (BD Pharmingen). The cells were acquired immediately on a FACSCANTO II multicolor flow cytometer and were analyzed with the DIVA software (BD Biosciences, Immunocytometry Systems, CA, USA).

Total DNA was isolated from frozen livers (left lobe) for Southern blot analysis, as previously described. 18 

Results are expressed as mean + SEM. All statistical analyses were performed in Prism 5 (GraphPad Software). Means between two groups were compared with two-tailed t test. Means among three or more groups were compared with oneway or two-way analysis of variance (ANOVA) with Bonferroni's post-hoc test. Some data were analyzed using Fisher's Least Significant Difference (LSD) test 

We first assessed whether IL-10 is produced upon acute HBV infection. We longitudinally analyzed the sera of a chimpanzee (A0A006) that had been inoculated with a monoclonal HBV inoculum of 10 10 GE of HBV DNA, as described. 13 Serum IL-10 was not detectable until CD8 + T cells accumulated in the liver and its appearance coincided with the onset of a necroinflammatory liver disease (Fig. 1A) , suggesting that this cytokine might have been produced by virus-specific CD8 T E upon hepatocellular Ag recognition. To test this hypothesis and to assess the role of IL-10 in liver immunopathology, we employed a well-established model of acute HBV pathogenesis, i.e. the adoptive transfer of HBV-specific CD8 + T E into HBV replication-competent transgenic mice. 14-16 Naïve CD8 + T cells from HBV nucleocapsid (Cor)specific TCR transgenic mice 17 (referred to as Cor93 cells) were differentiated in vitro into bona fide CD8 T E . Upon intravenous injection of 10 7 Cor93 CD8 T E into HBV replication-competent transgenic mice, the hepatic mRNA expression of the il10 gene increased sharply, reaching peak levels at 4-8 h after T cell transfer and mirroring the kinetics of the prototypical CD8 T E -derived pro-inflammatory and antiviral cytokine gene ifng (Fig. 1B) . Accordingly, IL-10 was also detected in the serum of these mice between 4 and 8 h after T cell transfer, again echoing the IFN-c kinetics (Fig. 1C) . Consistent with the hypothesis that IL-10 is produced by CD8 T E upon hepatocellular antigen recognition, we found that Cor93 T E cells stimulated for 4 h in vitro with the cognate Cor93 peptide produced both cytokines (Fig. 1D-G;  Fig. S1 ). Finally, to unambiguously identify the cellular source of the IL-10 detected in the liver of HBV replication-competent transgenic mice upon adoptive transfer of Cor93 CD8 T E , we genetically deleted the il10 gene in HBV replication-competent recipient mice, in Cor93 CD8 T E or in both. By using this approach, we could demonstrate that the transferred CD8 T E cells are the unique source of IL-10 in this experimental setting, as il10 expression was only detected when the transferred T cells were IL10-competent, regardless of the recipient genotype (Fig. 1H) .

To gain insight into the role of CD8 T E -derived IL-10 expression in liver immunopathology, we selectively blocked IL-10 receptor signaling by injecting HBV replication-competent transgenic mice with an anti-IL-10Ra Ab 2 h prior to Cor93 T E transfer. To our surprise, we found that IL-10 blockade decreased, rather than increased, liver damage by about 3-fold at the peak of the disease ( Fig. 2A) . Of note, anti-IL-10Ra Ab injection did not affect the total number of circulating Cor93 T E cells (data not shown), indicating that this treatment did not deplete the transferred T cells. Also, anti-IL-10Ra Ab treatment significantly reduced liver disease when HBV envelope-specific TCR transgenic 16, 17 or polyclonal CD8 T E 18 were injected into HBV replication-competent transgenic mice instead of Cor93 T E (data not shown). Moreover, consistent with IL-10 being produced exclusively by the transferred CD8 T E in this model, deletion of the il10 gene in HBV replication-competent recipient mice -which did not alter the Ag presentation capacity of hepatocytes (Fig. S2 ) -did not affect the severity of liver disease induced by Cor93 T E transfer (Fig. 2B) .

To explore the mechanisms underlying this IL-10-mediated increase in liver immunopathology, we quantified the number, function and viability of intrahepatic Cor93 CD8 T E 2, 8, and 24 h after adoptive transfer into HBV replication-competent transgenic mice that were or were not subjected to anti-IL-10Ra Ab treatment. IL-10R blockade significantly decreased the number of total and IFN-c + intrahepatic Cor93 CD8 T E cells recovered 24 h after injection ( Fig. 2C and D) . We next addressed if this reflected an increase in cell death. Indeed, the percentage of intrahepatic Cor93 CD8 T E that underwent apoptosis at 8 and 24 h after transfer was significantly increased by IL-10R blockade ( Fig. 2E; Fig. S3 ), and this was reflected by a lower disease severity ( Fig. 2F and G) . Although the number of intrahepatic Cor93 T E was reduced upon IL-10R blockade, these cells probably produced enough IFN-c to abolish viral replication (Fig. S4 ).

