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Gastroenterology. Author manuscript; available in PMC 2017 March 01. Published in final edited form as: Gastroenterology. 2016 March ; 150(3): 684–695.e5. doi:10.1053/j.gastro.2015.11.050.

HBV-specific and global T-cell dysfunction in chronic hepatitis B

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Jang-June Park1,2, David K. Wong3, Abdus S. Wahed4, William M. Lee5, Jordan J. Feld3, Norah Terrault6, Mandana Khalili6, Richard K. Sterling8, Kris V. Kowdley9, Natalie Bzowej7, Daryl T. Lau11, W. Ray Kim12, Coleman Smith13, Robert L. Carithers10, Keith W. Torrey1,2, James W. Keith1,2, Danielle L. Levine1,2, Daniel Traum1,2, Suzanne Ho1,2, Mary E. Valiga1,2, Geoffrey S. Johnson4, Edward Doo14, Anna S. F. Lok15, and Kyong-Mi Chang1,2 for the HBRN16 1Philadelphia

Corporal Michael J. Crescenz VA Medical Center, Philadelphia PA

2University

of Pennsylvania Perelman School of Medicine, Philadelphia PA

3University

of Toronto, Toronto, Ontario, Canada

4University

of Pittsburgh Graduate School of Public Health, Pittsburgh PA

5University

of Texas Southwestern, Dallas TX

6University

of California, San Francisco, San Francisco CA

7California

Pacific Medical Center, San Francisco CA

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8Virginia

Commonwealth University, Richmond VA

9Virginia

Mason Medical Center, Seattle WA

10University 11Beth

of Washington Medical Center, Seattle WA

Israel Deaconess Medical Center, Boston MA

12Mayo

Clinic, Rochester MN

13University 14National

of Minnesota, Minneapolis MN

Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda MD

15University

of Michigan, Ann Arbor MI

Abstract Author Manuscript

Background & Aims—T cells play a critical role in in viral infection. We examined whether Tcell effector and regulatory responses can define clinical stages of chronic hepatitis B (CHB). Methods—We enrolled 200 adults with CHB who participated in the NIH-supported Hepatitis B Research Network from 2011 through 2013 and 20 uninfected individuals (controls). Peripheral blood lymphocytes from these subjects were analyzed for T-cell responses (proliferation and production of interferon-γ and interleukin-10) to overlapping hepatitis B virus (HBV) peptides (preS, S, preC, core, and reverse transcriptase), influenza matrix peptides, and lipopolysaccharide. T-cell expression of regulatory markers FOXP3, programmed death-1 (PD1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA4) was examined by flow cytometry. Immune measures

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16The HBRN: Harvard Consortium: Raymond T. Chung, MD (Massachusetts General Hospital, Boston MA). Minnesota Alliance for Research in Chronic Hepatitis B Consortium Lewis R. Roberts, MB, ChB, PhD (Mayo Clinic Rochester, Rochester, MN). Saint Louis Midwest Hep B Consortium: Adrian M. Di Bisceglie, MD, (Saint Louis University School of Medicine, St Louis, MO), Mauricio Lisker-Melman, MD (Washington University, St. Louis, MO). University of Toronto Consortium: Harry L. A. Janssen, MD, PhD (Toronto Western & General Hospitals, Toronto, Ontario), Joshua Juan, MD (Toronto Western & General Hospitals, Park et al. Page 2 Toronto, Ontario), Colina Yim (Toronto Western & General Hospitals, Toronto, Ontario), Jenny Heathcote, MD (Toronto Western & General Hospitals, Toronto, Ontario). HBV CRN North Texas Consortium: Robert Perrillo, MD, (Baylor University Medical Center, Dallas, TX),compared Son Do, MDwith (University of Texas Southwestern, Dallas,physician-defined TX). Los Angeles Hepatitis B Consortium:immuneSteven-Huy B. Han, were clinical parameters, including immune-active, MD (David Geffen School of Medicine, UCLA, Los Angeles, CA), Tram T. Tran, MD (Cedars Sinai Medical Center, Los Angeles, CA). San Francisco Hepatitis B Research Group Consortium: Stewart L. Cooper, MD (California Pacific Medical Center, Research Institute & Sutter Pacific Medical Foundation, Division of Hepatology, San Francisco, CA). Michigan Hawaii Consortium: Robert J. Fontana, MD (University of Michigan, Ann Arbor, MI), Naoky Tsai, MD (The Queen’s Medical Center, Honolulu, HI). Chapel Hill, NC Consortium: Michael W. Fried, MD, (University of North Carolina at Chapel Hill, Chapel Hill, NC), Keyur Patel, M.D. (Duke University Medical Center, Durham, NC), Donna Evon, Ph.D. (University of North Carolina at Chapel Hill, Chapel Hill, NC). PNW/ Alaska Clinical Center Consortium: Margaret Shuhart, M.D. (Harborview Medical Center, Seattle WA), Chia C. Wang, MD (Harborview Medical Center, Seattle WA). Liver Diseases Branch, NIDDK: Marc G. Ghany, MD, MHsc (National Institutes of Health, Bethesda, MD) T. Jake Liang, MD (National Institutes of Health, Bethesda, MD). Data Coordinating Center: Steven Belle, PhD, MScHyg (Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA), Yona Cloonan, PhD (Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA) Central Pathology: David Kleiner, MD, PhD. (Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Disclosures: David K. Wong3 - Grant/Research Support: Gilead, BMS, Vertex, BI William M. Lee5 - Consulting: Eli Lilly, Novartis; Grant/Research Support: Gilead, Roche, Vertex, BI, Anadys, BMS, Merck Jordan J. Feld3 - Advisory Committees or Review Panels: Roche, Merck, Vertex, Gilead, Abbott, Tibotec, Theravance, Achillion Norah Terrault6 - Advisory Committees or Review Panels: Eisai, Biotest; Consulting: BMS; Grant/Research Support: Eisai, Biotest, Vertex, Gilead, AbbVie, Novartis Mandana Khalili6 Advisory Committees or Review Panels: Gilead Inc.; Grant/Research Support: Gilead Inc., BMS Inc, BMS Inc Richard K. Sterling8 - Advisory Committees or Review Panels: Merck, Vertex, Salix, Bayer, BMS, Abbott; Grant/Research Support: Merck, Roche/Genentech, Pfizer, Medtronic, Boehringer Ingelheim, Bayer, BMS, Abbott Kris V. Kowdley9 - Advisory Committees or Review Panels: Abbott, Gilead, Merck, Novartis, Vertex; Grant/Research Support: Abbott, Beckman, Boeringer Ingelheim, BMS, Gilead Sciences, Ikaria, Janssen, Merck, Mochida, Vertex Natalie Bzowej6 Daryl T. Lau11 - Advisory Committees or Review Panels: Gilead, BMS; Consulting:Roche; Grant/Research Support: Gilead, Merck W. Ray Kim12 - Advisory Committees or Review Panels: Salix; Consulting: Bristol Myers Squibb, Gilead Coleman Smith13 - Advisory Committees or Review Panels: Vertex, Gilead, Janssen; Grant/Research Support: Gilead, Abbvie,

