Accepted Manuscript The extent and duration of acute hepatitis in mice is dominated by the function rather than by the survival of effector CD8 T cells Michelle Vo, Lauren E. Holz, Yik Chun Wong, Kieran English, Volker Benseler, Claire McGuffog, Miyuki Azuma, Geoffrey W. McCaughan, David G. Bowen, Patrick Bertolino PII: DOI: Reference:

S0168-8278(16)00160-4 http://dx.doi.org/10.1016/j.jhep.2016.01.040 JHEPAT 6019

To appear in:

Journal of Hepatology

Received Date: Revised Date: Accepted Date:

25 June 2015 14 January 2016 26 January 2016

Please cite this article as: Vo, M., Holz, L.E., Wong, Y.C., English, K., Benseler, V., McGuffog, C., Azuma, M., McCaughan, G.W., Bowen, D.G., Bertolino, P., The extent and duration of acute hepatitis in mice is dominated by the function rather than by the survival of effector CD8 T cells, Journal of Hepatology (2016), doi: http://dx.doi.org/ 10.1016/j.jhep.2016.01.040

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The extent and duration of acute hepatitis in mice is dominated by the function rather than by the survival of effector CD8 T cells Michelle Vo,1,2 Lauren E. Holz,1,2,3 Yik Chun Wong,1,2 Kieran English,

1,2

Volker

Benseler,1,2,4 Claire McGuffog,1,2 Miyuki Azuma,4 Geoffrey W. McCaughan,2,5 David G. Bowen,1,2* Patrick Bertolino 1,2* 1

Liver Immunology Program, Centenary Institute, Newtown, NSW, Australia AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital Newtown, NSW, and Faculty of Medicine, University of Sydney, New South Wales, Australia 3 Current address: Department of Microbiology and Immunology, The Peter Doherty Institute, The University of Melbourne, Parkville, Victoria, Australia 4 Current address: Department of Surgery, University of Regensburg, Bavaria, Germany 4 Department of Molecular Immunology Graduate School, Tokyo Medical and Dental University Yushima, Tokyo, Japan 5 Liver Injury and Cancer Program, Centenary Institute, Newtown, NSW, Australia 2

*

Equal contribution

Electronic word count: 9,689 words Number of figures: 5 (+ 8 supplementary figures)

Corresponding authors: Patrick Bertolino, e-mail: [email protected], telephone: +612-9565-6186, fax: +612-9565-6101 and David Bowen, e-mail: [email protected], telephone: +612-9565-6264, fax: +612-9565-6101; Centenary Institute, AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital and Faculty of Medicine, University of Sydney, Camperdown, NSW 2050, Australia

Financial Support: This work was supported by the National Health and Medical Research Council (NHMRC) Australia (Program grant 571408). MV was supported by an Australian Postgraduate Award and PB by an NHMRC Senior Research Fellowship (511903).

Conflict of interest: None of the authors have a conflict of interest or financial disclosure to declare

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Author's contributions: MV (study concept and design, acquisition of data, data analysis and interpretation, drafting manuscript), LEH (acquisition of data, interpretation of data, critical revision of manuscript for important intellectual content), YCW (acquisition of data, interpretation of data, critical revision of manuscript for important intellectual content), KE (acquisition of data, interpretation of data), VB (interpretation of data), CM (animal husbandry, technical assistance), GWM (critical revision of manuscript for important intellectual content), DGB and PB (study concept and study design, interpretation of data, critical revision of manuscript for important intellectual content). The authors would like to thank the Centenary Institute Animal Facility and Advanced Cytometry Facility for their technical support, Dr Douglas Hilton (WEHI, Melbourne) for providing SOCS-1-deficient mice, and Drs Phillipe Bouillet and Andreas Strasser (WEHI, Melbourne) for providing Bim-deficient mice.

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List of Abbreviations: Alb

Albumin

B6

C57BL/6

CFSE

Carboxyfluorescein succinimidyl ester

CTL

Cytotoxic T lymphocyte

LN

Lymph node(s)

Met

Metallothionein

PD-1

Programmed cell death 1

PD-L1

Programmed death-ligand 1

RAG

Recombination-activating genes

SOCS-1

Suppressor of cytokine signaling-1

TCR

T cell receptor

Tg

Transgenic

wt

wild-type

Keywords: autoimmunity; transgenic; liver; tolerance

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Abstract Background and aims: Acute hepatitis is often mediated by cytotoxic T lymphocytes (CTLs); however, the intrinsic parameters that limit CTL-mediated liver injury are not well understood. Methods: To investigate whether acute liver damage is limited by molecules that decrease the lifespan or effector function of CTLs, we used a well-characterized transgenic (Tg) mouse model in which acute liver damage develops upon transfer of T cell receptor (TCR) Tg CD8 T cells. Recipient Tg mice were adoptively transferred with donor TCR Tg T cells deficient for either the pro-apoptotic molecule Bim, which regulates CTL survival, or suppressor of cytokine signaling-1 (SOCS-1), which controls expression of common gamma chain cytokines, or in the presence of anti-PD-L1 neutralizing antibodies. Results: Use of Bim deficient donor T cells and/or PD-L1 blockade increased the number of intrahepatic T cells without affecting the degree and kinetic of acute hepatitis. In contrast, SOCS-1-deficient T cells induced a heightened, prolonged acute hepatitis caused by their enhanced cytotoxic function and increased expansion. Although they inflicted more severe acute liver damage, SOCS-1-deficient T cells never precipitated chronic hepatitis and became exhausted. Conclusions: The degree of acute hepatitis is regulated by the function of CD8 T cells, but is not affected by changes in CTL lifespan. Although manipulation of the examined parameters affected acute hepatitis, persistent hepatitis did not ensue, indicating that, in the presence of high intrahepatic antigen load, changes in these factors in isolation were not sufficient to prevent T cell exhaustion and mediate progression to chronic hepatitis.

