Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection

Cellular Immunology xxx (2016) xxx–xxx

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Research paper

Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection Huaizhou Wang a,b, Beiying Wu a, Lei Li c, Liang Hu a, Jiafei Lin a, Cen Jiang a, Gang Cai a,⇑, Qian Shen b a

Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University Medical School, Shanghai, PR China Department of Experimental Diagnosis, Changhai Hospital, The Second Military Medical University, Shanghai, PR China c Department of Clinical Pathology, Ruijin Hospital, Shanghai Jiaotong University Medical School, Shanghai, PR China b

a r t i c l e

i n f o

Article history: Received 28 June 2016 Revised 3 October 2016 Accepted 6 October 2016 Available online xxxx Keywords: B and T lymphocyte attenuator Chronic virus infection Hepatitis B virus Regulatory CD8+ T cells

a b s t r a c t Similar to programmed death-1 (PD-1), B and T lymphocyte attenuator (BTLA) is a co-inhibitory molecule of the CD28 family. PD-1 is involved in T cell exhaustion during chronic viral infection. However, the role of BTLA in virus-specific T cells is poorly defined. Here we investigated the expression and function of BTLA in T cells from patients with chronic hepatitis B virus (HBV) infection. The phenotype of peripheral and intrahepatic HBV-specific T cells from 43 patients with chronic HBV infection was assessed by flow cytometry. Functional evaluation was analyzed by T cell expansion and cytokine secretion after different treatments. In chronic HBV patients, a subset of inefficient interferon-c producing antigen-specific CD8+ T cells recruited to the liver expressed high BTLA levels. The BTLA+ HBV-specific CD8+ T cell suppressive function was antigen-specific, at least in the induction phase, because they were only activated by a pool of HBV peptides but not with a pool of unrelated peptides. Suppression of T cell responses was restored by a BTLA signaling blockade and neutralizing IL-10, indicating that BTLA signaling-mediated IL-10 secretion plays a key role in suppression. This study provides important evidence that there is a subset of liver infiltrated virus-specific CD8+BTLA+ regulatory T cells in patients with chronic HBV infection. This subset of cells plays a pivotal role in controlling hepatic effector CD8+ T cell responses through BTLA signaling mediated regulatory factor IL-10 production. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Hepatitis B virus (HBV) can persist in adults and, particularly, child hosts to establish a lifelong liver disease [1]. The establishment of persistent HBV infection is related to the severe dysfunction of HBV-specific CD8+ T cells. The dysfunctional HBV-specific CD8+ T cells prevent resolution of the infection, and favors the onset of chronic liver immunopathology [1,2]. However, it is still unclear why the effector’s functions are impacted in the expanded HBV-specific CD8+ T populations. Recent studies indicate that Abbreviations: ALT, alanine aminotransferase; BTLA, B and T lymphocyte attenuator; CHB, chronic hepatitis B; CM, central memory; CMV, cytomegalovirus; CTL, cytotoxic T lymphocyte; DC, dendritic cell; EM, effector memory; HIV, human immunodeficiency virus; HBV, hepatitis B virus; HBcAg, hepatitis B virus core antigen; HBsAg, hepatitis B virus surface antigen; HCV, hepatitis C virus; IFN-, interferon-; IL-2, interleukin-2; LIL, liver infiltrated lymphocyte; PBMC, peripheral blood monouclear cell; PD-1, programmed death-1; TEMRA, CD45RA positive effector memory. ⇑ Corresponding author at: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University Medical School, Shanghai 200025, PR China. E-mail address: [email protected] (G. Cai).

exhaustion of antigen-specific T cells, caused by propagation with an excess of co-inhibitory signals (such as PD-1, CTLA-4 and Tim-3) and a lack of CD4+ T helper cells, contributes to HBV-specific CTL dysfunction [3–7]. Furthermore, different types of CD4+ and CD8+ regulatory T cells, which are potentially activated in an antigenspecific manner, suppress excess effector responses in an antigen-specific or -nonspecific manner [8–12]. The regulatory/effector T cell imbalance may contribute to a chronic low-grade inflammation which is critical to the survival of both the microbial agent and host. Although overexpressing PD-1 has been demonstrated to link impairment of CD8+ T cell functions in many types of chronic virus infection [4,5,13,14], the role of B and T lymphocytes attenuator (BTLA), which belongs to the same co-inhibitory family as PD-1, has not been well investigated in chronic viral infection. In this study, we found that patients with chronic HBV infection show a considerable enrichment of HBV-specific CD8+ T cells in the liver (in contrast to the periphery) that express the co-inhibitory receptor, BTLA. Importantly, we demonstrated that the BTLA+ HBVspecific CD8+ T cells take regulatory roles in the liver, partly

http://dx.doi.org/10.1016/j.cellimm.2016.10.002 0008-8749/Ó 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: H. Wang et al., Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.10.002

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H. Wang et al. / Cellular Immunology xxx (2016) xxx–xxx

depend on IL-10 secretion, and may have a major role in establishing chronic low-grade liver inflammation.

