Persistent infection with hepatitis B virus (HBV)

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1 Role of the Coinhibitory Receptor Cytotoxic T Lymphocyte Antigen-4 on Apoptosis-Prone CD8 T Cells in Persistent Hepatitis B Virus Infection Anna Schurich, 1 * Pooja Khanna, 1,2 * A. Ross Lopes, 1 Ki Jun Han, 1 Dimitra Peppa, 1 Lorenzo Micco, 1 Gaia Nebbia, 1 Patrick T.F. Kennedy, 3 Anna-Maria Geretti, 2 Geoffrey Dusheiko, 2 and Mala K. Maini 1,2 An excess of coinhibitory signals has been proposed to drive the T-cell exhaustion characteristic of persistent viral infections. In this study we examined the contribution of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) to CD8 T cell tolerance in chronic hepatitis B virus (HBV) infection (CHB). CD8 T cells in patients with CHB have an increased propensity to express the coinhibitory receptor CTLA-4 and this correlates with viral load. CTLA-4 is up-regulated on those HBV-specific CD8 T cells with the highest levels of the proapoptotic protein Bim, which we have previously shown mediates their premature attrition; abrogation of CTLA-4-mediated coinhibition can reduce Bim expression. Longitudinal study of CHB patients beginning antiviral therapy reveals that HBV DNA suppression induces transient reconstitution of HBV-specific CD8 T cells but does not reprogram their CTLA-4 hi Bim hi tolerogenic phenotype. Blocking CTLA-4 is able to increase the expansion of interferon gamma (IFN-c)-producing HBV-specific CD8 T cells in both the peripheral and intrahepatic compartments. The rescue of anti-hbv responses by either CTLA-4 or PD-L1 blockade is nonredundant. Conclusion: CTLA-4 is expressed by HBV-specific CD8 T cells with high levels of Bim and helps to drive this proapoptotic phenotype. CTLA-4 blockade could form one arm of a therapeutic approach to modulate the diverse patterns of coregulation of T-cell exhaustion in this heterogeneous disease. (HEPATOLOGY 2011;53: ) Persistent infection with hepatitis B virus (HBV) accounts for more than a million deaths a year from liver cirrhosis and hepatocellular carcinoma. Existing therapies usually suppress rather than cure infection, necessitating long-term use of antiviral agents Abbreviations: ALT, alanine transaminase; CHB, chronic hepatitis B virus infection; CTLA-4, cytotoxic T lymphocyte antigen-4; HBV, hepatitis B virus; OLP, overlapping peptides. From the 1 Division of Infection and Immunity, UCL, London, UK; 2 Centre for Hepatology, Royal Free Campus, UCL, London, UK; 3 Centre for Digestive Disease, Barts and the London School of Medicine and Dentistry, London, UK. Received October 28, 2010; accepted February 3, Funded by Medical Research Council Awards G and G (to M.K.M.) and by an unrestricted infrastructure grant from Roche and Schering Plough (to G.D.). *These authors contributed equally to this work. Address reprint requests to: Mala K. Maini, Department of Immunology and Molecular Pathology, Division of Infection and Immunity, Windeyer Building, 46 Cleveland Street, UCL, London, W1T 4JF. m.maini@ucl.ac.uk; fax: þ44 (0) Copyright VC 2011 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Dr. Dusheiko consults for, serves on the speaker s bureau of, and received grants from Bristol-Myers Squibb, Gilead, and Roche. Additional Supporting Information may be found in the online version of this article. with ongoing limitations of cost, viral resistance, and toxicity. There is therefore a pressing need to complement antiviral therapy with a targeted immunotherapeutic approach aiming to reverse the T-cell exhaustion characteristic of chronic HBV infection (CHB), thus restoring effective immune control and achieving sustained off-treatment responses. Antigen-specific CD8 T cells are critical for the control of HBV and are markedly diminished in patients with persistent infection. 1 We have identified Bimmediated apoptosis as a key mechanism resulting in the depletion of HBV-specific CD8 T cell responses in CHB. Interruption of this pathway reconstituted multispecific CD8 T-cell responses against HBV, confirming its functional relevance. 2 Virus-specific CD8 T cells have also been shown to be highly prone to apoptosis in patients with HCV infection. 3 We hypothesize that the proapoptotic defects in these hepatotropic viral infections are imposed by the tolerogenic liver environment they target. This is line with recent data showing that the premature death of T cells primed in the liver is Bim-dependent. 4 We postulate that in this setting, virus-specific T cells are driven towards apoptosis by 1494