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Introduction Acute liver injury has a variety of causes, but is often initiated by an immune response triggered by hepatotropic viruses and other pathogens targeting this organ. A robust and sustained immune response involving both the innate and adaptive immunity is critical to clear infections by viruses targeting the liver, such as the hepatitis B (HBV)[1] and C viruses (HCV).[2] Anti-viral cytotoxic CD8 T lymphocytes (CTLs) play a crucial role in mediating liver injury during infection and the induction and maintenance of broad vigorous virusspecific CD8 T cells responses is a positive predictor of spontaneous resolution of HCV infection.[3-5] Antibody-dependent depletion of CD8 T cells in a chimpanzee model of HCV infection led to prolonged viral infection.[6] The detection of CTLs in the periphery coincides with raised alanine transaminase (ALT) levels and manifests clinically as acute hepatitis.[7] Clearance of HCV is more common in symptomatic patients,[5] and it is likely that the degree of liver damage that ensues during the acute phase of infection is critical in influencing the outcome of infection.[8] Although it is known that CTLs kill target cells via granzyme and/or Fas/FasL interactions (classical CTL killing pathway), or via the secretion of hepatotoxic cytokines (bystander killing), the molecular processes limiting the action of CTLs and responsible for terminating acute liver damage mediated by CD8 T cells are not well understood. It is important to elucidate these processes to derive strategies to increase the chances of clearing persistent hepatotropic infections, and also to prevent too sustained or vigorous CD8 T cell responses that might lead to eventual development of significant fibrosis and cirrhosis, or in the more acute setting, to the development of acute liver failure. Several regulatory processes intrinsic to T cells could be important in limiting liver damage, including: induction of inhibitory receptors such as PD-1, Tim-3, LAG3, and CTLA4; regulation of CTL effector function (down-regulation of cytokine and effector molecules); and apoptosis of CTLs. Other

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parameters such as the number of CD8 T cell precursors specific for the virus,[9, 10] the affinity of the TCR recognizing viral epitopes,[11] the availability of CD4 T cell help,[12, 13] and the number of hepatocytes expressing antigen[14] have also been shown to influence long-term CD8 T cell outcome. In order to identify the T cell intrinsic parameters controlling the degree and kinetics of acute liver damage, we used the well-characterized Met-Kb transgenic (Tg) mouse model of immune mediated hepatitis in which acute liver injury is induced by TCR Tg CD8 T cells that are first activated in lymph nodes (LN) and subsequently recognize their cognate antigen in the liver, leading to hepatocellular injury.[15] By transferring donor Tg T cells deficient for genes that control T cell death (Bim) or regulate signaling of cytokines critical for effector T cell function (suppressor of cytokine signaling-1; SOCS-1), or by treating recipient mice with anti-PD-L1 blocking antibodies, we investigated the role of regulation of T cell death, regulation of CTL function and T cell inhibitory molecules, in limiting the degree and duration of liver damage, independently of TCR affinity, T cell help and antigen dose. Our results suggest that strategies that enhance T cell survival, promoted accumulation of CD8 T cells recognizing hepatocyte-expressed antigens in the liver, without altering the severity or tempo of liver damage. In contrast, augmenting the function of effector T cells prolonged acute hepatitis and increased the severity of liver damage. While these strategies had different effects on the fate and/or function of effector CD8 T cells, none prevented the resolution of acute hepatitis associated with the eventual development of functional exhaustion of T cells within in the liver. In the setting of high-level persistent antigen expression in the liver, the degree and duration of liver damage is thus predominantly regulated by genes controlling the function of effector

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cells rather than those affecting T cell lifespan. Persistence of liver damage is ultimately limited by functional T cell exhaustion. These data suggest that, to be effective, immunotherapies aimed at boosting the number of CTLs in patients with liver disease will need to be combined with strategies that enhance T cell effector function and, more importantly, interfere with T cell exhaustion.

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Materials and Methods Mice B10.BR, Des-TCR,[16] Des-SOCS-1w/-, Des-Bim-/-,[17, 18] Des-RAG-/-SOCS-1 -/- and DesRAG-/-SOCS-1 w/w, Met-Kb, [19] and Alb-Kb [20] mice were maintained at the Centenary Institute under specific pathogen-free conditions. Breeding strategy to generate Des-SOCS-1-/- and Des-RAG-1-/-SOCS-1 -/- mice SOCS-1-/- Tg mice were generated by inter-breeding Des-SOCS-1 w/- or Des-RAG-/-SOCS1w/- parents to produce Des-SOCS-1-/- and Des-RAG-/-SOCS-1 -/- progeny, respectively. SOCS-1-/- Tg progeny were sacrificed at 18 days of age (before they become ill) for adoptive transfer experiments. Adoptive transfer experiments Single cell suspensions of pooled LN cells were labeled with carboxyfluoroscein succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) as previously described.[15] 0.8 x 106 Des-RAG-/-SOCS-1 -/- or Des-RAG-/- or 5.0 X 106 Des, Des-SOCS-1-/- or Des-Bim-/lymphocytes were transferred into the tail veins of recipient mice. Immunofluorescent staining and flow cytometric analysis Liver, spleen, blood, and LN leukocyte cell suspensions were prepared as previously described.[15] Leukocyte counts were performed using AccuCount Beads (SpheroTech, Inc. Lake Forest, IL) with absolute cell numbers determined using flow cytometry. Monoclonal antibodies were purchased from Becton Dickinson (Franklin Lakes, NJ), Biolegend (San Diego, CA) or eBioscience (San Diego, CA). The clonotypic antibody recognizing the Tg Des-TCR was conjugated in house.[16] Surface and intracellular staining was performed as

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previously described.[18] Cells were stained with 0.1 µg/mL DAPI (Invitrogen) before acquisition on a BD LSR Fortessa flow cytometer or LSR-II (BD Biosciences), and were analyzed in FlowJo 9.4.9 (Tree Star). Prior to intracellular cytokine staining (Cytoperm/CytofixTM kit, Becton Dickinson), lymphocytes were stimulated with C57BL/6 splenocytes (which express the Des-specific alloantigen H-2Kb) or control syngeneic B10.BR splenocytes in vitro for 6 hours. When indicated, surface expression of CD107a, a surrogate marker for degranulation, was also assessed together with intracellular IFN-γ staining, as previously described. [14] Isolation and detection of liver-infiltrated immune cells via liver perfusion Infiltrated immune cells, together with hepatocytes and other cell types, were isolated from the liver by in situ retrograde perfusion with collagenase type IV as described previously. [21] Hepatocytes were removed via low-speed centrifugation (58g, 3min, 4°C). Cells recovered from the supernatant were then stained with antibodies and assessed by flow cytometry. To detect neutrophils and Ly6chi monocytes, cells were stained with antibodies against NK1.1, CD3ε, CD19, CD45, CD11b, Ly6C, Ly6G, F4/80, and MHC-II. Neutrophils were defined as NK1.1 - CD3ε- CD19 - CD45 + CD11b + Ly6G+ Ly6Cint. Ly6Chi monocytes were defined as NK1.1- CD3ε- CD19- Ly6G- CD45+ CD11b+ Ly6Chi Ly6G+ F4/80int. To identify different T cell subsets, cells were stained with antibodies against NK1.1, CD3ε, CD19, CD8α, CD4 and Des-TCR. CD4 T cells were defined as NK1.1- CD19 - CD3ε+ CD4 +. Recipient CD8 T cells were defined as NK1.1 - CD19- CD3ε+ CD8 + Des- while Des Tg CD8 T cells were identified as NK1.1- CD19 - CD3ε+ CD8+ Des+. In vivo CTL assay A 1:1 mix of antigen-expressing C57BL/6 and control B10.BR splenocytes were labeled with high (5µM) and low (0.5µM) concentrations of CFSE respectively. To determine in