2. Patients and methods 2.1. Study population Patients with HBV infection were studied according to the ethical guidelines of the 1975 Declaration of Helsinki and with a priori approval by the ethical committee of our hospitals. Patients with chronic HBV infection were diagnosed as previously reported5. Forty-three patients with chronic hepatitis B (Table 1) and 20 HLA-matched (HLA-A24+) healthy donors (age range, 22–56 years; M/F, 13/7) were enrolled in this study. All enrolled patients were negative for anti-hepatitis C virus, d virus, human immunodeficiency virus type 1 (HIV-1), HIV-2 antibodies, and other markers of viral or autoimmune hepatitis. No patients were being treated with Interferon (IFN)-a at the time of this study. Serum HBVDNA and HBV genotypes were determined by real-time polymerase chain reaction (RT-PCR) (TaqMan; Qiagen, Indianapolis, IN, USA) and sequencing of the RT and S genes was performed using a Trugene HBV genotyping kit (Siemens Healthcare Diagnostics Inc., Deerfield, IL, USA). All patients underwent percutaneous

needle liver biopsy. The tissue samples were processed for pathological diagnosis and immunological analysis. Histology was graded using the histological activity index (HAI): periportal necrosis, intralobular necrosis, or portal inflammation (values range from 0 to 10, 0 to 4, or 0–4, respectively; thus their sum provides the ‘‘grading” or the ‘‘inflammatory index”), as well as fibrosis (ranging from 0 to 4; this leads to the ‘‘staging” or the ‘‘fibrosis index”) and total score (grading + staging) [15] (Table 1). 2.2. Cell preparation Peripheral blood mononuclear cells (PBMCs) and liver infiltrating lymphocytes (LILs) were isolated as previously described [16]. CD8+ T cells were purified from PBMCs or LILs by immunomagnetic separation with mouse anti-CD8-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). BTLA+ and BTLA cells were purified from CD8+ LILs or PBMCs by staining with biotin labeled anti-BTLA and magnetic microbeads conjugated with antibiotin (Miltenyi Biotec). FACS analysis demonstrated more than 95% BTLA+ CTLs in the positively purified population, and less than 10% BTLA+ cells in the BTLA-depleted population. Immature dendritic cells (DCs) were derived from peripheral monocytes and purified by positive selection with CD14 monoclonal antibodies (mAb) coupled to magnetic beads (Miltenyi Biotec). Then, CD14+

Table 1 Clinical parameters of patients with chronic HBV infection. Pts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Age

48 56 35 24 48 40 29 28 22 48 34 65 58 28 28 34 42 39 61 44 19 64 50 37 38 59 66 23 61 45 54 37 58 44 55 50 38 35 48 37 53 35 34

Sex

M M F F M M F F M M M F M M M M M M M M M M M M M F F M F M M M M M F M M M M M M F M

ALT (U/mL)

42 60 55 43 115 82 62 305 324 82 74 44 164 50 22 89 43 66 234 110 111 99 130 134 86 35 59 145 29 33 123 126 64 105 80 87 26 66 215 107 25 97 87

HBV-DNA (U/mL)

9.55E+05 3.02E+05 5.50E+04 5.25E+03 5.70E+03 4.61E+06 5.01E+06 1.82E+04 1.58E+03 1.29E+04 5.01E+03 2.95E+05 5.89E+04 1.66E+06 4.47E+04 1.58E+04 1.26E+04 3.39E+03 6.76E+04 1.07E+06 7.94E+04 5.00E+07 1.74E+06 1.62E+04 1.51E+04 7.41E+03 2.88E+07 1.32E+06 5.75E+04 3.89E+05 7.24E+04 3.55E+04 5.00E+07 1.45E+06 1.51E+06 1.82E+05 5.00E+07 1.70E+03 4.68E+03 8.51E+06 1.26E+07 9.55E+05 6.46E+03