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Janssen, Salix, BMS; Speaking and Teaching: Merck, Vetex, Gilead, Bayer/Onyx, BMS Edward Doo14 Anna S. F. Lok15 - Advisory Committees or Review Panels: Gilead, Merck, GSK; Grant/Research Support: AbbVie, BMS, Gilead, Idenix Kyong-Mi Chang1, 2 - Stock Shareholder: BMS (spouse employment); Advisory Committees or Review Panels: Genentech, Alnylam, Arbutus The following people have nothing to disclose: Jang-June Park 1, 2, Abdus S.Wahed4, Robert L. Carithers10, Danielle L. Levine1, 2, James Keith1,2, Daniel Traum1, 2, Suzanne Ho1, 2, Mary E. Valiga1, 2

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Author Contributions: Jang-June Park1, 2 assay design and performance, data analysis, manuscript writing. David K. Wong3 study design, patient recruitment, data analysis and manuscript writing. Abdus S. Wahed4 study design, data analysis and manuscript writing William M. Lee5, patient recruitment, data analysis and manuscript writing Jordan J. Feld3 patient recruitment and data analysis Norah Terrault6 patient recruitment and manuscript writing Mandana Khalili6 patient recruitment and manuscript writing Richard K. Sterling8 patient recruitment and manuscript writing Kris V. Kowdley9 patient recruitment and manuscript writing Natalie Bzowej6 patient recruitment Daryl T. Lau11 study design, patient recruitment W. Ray Kim12 patient recruitment Coleman Smith13 study design, patient recruitment Robert L. Carithers10 patient recruitment Keith W. Torrey1, 2 assay optimization, performance and data analysis James Keith1, 2 assay performance and data analysis Danielle L. Levine1, 2 assay performance and data analysis Daniel Traum1, 2 assay design and performance and data analysis Suzanne Ho1, 2 assay performance and data analysis Mary E. Valiga1, 2 study coordination and data analysis Geoffrey Johnson4 data analysis Edward Doo14 study coordination and oversight Anna S. F. Lok15 study design, data review and manuscript writing Kyong-Mi Chang1, 2 study design, study coordination, data analysis and manuscript writing. All authors reviewed and approved the final manuscript

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tolerant, or inactive CHB phenotypes, in a blinded fashion.

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Results—Compared to controls, patients with CHB had weak T-cell proliferative, interferon-γ, and interleukin-10 responses to HBV, with increased frequency of circulating FOXP3+CD127− regulatory T cells and CD4+ T-cell expression of PD1 and CTLA4. T-cell measures did not clearly distinguish between clinical CHB phenotypes, although the HBV core-specific T-cell response was weaker in HBeAg+ than HBeAg− patients (% responders: 3% vs 23%, P=.00008). Although in vitro blockade of PD1 or CTLA4 increased T-cell responses to HBV, the effect was weaker in HBeAg+ than HBeAg− patients. Furthermore, T-cell responses to influenza and lipopolysaccharide were weaker in CHB patients than controls.

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Conclusion—HBV persists with virus-specific and global T-cell dysfunction mediated by multiple regulatory mechanisms including circulating HBeAg, but without distinct T-cell–based immune signatures for clinical phenotypes. These findings suggest additional T-cell independent or regulatory mechanisms of CHB pathogenesis that warrant further investigation. Keywords HBRN; LPS; IFN; IL10

Introduction

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Hepatitis B virus (HBV) is largely non-cytopathic, with host immune response playing a key role in liver injury and virus control1, 2. As such, clinical stages of chronic hepatitis B (CHB) are generally classified as immune active, immune tolerant or clinically inactive by serum alanine aminotransferase (ALT) activity and HBV DNA levels3. This nomenclature is based on the concept that these clinical measures reflect varying levels of host immune activation in response to HBV that mediate both liver injury and virus control4. For example, patients with immune active CHB display elevated ALT activity and active hepatic necroinflammation. By contrast, immune tolerant patients ‘tolerate’ high levels of HBV viremia without ALT elevation and respond less well to interferon-based immune modulatory therapy than immune active patients5. These immunologically conceptualized but clinically defined CHB phenotypes have been a cornerstone for clinical management of patients with CHB6, 7. However, the underlying mechanisms or immune correlates for clinical CHB phenotypes are not well defined.