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vivo killing capacity, target splenocytes were transferred into mice that had previously received Tg T cells (effectors). Loss of CFSE-high target-expressing cells (indicative of killing) was assessed by flow cytometry. In vivo cytotoxicity was calculated as percentage of specific killing = (1 – ratio of control mice/ratio of experimental mice) x 100, whereby the ratio refers to the percentage of CFSElow cells divided by the percentage of CFSEhigh cells. T cell proliferation assay Mice into which Tg T cells had been previously transferred were pulsed with BrdU for 4-6 hours (BD BrdU Flow kit). LN and livers were harvested, and lymphocytes isolated and stained with anti-CD8, anti-Des-TCR (surface staining) and anti-BrdU (intracellular) antibodies. BrdU incorporation was measured using flow cytometry. Serum ALT Measurement Mouse serum was separated from whole blood and ALT levels were determined using the Cobas® 8000 (Roche Diagnostics, Indianapolis, USA) by the Biochemistry Department, Royal Prince Alfred Hospital. Histological Analysis For paraffin sections, liver tissue was formalin fixed, paraffin embedded and 8 µm sections cut prior to H&E staining. For frozen sections, liver tissues were snap frozen over liquid nitrogen in Tissue-Tek OCT compound (Sukura, Finetechnical, Tokyo, Japan) and cryosectioned (8µm) prior to acetone fixation. For immunohistological staining, frozen tissue underwent blocking in 2% skim milk powder and an anti-CD8 antibody (clone YTS 169.4, rat IgG2b) was applied followed by horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rat immunoglobin secondary antibody (Dako A/G, Copenhagen,

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Denmark). Positive staining was visualized following the addition of diaminobenzidine and counter-stain with Mayers haematoxylin (Sigma-Aldrich, Saint Louis, Missouri). PD-L1 antibody treatment Neutralizing PD-L1 antibodies (clone MIH5, rat IgG2a[22]) were purified from cell culture supernatant using a protein G column (Pharmacia, Uppsala, Sweden). Purified control rat IgG was purchased from MP Biomedicals, Seven Hills, NSW, Australia. For experiments studying early time points, Met-Kb and B10.BR mice were treated with 250µg of MIH5 intraperitoneally (i.p.) at day 0 or day 1, and subsequently every 3 days until the completion of experiments, to inhibit the interaction of PD-1 with PD-L1. For experiments assessing late time points, anti-PD-L1 treatment began at day 20 following the transfer of Des-Bim-/- T cells, and continued every three days until experiments were completed. For all experiments using neutralizing antibodies in vivo, purified rat IgG was used as a control and was administered at the same dose and time as MIH5.

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Results Increased survival of Bim-deficient T cells activated in LN of Met-Kb mice promoted their accumulation in the liver To study the influence of potential molecular regulators of acute hepatitis, we used the MetKb Tg mouse model of immune-mediated hepatitis, in which liver damage is caused by CTLs activated in LN, [15] a situation akin to the damage caused by CTLs in viral hepatitis. Met-Kb mice express a Tg MHC class I molecule, H-2Kb, in both LN and liver. Adoptive transfer of syngeneic naïve Des T cells expressing a Tg TCR specifically recognizing the H2Kb molecule in association with self-peptide induce a severe, but self-limited acute hepatitis in Met-Kb hosts.[15] Serum ALT levels were highest at 5-6 days post-transfer, the peak of acute hepatitis, and rapidly returned to baseline levels after 4 days.[15] We have previously demonstrated that, while simultaneous primary activation of Des T cells occurred in both liver and LN of Met-Kb mice, liver injury was strictly dependent on CD8 T cells activated in the LN that migrated to the liver where they killed antigen-expressing hepatocytes.[15] To determine whether apoptosis of CTLs was critical in limiting liver damage and in preventing chronic hepatitis associated with ongoing immune-mediated injury, we generated a Tg Des mouse line deficient for the pro-apoptotic molecule Bim, a molecule which plays a critical role in CD8 T cells apoptosis.[18] Des-Bim-/- LN cells were adoptively transferred into Met-Kb or control syngeneic B10.BR (non-antigen expressing) hosts. At 30 days postadoptive transfer, the total number of intrahepatic Tg T cells harvested from Met-Kb recipient mice was 110-fold higher in Met-Kb mice into which Des-Bim-/- cells had been transferred than in recipients receiving Bim-sufficient Des T cells (Fig. 1A). Des-Bim-/- cells represented a higher proportion of intrahepatic lymphocytes (Fig. 1B), and were

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predominantly localized around portal tracts, however some were also located within the parenchyma (Fig. 1C). Interestingly, the intrahepatic Des-Bim-/- cells that had survived hepatitis displayed a CD69 high CD44 high phenotype (data not shown) with down-regulated levels of the Des-TCR (Fig. 1B), suggesting the Tg T cells were in a state of continual activation in the presence of antigen. The accumulation of Bim-deficient T cells was not preceded by increased numbers of other leukocytes within the liver. At day 5 post-T cell transfer, the peak of biochemical hepatitis, the numbers of monocytes, recipient CD4 and CD8 T cells, and neutrophils in the livers of Met-Kb recipient mice receiving Bim-deficient T cells did not differ significantly from those receiving Bim-sufficient T cells (Supplementary Fig. 1). These results suggest that the effect of Bim in the accumulation of donor Tg CD8 T cells was a direct effect on their survival. To determine the relative contributions of the liver and LN to the increase in donor CD8 T cells, Des-Bim-/- cells were adoptively transferred into Alb-Kb mice, in which H-2Kb expression is restricted to the liver.[18] Confirming our previous study showing that liveractivated T cells died by Bim-dependent apoptosis, [18] the total number of Des-Bim-/- CD8 T cells recovered from Alb-Kb recipients was increased in comparison to Alb-Kb mice into which Bim-sufficient Des CD8 T cells had been transferred. However, this was only a 5-fold increase (Fig. 1A), suggesting that the majority of Tg CD8 T cells recovered from Met-Kb livers had undergone initial activation in the LN. These findings reveal that the vast majority of Des-Bim-/- T cells surviving in Met-Kb mice at 30 days were initially activated in the LN and that, like CD8 T cells activated intrahepatically, CD8 T cells activated in LN died by Bim-dependent apoptosis.