Genotype

B B C C B C B B B B C C B C C C B B B B B C C B C C B C B C B B B C B C C B B B C C B

HAI Total score

Grading

Staging

3 3 4 2 6 7 3 2 2 5 7 4 8 3 3 10 9 3 4 2 2 4 8 3 3 2 5 7 4 2 3 2 2 2 5 3 3 6 4 3 2 5 8

3 3 3 2 5 5 3 2 2 4 5 3 7 3 3 8 7 3 3 2 2 3 6 3 3 2 4 5 3 2 2 2 2 2 4 3 3 5 3 3 2 4 7

0 0 1 0 1 2 0 0 0 1 2 1 1 0 0 2 2 0 1 0 0 1 2 0 0 0 1 2 1 0 1 0 0 0 1 0 0 1 1 0 0 1 1

Please cite this article in press as: H. Wang et al., Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.10.002

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cells were induced to differentiate into immature DCs by a 24-h stimulation with lipopolysaccharide (Sigma-Aldrich) as previously described [17]. 2.3. Cytofluorimetric assays PBMCs and LILs were stained with a combination of tetramers and different mAbs. Tetramers included PE-labeled HLA-A2402 tetramers in complex with HBV core (EYLVSFGVW) and pol (KYTSFPWLL) proteins (Proimmune Limited, Oxford, UK). To increase the percentage of HBV-specific CD8+ T cells and save samples, tetramers were pooled according to their HLA genotype. Tetramers and surface antigens were stained according to the manufacturer’s instructions. Cells were acquired with a FACSCantoII flow cytometer (Becton Dickinson, San Jose, CA, USA), and analyzed using Diva software (Becton Dickinson). Negative controls were obtained by staining cells with an unrelated isotypematched mAb. 2.4. Functional assays In functional experiments, PBMCs or LILs previously stained with tetramer and different mAbs were unstimulated or

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stimulated for 6 h at 37 °C with peptide (10 lg/mL) plus anti-CD28 mAb (2 lg/mL) (BD Pharmingen); PMA (50 ng/mL) plus ionomycin (1 lg/mL) (Sigma-Aldrich, Shanghai, China); or peptide plus autologous irradiated PBMCs as APCs. At the beginning of the second hour, 10 lg/mL brefeldin-A (Sigma-Aldrich) was added. Cells were washed, fixed, and permeabilized using Cytofix/Cytoperm solution (BD Pharmingen), and intracellular cytokines stained with FITC-labeled anti-IFN-c and PerCP-Cy5.5 labeled anti-IL-10 for 30 min at 4 °C. Similar assays were performed using short-term BTLA+CD8+ or BTLA CD8+ lines derived from PBMC or LILs (after 7 days of stimulation with peptide plus anti-CD28 mAb or 3 days of preculture with IL-2). 2.5. Suppressor assays In the antigen-nonspecific assays, PBMCs (responder cells) were cultured in 96-well round-plates for 6 days, in the presence or absence of 4–5  104 cells/well of highly purified CD8+BTLA+ or CD8+BTLA LILs as suppressor cells in the presence of both soluble anti-CD3 and anti-CD28 mAbs (BD Pharmingen), at a responder:suppressor ratio of 2:1. Because of the paucity of cells, we pooled LILs from two or three liver biopsies derived from

Fig. 1. Expression of BTLA on peripheral HBV-specific CTLs. PBMCs from a representative patient (Pt. 6) were stained first with indicated PE-labeled tetramers, and then with mAbs to CD8, CD27, CD45RA and BTLA. (A) Dot plot analyses showing CD8+tetramer+ cell percentage. (B) Dot plots, corresponding to those in A, are gated on tetramer+CD8+ (HBV-specific CTLs) or tetramer CD8+ (CTLs), and show their differentiated stages. (C) Histogram plots, corresponding to those in B, show BTLA expression on different differentiated stages (CM and EM) and all gated cells (Total). Results are expressed as percentage of cells, indicated in each gate.