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HBV is believed to be a ‘stealth’ virus that is not readily sensed by the innate immune defense8. By contrast, a critical role for T-cells was shown in animal models of HBV infection and/or replication2, 9. Successful HBV clearance in patients is associated with robust and broad HBV-specific proliferative and IFNγ+ effector T-cell responses compared to weak, dysfunctional responses in CHB10. Multiple inhibitory pathways have been implicated for HBV-specific T-cell dysfunction in CHB including: 1) extrinsic regulation through regulatory T-cells, cytokines and serum factors; 2) intrinsic regulation through coinhibitory molecules such as programmed death-1 (PD-1) or cytotoxic T lymphocyteassociated antigen-4 (CTLA-4); and 3) deletion of virus-specific T-cells11–20. Suppressive CD4+CD25+FoxP3+ regulatory T-cells (Tregs) were induced in patients with CHB in direct correlation with disease progression in some21, 22 but not all studies23, 24. Furthermore,

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HBV-specific T-cell dysfunction in CHB was associated with increased T-cell expression of PD-118–20. Importantly, antibody-mediated blockade of these inhibitory receptors restored HBV-specific effector T-cell function in vitro, raising hope for potential therapeutic application18–20. In this study, we hypothesized that clinical phases of CHB represent the balance between immune effector and regulatory factors that impact HBV-specific T-cells. We looked for virus-specific effector T-cell function (proliferation, IFNγ) relative to regulatory parameters such as virus-specific IL-10 response, FoxP3+ Treg frequency and T-cell expression of PD-1 and CTLA-4 in peripheral blood of CHB participants enrolled into the National Institutes of Health (NIH)-funded Hepatitis B Research Network (HBRN) Immunology Study. We also examined whether circulating hepatitis Be antigen (HBeAg) impacted antigen-specific T cell tolerance and responses to immune inhibitory blockade in-vitro.

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Methods Patient Recruitment Between January 2011 and December 2013, 200 of 1763 participants with CHB enrolled into the NIH-funded HBRN Adult Cohort Study were recruited into the ancillary HBRN Immunology Cohort Study (‘Immunology Cohort Study’) from eight participating clinical centers in Toronto, Dallas, San Francisco, Richmond, Seattle, Minnesota, Boston and Chapel Hill (see Supplementary Methods #S1 for individual institutions).

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The Adult Cohort Study protocol has been described elsewhere25. In brief, the Adult Cohort Study enrolled hepatitis B surface antigen (HBsAg)-positive patients that were 18 years or older without evidence for hepatic decompensation, hepatocellular carcinoma, liver transplant or human immunodeficiency virus (HIV) infection, and not receiving antiviral therapy. The inclusion criteria for Immunology Cohort Study were: 1) enrollment in the Adult Cohort Study; 2) informed consent for the Immunology Cohort Study; 3) absence of active conditions that preclude large volume research blood draws; 4) absence of active autoimmune disease, medications or co-morbid illnesses that may impact immune response. Twenty HBV-uninfected healthy control subjects including 17 prior HBV vaccinees (vaccinated 0–33 years from enrollment) were recruited from two HBRN clinical centers (Toronto, Dallas) and the HBRN Immunology Center in Philadelphia, with no known liver disease, conditions that preclude large volume research blood draws, active autoimmune disease or immunosuppression. All subjects were screened for HBsAg, antibody to hepatitis B core antigen (anti-HBc), antibody to HBsAg (anti-HBs), antibody to hepatitis C virus (anti-HCV), antibody to HIV (anti-HIV) and liver enzymes. Serum HBV DNA was quantified by real-time PCR assays at each clinical center. Participation in the Immunology Cohort Study involved 50ml blood draws at weeks 12 and 24 (or 48 in case of missed blood draw) in the 1st year of enrollment into the Adult Cohort Study. Participants with ALT flares (ALT 10 times the upper limit of normal) underwent 50ml blood draws within 1–2 weeks of flare, at 4 weeks from the ALT flare and after flare resolution. Blood samples were collected in EDTA-coated tubes, shipped overnight in ambient air to the Immunology Center and processed within 24 hours.

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CHB Phenotype Groups

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Baseline CHB phenotype was assigned by investigators from each clinical center based on available history and baseline laboratory results at the time of Immunology Cohort Study enrollment, using phenotype characteristics described in Supplementary Table S1. The 200 HBRN participants included 21 (11%) immune tolerant (IT), 60 (30%) HBeAg+ immune active (IA+), 67 (34%) HBeAg− immune active (IA−), 48 (24%) inactive carriers (IC), and 4 (2%) subjects with ‘indeterminate’ phenotype (Supplementary Table S2). Cross-sectional immune comparisons were made with the first available immune assay results. All assays were performed with the investigators at the HBRN Immunology Center blinded to the knowledge of CHB phenotype. Clinical and laboratory results were available only to the statistician at the Data Coordinating Center. Viral Peptides and Controls