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Intrahepatic accumulation of Bim-deficient CD8 T cells was not associated with increased liver damage. Since T cells activated in the lymphoid tissues of Met-Kb mice developed into efficient CTLs, [15] we next investigated if the substantial accumulation of Des-Bim-/- Tg CTLs in Met-Kb hosts increased or prolonged liver damage. Surprisingly, hepatitis mediated by DesBim-/- cells remained self-limiting, and exhibited the same kinetic and intensity as acute hepatitis induced by Bim-sufficient Des T cells (Fig. 1D). Thus, the higher number of infiltrating T cells in the liver was not associated with increased liver injury, suggesting that they were silenced. The total number of CD4+CD25 +FoxP3 + (that includes T cells with regulatory function) in the recipient Met-Kb liver was not increased at 30 days post T cell transfer (data not shown), suggesting that silencing of the increased number of Bim-deficient T cells was not mediated by Tregs. To investigate the alternative possibility that Bim-deficient cells were silenced because they became functionally exhausted, we analyzed whether these cells expressed PD-1 and Tim-3, two markers associated with T cell exhaustion. A significant proportion of Bim-deficient CD8 T cells isolated from the livers and LNs of recipient Met-Kb mice at days 5, 9 or 15 after adoptive transfer expressed PD-1 and/or Tim-3 (Supplementary Fig. 2). Interestingly this proportion tended to be lower than for Bim-sufficient CD8 T cells at all time points in both liver and LNs (Supplementary Fig. 2). Despite this, exhaustion of intrahepatic Bimdeficient donor T cells was confirmed at the functional level after the peak of acute hepatitis In contrast to earlier time points, Bim-deficient CD8 T cells isolated from the liver 15 days after adoptive transfer failed to degranulate and secrete IFN-γ following ex vivo restimulation with Ag-expressing splenocytes, as assessed by CD107a cell surface staining

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and intracellular IFN-γ staining, respectively (Supplementary Fig. 2). As expected from previous results showing that functional Des T cells were generated in the LNs rather than the livers of Met-Kb recipients, the number of functional Bim-sufficient donor CD8 T cells in the LNs was highest on day 5, and subsequently decreased over time (Supplementary Fig. 2). This decrease in LNs was less marked for Bim-deficient T cells (Supplementary Fig. 2), a result consistent with the pro-apoptotic role of Bim. These results suggest that, as previously shown for Bim-sufficient Des CD8 T cells [23, 24], Bim-deficient CD8 T cells generated in the LNs acquired effector function and induced acute liver damage. However, in the presence of persisting high antigen levels, they eventually became exhausted. Thus, although Bim regulates the survival of CD8 T cells specific for hepatically-expressed antigen, deficiency of this factor does not prevent T cell exhaustion within the liver.

Blocking PD-1/PD-L1 interactions increased T cell numbers without augmenting the severity of acute liver damage or leading to chronic hepatitis. PD-1/PD-L1 blockade is currently used in the clinic to boost the function of exhausted CD8 T cells in a range of solid tumors, has been explored in chronic HCV infection [25], and is under investigation for treatment of hepatocellular carcinoma (HCC).[26] It was therefore important to test the role of this molecule in our model. PD-1 was expressed at similar low levels on donor Des-RAG-/- T cells following 24h of activation in the liver and LN of Met-Kb mice, but was not expressed at any stage on T cells transferred into B10.BR mice (Fig. 2A). At 48h post-activation, liver-activated CD8 T cells expressed more PD-1 compared to LN-activated T cells (p < 0.05) (Fig. 2A, B). Similar levels of expression were detected on liver- and LN-activated CD8 T cells less than a day

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later, probably due to death of liver-activated T cells and recirculation of LN-activated T cells to the liver.[18, 27] Over time, all Des-RAG-/- T cells activated in Met-Kb mice became PD-1high and expression tended to increase with time (Fig. 2B), a result consistent with the phenotype of intrahepatic CD8 T cells reported in different models when antigen persists.[28, 29] PD-L1, the main PD-1 ligand, was also expressed at very low levels in the hepatic parenchyma of Met-Kb mice (Fig. 2C), but not in B10.BR mice (data not shown) at day 15 post-T cell transfer. Interestingly, PD-L1 expression was positively correlated with the number of infiltrating cells, and was increased in association with the more marked infiltrates of Des-Bim-/- T cells. In these livers, PD-L1 expression was found in the vicinity of infiltrating Des-Bim-/- T cells in the parenchymal regions and also in portal regions (Fig. 2C). To examine the possibility that PD-1/PD-L1 interactions contributed to the induction of tolerance in liver-activated T cells and/or to the attenuation of acute hepatitis, Met-Kb and B10.BR mice, into which wild-type Des T cells had been transferred, were treated with neutralizing anti-PD-L1 or control antibodies (rat IgG). Treatment of Met-Kb mice with antiPD-L1 every three days, beginning from day one after adoptive transfer of T cells, led to increased numbers of donor Des T cells in the liver at day 15, in comparison to control rat IgG-treated mice (Fig. 2D), suggesting that PD-L1 blockade increased T cell proliferation and/or survival. Despite accumulation of the Des T cell progeny in the liver, treatment with anti-PD-L1 did not have a significant effect on serum ALT levels (Fig. 2E). Interestingly, commencing administration of the anti-PD-L1 antibody one day earlier (day 0) decreased the number of activated T cells and inhibited hepatitis (data not shown), suggesting anti-PDL1 interfered with T cell priming. Consistent with the failure of anti-PD-L1 to change the outcome of acute hepatitis, the phenotype of hepatocyte-activated T cells was not affected by anti-PD-L1 when treatment started at day 1, as all cells proliferated at the same rate,

