Journal of Clinical Virology 61 (2014) 40–46
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CD8+ T cell responses specific for hepatitis B virus core protein in patients with chronic hepatitis B virus infection Wei Cao, Zhifeng Qiu, Ting Zhu, Yanling Li, Yang Han, Taisheng Li * Department of Infectious Diseases, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, 1# Shuaifu Yuan, Dongcheng District, Beijing 100730, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 March 2014 Received in revised form 3 June 2014 Accepted 22 June 2014
Background: Chronic hepatitis B virus (HBV) infection includes a set of heterogeneous clinical patterns, and core-protein-specific T cell response is important for virus control and disease progression, yet is not well elucidated. Objectives: To analyze the phenotypic and functional profiles of HBV-core-protein-specific CD8+ T cells in different clinical patterns of chronic HBV infection. Study design: A total of 46 HBV patients were recruited and classified according to their clinical status. CD8+ T cell responses in different patterns of chronic HBV infections were tested with flow cytometry using overlapping 15-mer peptides covering HBV core protein. Meanwhile, the CCR7/CD27 phenotypes of these CD8+ T cells were also determined. Results: Frequencies of gamma interferon (IFN-g) positive CD8+ T cells in inactive HBV surface antigen (HBsAg) carriers in response to the core protein peptide pools were generally stronger than those of chronic HBV carriers and resolved individuals, especially with regards to peptide pool C13–C24. Moreover, phenotypic studies further highlighted the group of CD8+ CCR7–CD27+ T memory cells, which showed significantly higher levels of IFN-g secretion in inactive HBsAg carriers than those in chronic hepatitis B patients, chronic HBV carriers and resolved individuals. Conclusions: Core-protein-specific T cell response plays an important role in chronic HBV infection. Inactive HBsAg carriers showed a much stronger core-protein-specific cytotoxic T cell response than other types of chronically infected patients. CD8+ CCR7–CD27+ T memory lymphocytes may be crucial in the immune pathogenesis of chronic HBV infection. ã 2014 Elsevier B.V. All rights reserved.
Keywords: CCR7–CD27+ T memory cell HBV core protein Inactive HBsAg carrier Specific T cell response
1. Background Hepatitis B virus (HBV) infection is a global health problem. Approximately 350 million people worldwide have persistent HBV infection, which leads to increased risk of cirrhosis or hepatocellular carcinoma. Although mechanisms for the chronicity of HBV infection are not well elucidated, there is consensus that pathogenesis of HBV infection is largely immune mediated, and
Abbreviations: HBsAg, hepatitis B surface antigen; IFN-g, gamma interferon; CTL, cytotoxic T lymphocytes; HLA, human leucocyte antigen; ALT, alanine aminotransferase; (anti-) HBe, (antibodies to) hepatitis B e antigen; (anti-) HBcAg, (antibodies to) hepatitis B core antigen; anti-HBs, antibodies to hepatitis B surface antigen; HIV-1, human immunodeficiency virus type 1; CMV, cytomegalovirus; PBMC, peripheral blood mononuclear cell; IL-2, interleukin-2; IQR, interquartile range; AST, aspartate transaminase; CHB, chronic hepatitis B patient; CHBC, chronic HBV carrier; IHBC, inactive HBsAg carrier; RS, resolved individual. * Corresponding author. Tel.: +86 10 69155086; fax: +86 10 69155046. E-mail address:
[email protected] (T. Li). http://dx.doi.org/10.1016/j.jcv.2014.06.022 1386-6532/ ã 2014 Elsevier B.V. All rights reserved.