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Four sets of 155 genotype-specific overlapping 15-mer peptides (genotypes A–D) were synthesized by Mimotopes (Victoria, Australia). Peptide sequences were determined by aligning two or more published HBV S, Core or Polymerase sequences from each HBV genotype (see Supplementary Methods #S2 and Table S3 for published HBV sequences used and strategy for peptide pools). T-cell response to influenza virus was measured using 41 overlapping 15-mers spanning the matrix M1 protein (residues 1–252) based on A/PR/ 8/34 (H1N1) virus26. Additional controls included Lipopolysaccharide (LPS) and phytohemagglutinin (PHA) (Sigma Aldrich, St Louis MO). Antibodies Fluorescent and blocking antibodies are listed in Supplementary Method #S3

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Peripheral blood mononuclear cells (PBMC) PBMC were isolated by Ficoll-Histopaque (Sigma Chemical Co., St Louis, MO)27, 28 and used directly or cryopreserved. Lymphoproliferation (LPR) Response Assay Virus-specific T-cell proliferation was measured as described previously28–30 and in Supplementary Methods #S4 with the cutoff for a positive response defined as at least 3.0 stimulation index (SI). PD-1/CTLA-4 Blockade Assay

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Effect of PD-1 or CTLA-4 blockade was examined with and without anti-human PD-L1 (10μg/ml) or anti-human CTLA-4 (10μg/ml)13, 31 in 7-day LPR assay with PBMC stimulated with HBV peptides, control Flu peptides and PHA. IFNγ and IL-10 ELISPOT Assay IFNγ and IL-10 ELISPOT assays were performed as described28, 30, 32 and in Supplementary Methods #S5. The cutoff for a positive response for each condition was calculated as 2 standard deviations above background in media control wells for all subjects as follows: 62 IFNγ spot forming units (SFU)/106 PBMC and 122 IL-10 SFU/106 PBMC.

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Multi-parameter Flowcytometry

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Cells stained with fluorescent antibodies were acquired with FACSCanto (BD Biosciences, San Jose, CA) and analyzed with FlowJo (Tree Star Inc., San Carlos, CA).13, 31 Statistical Analysis Clinical and demographic characteristics are summarized using medians, 25th, and 75th percentiles for continuous variables and using frequencies and percentages for categorical variables. These characteristics were compared across phenotype groups by Kruskal-Wallis (continuous) or Chi-square tests; where appropriate, exact tests were used, with further details in Supplementary Methods #S6.

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Baseline patient characteristics

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Chronic hepatitis B is associated with HBV-specific T-cell suppression as well as global immune regulatory mechanisms that extend beyond HBV

Baseline characteristics of the HBRN immunology study participants (shown in Supplementary Table S2) resembled those reported in the entire HBRN cohort25 with median age of 42 years, equal male/female distribution (55%:45%), Asian predominance (83%) and high prevalence of genotypes B (47%) and C (30%), followed by genotypes A (9%) and D (6%). The CHB phenotype groups displayed expected differences in liver function parameters, HBV DNA titers (e.g. higher HBV DNA for IT patients; higher ALT and fibrosis scores FIB-4 and APRI for IA patients) and age (e.g. lower age for IT than other groups). While patients currently on antiviral therapy were excluded, a minority (14%) had prior history of antiviral therapy without a difference between CHB phenotype groups (p=0.85).

HBV-specific effector T-cell responses in PBMC were measured by lymphoproliferation (LPR) and IFNγ Elispot assays. As expected, HBV-specific T-cell responses were not readily detected in CHB patients. For example, each HBV peptide pool was immunogenic in less than 20% of CHB patients (Figure 1A), with median stimulation index (SI) and IFNγ responses well below the cutoff values for a positive response (SI 3.0 for LPR and 62 SFU/M for IFNγ response). Overall, only 34% and 21% of CHB patients showed a positive LPR or IFNγ response to at least one HBV peptide pool, respectively. There was no significant difference in HBV-specific T-cell responses between patients with and without prior antiviral therapy (%LPR responders: 44% vs 33%, p=0.37; %IFNγ responders: 20% vs 21%, p>0.99).

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Among 6 HBV peptide pools, the RT1/RT2 peptide pools were the most immunogenic for LPR response (RT1 15%, RT2 16%), followed by HBV Core (14%), PreC (8%), S (8%) and PreS (6%). For IFNγ response, RT1 and Core peptides were the most immunogenic (both at 11%), followed by RT2 (8%), S (8%), PreC (2.7%) and PreS (0.5%). Overall, HBV RT1/RT2 peptides were most frequently immunogenic in CHB patients, followed by Core/ PreC and S/PreS peptides (22.6% vs 18.4% vs 12.6%, p=0.038). T-cell response to HBV S was weaker in CHB patients than uninfected HBV vaccinees. T-cell responses to HBV S

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(and other HBV peptides) in CHB patients were also weaker than Flu-specific T-cell response in the same CHB patients, consistent with HBV-specific T-cell dysfunction in CHB. The magnitude of Flu-specific LPR response was significantly weaker in CHB patients than in HBV vaccinees (median SI: 1.9 vs 7.0, p=0.023). CHB patients also displayed weaker IFNγ (but not LPR) responses to TLR4 agonist LPS compared to uninfected vaccinees (%responders: 36% vs 71%, p=0.0077; median IFNγ SFU/M: 33 vs 139, p=0.0018), whereas LPR response to T-cell mitogen PHA was comparable (SI 453 vs 349, p=0.07). Furthermore, HBV-specific T-cell responsiveness in CHB patients correlated with their concurrent responses to Flu and LPS (Figure 1B/C). Collectively, these findings suggest CHB-associated induction of global immune regulatory mechanisms that extend beyond HBV-specific T-cells.