2 HEPATOLOGY, Vol. 53, No. 5, 2011 SCHURICH ET AL Table 1. Patient Clinical Details Number Gender (m) Age HBV DNA (IU/ml) ALT (U/L) eag1 CHB VL<2000 IU/ml (21-58) 116 (1-1600) 27 (10-103) 0 CHB VL>2000 IU/ml (23-67) 700,000 ( x10 6 ) 57 (15-261) 28 Resolved , 35, 71 n/a n/a n/a Healthy (26-56) n/a n/a n/a Abbreviations: VL ¼ viral load; n/a ¼ not applicable. *Median and range shown. persistent high-dose antigen with insufficient costimulatory signals, outweighed by an excess of coinhibitory signals. One such coinhibitory signal is mediated through the PD-1 pathway, which has been shown to be critical for engendering intrahepatic tolerance 5 and to contribute to the failure of the T-cell response in CHB. 6,7 Blockade of the PD-1 pathway only results in a partial recovery of some HBV CD8 specificities in a proportion of patients with CHB, 6 implicating a role for additional mediators. Tolerogenic antigen presentation can be mediated through the combination of both PD-1 and cytotoxic T lymphocyte antigen-4 (CTLA- 4) 8 ; the latter coinhibitory receptor has recently been shown to act synergistically with PD-1 to promote T-cell exhaustion in HCV infection. 9 The crucial role of CTLA-4 in peripheral tolerance has been demonstrated in CTLA-4 knockout mice that develop a lethal lymphoproliferative disorder. 10 Conversely, CTLA-4 expression inhibits clearance of a number of pathogens and in particular correlates with reversible immune dysfunction and disease progression in human immunodeficiency virus (HIV) infection. 11 A role for CTLA-4 in the pathogenesis of HBV disease has been suggested by a study linking single nucleotide polymorphisms of CTLA-4 to the outcome of HBV infection. 12 In this study we examined the propensity of CD8 T cells from patients with CHB to up-regulate CTLA-4. We found a link between the expression of CTLA-4 and the proapoptotic mediator Bim in HBV-specific CD8. Longitudinal study of a cohort of CHB patients commencing antiviral therapy showed that viral load reduction did not reduce CTLA-4 or Bim levels in antiviral T cells. We therefore explored the potential to manipulate this coinhibitory pathway in vitro to restore expansion of HBV-specific CD8 T cells. Patients and Methods Patients and Controls. The study was approved by the local Ethical Committees and written informed consent was obtained from all patients. A total of 86 patients with CHB, three patients with resolved HBV infection, and 23 healthy volunteers participated in the study; there were no significant differences in their demographics (Table 1). All participants were HCV and HIV seronegative and cytomegalovirus (CMV) seropositive. Patients with CHB were stratified by HBV DNA levels above or below 2,000 IU/mL (determined by real-time polymerase chain reaction [PCR]), according to European Association for the Study of the Liver (EASL) guidelines. 13 All CHB patients were treatment-naïve at recruitment; a subgroup of seven patients was followed longitudinally after commencing lamivudine and adefovir (Table 2). Hepatitis B s-antigen (HBsAg) was quantitated with the Architect assay. Paired peripheral blood and liver biopsy specimens (surplus to diagnostic requirements) were obtained from eight patients with CHB (Table 1). Peripheral Blood Mononuclear Cells (PBMCs) Isolation, Culture, and Analysis of Virus-Specific CD8 T Cells. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation and cultured on anti-cd3 monoclonal antibody (mab)-coated plates (1 lg/ml) or in medium alone for 16 hours before analysis of CTLA-4 in total CD8 T cells. For detection of intracellular CTLA-4 on virus-specific CD8 T cells ex vivo, cells were stained with human leukocyte antigen A2 (HLA-A2)/c18-27, HLA-A2/e , HLA-A2/e , and HLA-A2/e HBV dextramers (Immudex) before stimulation with HBV-specific peptides for 4 hours in the presence of Brefeldin A. CMV-specific CD8 T cells were detected by HLA-A2/NLVPMVAYV pentamers (Proimmune). For functional detection of virus-specific CD8 T cells, cells were stimulated with HBV or control viral peptide and cultured for 10 days, supplemented with 20 U/mL IL-2 at 0 and 4 days, restimulated with 1 lm peptide for 16 hours in the presence of 1 lg/ml Brefeldin A (Sigma-Aldrich), and identified by intracellular staining for IFN-c. To examine the effect of blocking inhibitory pathways, purified, NA/LE monoclonal antibodies against CTLA-4 (BD Biosciences), PD-L1, PD-L2 (ebioscience), or control IgG (BD-Biosciences) were added at 5 lg/ml with

3 1496 SCHURICH ET AL. HEPATOLOGY, May 2011 Table 2. Clinical Characteristics of Patients Followed-Up on Treatment Gender Pretreatment Viral Load Pretreatment ALT HBeAg HLA-A2 Status Duration of Treatment at Last Sample (Months) No. of Timepoints Sampled Patient 1 Male 339, Pos Pos 14 8 Patient 2 Male 70, Neg Neg 18 5 Patient 3 Male Neg Neg 13 4 Patient 4 Male 418, Neg Neg 18 6 Patient 5 Male 16,000, Pos Pos 14 6 Patient 6 Female 1,548, Pos Pos 16 7 Patient 7 Female 264, Neg Neg 15 6 peptide at onset of culture. Responses were analyzed as described above. Intrahepatic Lymphocyte Isolation, Culture, and Functional Assay. Liver sections from biopsies were homogenized and filtered. Cell suspensions were taken into culture ( cells per well) with the addition of autologous feeder cells ( ), derived from PBMC irradiated with 40 Gy. Cells were stimulated with overlapping peptides (OLPs) spanning core in the presence of CTLA-4 blocking mab or control IgG and restimulated after 10 days as described for PBMC. Peptides. HLA-A2 patients were stimulated with a pool of 15mer peptides overlapping by 10 residues (OLP) spanning core of HBV genotype D or OLP spanning the pp65 protein of CMV. HLA-A2 þ individuals were stimulated with a panel of peptides representing immunodominant HLA-A2 restricted epitopes from HBV (envelope: FLLTRILTI, WLSLLVPFV, LLVPFVQWFV, GLSPTVWLSV; core: FLPSDFFPSV; polymerase: GLSRYVARL, KLHLYSHPI), CMV pp65 (NLVPMVATV) or Epstein-Barr virus (EBV) BMLF1 (GLCTLVAML) (Proimmune). Flow-Cytometric Analysis of Total and Virus- Specific CD8 T Cells. For analysis of total CD8, cells were surface-stained with mab CD3 PerCP-Cy5.5, CD8 APC, fixed and permeabilized for intracellular staining with CTLA-4 PE (BD Biosciences). Background CTLA-4 expression calculated on unstimulated cells was subtracted. Virus-specific cells were analyzed by 8-color flow-cytometry. PBMCs were surfacestained with saturating concentrations of mab anti- CD3 PE-Cy7, CD8 Alexa700, PD-1 PErCPeFluor710, and CD4 APC-Cy7 (ebioscience) in the presence of fixable live/dead stain (Invitrogen). Cells were fixed and permeabilized followed by intracellular staining for CTLA-4 PE, IFN-c APC (BD Biosciences) and Bim unconjugated (Alexis Biochemicals) detected with goat anti-rat IgG2a FITC (Bethyl Laboratories). Cells were acquired on a LSRII (BD Biosciences) and analyzed using Flowjo. Statistical Analysis. Data were analyzed using the nonparametric Mann-Whitney, Wilcoxon matched pairs test or Spearman correlation coefficient as appropriate (*P < 0.05; **P < 0.005; ***P < ). Results CD8 T Cells in CHB Have an Enhanced Propensity to Up-Regulate CTLA-4. To investigate whether the coinhibitory receptor CTLA-4 plays a role in CHB, we initially analyzed expression levels in mitogen-activated CD8 T cells from a cohort of 12 healthy controls and 36 patients with CHB (Table 1). CTLA- 4 is not constitutively expressed on effector T cells but is rapidly up-regulated upon their activation. 