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maintained low CD25 expression and did not produce IL-2 or IFN-γ (Supplementary Fig. 3). PD-1 expression was slightly decreased on hepatocyte-activated T cells isolated from the anti-PD-L1-treated mice in comparison to control mice, but its expression was high in both groups (Supplementary Fig. 3), providing further evidence that PD-1 expression was not sufficient to control the lack of effector function in these cells. Anti-PD-L1 treatment from day 1 post-transfer did not alter the naïve phenotype of donor T cells transferred into B10.BR mice (data not shown). To further test whether PD-1/PD-L1 interactions prevented Bim-deficient Des cells from mediating hepatitis, Met-Kb mice into which Bim-deficient Des T cells had been adoptively transferred were treated with anti-PD-L1 from day 1. The phenotype of the donor T cells was not influenced by the expression of Bim or antibody treatment at day 2 post-transfer, as all Bim-deficient Des T cells transferred into B10.BR mice maintained a naïve CFSEhigh CD44low PD-1 low CD25 low phenotype, whereas all hepatocyte-activated Des T cells in Met-Kb recipients proliferated and expressed low levels of CD25 on their surface (Supplementary Fig. 4). Anti-PD-L1 treatment beginning at day 1 post-transfer did not decrease PD-1 expression on hepatocyte-activated Bim-deficient Des T cells (Supplementary Fig. 4). Blocking PD-1/PD-L1 interactions also induced a significant increase in donor Bimdeficient Des T cell numbers at day 15 in comparison to Met-Kb mice treated with control rat IgG (Fig. 2D), but did not interfere with the severity, kinetic, or the transient nature of acute hepatitis (Fig. 2E). In a similar manner to wild-type Des cells, a large proportion of these Bim-deficient Des T cells were found in the liver (Fig. 2D). Histological analysis of the liver correlated with T cell survival data, with large numbers of T cells detected in the portal and parenchymal regions of Met-Kb mice receiving Des-Bim-/- T cells (data not shown). To test whether the function of Bim-deficient T cells could be restored at a late time point (when T cells were silenced), we commenced treatment of recipient mice with anti-

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PD-L1 or control rat IgG at day 20 post-transfer of Bim-deficient T cells, administering antibody every three days till day 45. Late treatment with anti-PD-L1 altered the cell survival and/or proliferation of donor T cells, such that greater numbers of Des-Bim-/- T cells could be found in Met-Kb mice receiving anti-PD-L1 antibodies compared to those receiving control IgG (data not shown). However, anti-PD-L1 treatment of recipient mice did not enhance the CTL activity of donor Des-Bim-/- cells at day 38 (Supplementary Fig. 5), and hepatitis was not triggered as assessed by serum ALT (data not shown). Collectively, these results demonstrate that inhibition of PD-1/PD-L1 interactions promoted the proliferation and/or survival of self-reactive Bim-deficient T cells, suggesting that these two molecules increased T cell survival via distinct pathways. Most importantly, although activated Bim-deficient CD8 T cells accumulated and reached impressive numbers in the liver of mice treated with anti-PD-L1, their function was not restored, suggesting that, in a high and persisting antigen load setting, anti-PD-L1 blockade was unable to overcome the robust silencing imposed on T cells within this organ, even when the lifespan of liverreactive T cell was prolonged.

Regulation of cytokine signaling by SOCS-1 in liver-reactive CD8 T cells controls the kinetics and severity of acute hepatitis As regulation of T cell survival did not control acute liver damage, we next examined the possibility that liver injury was regulated at the functional level. The cytotoxic function of CTLs is acquired early after primary activation, and is influenced by TCR affinity and CD4 T cell help, as well as cytokines expressed by the priming antigen-presenting cells (recently reviewed in [30]). Cytokines are also likely to be important in maintaining CTL function upon secondary activation. Decreasing cytokine expression by T cells themselves, could

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thus be another strategy to attenuate and prevent excessive acute liver injury or evolution to chronic hepatitis. To alter cytokine expression by T cells, we generated Des Tg mice deficient in the suppressor of cytokine signaling-1 (SOCS-1) gene, a negative regulator of cytokines critical for CD8 T cell proliferation and effector function. SOCS-1 negatively regulates the strength and signaling duration of the Janus family of tyrosine kinases (JAK) and the signal transducers and activators of transcription proteins (STAT), commonly referred to as the JAK/STAT pathway.[31-33] SOCS-1 is both induced and regulated by a broad spectrum of cytokines in vitro and in vivo.[34] It is particularly important for negatively regulating IFN-γ signaling following receptor/cytokine engagement,[35] as well as cytokines that signal through the common γ chain: IL-2, IL-7, IL-15 and IL-21.[36] Consistent with published data on SOCS-1-/- mice,[31-33] Des-SOCS-1-/- mice displayed retarded growth and most mice did not survive beyond 22 days (Fig. 3A, B). Donor LN cells were therefore harvested from 18 day old Des-SOCS-1-/- mice, before they became sick, and were adoptively transferred into Met-Kb and syngenic control B10.BR hosts. Unlike DesBim-/- T cells, adoptive transfer of equivalent numbers of Des-SOCS-1 -/- T cells intensified the severity of hepatitis observed in Met-Kb mice compared to SOCS-1 sufficient Des T cells (Fig. 3C). Liver damage was not only more severe as assessed by ALT release, but also peaked later, at day 9 (Figs. 3C), rather than at day 5, as observed in Met-Kb mice that received Des-Bim-/- T cells (Fig. 1C) or wild-type Des T cells (Figs. 1C, 3C). Hepatitis induced by Des-SOCS-1 -/- T cells rapidly waned, with serum ALT returning to normal levels by day 15, suggesting that the absence of SOCS-1 in T cells was not sufficient to promote chronic liver damage.

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Prior to adoptive transfer, CD8 T cells isolated from LN of SOCS-sufficient and SOCSdeficient Des T cells were CD62Lhigh. However, consistent with previous reports, DesSOCS-1 -/- mice expressed lower levels of Des-TCR (Supplementary Fig. 6A) and displayed an activated CD44high phenotype compared to their SOCS-1-sufficient counterparts (Supplementary Fig. 6B), due to their increased susceptibility to undergo IL-15 driven homeostatic proliferation.[36-38] To exclude the possibility that the heightened hepatitis induced by Des-SOCS-1 -/- T cells was caused by the pre-activated phenotype of SOCSdeficient Des T cells, Des-SOCS-1 -/- mice were further bred onto the RAG-1 deficient background. Des-SOCS-1 -/-RAG-/- mice do not rearrange the endogenous TCR alpha chain typically responsible for T cell cross-reactivity to environmental antigens observed in RAGsufficient TCR transgenic animals.[39] Due to restriction in their TCR repertoire to solely the H-2Kb-reactive Des-TCR, Des-SOCS-1 -/-RAG-/- mice survived to adulthood (>12 months) and CD8 T cells isolated from the LN of 18-day old mice displayed a naïve CD44low phenotype similar to their naïve SOCS sufficient counterparts (Supplementary Fig. 6B). LN cells from Des-SOCS-1-/-RAG-/- mice adoptively transferred into Met-Kb mice also potentiated the severity of acute liver damage compared to control Des-RAG-/- T cells (Fig. 3D), suggesting that the increased ALT levels and altered hepatitis kinetics were driven by SOCS-1 deficiency, irrespective of the activation status of the T cells prior to encounter with the antigen recognized by the transgenic TCR.[40] Collectively, these results suggest that SOCS-1 is a critical regulator of acute liver damage mediated by CD8 T cells.