Please cite this article in press as: H. Wang et al., Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.10.002

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H. Wang et al. / Cellular Immunology xxx (2016) xxx–xxx

different patients. In some wells, BTLA-Ig (R&D) neutralizing anti-IL-10 (JES3-9D7, IgG1), or IgG1 isotype controls were added at the beginning of the assay. To evaluate the antigen specificity of suppressor cells, PBMCs were co-cultured with autologous BTLA+ or BTLA LILs (derived from single biopsies) in the presence or absence of both recombinant core protein (Sangon, Shanghai, China) as well as HLA-A2-related peptides. In some wells, BTLA-Ig (R&D) neutralizing anti-IL-10 (JES3-9D7, IgG1), or IgG1 isotype controls were added at the beginning of the assay. Eighteen hours before cultures were harvested, 1 lCi [3H]thymidine (Amersham, Buckinghamshire, UK) was added and the cellular radioactivity was determined by the level of [3H]thymidine incorporation.

2.6. Statistical analysis For quantitative comparisons, Student’s t-test (2-sample 2tailed comparison) or paired t-test was performed with Prism 5.0 (GraphPad Software, Inc.) along with an F test to compare variance. For correlation analysis between BTLA+ cells and clinical indexes, Pearson product-moment correlation coefficient was calculated. Values of P < 0.05 were considered statistically significant.

3. Results 3.1. Downregulation of BTLA expression on peripheral HBV-specific CTLs In all healthy donors, the proportion of CD8+ class I HLApeptide tetrameric complex+ (tetramer+) cells in peripheral CD8+ T cells was lower than 0.01% (data not shown). Thus 0.01% was considered as the sensitivity threshold in this study. The portion of tetramer+ cells was higher than 0.01% in 9 of 43 patient samples. In these patient samples, we found that BTLA expression level was greatly reduced in tetramer+ cells, compared to total CD8+ T cells. (Fig. 1). Since the expression level of BTLA is related to the lymphocyte stage of differentiation [18], we analyzed CD45RA and CD27 expression in the tetramer+ cell population. We found that these cells expressed a central memory (CM) phenotype, as CD45RA CD27+ (Fig. 1). Moreover, BTLA level was significantly increased in the CM subsets than on effector memory (EM) (CD45RA CD27 ) subsets (Fig. 1). Because of the very low percentage of peripheral tetramer+ cells, we were only able to test the T cell effector function in four patient samples which a relative higher percentage of HBVspecific CTLs in the peripheral blood (Fig. 2A). Interestingly, the

Fig. 2. Peripheral virus-specific BTLA+ CTLs efficiently differentiate upon antigen-driven proliferation in vitro. (A) Dot plot analyses showing CD8+tetramer+ cell percentage in PBMCs from four patients (Pt. 6, 15, 30 and 34 in Table 2) selected for their elevated frequencies of peripheral virus-specific CD8+ T cells, as detected by the indicated tetramers. (B) Representative functional analyses of CD8+tetramer+ cells, corresponding to A, show IFN-c production of virus-specific CTLs after 6-h stimulation with viral peptides or PMA/IO. The cells are gated CD8+tetramer+ cells. (C) PBMCs from three patients (Pt. 6, 30 and 34) were stimulated with viral peptides plus anti-CD28 for 7 days. Cells were then stained with corresponding tetramer and mAbs to CD8, restimulated with autologous APCs and peptide for 6 h, and stained for intracellular IFN-c. Dot plots are gated on tetramer+CD8+ cells. (D) Both BTLA+ and BTLA cells purified from PBMCs from two patients (Pt. 6 and 30) were cultured with the relevant peptides and autologous DCs for 7 days. Cell staining was performed as in C. Dot plots are gated on tetramer+CD8+ cells. Results are expressed as percentage of cells.

Please cite this article in press as: H. Wang et al., Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.10.002

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ex vivo antigen-specific effector function was greatly impaired in these CTLs, determined by the IFN-c production in response to viral peptides stimulation (Fig. 2B). However, PMA and ionomycin stimulation-induced IFN-c was not affected. (Fig. 2B). Additionally, after sustained antigen stimulation, they proliferated and produced large amounts of IFN-c (Fig. 2C). Cytokines were mainly produced by the BTLA+tetramer+ cells (Fig. 2D) suggesting that most BTLA+ cells were of a CM subtype, which have the potential to differentiate into potent effector cells after in vitro stimulation. 3.2. Hepatic expansion of an inefficient effector BTLA+ HBV-specific CTL population To study the phenotype of HBV-specific CTLs in infected livers, we compared LILs, which are isolated from the liver tissues, to the peripheral CD8+ T cells of patients with chronically infected with HBV. Because of the low number of LILs obtained from each biopsy, HBV-specific CTLs were stained with a pool of HLA-A24/HBV tetramers with anti-CD8 monoclonal antibody. The frequency of virus-specific CD8+ LILs, as detected by the tetramer pool, was significantly higher than the corresponding sum of the peripheral cells, which were detected by single tetramers (Table 2 and Figs. 3 and 4A). Compared to CTLs from