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Antiviral T-cell responses do not provide distinct immune signatures for clinically defined CHB phenotypes

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As for antiviral effector T-cell responses relative to CHB phenotype, LPR and IFNγ responses to individual HBV peptide pools were detected in less than 25% of CHB patients in any of the CHB phenotype groups (Figure 2A). Significant differences were detected for LPR response to HBV Core (p=0.002) and IFNγ response to HBV RT1 (p=0.05) as well as LPR response to LPS (p=0.01). Although HBV-specific LPR and IFNγ responses were least frequent in IT group, these differences did not reach statistical significance (Figure 2B). Collectively, the pattern of antiviral T-cell responses did not clearly distinguish between CHB phenotypes since they were generally weak across all CHB phenotypes. Multi-variable analysis showed that race, gender, age, ALT and HBV DNA did not have significant independent effects on HBV-specific T-cell responsiveness. Further comparison of CHB patients with and without multi-specific LPR responses (i.e. responses to 2 or more HBV regions) showed differential HBV DNA levels in initial univariate analysis (3.5 vs 5.4 log IU/ml, p=0.01). However, this difference did not remain significant upon multi-variable analysis (p=0.24). HBV-specific effector T-cell dysfunction is associated with circulating HBeAg status but not virus-specific IL-10+ Tr1 response

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We asked if HBV-specific effector T-cell dysfunction in CHB reflects the induction of virus-specific IL-10+ Tr1 response as in chronic hepatitis C32. As shown in Figure 3A, there was minimal IL-10 response to any HBV or Flu peptides in CHB patients (Figure 3A) despite robust IL-10 responses to LPS. HBV-specific Tr1 response was weak in all groups including IT patients (Figure 3B). Moreover, sum HBV-specific IL-10 response correlated positively with sum HBV-specific IFNγ (but not LPR) response (Figure 3C). Thus, both Th1 and Tr1 responses were suppressed in CHB without an IL-10+ Tr1 deviation. Since HBeAg was shown to be tolerogenic for HBV core-specific T-cells in transgenic mouse models33–35, we examined if circulating HBeAg status can influence HBV-specific T-cell responses in CHB. As shown in Table 1 and Figure 2A, LPR, IFNγ and IL-10 responses to HBV Core were significantly weaker in HBeAg+ (IT and IA+) than HBeAg−

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(IA− and IC) patients. Significant differences were also seen for IFNγ/IL-10 responses to HBV RT1 and LPR response to LPS. Thus, HBeAg+ status was associated with weaker Tcell responses to HBV Core, HBV RT-1 and LPS, without increased regulatory IL-10 response. The association between HBeAg status and LPR response to HBV Core persisted in multi-variable analyses (p=0.0409). Multiple immune regulatory pathways are induced in circulating T-cells from patients with CHB regardless of clinical phenotype

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Evaluation of additional T-cell regulatory pathways showed increased FoxP3+ as well as FoxP3+CD127lo CD4 T-cell frequency in CHB patients than uninfected controls (Table 2A). FoxP3+CD4 T-cells from CHB patients displayed high CTLA-4 and low CD127 expression levels that were consistent with FoxP3+CD4 Tregs from uninfected controls but distinct from FoxP3−CD4 T-cells (Supplementary Figure S1). PD-1 and CTLA-4 expression levels in CD4 (but not CD8) T-cells were also greater in CHB patients than uninfected controls (Table 2A). FoxP3, PD-1 and CTLA-4 expression in CD4 T-cells correlated positively with each other (Figure 4A) suggesting that they are induced together. However, these regulatory pathways were not preferentially induced in IT patients and were in fact higher in IA+ patients with marginal but statistically significant differences for %FoxP3+CD127lo/CD4 T-cells and %CTLA4+/CD4 T-cells (Table 2B). Notably, expression levels of these pathways did not correlate with ALT or HBV DNA levels (Figure 4B). Antiviral effector T-cell function is enhanced by in vitro PD-1 or CTLA-4 blockade for both CHB and uninfected controls

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The relevance of PD-1 and CTLA-4 inhibitory pathways for T-cell function was examined in 19 CHB participants, by measuring LPR responses in PBMC cultured for 1 week with HBV, Flu and control PHA in the presence of blocking antibodies (αPDL1 or αCTLA-4) or isotype antibodies.13, 31 As shown in Figure 5A, αPDL1 and αCTLA4 significantly enhanced background stimulation as measured by 3H thymidine uptake in counts per minute (cpm). However, there was further enhancement of antigen-specific stimulation index despite correction for the increased background cpm. As shown in Figure 5B and Supplementary Table S4, positive responses to HBV S, C and RT2 were unmasked in 11– 26% of patients by αPDL1 and αCTLA4 blockade (highlighted as red lines). PD-1 and CTLA-4 blockade also enhanced LPR responses to Flu in CHB patients as well as LPR responses to HBV S and Flu in uninfected controls. Furthermore, PD-1 and CTLA-4 blockade significantly enhanced LPR response to T-cell mitogen PHA in CHB but not uninfected subjects (Supplementary Figure S2).