that CD8+ cytotoxic T lymphocytes (CTLs) play crucial roles in viral control and liver inflammation [1,2]. In chronic HBV infection, virus-specific CTL responses are rather weak and limited compared with the robust responses in acute infection, which may lead to viral persistence and disease progression [3,4]. However, current studies are more focused on the dichotomy between T cell responses in acute and chronic infections, obscuring the diversity within chronic HBV infection itself. Chronic HBV infection is highly heterogeneous with variable levels of virus replication and liver inflammation. Active chronic hepatitis B can progress into cirrhosis or carcinoma if left untreated, while HBV carriers, in the absence of liver inflammation, could be asymptomatic for life. However, in most cases HBV-specific T cell impairments are analyzed with a few human leucocyte antigen (HLA)-restricted HBV epitopes [5,6,7], which is limited by HLA typing and host ethnicity [8,9]. Available results are controversial, and there is still no conclusion with regard to the exact role these T cells take in chronic HBV infection [10]. To overcome these limitations, peptide pools covering the whole HBV genome or part of it, instead of single
W. Cao et al. / Journal of Clinical Virology 61 (2014) 40–46
peptides, have been used to study HBV-specific T cell functions in recent years [11,12]. It is known that pathogen-specific CTLs may differ in major phenotypes and functions with different viral infections [13,14]. However, studies of HBV-specific T phenotypes and functions in chronic HBV infection are very incomplete. A series of surface molecules have been known as markers of CD8+ T cell differentiation. CCR7 is a chemokine receptor involved in lymphocyte recirculation to secondary lymphoid tissues [15,16]. CD27, a member of the tumor necrosis factor receptor (TNFR) family, is a co-receptor in T cell regulation [17]. Both molecules are downregulated with T cell differentiation towards the effector phase. And the chronological expression of CCR7+ CD27+, CCR7–CD27+ and CCR7–CD27- can represent the early, median to late stages of CD8+ T memory cell differentiation. Simultaneous analysis of the phenotypes and intracellular cytokine production of CD8+ T cells in response to HBV core peptide pools in chronic HBV infections, will help to better understand the pathogenesis of HBV chronicity. 2. Objectives The present study aims to provide initial assessment of HBV core-protein-specific CD8+ T cell function between subtypes of chronic HBV infections by multiparameter flow cytometry, to further identify the contributory factors of disease outcomes.
3. Study design
41
3.2. Virological assessment Serum HBV DNA levels were assessed by real-time fluorescent quantitative polymerase chain reaction method (real-time-PCR) using real-time-PCR system (ABI Prism 7500, ABI) with a detection limit of approximately 103 viral copies ml 1. The experimental procedures were performed in strict accordance with the reagent kit (Da An Gene Co., Ltd.) package insert. HBsAg, anti-HBs, total and immunoglobulin M anti-HBc, HBeAg and anti-HBe were determined by commercial enzyme-linked immunosorbent assay (ELISA). 3.3. Synthetic HBV core protein peptide pools Core protein of Hepatitis B virus genotype C (serotype adw2) consisting of 183 residues was selected (Entrez Protein Locus: AAP06598). A panel of 35 15-mer peptides overlapped by 10 residues and covering the whole core protein sequence were constructed (C1, C2, . . . ,C34, C35) and synthesized by the Chinese Peptide Company, Co., Ltd. (Hangzhou, China). The purity of each peptide was determined to be greater than 80% by high-pressure liquid chromatography analysis. These 15-mer peptides were further pooled in three mixtures, C1–C12, C13–C24 and C25–C35, covering the core amino acids 1–70, 61–130 and 121–183, respectively. Detailed information of the synthetic peptide sequences is provided in Appendix A. 3.4. Isolation, stimulation and staining of peripheral blood mononuclear cells (PBMCs)
3.1. Patients Between October 2009 and April 2011, a total of 46 patients with chronic HBV infection were enrolled from outpatient clinic of infectious diseases, Peking Union Medical College Hospital. These patients were divided into four groups according to their clinical and virological assessments, the chronic hepatitis B patients (13 patients, CHBs), chronic HBV carriers (10, CHBCs), inactive HBsAg carriers (10, IHBCs) and resolved individuals (13, RS’s). Standards of patient selection and classification were based on the 2009 AASLD (American Association for the Study of Liver Diseases) guidelines on chronic hepatitis B (Table 1) [18]. All of the subjects were negative for antibodies to hepatitis C virus, human immunodeficiency virus type 1 (HIV-1), and for other markers of viral or autoimmune hepatitis. None of these patients had received any antiviral therapy before. All subjects provided written informed consent.