peripheral blood, most tetramer+ LILs had lost CD27 expression, and some had increased CD45RA expression. Thus, tetramer+ LILs contained mainly two subsets: EM (CD45RA CD27 ) and effector memory CD45RA+ (TEMRA, CD45RA+CD27 ) (Fig. 3). Despite the acquisition of the EM and TEMRA phenotype, only a minority of LILs acquired prompt IFN-c production capacity after a few days in the presence of a pool of corresponding viral peptides. However, significant amounts of tetramer+ LILs from patients with chronic HBV infection maintained a high expression of BTLA (Figs. 3 and 4A), and its expression was negatively correlated to IFN-c production of tetramer+ LILs (Fig. 4 B), but positively correlated to HBV-DNA load (Fig. 4F). Additionally, tetramer+BTLA+CD8+ LIL frequencies were negatively correlated to the total HAI score (Fig. 4C) and serum ALT level of chronically HBV-infected patients (Fig. 4E). These data suggested a regulatory role for BTLA on the specific CTLs-mediated antiviral immune response in the liver. 3.3. Hepatic expansion of an IL-10-producing HBV-specific BTLA+ CTL population Previous studies have demonstrated that IL-10 secreted by LILs protect from antivirus immune response-mediated hepatic tissue damage by suppressing the effects of IFN-c [10]. Moreover, we

Table 2 Peripheral and intrahepatic CD8+tetramer+ cells and their functional properties in patients with chronic HBV infection. Pts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Blood

Liver

% core

% core+BTLA+

% pol

% pol+BTLA+

% Mix tetr

%IFN-g cells

% IL-10+ cells

% BTLA+ cells

0.02 N.D. N.D. N.D. 0.02 0.5 N.D. N.D. N.D. N.D. 0.02 N.D. N.D. N.D. 0.12 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 0.04 N.D. N.D. N.D. N.D. 0.15 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

32 N.D. N.D. N.D. 45 31 N.D. N.D. N.D. N.D. 55 N.D. N.D. N.D. 48 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 29 N.D. N.D. N.D. N.D. 41 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

0.04 N.D. N.D. N.D. N.D. 0.05 N.D. N.D. N.D. N.D. 0.03 N.D. 0.13 N.D. 0.04 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 0.14 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

54 N.D. N.D. N.D. N.D. 28 N.D. N.D. N.D. N.D. 35 N.D. 47 N.D. 30 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 23 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

2.5 3.1 1.5 2.3 1.4 0.7 2.2 0.5 1.1 1.7 2 0.2 0.5 8 3 8 4.8 4.7 1 3.4 0.7 1.7 0.1 5.5 2.2 0.1 2.5 4 3.5 1.5 1.2 2.3 0.1 1.8 2 1.1 1.5 2.6 1.7 2 1.3 0.4 0.3

1 0.85 0.52 0.75 1.32 1.56 0.85 0.43 0.37 1.36 2.24 0.88 1.77 0.7 0.52 1.9 2.3 0.7 1 0.5 0.32 1.2 1.4 0.8 0.5 0.4 0.7 1.2 0.5 0.6 0.3 0.7 0.5 0.2 0.9 0.8 0.3 1.5 2.1 0.8 0.5 1.7 2.2

N.D. 13.3 6.7 5.2 0 4.6 9.7 N.D. 0 N.D. N.D. 5.7 4.8 22.1 16.8 3.4 10.5 8.8 1.4 2.9 N.D. 4 N.D. 15.3 11 N.D. 13.7 4.3 8.6 24.6 22.3 5.3 7.8 3 N.D. 4.3 13.5 N.D. 5.4 3.9 13.2 4.2 2.8

75 63 55 60 46 55 70 46 53 37 41 48 28 63 71 34 29 52 38 63 45 50 35 67 58 70 60 45 57 82 76 48 55 60 43 40 62 38 46 58 76 47 31

Please cite this article in press as: H. Wang et al., Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.10.002