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Since HBeAg status affected HBV-specific LPR responses, we examined the impact of HBeAg status on responses to PD-1 or CTLA-4 blockade. Significant differences in HBVspecific LPR responses became apparent between HBeAg+ and HBeAg− groups upon PD-1 or CTLA-4 blockade (Figure 5C). These differences did not extend to Flu or PHA (data not shown). Collectively, these findings are consistent with immune regulatory impact of PD-1 and CTLA-4 as well as HBeAg status in CHB.

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Discussion

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T-cells mediate both liver injury and viral clearance in animal models of HBV infection1, 2, 9. While CD4 T-cells play a key regulatory role, CD8 T-cells directly eliminate virus-infected cells through cytolytic and noncytolytic mechanisms36, 37. In CHB, HBVspecific effector T-cells are functionally exhausted by continued antigen exposure and multiple regulatory pathways.12–20,16, 18–20, 38–40 Unproductive encounters between exhausted virus-specific T-cells and infected hepatocytes can recruit non-specific inflammatory cells that induce cellular damage. Yet, these ‘exhausted’ T-cells exert at least partial virus control, as shown by increased HBV DNA levels upon immunosuppression41–43. HBV-specific T-cell dysfunction can be reversed in-vitro by blocking one or more inhibitory receptors including PD-1 and CTLA-416,18–20. Here, we examined several T-cell effector and regulatory parameters in CHB patients to define their relevance in HBV persistence and to identify potential immune signatures for clinically defined CHB phenotypes in a unique North American cohort of CHB participants enrolled into the NIH-sponsored HBRN.

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HBV persistence was associated with virus-specific effector T-cell suppression and increased circulating FoxP3+CD127lo CD4 T-cells as a marker of Tregs in our study, as previously reported in chronic hepatitis B and C22, 23, 30, 44 More interestingly, however, our findings also suggest that immune dysregulation in CHB extended beyond HBV-specific Tcells. First, PD-1 and CTLA-4 expression was higher in total CD4 T-cells from CHB patients than those from uninfected controls in our study. These findings agree with increased PD-1 expression reported in total CD4 and CD8 T-cells from young CHB patients,45 whereas most studies reported focused PD-1 and/or CTLA-4 induction on exhausted virus-specific T-cells.13, 18, 31 Second, consistent with global immune regulatory induction in CHB, T-cell responses to influenza and TLR4 agonist LPS were also weaker in CHB than uninfected subjects in direct correlation with sum HBV-specific T cell responses. This difference was not due to advanced liver cirrhosis, since patients with decompensated cirrhosis were excluded in our study. It was not due to increased HBV-specific IL10+ regulatory Tr1 response in CHB, unlike chronic hepatitis C in which HCV-specific IL10+ Tr1 response is increased.32

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Although CHB patients do not manifest clinically evident immune deficit, our study of a large and unique North American CHB cohort are consistent with generalized immune dysregulation in CHB that may impact host responses to other antigens, pathogens and vaccines.17, 46 Global CD8 T-cell dysfunction with downregulated T-cell receptor signaling molecule CD3z and arginine depletion has been reported in CHB17 whereas transient suppression of T-cell responses to EBV, CMV and Flu was detected during acute hepatitis B with increased arginase and IL-10 levels46. PD-1 and CTLA-4 may play a role in this process, given enhanced LPR responses to Flu and PHA as well as HBV upon PD-1 or CTLA-4 blockade in our study. Suppressed LPS response in our cohort further suggests that CHB may even impact innate TLR4-associated responses. These findings warrant further studies examining the impact of CHB on immune responses to other viral and non-viral pathogens.

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T-cell-based effector and regulatory parameters did not correlate significantly with clinical parameters or provide distinct immune signatures for CHB phenotype groups. For example, HBV-specific effector T-cell response in PBMC was no greater in immune active patients. Conversely, the induction of immune regulatory pathways was no greater in immune tolerant patients. In fact, immune regulatory markers were more induced in immune active patients (e.g. %FoxP3+CD127or %CTLA-4 in CD4 T-cells) suggesting a compensatory induction due to inflammation. Nevertheless, inactive carriers with minimal liver disease and viremia also displayed HBV-specific immune dysregulation. Our concept of T-cell mediated control of virus and disease is challenged by these findings of weak HBV-specific T-cell responses in CHB regardless of viremia, ALT activity or T-cell regulatory markers.

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Our findings are not related to CHB phenotype classifications, since patient sub-grouping by concurrent HBV DNA and ALT activity yielded similar results (data not shown). We acknowledge our limitation in studying peripheral blood rather than the liver compartment, due to impracticality of performing an invasive liver biopsy for immune analyses. However, liver compartment can be subject to oscillations in intrahepatic T-cell function and phenotype following antigenic stimulation in-vivo.47 Furthermore, the induction of multiple, inter-related immune regulatory pathways in circulating T-cells suggests that the peripheral compartment is impacted in CHB.

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We propose that these immune regulatory pathways are induced to dampen ongoing immune activation and inflammation in-vivo. This may mask the underlying antiviral effector T-cell responses in a ‘zero-sum game’ that prevents our ability to detect meaningful differences between CHB phenotype groups. Indeed, effector T-cell responses were unmasked upon PD-1 or CTLA-4 blockade in-vitro. The balance between antiviral effector and regulatory responses may also fluctuate in-vivo 47 with potential antiviral or pathogenic consequences. Although beyond the scope of the current study, systematic comparison of antiviral effector T-cell responses unmasked in-vitro by inhibitory receptor blockade might help distinguish between CHB phenotype groups and inform potential responses to immunotherapeutic application with immune inhibitory blockade in-vivo.