SOCS-1 regulates liver damage by enhancing the cytolytic function of donor liver-reactive T cells following their primary activation in lymphoid tissues

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The role of SOCS-1 in regulating the degree of acute hepatitis might be caused by a direct effect of this molecule on the effector function of donor CD8 T cells or by increasing the number of infiltrating and potentially pathogenic cells recruited to this organ following the larger release of cytokines by T cells (indirect effect). An indirect effect of SOCS-1 seems however unlikely, as the numbers of monocytes, neutrophils, and non-Des CD8 T cells isolated from the livers of Met-Kb mice receiving SOCS-1 deficient Tg T cells were actually lower than those isolated from the livers of mice receiving SOCS-1 sufficient Tg T cells, while CD4 T cell numbers did not differ significantly (Supplementary Fig. 7). These results suggest that SOCS-1 had a direct effect on liver-reactive Tg CD8 T cells by potentiating their ability to induce liver damage. As hepatitis in Met-Kb mice was strictly dependent on T cell activation in LN, it was important to explore whether SOCS-1 deficiency increased the intra-nodal activation and/or proliferation of TCR transgenic T cells as the basis of the observed effect on the extent of hepatitis. To test this possibility, CFSE-labeled Des-SOCS-1 -/-RAG-/- T cells were adoptively transferred into Met-Kb mice and 1-2 days later, LN were retrieved to assess cell numbers and proliferation. At day 1, donor Tg T cells up-regulated CD44, down-regulated CD62L, and demonstrated increased FSC in both groups (Fig. 4A), suggesting that they underwent similar activation and blast formation. SOCS-1-deficient and sufficient Des T cells also underwent similar proliferation in the LN of Met-Kb recipients, as assessed by comparable dilution of CFSE intensity at day 2 (Fig. 4B). Quantification of donor T cell numbers in LN at day 1 (Fig. 4C) and over the course of hepatitis demonstrated that SOCS1 deficiency never enhanced the number of Des T cell in the LN (Fig. 4D), suggesting that the severe hepatitis caused by Des-SOCS-1 -/-RAG-/- T cells was not the consequence of increased homing to LN, or a priming advantage during initial activation in LN.

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SOCS-1 regulated both the proliferation/survival and effector function of effector CD8 T cell function upon secondary activation. As SOCS-1 did not lead to differential expression of makers of activation or regulate the proliferation of CD8 T cells during primary activation in LN, we next examined whether it regulated the number and function of CTLs upon secondary activation, i.e. when LNactivated T cells left lymphoid tissues and were re-stimulated in the liver after day 3-4. At day 5, the number of intrahepatic SOCS-1 deficient Des-RAG-/- T cells was significantly lower than the number of intrahepatic SOCS-1 sufficient Des-RAG-/- T cells (Fig. 5A). However, the most important significant statistical difference between SOCS-1-deficient and SOCS-1-sufficient Des-RAG-/- T cell numbers was observed after day 5: while the number of intrahepatic SOCS-1-sufficient Tg T cells gradually fell after day 5, the number of intrahepatic SOCS-1-deficient Tg T cells continued to increase and peaked at day 9 (Fig. 5A), a result consistent with the increased and delayed hepatitis developed by these recipient mice (Fig. 3C,D). SOCS-1-deficient Tg T cells expressed high levels of the high affinity trimeric IL-2 receptor (Fig. 5B) and exhibited a statistically significant higher proliferation rate than SOCS-1-sufficient Tg T cells in livers of Met-Kb hosts at day 5 (Fig. 5C), suggesting that SOCS-1 deficiency enhanced the sensitivity of liver-infiltrating CTLs to IL2-mediated survival and/or proliferation. SOCS-1 deficient Des-RAG-/- T cells isolated from the LN at 5 and 9 days post-transfer displayed sustained IFN-γ secretion compared to their SOCS-1 sufficient counterparts (Fig. 5D and E). Interestingly, this was only observed in SOCS-1-/- Tg T cells isolated from the LN. Although we cannot exclude the possibility that SOCS deficiency promoted retention of functional T cells in LN, we favor the alternate possibility that SOCS-1 deficient T cells boosted their IFN-γ secretion when they re-circulated to the LN. In addition to increased

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IFN-γ secretion, a higher proportion of SOCS-1 deficient T cells from LNs degranulated following restimulation with antigen-expressing splenocytes ex vivo, indicative of augmented CTL function (Fig. 5 E). In vivo CTL assays confirmed the enhanced in vivo cytotoxicity of SOCS-1-deficient Des-RAG-/- T cells compared to their SOCS-1 sufficient counterparts (Fig 5F). The number of donor T cells infiltrating the liver started to decrease after day 9 (Fig. 5A). Although SOCS-1-deficient Des-RAG-/- CD8 T cells could be readily detected in the livers of Met-Kb mice at day 35, long after the resolution of acute hepatitis (Fig 5G, top panels), significant liver damage was not observed at this time point as assessed by serum ALT levels (Fig. 3D) and the presence of normal histology (Fig. 5G, bottom panel). These results suggest that the effect of SOCS-1 deficiency on the ability of T cells recognizing hepatocyte-expressed antigen to mediate hepatitis was short-lived and that SOCS-1-deficient T cells that remained in the liver after acute hepatitis became silenced. To investigate whether the silencing of persisting liver-reactive SOCS-1-deficient T cells was caused by exhaustion, similar to mechanisms observed in Bim-sufficient T cells, we assessed the expression of PD-1 and Tim-3 by SOCS-1 deficient T cells in both liver and LNs of recipient mice at different time points. Between 5 and 9 days after adoptive T cell transfer, donor CD8 T cells were observed to up-regulate PD-1 and Tim-3, irrespective of SOCS-1 expression (Supplementary Fig. 8 and data not shown). Although variations in the proportion of PD-1 expressing donor T cells were observed between SOCS-1 deficient and sufficient donor T cells at some time points, transferred liver-reactive Tg T cells expressed PD-1 irrespective of SOCS-1 expression, with a higher proportion of PD-1 positive cells seen in the liver than in LN. Interestingly, a higher proportion of donor SOCS-1-deficient Des T cells in the LN were Tim-3+ in comparison to their SOCS-1-sufficient counterparts. All Tim-3+ cells co-expressed PD-1 (Supplementary Fig. 8).