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Fig. 3. Hepatic expansion of an effector BTLA+ HBV-specific CTL population. Liver infiltrating lymphocytes from two representative patients were stained first with indicated PE-labeled tetramers, and then with mAbs to CD8, CD27, CD45RA and BTLA. (A) Dot plot analyses showing CD8+tetramer+ cell percentage. (B) Dot plots, corresponding to those in A, are gated on tetramer+CD8+ [R1 (HBV-specific CTLs)], and show their differentiated stages. (C) Histogram plots, corresponding to those in B, show BTLA expression on different subsets [R2 (CD45RA CD27 ) and R3 (CD45RA+CD27 ) ] and all gated cells [R1 (Total)]. Results are expressed as percentage of cells, indicated in each gate.

and others previously demonstrated that activation of the BTLA signaling pathway induced prompt IL-10 secretion [19,20]. Thus, we determined whether the regulatory role of BTLA was related to the induction of IL-10 production. The tetramer+IFN-c+ LIL frequency was inversely related to tetramer+BTLA+CD8+ LIL frequency (Fig. 4B). In contrast, we observed that tetramer+IL-10+ LIL frequency was directly related to the frequency of tetramer+BTLA+CD8+ LILs (Fig. 4D). Additionally, after seven days of stimulation with a pool of peptides plus anti-CD28 mAb, BTLA+ LILs secreted significant amounts of IL-10 which were detected in the culture medium from BTLA+ LILs, but not from BTLA+ cell-depleted LILs (Fig. 5). Furthermore, blocking the BTLA signaling pathway with BTLA-Ig neutralization antibody greatly decreased IL-10 production from corresponding peptides plus anti-CD28 mAbs-stimulated LILs (Fig. 5). Taken together, our data indicated that BLTA-mediated pathway is required for the IL-10 production in LILs. 3.4. Suppressor activity of intrahepatic BTLA-expressing CD8+ T cells In order to investigate the regulatory role of IL-10-producing BTLA+CD8+ LILs, we developed a simple and reliable non-antigenspecific suppressor assay by co-culturing BTLA+ LILs from different donors and PBMCs in the presence of anti-CD3 and anti-CD28 mAbs. anti-CD3 plus anti-CD28 mAb stimulation simultaneously induce the proliferation of PBMCs and the regulatory effects of in vivo-differentiated BTLA+ CD8+ LILs, which can suppress PBMC proliferation. Because of the limiting cell number of LILs, BTLA+ LILs from two to three biopsies were pooled together for each individual experiment. In the majority of experiments, co-culture with BTLA+CD8+ LILs greatly suppressed PBMC proliferation. This strongly supports the assertion that BTLA+CD8+ LILs have a major regulatory role on monocytes proliferation (Fig. 6). As expected, similar results were obtained using either autologous or allogeneic

PBMCs as responders in this non-antigen-suppression system. The addition of either neutralizing mAbs to IL-10 or BTLA-Ig markedly abrogated the suppression of BTLA+ LILs (Fig. 6), which confirmed the regulatory role of the BTLA signaling pathway-IL-10 axis. Since IL-10 producing BTLA+ subsets were present within PBMCs, we performed control experiments where PBMCs were exposed to the same proteins without LILs. Neither BTLA-Ig nor IL-10 mAbs treatment failed to suppress PBMCs proliferation, ruling out the presence of IL-10-producing regulatory cells in the periphery. 3.5. Antigen-specificity of regulatory BTLA-expressing CD8+ LILs After confirming the regulatory role of BTLA-expressing CD8+ LILs, we further determined the antigen-specificity of these regulatory cells. BTLA+ LILs derived from single biopsies were co-cultured with autologous PBMCs in the presence of both the relevant HLAA24-related HBV peptides and soluble form of the recombinant HBV core protein. Antigen presenting cells in PBMCs could present soluble recombinant HBV core proteins to self (CD4+) T cells and HLA-A24-related HBV peptides to the in vivo-differentiated antigen-specific CD8+ T cells, which in turn inhibit PBMC proliferation to antigenic HBV stimuli in the presence with the autologous LIL population. Six independent experiments were performed to determine the capacity of PBMCs to proliferate in response to soluble HBV core proteins. LILs from HLA-A24+ patients suppressed autologous antigen-specific PBMC responses only upon antigenspecific activation with a pool of HLA-A24-related peptides, but not with a pool of unrelated HLA-A24-related HCV peptides (Fig. 7A and B). Importantly, blocking BTLA pathways significantly reduced the antigen-specific suppression of LILs (Fig. 7A). These data clearly support the assertion that the suppression function is antigen-specific, at least in the induction phase [21,22,9]. Notably, BTLA-mediated signaling played key roles in the suppression function.