PBMCs were isolated from 10 ml fresh heparinized blood each by Ficoll-Hypaque density gradient centrifugation, purified and resuspended at the concentration of 3.0 106 ml 1 in RPMI 1640–10% fetal calf serum (FCS). PBMC stimulation was performed with the three synthetic core peptide pools (single peptide 2.5 mg ml 1) or with PMA (25 ng ml 1) plus ionomycin (1 mg ml 1) as a positive control at 37 C for 18–22 h, with brefeldin A (10 mg ml 1, Sigma–Aldrich, U.S.A) present for the last 4 h of stimulation. Negative controls were also prepared as abovementioned except for the stimuli. Post-stimulation PBMCs were washed, surface-stained with anti-CD27 fluorescein isothiocyanate (FITC), anti-CCR7 phycoerythrin (PE)-Cy7 conjugated, and anti-CD8 allophycocyanin (APC)Cy7 antibodies (BD Pharmigen, U.S.A), permeabilized, and fixed with Cytofix/Cytoperm (BD Pharmigen, U.S.A) according to the manufacturer’s instructions. Then, anti-IL-2 PE and anti-IFN-g
Table 1 Baseline characteristics of patients with chronic HBV infection. Groups
Chronic hepatitis B patients (CHBs)
Chronic HBV carriers (CHBCs)
Inactive HBsAg carriers (IHBCs)
Resolved individuals (RS’s)
No. Male/Female Age
13 8/5 44.2 10.5
10 3/7 32.7 9.1
10 6/4 37.8 8.1
13 7/6 25.5 0.7
+ +
+ +
+
Serum viral markers HBsAg HBeAg Anti-HBs Anti-HBe Anti-HBc HBV DNA log copies ml 1 ALT level U l a
1
+ + + 6.63 1.01
+ 6.46 2.03
Undetectable
+ Undetectable
262.6 81.2
Normal rangea
Normal rangea
Normal rangea
Normal range: ALT 5–40 Ul 1.
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W. Cao et al. / Journal of Clinical Virology 61 (2014) 40–46
Alexa-647 (BD Pharmigen, U.S.A) antibodies were added and kept for 15 min, and the cells were washed twice and analyzed by flow cytometry.
peptide pools were generally very low, and not distinguishable between groups (Table 2, Fig. 2). 4.2. CCR7/CD27 phenotype analysis of peripheral CD8+ T cells
3.5. Statistical analysis Normal variables were summarized as means and standard deviations, and non-normal variables as medians and interquartile range (IQR). Normal data were compared by Student t-test or one-way ANOVA adjusted for multiple comparisons, as appropriate. Multiple comparisons of non-normal data were carried out by Kruskal–Wallis test. All tests were two-sided, and a P-value 0.05 was considered significant. P < 0.009 was considered significant for further comparisons within samples. Associations between variables were assessed using Spearman’s rank correlations. All statistical procedures were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). 4. Results 4.1. HBV-specific CD8+ T cell responses to HBV core peptide pools Frequencies of IFN-g positive and/or interleukin-2 (IL-2) positive CD8+ T cells in response to core peptide pools were measured in each patient group (Fig. 1A, Table 2). The total frequencies of IFN-g + CD8+ T cells upon stimulation of the core peptide pools (C1-C35) in CHBs, CHBCs, IHBCs and RS’s were 0.135% (0.038%, 0.225%), 0.023% (0.003%, 0.198%), 0.136% (0.077%, 0.180%) and 0.027% (0.011%, 0.095%), respectively. IHBCs showed stronger responses to the core peptide pools than the other groups (P < 0.009 v.s. CHBCs and RS’s), while responses of CHBs were moderately but not statistically elevated. With regard to each single peptide pool, IHBCs held much stronger responses to peptide pool C13–C24 than CHBCs and RS’s, with the frequencies of IFN-g + CD8+ T cells 0.068% (0.029%, 0.151%) (P < 0.009) (Fig. 2). IHBCs also displayed stronger IFN-g responses to peptide pool C1– C12 compared with CHBCs and RS’s, though not statistically significant. However, no marked difference was observed in response to peptide pool C25–C35 between the four groups. IL-2 production and dual cytokine production in response to the