To explore if CD8 T E -derived IL-10 directly decreased Ag-induced apoptosis, we exposed purified Cor93 CD8 T E to Ag in the presence or absence of IL-10R blockade or exogenous IL-10. As shown in Fig. 3A , Ag exposure for 8 h triggered Cor93 CD8 T E apoptosis.

Notably, IL-10R blockade increased the percentage of Cor93 CD8 T E that became apoptotic, whereas the addition of exogenous recombinant IL-10 decreased Cor93 CD8 T E apoptosis ( Fig. 3A and Fig. S5A) . We next set out to determine the mechanism whereby IL-10 prevents CD8 T E from dying. Since IL-10 has been proposed to enhance the growth of activated CD8 + T cells in the presence of IL-2, [19] [20] [21] we reasoned that CD8 T E -derived IL-10 might increase IL-2 responsiveness by, for instance, modulating IL-2 receptor expression. Indeed, Ag-induced IL-2Ra (CD25) upregulation on CD8 T E was lower upon IL-10R blockade, whereas the addition of exogenous recombinant IL-10 increased CD25 upregulation (Fig. 3B and Fig. S5B ). Accordingly, blocking IL-10R signaling decreased the capacity of CD8 T E to respond to IL-2 (as assessed by STAT5 phosphorylation), and IL-10 treatment increased IL-2 sensitivity ( Fig. 3C; Fig. S5C ). Finally, we explored whether the capacity of CD8 T E -derived IL-10 to act in an autocrine/paracrine fashion to rescue CD8 T E from Ag-triggered apoptosis was restricted to murine CD8 T E or it extended to HBV-specific CD8 T E isolated from acutely infected patients. To this end, PBMCs from 5 HLA-A201 + patients with acute hepatitis B, in whom HBV-specific CD8 + T cells can be specifically visualized by HBV-specific dextramers 22 (Fig. S6 ), were stimulated with the cognate Cor18 peptide in the presence or absence of anti-IL-10Ra Abs or exogenous IL-10. The results mirrored those obtained with murine CD8 T E , in that IL-10R blockade increased Ag-induced apoptosis, whereas the addition of recombinant IL-10 partially rescued CD8 T E from cell death (Fig. 3D ).

In conclusion, our results indicate that CD8 T E -derived IL-10 acts in an autocrine/paracrine fashion to increase IL-2 responsiveness, rescuing CD8 T E from Ag-induced apoptosis. Although the net contribution that IL-10 plays during a natural HBV infection -where this cytokine may be produced by additional cell types 23,24 -remains to be determined, the results described herein suggest that IL-10 may promote rather than suppress liver immunopathology. with medium (white), anti-IL-10Ra (20 lg/ml, light blue), or rIL-10 (200 ng/ml, dark blue). After 1 h Cor18-27 peptide (1 lg/ml) was added (+Cor18) or not (Ctrl).

The percentage of Annexin V+ (apoptotic) cells was determined on Core 18-27 dextramer + cells 5 h later. Results are representative of 2 independent experiments. Results are expressed as mean + SEM. *p .05, **p .01, ***p .001. Means between two groups were compared with two-tailed t test. Means among three or more groups were compared with one-way or two-way analysis of variance with Bonferroni's post-hoc test. Data in Fig. 3D were analyzed using Fisher's Least Significant Difference (LSD) test.

European Molecular Biology Organization (EMBO) Young Investigator Program (to M.I.), EMBO Long-Term Fellowships ALTF 694-2016 (to A.B.) and ALTF 1018-2016 (to D.I.), a Marie Curie Intra-European Fellowship (IEF) for Career Development 327336

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Mouse models of hepatitis B virus pathogenesis

Immune outcomes in the liver: Is CD8 T cell fate determined by the environment?

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CD8(+) T cell control of hepatitis B virus replication: direct comparison between cytolytic and noncytolytic functions

Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10

Autocrine regulation of pulmonary inflammation by effector T-cell derived IL-10 during infection with respiratory syncytial virus

Shifting hierarchies of interleukin-10-producing T cell populations in the central nervous system during acute and persistent viral encephalomyelitis

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CD8+ Treg cells suppress CD8

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Immunosurveillance of the liver by intravascular effector CD8(+) T cells

CD40 activation rescues antiviral CD8 + T cells from PD-1-mediated exhaustion

Platelets mediate cytotoxic T lymphocyte-induced liver damage

IL-10: a novel cytotoxic T cell differentiation factor

Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells

The light and the dark sides of Interleukin-10 in immune-mediated diseases and cancer

Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection

Interleukin-10 and the interleukin-10 receptor

The regulation of IL-10 expression

Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli

Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes

Bisphosphonates target B cells to enhance humoral immune responses

We thank L. Giustini, M. Mainetti and M. Raso for technical support; M. Silva for secretarial assistance and the members of the Iannacone laboratory for helpful discussions. Flow cytometry was carried out at FRACTAL, a flow cytometry resource and advanced cytometry technical applications laboratory established by the San Raffaele Scientific Institute.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2017.04. 020.

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.Please refer to the accompanying ICMJE disclosure forms for further details.