Please cite this article in press as: H. Wang et al., Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.10.002

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Fig. 4. Correlations among immunological and clinical parameters. (A) The percentage of HBV-specific cells in liver was greater than in blood. The percent of tetramer+CD8+ cells in blood was the sum of both HLA-peptide-labeled cells. (B) The percentage of BTLA+tetramer+CD8+ LILs was inversely related to the percentage of IFN-c+tetramer+CD8+ LILs. (C) Frequencies of BTLA+tetramer+CD8+ LILs were inversely related to total HAI score. (D) Frequencies of BTLA+tetramer+CD8+ LILs were related to the percentage of IL10-producing tetramer+CD8+ LILs. (E) Frequencies of BTLA+tetramer+CD8+ LILs were inversely related to serum ALT levels. (F) Frequencies of BTLA+tetramer+CD8+ LILs were related to serum HBV-DNA levels. Statistical analysis was performed by the Spearman correlation test.

4. Discussion This study provided evidence that patients with chronic HBV infection show concomitant and marked intrahepatic expansion of BTLA+ virus-specific CD8+ T cells. These cells are capable of reducing effector functions or suppressing antiviral inflammatory responses by producing IL-10. BTLA is a co-inhibitory receptor of the immunoglobin superfamily, but propagates signaling from herpes virus entry mediator (HVEM), a member of the TNF-TNFR superfamily [23,24]. In this study, we discovered that BTLA is predominately expressed on HBV-specific EM cells rather than total CD8+ EM cells in peripheral blood. Furthermore, BLTA expression level is much higher in HBV-nonspecific CD8+ T cells, compared to HBV-specific CD8+ T cells (the majority of them were CD45RA ). This finding is consistent with a previous study showing decreased BTLA expression during T cell differentiation [18]. Additionally, in response to sustained antigen stimulation, CD8+BTLA+tetramer+ cells markedly proliferated and produced significantly greater amounts of IFN-c than BTLA cells. This suggests that peripheral BTLA+ cells represented a population (such as the EM subset) with the potential to differentiate into effector cells efficiently. It has been reported that patients with acute resolving HBV infection showed a predominant expansion of activated HBV-specific EM cells and a low number of central memory cells in the peripheral blood [25]. However, during persistent infection, EM cells might maintain homeostasis and be instrumental in providing continuous waves of effector memory CTLs homing to the liver in response

to continuous antigen stimulation [26,27]. The expression of coinhibitory receptors, such as BTLA, represents a mechanism by which preventing the excessive differentiation of CTLs from the CM stage to EM stage occurs. In this study, we found that comparing to in vitro differentiated EM CTLs, EM CTLs homing to the liver exerted poor effector functions in vivo, This is in agreement with a previous study, which showed the majority of intrahepatic HBV-specific CTLs were characterized by their inability to function as fully differentiated effectors [16]. Despite the acquisition of an effector tissue-homing membrane phenotype, intrahepatic HBV-specific CTLs from some patients still expressed considerable levels of BTLA. In previous studies about patients with chronic virus infection or tumors, multiple co-inhibitory molecules, such as PD-1, TAG-3, Tim-3 and CD160, were upregulated on virus- or tumor antigen(TA)-specific CTLs [4–7,16,28,29], but their effects on the immune system were poorly understood. For instance, PD-1 was shown to be a representative marker of exhausted CTLs in many studies of chronic virus-infected patients, including HBV [16], HCV [30] and CMV [18], while another study observed an accumulation of regulatory T cells in liver tissues with high PD-1 expression that inhibited regulatory T cells expansion [31]. In addition, a study on tumor antigen-specific CTLs in patients with melanoma demonstrated that BTLA+PD-1+Tim-3+ and BTLA+PD-1+Tim-3 tumor tissue infiltrating TA-specific CTLs represented two distinct populations, suggesting different effects of those inhibitory receptors on TA-specific CTLs [28]. Although both BTLA and PD-1 belong to the same

Please cite this article in press as: H. Wang et al., Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.10.002

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Fig. 5. BTLA+ LILs produce marked amounts of IL-10. Fresh hepatic BTLA+ and BTLA cells purified from LILs of six patients were stimulated with anti-CD28 mAbs and corresponding peptides in the presence of BTLA-Ig or control IgG for 7 days. Then, cells were intracellularly stained with mAbs to IL-10 after anti-CD8 mAb stimulation and tetramer labeling. Dot plots are gated on tetramer+CD8+ cells. Results are expressed as percentage of cells.