Frequencies of peripheral CD8+ CCR7+ T cells in CHBs, CHBCs, IHBCs and RS’s were 34.1% 14.3%, 49.4% 17.8%, 48.0% 16.0% and 57.8% 20.4%, respectively. Frequencies of peripheral CD8+ CD27+ T cells were 48.2% 16.1% for CHBs, 64.2% 16.1% for CHBCs, 65.2% 14.0% for IHBCs, and 76.2% 14.7% for RS’s. CHBs showed the lowest levels of CD27 and CCR7 expression (Fig. 3). More specifically, frequencies of peripheral CD8+ CCR7+ CD27+ T cells in CHBs, CHBCs, IHBCs and RS’s were 32.5% 14.7%, 48.2% 18.0%, 46.5% 15.8% and 57.1% 20.5%, respectively. Frequencies of dual negative CD8+ T cells in CHBs, CHBCs, IHBCs and RS’s were 50.3% 15.6%, 34.6% 15.8%, 33.3% 13.7% and 23.1% 14.6%, respectively. Again, CHBs showed a marked lower level of CD8+ CCR7+ CD27+ T cells, with elevated proportion of CD8 + CCR7–CD27- T cells. No significant differences have been observed between the CD8+ CCR7–CD27+ phenotypes in each group (Fig. 3). 4.3. Phenotypic and functional profiles of core-protein-specific CD8+ T cells The intracellular cytokine (IFN-g and/or IL-2) productions of various CCR7/CD27 phenotypes were also compared (Fig. 1B,C). And the major differences existed in cytokine responses to peptide pool C13–C24. Frequencies of IFN-g + CD8+ CCR7–CD27+ T cells in IHBCs were 0.103% (0.063%, 0.234%), markedly higher than those in the other three groups (P < 0.009). Frequencies of IFN-g + CD8+ CCR7–CD27- T cells in IHBCs and CHBCs were 0.034% (0.008%, 0.131%) and 0.033% (0%, 0.110%), respectively, both higher than those in RS’s (P < 0.009) (Fig. 4). No significant difference was observed with the C1–C12 and C25–C35 pools, in terms of the responsive cytokine production of each phenotype in different groups (data not shown here). A correlation analysis between the level of CCR7 or CD27 expression and intracellular IFN-g production was also done. Surface expression of both molecules was inversely correlated with
Fig. 1. Intracellular cytokine analysis by flow cytometry. (A) IFN-g and IL-2 production by the CD8+ T cell population; (B) The CCR7/CD27 phenotypes of CD8+ T cells; and (C) IFN-g and IL-2 production in four phenotypes of CD8+ T cells: CCR7+ CD27-, CCR7+ CD27+, CCR7–CD27-, CCR7–CD27+.
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Table 2 HBV-core-protein-specific CD8+ T cell responses in chronic HBV infection by cytokine production. Cytokine production
Chronic hepatitis B patients (CHBs)
Chronic HBV carriers (CHBCs)
Inactive HBsAg carriers (IHBCs)
Resolved individuals (RS’s)
Peptide Pool C1–C12 IFN-g + CD8+ T% IL-2 + CD8+ T % IL-2&IFN-g + CD8+ T%
0.037 (0.02, 0.062) 0.015 (0, 0.042) 0 (0, 0)
0.012 (0, 0.027) 0 (0, 0.030) 0 (0, 0)
0.038 (0.014, 0.072) 0.012 (0, 0.017) 0 (0, 0)
0.012 (0.003, 0.027) 0.004 (0.003, 0.013) 0 (0, 0004)
Peptide Pool C13–C24 IFN-g + CD8+ T% IL-2 + CD8+ T% IL-2&IFN-g + CD8+ T%
0.042 (0, 0.076) 0.018 (0, 0.026) 0 (0, 0)
0 (0, 0.032) 0.023 (0.003, 0.110) 0 (0, 0)
0.068 (0.029, 0.151)a 0.014(0.003, 0.062) 0 (0, 0)
0.005 (0, 0.01) 0.02 (0, 0.061) 0 (0, 0.008)
Peptide Pool C25–C35 IFN-g + CD8+ T% IL-2 + CD8+ T% IL-2&IFN-g + CD8+ T%
0.025 (0, 0.061) 0.004 (0, 0.025) 0 (0, 0)
0 (0, 0.052) 0 (0, 0.007) 0 (0, 0.006)
0 (0, 0.029) 0.003 (0, 0.038) 0 (0, 0)
0.019 (0, 0.032) 0.007 (0, 0.018) 0 (0, 0)
Peptide Pools C1–C35 IFN-g + CD8+ T% IL-2 + CD8+ T%
0.135 (0.038, 0.225) 0.045 (0, 0.103)
0.023 (0.003, 0.198) 0.036(0.009, 0.154)
0.023 (0.011, 0.086) 0.043 (0.019, 0.08)
IL-2&IFN-g + CD8+ T%
0 (0, 0)
0.004 (0, 0.025)
0.136 (0.077, 0.180)b 0.031 (0.013, 0.122) 0.004 (0,0.016)
0.008 (0.004, 0.016)
Cytokine production profiles of CD8+ T cells were measured in responses to core peptide pools as stated above, and were presented in the table as medians and IQR. IHBC showed much stronger IFN-g response to the whole core peptide pools C1–C35 than the other groups (aP < 0.009 v.s. CHBCs and RS’s). They displayed stronger IFN-g response to peptide pool C13–C24 than CHBCs and RS’s (bP < 0.009, Fig. 2), and also stronger response to C1–C12, though not statistically significant. Responses to C25–C35 showed no difference between the four groups. IL-2 production or dual cytokine production were low and not distinguishable between different groups.