14 CTLA- 4 up-regulation was significantly augmented on global CD8 T cells from patients with CHB compared to healthy controls, with a trend to increasing expression of CTLA-4 with higher HBV DNA (Fig. 1A). In line with this increased propensity for global CD8 T cells to up-regulate CTLA-4 upon stimulation, CD8 T cells increased CTLA-4 expression more in CHB than in healthy controls upon stimulation with peptides representing immunodominant HLA-A2-restricted epitopes from CMV and EBV (Supporting Fig. S1). CTLA-4 expression was also increased upon anti-cd3 stimulation of CD4 T cells from CHB compared to healthy controls (Supporting Fig. S2). Next we analyzed CTLA-4 expression in HBVspecific CD8 T cells following recognition of their cognate peptide. HBV-specific cells were first identified by IFN-c production following expansion in vitro with peptides representing immunodominant HLA-A2 restricted epitopes or spanning HBV core and examined for their intracellular expression of CTLA-4 (Fig. 1B). A high percentage of HBV-specific CD8 T cells expressed CTLA-4 and the level of CTLA-4 expression (mean fluorescence intensity [MFI]) was increased compared to the total CD8 T-cell population (Fig. 1B, summary data). These CTLA-4-expressing CD8 T cells expressed less IFN-c than their CTLA-4-negative

4 HEPATOLOGY, Vol. 53, No. 5, 2011 SCHURICH ET AL Fig. 1. CD8 T cells in CHB have an enhanced propensity to up-regulate CTLA-4. (A) Representative FACS plots and cumulative data of intracellular CTLA-4 expression in total CD8 T cells upon mitogenic stimulation from healthy controls (n ¼ 12) and HBV-infected individuals with low (n ¼ 10) and high (n ¼ 23) viral load (VL). Final values of CTLA-4 expression were calculated after subtraction of background expression in unstimulated samples. (B) Representative FACS plots and summary data of CTLA-4 expression in HBVspecific CD8 T cells identified by ICS for IFNc after 10 days expansion using peptides representing immunodominant HLA-A2 restricted epitopes in 23 patients with CHB. (C) Comparison of CTLA-4 MFI in total CD8 T cells versus HBV-specific CD8 T cells directly ex vivo in 11 patients with CHB. (D) CTLA-4 MFI of HBV-specific CD8 from resolved compared to CHB-derived cells identified with multimers ex vivo and correlated with viral load for CHB samples. (E) Representative FACS plots and summary data comparing CTLA-4 expression in multimer positive CD8 versus IFN-cþ CD8 stimulated ex vivo for 4 hours. counterparts (P < 0.05, data not shown). The expression of CTLA-4 was also significantly increased after 4-hour peptide stimulation of HBV-specific CD8 T cells identified by HLA-peptide multimer staining ex vivo compared to total CD8 T cells (Fig. 1C). The expression of CTLA-4 on ex vivo HLA/peptide multimer stained HBV-specific CD8 T cells was examined in patients with different outcomes of HBV infection and was found to be significantly higher in patients with viral load (VL) greater than 2,000 IU/mL than in those with resolved infection or VL <2,000 IU/mL (Fig. 1D). The high standard deviation of CTLA-4 expression in the group with VL >2,000 IU/mL reflected the heterogeneity of this group, with CTLA-4 correlating with both viral load (Fig. 1E, r ¼ 0.6*) and sag (Supporting Fig. S3, r ¼ 0.85**). Those CD8 T cells able to produce IFN-c in response to 4-hour peptide stimulation expressed lower levels of CTLA-4 than populations staining ex vivo with HLA-A2/peptide multimers but unable to produce IFN-c (Fig. 1E), suggesting that CTLA-4 may impair effector function of HBV-specific CD8 T cells. CTLA-4-Expressing HBV-Specific CD8 T Cells Have High Levels of the Proapoptotic Mediator Bim. We have previously demonstrated increased levels of the proapoptotic protein Bim in HBV-specific CD8 T cells from patients with CHB. 2 To probe a potential role for CTLA-4 in driving Bim-mediated attrition of the antiviral response, we examined the intracellular expression of Bim in CTLA-4 þ and CTLA-4 HBV-specific