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At day 15, the proportion of Tim-3+ cells in liver and LNs decreased similarly in both groups, irrespective of their expression of SOCS-1 (Supplementary Fig. 8). Although the percentages of PD-1 + cells decreased in LNs, they remained high in the liver, a result consistent with exhaustion at this time point. The exhausted phenotype of T cells in the liver was confirmed at a functional level. At day 15, the total number of SOCS-1-deficient Des T cells displaying effector function (CD107a+ and/or IFN-γ+) decreased dramatically in recipient mice. This decrease occurred in both liver and LNs, with both a fall in the total number of cells and in the percentage of functional cells observed (Supplementary Fig. 8). These data suggested that although SOCS-1 does not regulate the initial activation and proliferation of Tg T cells in LN (primary activation), it plays a critical role in limiting cytokine-dependent proliferation/survival, number, and function of CTLs upon secondary activation. However, this effect of SOCS-1 is short-lived and despite their enhanced effector function, SOCS-1 deficient T cells became functionally exhausted eventually.

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Discussion By examining the ability of Tg Des T cells deficient for Bim or SOCS-1 to induce hepatitis and the role of PD-1/PD-L1 interactions in a well-characterized mouse model of acute immune-mediated hepatitis, this study demonstrated that the regulation of effector function was more critical than the regulation of the lifespan of effector CD8 T cells in limiting the tempo and degree of acute liver damage. However none of these factors were able to interfere with the dominant exhaustion process occurring in the liver in the presence of ongoing high antigen load setting, and hence chronic hepatitis did not ensue. Hepatocytes can be injured by many agents, including alcohol, toxins, chemicals and immune cells. Immune-mediated hepatocellular injury can be mediated by soluble factors such as inflammatory cytokines, including IFN-γ and TNF-α, that kill hepatocytes in a bystander manner,[41] or by direct CTL killing.[42] CTLs kill their targets in an antigenspecific manner by delivering lethal granzymes to the target cells [43] and/or by Fas/FasL interactions [44]. Although the mediators of effector T cell-dependent liver damage have been well characterized, the intrinsic T cell parameters that regulate CTL-mediated liver damage have not been previously investigated. This is an important unanswered question, as prolonged and uncontrolled and/or excessive CTL-mediated hepatocyte killing might lead to chronic or fulminant hepatitis, respectively. By using a Tg model of acute hepatitis mediated by a monoclonal population of CTLs of known specificity activated in LN, we manipulated intrinsic CD8 T cell factors that might regulate acute hepatitis, namely their susceptibility to apoptosis (via Bim), responsiveness to activation (via PD-1/PD-L1 interactions) and cytokine regulation/effector T cell function (via SOCS-1). The transfer of CD8 Tg T cells deficient for the pro-apoptotic molecule Bim was clearly associated with prolonged survival of these liver-reactive T cells in recipient mice,

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suggesting that Bim was a critical regulator of T cells survival. The five fold lower intrahepatic numbers of Bim-deficient Tg T cells observed in Alb-Kb compared to Met-Kb mice demonstrated that the vast majority of T cells that accumulated in the liver of Met-Kb mice at 30 days post transfer were originally activated in the LN. Surprisingly, one of the remarkable finding of this study was that the striking accumulation of Bim-deficient Des T cells in the livers of Met-Kb mice was not sufficient to intensify or even prolong the severity and kinetic of acute hepatitis nor induce chronic liver damage. This observation extends the conclusions of our previous studies showing that Bim-deficient Tg T cells activated solely by intrahepatic bone-marrow derived cells were silenced after inducing acute liver damage [23]. These results also confirm previous reports that Bim regulates survival without altering the effector function and autoimmune potential of T cells.[18] Interestingly, inhibiting the binding of the inhibitory receptor PD-1 with its ligand PD-L1 early post-activation had a similar effect to Bim deficiency in hepatocyte-reactive CD8 T cells, resulting in increased accumulation of antigen-specific T cells in the liver without affecting the kinetic and severity of acute liver injury (Figs 2D and E). This suggests that PD-1/PD-L1 interactions in the earlier phase post-activation are important in inhibiting T cell expansion and/or survival, rather than in dampening CD8 T cell effector function. This effect of PD-1/PD-L1 on survival seems to be distinct from the pro-apoptotic effect of Bim, as anti-PD-L1 treatment resulted in a pronounced accumulation of Bim-deficient T cells in the liver (Fig 2D). Consistent with this, recent data suggests that PD-1 appears to promote T cell survival via induction of the transcription factor FoxO1, which regulates PD-1 expression in a positive feedback loop.[45] The more marked hepatitis observed following adoptive transfer of hepatocyte-reactive T cells deficient for SOCS-1, a molecule that regulates signaling of several cytokines binding to the common γ chain, including IL-2,[36] suggests that cytokine expression by T cells

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changed the effector function of T cells with an associated increase in the severity of hepatitis. The similar T cell numbers in the Met-Kb LN during the first days after transfer regardless of SOCS-1 expression (Fig. 4D) suggests that the effect of SOCS-1 on hepatitis did not occur during primary activation in LN. Instead, SOCS-1-deficient Tg T cells acquired enhanced function after initial LN priming at day 5. This seemed to occur not only via increased expansion of CD8 T cells in the liver, probably due to their increased sensitivity to IL-2, but also by enhanced cytotoxic function. It is not clear whether this enhanced effector function was programmed during primary activation in LN or, alternatively, acquired upon secondary re-stimulation in the liver. However, the high expression of CD25 by SOCS-1-deficient, but not SOCS-1-sufficient, CD8 T cells in the Met-Kb recipient liver (Fig. 5B), suggests that the absence of SOCS-1 regulation extended cytokine expression in T cells and favored higher proliferation within the liver and enhanced effector function. This data is consistent with the known regulatory role of SOCS-1.[36, 37] SOCS-1 gene transcription is induced in response to initial IL-2R/IL-2 binding that occurs just after T cell activation. SOCS-1 inhibits JAK/STAT signaling that lie downstream of the IL-2R,[31-33] acting in a negative feedback loop to regulate the IL-2 signaling pathway.[36] SOCS-1 deficiency is thus thought to augment the “responsiveness” of T cells to cytokines at a later stage of the immune response when these molecules accumulate. Although we were able to prolong effector function and alter the outcome of hepatitis by ablating SOCS-1 in donor T cells, liver damage was still controlled and did not evolve into chronic hepatitis in this model. SOCS-1 and SOCS-3 have been reported to have redundant inhibitory activities. Thus it is possible that other cytokine inhibitory mechanisms such as SOCS-3 compensate for SOCS-1 deficiency to restrict acute hepatitis. Regardless of cytokine regulation, most Tg SOCS-1-/- T cells were also deleted following early increases in survival and expansion, suggesting that like SOCS-1-sufficient Tg T cells, SOCS-1-deficient