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To our knowledge, this study is the first to show that circulating HBeAg status significantly impacted T-cell responses to HBV in HBV-infected patients, beyond transgenic mouse models33–35. Specifically, LPR, IFNγ and IL-10 responses to HBV Core were all markedly suppressed in HBeAg+ patients compared to HBeAg− patients. One potential interpretation for these results is a causal role for HBV Core-specific T-cell response in HBeAg loss. However, since HBeAg and HBcAg share the same 181 amino acid sequence within the Core open reading frame (also spanned by our Core peptide pool), it is more likely that HBV Core-specific T-cells are tolerized by continued exposure to circulating HBeAg, as shown in HBV transgenic mice33–35. Interestingly, restoration of T-cell function in response to PD-1 or CTLA-4 blockade was greater in HBeAg− patients (for S and RT2 as well as Core), suggesting a deeper level of tolerance or exhaustion in HBeAg+ patients. The tolerogenic capacity of circulating HBeAg may extend to other HBV antigens. In this regard, poor T-cell response to HBV S in CHB patients (only 8% LPR responders) could reflect abundant circulating HBsAg. Although speculative, it is interesting to consider a T-

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cell tolerance mechanism involving monocyte-mediated cross-presentation in-vivo.48 The tolerizing effect of circulating antigen is relevant in our quest to cure CHB, particularly if decline in HBsAg titers associated with treatment response49–52 leads to more durable immune-mediated virus control or cure. Future studies examining quantitative HBeAg and HBsAg levels relative to host immune responses would be informative.

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In conclusion, direct measurement of HBV-specific and global T-cell function and phenotype did not provide distinct immune signatures for clinical CHB phenotypes. However, multiple immune regulatory pathways were induced in CHB with reversible suppression of both HBV-specific and global effector T-cell function. Circulating HBeAg status also contributed to antigen-specific T-cell tolerance and functional responses to PD-1 or CTLA-4 blockade in-vitro. While these findings challenge the relevance of T-cells in virus control and pathogenesis in chronic hepatitis B, future studies are needed that explore effector T-cell responses that are masked by immune inhibitory pathways as well as T-cell independent mechanisms.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

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This study was supported by NIH Grants UO-1DK082866 and R01-AI-47519; the Philadelphia VA Medical Research; NIH/NIDDK Center of Molecular Studies in Digestive and Liver Diseases P30DK50306 and its Molecular Biology and Cell Culture Core Facilities; the NIH Public Health Service Research Grant M01-RR00040. This material is based upon work supported in part by the Office of Research and Development, Department of Veterans Affairs and with the resources and the use of facilities at the Philadelphia VA Medical Center. The contents of this work do not represent the views of the Department of Veterans Affairs or the United States Government. Funding: The HBRN was funded by a U01 grant from the National Institute of Diabetes and Digestive and Kidney Diseases to the following investigators Lewis R. Roberts, MB, ChB, PhD (DK 082843), Anna Suk-Fong Lok, MD (DK082863), Steven H. Belle, PhD, MScHyg (DK082864), Kyong-Mi Chang, MD (DK082866), Michael W. Fried, MD (DK082867), Adrian M. Di Bisceglie, MD (DK082871), William M. Lee, MD (U01 DK082872), Harry L. A. Janssen, MD, PhD (DK082874), Daryl T-Y Lau, MD, MPH (DK082919), Richard K. Sterling, MD, MSc (DK082923), Steven-Huy B. Han, MD (DK082927), Robert C. Carithers, MD (DK082943), Norah A. Terrault, MD, MPH (U01 DK082944), an interagency agreement with NIDDK: Lilia M. Ganova-Raeva, PhD (ADK-3002-001) and support from the intramural program, NIDDK, NIH: Marc G. Ghany, MD. Additional funding to support this study was provided to Kyong-Mi Chang, MD, the Immunology Center, (NIH/NIDDK Center of Molecular Studies in Digestive and Liver Diseases P30DK50306, NIH Public Health Service Research Grant M01RR00040, VA Merit Review BX000649), Richard K. Sterling, MD, MSc (UL1TR000058, NCATS (National Center for Advancing Translational Sciences, NIH), Norah A. Terrault, MD, MPH (CTSA Grant Number UL1TR000004), Michael W. Fried, MD (CTSA Grant Number UL1TR001111), and Anna Suk-Fong Lok (CTSA Grant Number UL1RR024986.) Additional support was provided by Gilead Sciences, Inc. and Roche Molecular Systems via a CRADA through the NIDDK.

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In addition to the authors and the research participants, the HBRN would like to acknowledge the contributions of the following: Harvard Consortium: Nezam Afdhal, MD, Asad Javaid, MBBS, Jianghe Niu, Johanna Han, Imad Nasser, MD (Beth Israel Deaconess Medical Center, Boston, MA). Minnesota Alliance for Research in Chronic Hepatitis B Alisha C. Stahler, Linda Stadheim, RN (Mayo Clinic Rochester, Rochester, MN), Mohamed Hassan, MD (University of Minnesota, Minneapolis, MN). Saint Louis Midwest Hep B Consortium: Debra L. King, RN, Rosemary A. Nagy, MBA, RD, LD, Jacki Cerkoski, RN MSN (Saint Louis University School of Medicine, St Louis, MO). University of Toronto Consortium: Victor Lo, MASc (Toronto Western & General Hospitals, Toronto, Ontario), Danie La, RN (Toronto Western & General Hospitals, Toronto, Ontario), Lucie Liu (Toronto Western & General Hospitals, Toronto, Ontario). HBV CRN North Texas Consortium: Stacey Minshall, RN, BSN (Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas), Sheila Bass (University of Texas Southwestern, Dallas, TX). Los Angeles Hepatitis B Consortium: Samuel