5 1498 SCHURICH ET AL. HEPATOLOGY, May 2011 Fig. 2. CTLA-4-expressing HBV-specific CD8 T cells have high levels of the proapoptotic mediator Bim. (A) FACS plot showing HBV-specific cells CD8 T cells, costained for Bim and CTLA-4. Representative histograms showing expression of Bim on CTLA-4 þ and CTLA-4 fractions of the HBV-specific IFN-cþ CD8 T cell response. (B) Cumulative data evaluating differential expression of Bim on CTLA-4-expressing HBV-specific CD8 T cells (n ¼ 11). (C) Bim MFI (shown as a fold change above that in global CD8 T cells) in CMV-specific CD8 T cells in healthy controls and in CMV and HBV-specific CD8 T cells in patients with CHB identified by multimer staining after 4-hour peptide stimulation. (D) Representative plots and summary data of staining with fixable live/dead stain (Invitrogen) of global CD8 T cells compared to CMV or HBV multimer stained CD8 T cells. (E) Peptide-stimulated PBMCs were cultured in the presence of CTLA-4 blocking mab or control IgG for 10 days. Representative experiment showing a reduction in Bim expression upon blocking CTLA-4. Summary data showing Bim levels calculated as fold change comparing IFN-cþ HBV-specific CD8 T cells cultured in the absence or presence of CTLA-4 blocking and correlated with patient ALT levels. CD8 T cells. HBV-specific CD8 T cells expressing CTLA-4 had much higher levels of Bim than CTLA- 4-negative cells (Fig. 2A). In the 11 patients with CHB examined, we consistently found increased amounts of Bim in HBV-specific CD8 T cells expressing CTLA-4 than in their CTLA-4-negative counterparts (Fig. 2B). Levels of Bim were significantly higher in CMV-specific CD8 T cells from patients with CHB than healthy controls (Fig. 2C) in line with their higher CTLA-4 expression (Supporting Fig. S1), but were further increased in HBV-specific CD8 T cells (Fig. 2C). The propensity of the Bim hi HBV-specific CD8 T cells to undergo apoptosis was reflected in the much higher proportion of these cells falling within the dead gate with fixable live/dead stain (Fig. 2D). To determine whether CTLA-4 can actually drive the up-regulation of Bim in HBV-specific CD8 T cells, we tested the impact of blocking the coinhibitory receptor CTLA-4. After 10 days culture, intracellular levels of Bim expression in HBV-specific CD8 T cells could be reduced in the presence of a CTLA-4 blocking mab (Fig. 2E, representative histogram). The ability to reduce Bim expression by CTLA-4 blockade showed a negative correlation with disease activity as measured by serum ALT (Fig. 2E). This suggested that CTLA-4 signaling contributes to the up-regulation of

6 HEPATOLOGY, Vol. 53, No. 5, 2011 SCHURICH ET AL Bim in CHB, but that in the presence of active liver inflammation other factors dominate over CTLA-4 in the induction of their proapoptotic propensity. Antiviral Therapy Is Unable to Reverse the CTLA-4 hi Bim hi Phenotype of HBV-Specific CD8 T Cells. We next asked whether potent antiviral therapy has the capacity to reverse this tolerogenic phenotype of HBV-specific CD8 T cells. We followed seven treatment-naive patients with CHB for more than a year after beginning antiviral therapy with a combination of lamivudine and adefovir (Table 2). Patients were sampled on five to eight occasions during the study to allow temporal dissection of the immunological changes induced by the rapid HBV DNA decline on therapy. Figure 3A shows the temporal correlation observed between viral load, HBsAg, expansion of HBV-specific CD8 T cells, and levels of CTLA-4 and Bim expressed in HBV-specific CD8 T cells in two representative patients with CHB (Patient 1, HLA-A2 þ ; Patient 2 HLA-A2 ). In both patients there was a small recovery in the number of IFN-c-producing HBV-specific CD8 T cells that could be expanded, peaking 4 to 7 months after viral load became undetectable (7-12 months after the commencement of antiviral therapy, Fig. 3A). However, this reconstitution was short-lived, with the number of HBV-specific CD8 T cells returning to around or below baseline by the last timepoint of sampling (13-18 months after treatment commencement, Fig. 3A,B, and Supporting Fig. S4a). The increase in HBV-specific CD8 T cells was both low-level and transient, irrespective of whether epitope responses detected were only HLA-A2 restricted or of any HLA restriction (Fig. 3A). In line with their brief survival, HBV-specific CD8 T cells continued to express high levels of CTLA-4 and Bim in the face of a durable reduction in viral load (Fig. 3A,B), with variable fluctuations (Fig. 3A) and no sustained decrease in expression up to 18 months of follow-up (Fig. 3B, Supporting S4b,c). The maintenance of a tolerogenic phenotype and lack of