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Tg T cells also died by Bim-dependent apoptosis. It would be interesting to know whether Tg T cells deficient for both SOCS-1 and Bim, and thus displaying enhanced survival as well as dysregulated effector function, would induce fulminant hepatitis or become exhausted following transfer into Met-Kb mice. Unfortunately SOCS-1-/- Bim-/- Des-TCR Tg mice could not be bred despite our several attempts to generate this line. The exact mechanisms responsible for T cell silencing after acute hepatitis in these different models remain unclear. However, our recent study showing that CTLs become exhausted in the presence of a persisting high intrahepatic antigen load [14]. Together with the results from this study showing that Bim- and SOCS-1-deficient T cells express PD-1 and Tim-3 and become functionally exhausted, it would appear that T cell exhaustion occurs in the face of ongoing high levels of intrahepatic antigen expression, regardless of enhanced T cell function or resistance to apoptosis. Based on these findings, we predict that strategies that tend to increase the survival of antigen-specific T cells would fail to interfere with robust T cell exhaustion mechanisms that occur in the liver in this setting. This data is highly relevant for therapies that attempt to restore T cell effector function in patients chronically infected with HBV or HCV. It is thought that in these clinical settings, persisting high antigen load might play an important role in evolution towards chronic infection by promoting T cell exhaustion; similar mechanisms may also play a role in promoting resistance of hepatocellular carcinoma to tumor-specific CTL responses.[46] The relatively limited efficacy of PD-1 inhibition in HCV infection in vivo [25, 47], suggests that other parameters need to be taken into consideration. The compensatory roles of other inhibitory molecules, including Tim-3, TGIT, and CTLA4, have been proposed as key to understand these observations. [48] An alternative, non-exclusive possibility suggested by our findings is that T cell responses are difficult to restore or enhance in the presence of high antigen load (e.g. large tumor burden in HCC or high levels of intrahepatic antigen

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expression in HBV and HCV). This study and previous results [14] would predict that inhibition of PD-1/PD-L1 interactions would be most effective in combination with other treatments that interfere with T cell exhaustion or in the presence of lower antigen loads, such as lowered tumor burden following resection in HCC or in combination with antiviral therapy to lower viral titers in chronic HBV and HCV. In conclusion, this study demonstrates that a highly efficient population of CTLs that secrete hepatotoxic cytokines is critical to potentiate severe acute hepatitis. Although promoting survival of CD8 T cells enhanced their clonal expansion and accumulation in the liver, it was not sufficient to amplify liver damage or mediate chronic hepatitis. This would suggest that once activated, in the presence of high levels of hepatocyte-expressed antigen, CD8 T cells have a limited window of opportunity during which effector function is mediated, regardless of their lifespan. Past this phase, if antigen is not cleared in the liver, effector CD8 T cells become exhausted. This pathway might have been selected during evolution to avoid irreversible damage to the liver and prevent death by fulminant hepatitis. The adverse effect of this protection mechanism is, however, the persistence of viruses (e.g. HBV and HCV) that tend to spread rapidly and chronically infect the host by promoting silencing of the anti-viral T cell response. Collectively, these results assist in our understanding of why T cells become exhausted after acute hepatitis in the presence of ongoing high-level antigen expression, and why the extent of viral antigen-specific T cell infiltrates do not always correlate with ALT levels in patients chronically infected with HBV and HCV.[49] This current study predicts that anti-viral immunity strategies that boost the priming or effector function of CTLs would be more effective than those that increase CTL survival. The combination of treatments that increase CD8 T cell survival and function and interfere with T cell exhaustion might also improve the efficacy of these therapies.

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Figure Legends Figure 1. Des-Bim-/- T cells accumulated in the livers of Met-Kb mice but did not mediate more liver damage than their Bim-sufficient counterparts. Equal numbers of Des and Des-Bim-/- LN cells were adoptively transferred into Met-Kb, Alb-Kb and B10.BR recipients; (A) recipient mice were harvested at 30 days post-transfer and the absolute number of viable liver-infiltrating Tg T cells counted; (B) Des-TCR and CD8 expression levels on viable CD8 + Des-TCR+ cells isolated from the livers of Met-Kb and B10.BR hosts 30 days post-transfer; (C) Anti-CD8 antibody stained cryosections of livers from Met-Kb recipients of Bim-sufficient and Bim-/- Des T cells. Magnification 200x; (D) serum ALT levels in Met-Kb and control B10.BR hosts at 3, 5, 7 and 9 days posttransfer. Plots represent means ± S.E.M.

Figure 2. Inhibiting PD-1/PD-L1 interactions promoted the accumulation of Tg T cells without enhancing acute liver damage. (A) PD-1 expression on liver and LN lymphocytes (gated on Des-TCR+CD8+DAPI- lymphocytes) at 24 and 48 h after transfer of Des-RAG-/- T cells into Met-Kb and B10.BR mice (n=-3 per group). Data representative of 3 independent experiments (for day 1) and 2 independent experiments for day 2. (B) Kinetics of relative expression level of PD-1 on donor T cells (calculated by dividing the MFI of the donor CD8 cells in Met-Kb mice by the MFI of donor cells in B10.BR mice). Autofluorescence values for lymphocytes from the blood, livers, LN, and spleens of B10.BR mice were comparable. Results displayed represent the means ± S.E.M. (n= 3-6 mice per timepoint, * = p

Effector T cell function rather than survival determines extent and duration of hepatitis in mice.

Acute hepatitis is often mediated by cytotoxic T lymphocytes (CTLs); however, the intrinsic parameters that limit CTL-mediated liver injury are not we...
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