levels of intracellular IFN-g production (CCR7, r = 0.445, P < 0.001; CD27, r = 0.471, P < 0.001, figure not shown). 5. Discussion Hypo-responsiveness of HBV-specific T cells has been considered an important determinant of virus persistence in chronic HBV infection. How they differ and affect the prognosis in chronic HBV infection is still a mystery. In the present study, we explored coreprotein-specific CD8+ T cell responses and their phenotypic profiles in chronic HBV patients with divergent outcomes. Of the chronic HBV patients, IHBCs presented the strongest coreprotein-specific CTL response. Further analysis showed a distinctive phenotypic T cell responsiveness, indicating CD8+
CCR7–CD27+ T subsets with stronger response might play an important role in viral control and disease quiescence. The overall core-protein-specific T cell response in chronic HBV infection turned out to be quite weak, with a preference of IFN-g production over IL-2, which may indicate an altered or impaired cytokine profiles in chronic HBV infection [19–21]. Still, a generally stronger CTL response was observed in inactive HBsAg carriers, in response to both the peptide pool C13–C24 and the whole mixed peptides, which indicated possible better preservation of T cell functions in this group. The IHBC state is determined by the presence of the surface antigen but with an undetectable HBV DNA in PCR-based assays and repeatedly normal liver function. On biopsy, minimal or absence of liver disease activity could be observed. We did not assess the pathological status of our
Fig. 2. HBV-specific CD8+ T cell responses (IFN-g) to peptide pools of HBV core protein. Frequencies of IFN-g positive CD8+ T cells in response to peptide pools C1–C12 and C13–C24 in IHBCs were higher than those in CHBCs and RS’s (P < 0.009). Frequencies of IFN-g positive CD8+ T cells in response to the whole peptide library in IHBCs were also higher than those in CHBCs and RS’s (P < 0.009).
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Fig. 3. CCR7/CD27 phenotype analysis of peripheral CD8+ T cells. Frequencies of peripheral CD8+ CCR7+ CD27+, CD8+ CCR7–CD27-, CD8+ CCR7–CD27+, CD8+ CCR7+ and CD8+ CD27+ T cells in patients with chronic HBV infection were shown, with P values marked in the figure. Levels of CCR7 and CD27 expression in CHBs were much lower than those in other patients.
Fig. 4. C13–C24 specific CD8+ T cell response in different CCR7/CD27 phenotypes. Responses of CD8+ CCR7-CD27+ T cells to peptide pool C13–C24 in IHBCs were significantly higher than those in other infected patients (P < 0.009). Frequencies of IFN-g positive CD8+ CCR7–CD27- T cells to the same peptide pool in IHBCs and CHBs were both higher than those in RS’s (P < 0.009).