durable reconstitution of HBV-specific CD8 T cells correlated with the residual production of HBsAg (Fig. 3A,B, Supporting S4d), indicative of continuing intrahepatic production of HBV replicative intermediates. Blocking CTLA-4 Can Rescue Functional HBV- Specific CD8 T Cells. Because antiviral therapy alone was unable to reconstitute a durable antiviral T-cell response, we investigated the potential to augment this by blocking the CTLA-4 pathway in vitro. Virusspecific CD8 T cells were expanded in culture with cognate peptide in the presence of a CTLA-4 blocking or control IgG mab and functional responses assessed by IFN-c staining (Fig. 4A). CTLA-4 blockade resulted in significantly greater fold increases for HBVspecific CD8 T cell responses than for responses directed against control viruses (CMV, EBV) in patients with CHB (Fig. 4B). In more than half of the 33 patients tested, the addition of a CTLA-4 blocking mab was able to increase the percent of functional HBV-specific CD8 T cell responses expanded in vitro by 2 to 5-fold (Fig. 4B,C). It was possible that we had underestimated the contribution of the CTLA-4 pathway by only analyzing the impact of blocking this coinhibitory receptor on circulating HBV-specific CD8 T cells. We therefore compared the increase in HBV core-specific CD8 T-cell responses achieved following abrogation of CTLA-4 signaling in the peripheral and intrahepatic compartments (Fig. 4D). Sufficient intrahepatic lymphocytes were expanded for analysis from seven of eight patients from whom paired liver biopsies and PBMCs were available. Two patients with relatively low HBV load had a greatly enriched population of HBV corespecific CD8 T cells expanded from the liver compared to the periphery, as described, 1 and these intrahepatic responses were not amenable to additional expansion upon CTLA-4 blockade. By contrast, in three of the four patients with higher viral load, enhanced responses to HBV core peptides were seen upon CTLA-4 blockade in liver-infiltrating compared to circulating lymphocytes (Fig. 4E). These data, although limited to CD8 responses directed against core epitopes, underscore the potential importance of coinhibition by CTLA-4 in the liver, the site of HBV replication. The lack of rescue of HBV-specific CD8 T cell responses upon CTLA-4 blockade in some patients with CHB pointed to coregulation by additional coinhibitory pathways. We therefore questioned whether CTLA-4 might complement the PD-1 pathway, interruption of which has been shown to increase some CD8 T-cell responses in CHB. 6,7 CTLA-4 and PD-1 expression were examined on CD8 stained with HBV multimers ex vivo and showed some correlation because both increased with viral load; however, there were also examples of differential expression of these two inhibitory receptors (Fig. 5A,B), suggesting that they may tolerize T cells in a complementary manner in CHB. Consistent with this, we observed that the proportion of patients responding to single blockade of either CTLA-4 or PD-1 was similar and that responders to these two approaches were largely nonoverlapping, such that only three of 18 patients failed to respond to one of these blocking strategies (Fig. 5C).

7 1500 SCHURICH ET AL. HEPATOLOGY, May 2011 Fig. 3. No reduction in CTLA-4 and Bim in HBV-specific CD8 T cells upon viral load reduction. (A) Temporal analysis of HBV DNA, HBsAg, the frequency of HBV-specific CD8 T cells, and their expression of CTLA-4 and Bim in a representative HLA-A2 þ and HLA-A2 patient. (B) Bar graphs summarizing the fold change in the magnitude of HBV-specific CD8 T cells, their expression of Bim and CTLA-4, and HBsAg levels in seven patients after months of antiviral therapy. Timepoints where serum sag could not be analyzed are asterisked. In addition, dual CTLA-4 and PD-1 blockade had a synergistic effect on HBV-specific CD8 T-cell reconstitution in six of the 18 patients tested (Fig. 5C), including a patient in whom responses were diminished by single blockade of either pathway (Fig. 5D). Taken together, these data show nonredundant roles for the CTLA-4 and PD-1 pathways in driving T-cell exhaustion in CHB.