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French, MD, Velma Peacock, RN (David Geffen School of Med, UCLA, Los Angeles, CA). San Francisco Hepatitis B Research Group Consortium: Ashley Ungermann, MS, Claudia Ayala, MS, Emma Olson, BS, Ivy Lau, BS (University of California-San Francisco), Veronika Podolskaya, BS, NCPT, Nata DeVole, RN (California Pacific Medical Center, Research Institute). Michigan Hawaii Consortium: Barbara McKenna, MD, Kelly Oberhelman, PAC, Sravanthi Kaza, Bpharm, Cassandra Rodd, BS (University of Michigan, Ann Arbor, MI), Leslie Huddleston, NP, Peter Poerzgen, PhD (The Queen’s Medical Center, Honolulu, HI). Chapel Hill, NC Consortium: Jama M. Darling, M.D., A. Sidney Barritt, M.D., Tiffany Marsh, BA, Vikki Metheny, ANP, Danielle Cardona, PAC (University of North Carolina at Chapel Hill, Chapel Hill, NC). Virginia Commonwealth University Medical Center Velimir A. Luketic, MD, Paula G Smith, RN, BSN, Charlotte Hofmann, RN (Virginia Commonwealth University Health System, Richmond, VA). PNW/Alaska Clinical Center Consortium: Terri Mathisen, RN, BSN, Susan Strom, MPH (University of Washington Medical Center, Seattle WA) Jody Mooney, Lupita CardonaGonzalez (Virginia Mason Medical Center, Seattle WA). Liver Diseases Branch, NIDDK, NIH: Jay H. Hoofnagle, MD, Averell H. Sherker, MD, Rebecca J. Torrance, RN, MS, Sherry R, Hall, MS, Nancy Fryzek, RN, BSN, Elenita Rivera, BSN, Nevitt Morris, Vanessa Haynes-Williams. Immunology Center: Philadelphia VA Medical Center Medical Research, Keith Torrey, BS, Michael Betts, PhD (University of Pennsylvania, Philadelphia, PA), Luis J. Montaner, DVM, DPhil (Wistar Institute, Philadelphia, PA). Data Coordinating Center: Michelle Danielson, PhD, Tamara Haller, Geoffrey Johnson, MS, Stephanie Kelley, MS, Sharon Lawlor, MBA, Joan M. MacGregor, MS, Andrew Pelesko, BS, Donna Stoliker, Ella Zadorozny, MS (Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA).

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Abbreviations

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CHB

Chronic hepatitis B

HBV

hepatitis B virus

HBRN

Hepatitis B Research Network

IFN

interferon

PD-1

programmed death-1

PDL1

programmed death ligand-1

CTLA-4

cytotoxic T lymphocyte-associated antigen-4

ALT

alanine aminotransferase

anti-HBc

antibody to hepatitis B core antigen

anti-HBs

antibody to HBsAg

HBsAg

hepatitis B surface antigen

HBeAg

hepatitis B e antigen

anti-HIV

antibody to human immunodeficiency virus

anti-HCV

antibody to hepatitis C virus

Tregs

regulatory T-cells

IL

interleukin

IT

immune tolerant

IA+

HBeAg+ immune active

IA−

HBeAg− immune active

IC

inactive carriers

RT

reverse transcriptase

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LPS

lipopolysaccharide

PHA

phytohemagglutinin

PBMC

peripheral blood mononuclear cells

LPR

lymphoproliferation

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Figure 1. Effector T-cell responses to HBV and control conditions in subjects with chronic hepatitis B and uninfected HBV S vaccine recipients

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(A) Comparison of lymphoproliferative and IFNγ responses in CHB patients (gray bars) and uninfected vaccinees (white bars). The antiviral effector T-cell responses from 200 chronic hepatitis B (CHB) participants and 17 vaccinees were examined by stimulating PBMC with HBV and flu matrix peptide pools, lipopolysaccharide (LPS) and phytohemagglutinin (PHA) in lymphoproliferation (LPR) assay with 3H-thymidine uptake and in IFNγ Elispot assay. LPR response is shown as median stimulation index (SI) and % positive LPR responder based on SI cutoff of 3.0. IFNγ response in Elispot assays is shown as median IFNγ spot forming units (SFU) per million PBMC (SFU/M) and % positive IFNγ responders with a cutoff of 62 SFU/M PBMC (2 standard deviations above background in media control wells for all subjects). (B) Sum HBV-specific LPR response (combined SI for all 6 HBV peptide pools) is correlated to LPR responses to Flu and LPS. (C) Sum HBV-specific IFNγ response (combined IFNγ responses for all 6 HBV peptide pools) is compared with IFNγ responses to Flu and LPS. Statistical significance was determined by Wilcoxon’s tests (A, top panel), Fisher’s Exact or Chi-Square (A, bottom panel), and Spearman rank correlation test for correlation coefficient and p-values (B and C). P-values 0.99

0.00018

0.403

0.0535

0.0298

*p

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Table 1 Park et al. Page 21

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4.4(3.4, 5.7)

IC (n=46)

KWT (Kruskal Wallis Test), p