Fig. 6. Hepatic BTLA+CD8+ T cells have a regulatory function through BTLA signaling/ IL-10 axis. Highly purified CD8+ LILs pooled from two to three biopsies were cocultured with PBMCs plus anti-CD3 and anti-CD28 mAbs in the presence of antiIL-10 mAbs or BTLA-Ig. Control cultures in which PBMCs were stimulated with the same mAbs and soluble proteins but in the absence of CD8+ LILs were also prepared. Each symbol represents an individual experiment performed with a single LIL pool derived from two biopsies. In all experiments, 1 lC [3H]thymidine was added to cultures after 6 days and the radioactivity incorporated by cells was determined after 18 h. Statistical analysis was performed by Student’s t-test for paired data.

co-inhibitory receptor superfamily, BTLA is different from PD-1 in many respects [18]. The roles of BTLA in reduced-function antigen-specific CTLs infiltrating target tissues during chronic virus infection and tumors were poorly understood. In this study, the proportion of BTLA+ LILs was inversely related to the HAI score of patients, but was associated with the HBV-DNA load in serum. This is in contrast to exhausted HBV-specific LILs with high PD-1 expression that were inversely related to HBV-DNA load. Importantly, BTLA+ HBV-specific LILs produced marked amounts of IL10 after stimulation with peptide plus anti-CD28 mAbs. Thus, BTLA+ HBV-specific LILs represent regulatory CD8+ T cells that inhibit effector CTL-mediated long-lasting tissue damage. As to the mechanisms by which BTLA+ HBV-specific LILs play a regulatory role on effector responses in individuals chronically infected with HBV, we hypothesized that IL-10 is a key mediator. Indeed, we found that BTLA+ HBV-specific LILs produced IL-10 after specific antigen stimulation. Furthermore, IL-10 neutralizing mAbs treatment could reduce the suppression response. It suggests that IL-10-producing BTLA+ HBV-specific LILs play a major role in controlling chronic liver immunopathology. In addition, our data indicated that regulatory BTLA+ virus-specific LILs were antigenspecific during the induction phase of the response but that they exerted suppression in a non-antigen-specific manner via IL-10 in the effector phase. The regulatory function of BTLA+ HBV-specific cells seemed to be limited in the liver tissue of chronically HBV-infected patients, as their peripheral counterparts did not express large amounts of IL-10. In the peripheral blood, BTLA+ cells were mainly CM cells. During persistent antigen stimulation, BTLA expression levels were down-regulated in these cells as they differentiated into EM cells [18]. However, in liver tissues, the expression of BTLA was maintained on antigen-specific CTLs during the EM stage, and IL-10

Please cite this article in press as: H. Wang et al., Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.10.002

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Fig. 7. HBV antigens induce regulatory CD8+ T cells. (A) Five independent experiments showing BTLA+ LILs from five patients suppress HBV-specific proliferation by autologous PBMCs, and that suppression is reversed by blocking BTLA signaling. (B) Four control experiments showing BTLA+ LILs from four patients suppress HBV-specific proliferation by autologous PBMCs only when stimulated with HBV peptides but not with irrelevant HLA-A24–related HCV peptides. In all experiments, 1 lC [3H]thymidine was added to cultures after 6 days and the radioactivity incorporated by cells was determined after 18 h. Statistical analysis was performed by Student’s t-test for paired data.

could be induced continuously through the activation of BTLAmediated signals. The mechanism(s) involved in maintaining BTLA expression on EM CD8+ LILs are still unclear. We believe that the complicated microenvironment of HBV-infected liver tissues might contribute to this. In conclusion, we here report that IL-10-producing HBV-specific BTLA+ LILs play a role in controlling the magnitude of immune responses by sustaining a long-lasting virus/host symbiosis. Because of its capacity to generate escape mutants [32,33], or to exert immunosuppressive effects via certain viral proteins [34,35], virus was not completely cleared by CTLs in chronic HBV-infected patients. Our data suggest that in cases where the virus avoids effector CTL responses during the early phase of infection, induced expression of BTLA on tissue-homing HBV-specific CTLs can exert effects to minimize chronic pathological responses by effector CTLs. The equilibrium between effector CTLs and regulatory T cells establishes a status of long-lasting low-level inflammation which is instrumental for the survival of both the microbial agent and the host. Understanding the role of regulatory cells in antiviral immunity will provide us an important platform for the design of innovative strategies to re-engineer antiviral immune responses in persistent infections.

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