8 HEPATOLOGY, Vol. 53, No. 5, 2011 SCHURICH ET AL Fig. 4. Impact of CTLA-4 blockade on HBV-specific responses expanded from peripheral and intrahepatic compartments. (A) Representative example of expansion of HBV-specific CD8 T cells in the absence or presence of CTLA-4 blockade. (B) Comparison of the impact of CTLA-4 blockade on HBV versus CMV/EBV-specific CD8 T-cell responses. (C) Summary of the impact of CTLA-4 blockade for 33 patients with CHB. A2þ ¼HLA-A2 þ patients stimulated with peptides representing HLA-A2 restricted epitopes; A2 ¼ HLA-A2 patients stimulated with overlapping peptides spanning HBV core. (D) Representative plots and (E) summary data showing the effect of CTLA-4 blockade on intrahepatic lymphocytes (IHL) compared to PBMC for seven patients. Discussion Successful T-cell activation requires a TCR-mediated signal accompanied by a costimulatory signal through receptors such as CD28. However, T cells also receive inhibitory signals through a number of coreceptors that temper the immune response and maintain peripheral tolerance. One such coinhibitory receptor is CTLA-4 (CD152), a member of the CD28-B7 family, which binds to the same ligands as its costimulatory counterpart CD28. CTLA-4 is not constitutively expressed on effector T cells but is rapidly induced upon TCR engagement. 14 In patients with CHB, we found a greater propensity for the induction of CTLA- 4 upon TCR stimulation with either mitogen or cognate peptide, with CTLA-4 induction in HBV-specific CD8 T cells correlating strongly with viral load. These data are in line with recent demonstrations that CTLA-4 plays a critical role in the effector T-cell compartment in addition to its contribution to regulatory T-cell function. 15,16 CTLA-4 mediated inhibition may depend on the fact that it shares ligands (B7-1 and B7-2) with CD28 but has higher avidity; when the supply of these ligands is limited, CTLA-4-mediated inhibitory signaling could override CD28-mediated positive costimulation. In CHB, the ability of CTLA-4 to outcompete CD28 may be favored not only by the increase in CTLA-4, but also by the reduced levels of CD28 on CD8 T cells 17 and by the scarcity of B7 ligands on hepatocytes and other intrahepatic cells with antigen-presenting capability. 18 We postulated that CTLA-4-mediated coinhibition may be one of the pathways that drives T cells encountering their antigen in the liver towards Bimdependent apoptosis. In support of this, we found the highest intracellular levels of Bim in CTLA-4 hi HBVspecific CD8 T cells. We speculate that CTLA-4 signaling may induce Bim by its capacity to reduce availability of IL-2 14 while increasing cell-intrinsic transforming growth factor beta (TGF-b), 19 which is up-regulated at the transcriptional level in HBVspecific CD8 T cells 2 and can promote Bim-dependent attrition of LCMV-specific T cells. 20 In most patients with CHB without evidence of liver inflammation, blocking the CTLA-4 receptor was able to reduce Bim expression. However, in patients with CHB-related liver inflammation the lack of reduction in Bim achieved by CTLA-4 blockade invoked a dominant role for other factors in driving this proapoptotic phenotype. We have recently described a signaling defect reducing cell-autonomous production of IL-2 in patients with CHB-related liver inflammation 17 that may limit the efficacy of CTLA-4 blockade in such patients. In addition, as discussed below, a number of different coinhibitory pathways may play nonredundant roles in T-cell exhaustion in CHB. To explore the therapeutic potential to reprogram the tolerogenic phenotype of HBV-specific CD8 T cells we examined the impact of antiviral therapy. A previous study of CD8 T-cell reconstitution on antiviral monotherapy had suggested that viral load

9 1502 SCHURICH ET AL. HEPATOLOGY, May 2011 Fig. 5. Complementary roles for CTLA-4 and PD-1 pathways in rescuing HBV-specific responses. (A) Costaining with CTLA-4 and PD-1 of CD8 T cells, gated on those binding HBV HLA-A2/peptide multimers ex vivo. (B) Differential expression of CTLA-4 and PD-1 on HBV-specific CD8 T cell responses identified ex vivo from 13 patients (r ¼ 0.5, NS). Fold change CTLA-4 and PD-1 compared to levels for global CD8 T cells was calculated to control for between-experiment variability in baseline MFI. (C) Summary of IFNcþ CD8 T cell responses to HBV peptides following single blockade of CTLA-4 or PD-1 pathways or dual blockade compared to those seen with control IgG. (D) FACS plots comparing recovery of HBV-specific CD8 T-cell responses upon single or dual CTLA-4/PD-1 blockade. reduction resulted in some increase in cytolytic responses against HLA-A2 restricted HBV epitopes, 21 but that these were of limited lifespan. 22 Our data confirmed that, even when patients were treated with combination antiviral therapy and virus-specific responses were detected (regardless of HLA-restriction), the small increases in IFN-c-producing HBV-specific CD8 were short-lived. In line with their transient reconstitution, these populations continued to express high levels of CTLA-4 and Bim. The lack of reversal of their proapoptotic phenotype in the face of effective suppression of circulating HBV DNA may reflect the inability of antiviral therapy to adequately switch off intrahepatic production of covalently closed circular (cccdna), manifested in high residual serum HBsAg levels. Patients in this study were only followed for a maximumof18monthsaftertheinitiationoftherapy;it will be important in future studies to assess whether there is more effective T-cell reprogramming in at least a subset of patients after more prolonged treatment. The lack of sustained off-treatment responses generally seen in CHB, accompanied by the ineffective T-cell reprogramming that we observed, point to the need for a more directed therapeutic approach. We therefore investigated the potential to rescue HBVspecific CD8 T cell responses in vitro, using the approach of mabs blocking the CTLA-4 receptor already used in human cancer trials. 16 The fact that this was able to increase the expansion of functional HBV-specific responses in a number of patients supports a role for CTLA-4 in T-cell exhaustion in CHB. However, in some cases in the large cohort examined, a lack of detectable T-cell reconstitution upon CTLA-4 blockade is likely to reflect a dominant role for other coinhibitory pathways. This is supported by our data showing nonredundant roles for the CTLA-4 and PD-1 pathways in the T-cell tolerance of CHB, with a similar number of patients only responding to blockade of one or other pathway and some responding synergistically to dual blockade. Complementary roles for

10 HEPATOLOGY, Vol. 53, No. 5, 2011 SCHURICH ET AL different coinhibitory pathways have been recently highlighted in the LCMV model, 23 in HCV, 9 and in HIV, where another coinhibitory molecule, Tim-3, was found to be expressed on largely nonoverlapping T-cell populations to those expressing PD It remains to be determined whether the contribution of different coinhibitors is stochastic or is predictable from the baseline expression of these receptors on HBVspecific T cells in different patients, such that the selection of blocking strategies could be individually tailored. Our findings suggest that whereas CTLA-4 may promote exhaustion of HBV-specific CD8, it may also serve as a brake on liver inflammation through its increased expression on CD8 of other specificities. Recent work has highlighted the critical role for CTLA-4-expressing antigen-specific effector T cells in regulating peripheral tolerance after secondary encounter with antigen in target tissues. 15 Restoration of effective antiviral immunity through blockade of CTLA-4 may therefore be at the expense of control of collateral tissue damage, emphasizing the need for a targeted therapeutic approach. 25 In summary, we demonstrate a contributory role for CTLA-4 in driving Bim-dependent apoptosis of the antiviral response in CHB. We show that blockade of the nonredundant coinhibitory receptors CTLA-4 and PD-1 can be complementary in the attempt to reconstitute an effective HBV-specific CD8 T cell response. Our data add to the body of evidence that multiple mechanisms can perpetuate the deletion of HBVspecific CD8 T cells and highlight a new pathway that could be targeted to restore the balance of signals to favor effective viral control. References 1. 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