W. Cao et al. / Journal of Clinical Virology 61 (2014) 40–46
outpatients. However, all the recruited IHBCs completely met the clinical standards, and had been regularly followed in the clinic for decades before being considered for this study. This non-/or verylow-replicative immune tolerant phase with cessation of HBV activation and histological remission of liver disease could be permanent [22,23]. Reported long-term follow-up of these patients suggested relative benign prognosis with very low risk of disease progression [24–26]. However, little was known about their cellular immune functions. We found for the first time that the average level of HBV core-protein-specific CTL response in IHBCs is much higher than those in other chronic HBV patients, indicating its important role in disease control. Many studies showed an inverse correlation between the CTL intensity and the viraemia levels [5,12,21]. In IHBCs, the existing low level of viral antigen may present as an effective stimulus to the immune system, which relatively preserves the T cell function. In contrast, overload or absence of viraemia often fails to induce potent T cell response, just as situations in chronic carriers or resolved individuals, with the latter the weakest T response. Nevertheless, 20–30% of IHBCs may end up with spontaneous reactivation of hepatitis B after a long period of quiescence [25,27]. In our study, we did notice variations in response levels of inactive carriers. Further follow-up is needed to decide whether this hyporesponsiveness predicts a later disease reactivation. Use of mixed peptide pools of HBV core protein allows identification of immunogenic regions in this protein regardless of the specific HLA-I typing. Most of the previous studies in this field were based on HLA-A2 restricted epitopes, which were more common in Caucasian patients. However, a large population of chronic HBV infection lives in Asia, with quite different HLA-I profiles, such as HLA-A11, -A24, -A33 and -A30 [8,9,28–30]. Our results indicated possible epitopes in peptide pools C1–C12 and C13–C24 (core 1–70 and core 61–130, respectively), especially core 61–130, due to the stronger T cell response in these regions. In addition to those previously well-defined HLA-A2 and -A24 restricted epitopes, there are probably epitopes restricted by other common Asian HLA-I molecules existing in this area. Further exploration of these epitopes would enhance the understanding of immune responses in this large HBV infected population. Further phenotypic studies showed a marked difference in CCR7/CD27 expression of peripheral CD8+ T cells in chronic HBV infections. In CHBs, frequencies of CD8+ CCR7–CD27- T cells were the highest. This elevated proportion of peripheral effector T type indicated a persistently activated immune system of CHBs, though hyporesponsiveness of these cells may exist. By contrast, levels of CCR7 and CD27 expression in both CHBCs and IHBCs were comparable with those in RS’s, demonstrating relative immune quiescence of these patients. Further correlation analysis showed that CCR7 and CD27 expression levels were inversely correlated with the CTL responses, which was in consistence with previous reports [31]. Comparison of the cytokine production between different groups highlighted the population of CD8+ CCR7–CD27+ T cells. In the better responsive IHBC group, CD8+ CCR7–CD27+ T cells showed stronger response to peptide pool C13–C24 as well as to the whole peptide pools. In contrast, the effector CD8+ CCR7– CD27- T responses in CHBs and IHBCs were comparable to each other. These data suggested that CD8+ CCR7–CD27+ T cells may play an important part in the control of virus replication in IHBCs, further contributing to the benign outcome. Studies of CD8
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+ CCR7–CD27+ T cell population in healthy subjects have demonstrated that this population belongs to a stage between naïve and effector CD8+ T cells, with cytotoxic function but weaker than that of the effector type, hence the name ‘preeffector cells’ [13,31]. However, the exact role of this population in infections has been poorly defined. It has been noted that phenotypic diversity in pathogen-specific CD8+ T cells gradually evolved with disease chronicity. For example, the major responsive CD8+ T cell subsets in vivo were reported the early type CCR7 CD27+ for chronic EBV and HCV infection, CCR7–CD27+ for chronic HIV infection, and CCR7–CD27- for chronic cytomegalovirus (CMV) infection [14]. Likewise, we assumed that a distinctive highly responsive CD8+ T subset also exists in chronic HBV infection, probably CD8+ CCR7–CD27+ T cells. However, more studies are needed with a larger population and better characterization of these cells. Another interesting finding is that CD8+ CCR7–CD27- T cells, traditionally regarded as the major cytotoxic effector, showed no difference in responses to core peptide pools between CHBs and IHBCs, indicating the limited impact of these cells on the outcomes of chronic HBV infection. In conclusion, defective specific T cell function is associated with HBV persistence and chronicity. Clinical features of inactive HBsAg carriers make themselves a promising study population in the perspective of T function maintenance and long-term disease control. Our findings demonstrated a stronger core-protein-specific CD8+ T cell response in inactive HBsAg carriers. The low but adequate amount of circulating antigen in inactive carriers probably served as effective stimulus for CTLs, thus avoiding the viraemic exhaustion of specific Tcells. And the highlighted CD8+ CCR7–CD27+ T cell subsets in these patients provided more insights into the cytokine strategies for functional T recovery. Therefore, virological control in chronic HBV infection is a necessary but not the only step in specific T function reconstitution [9,32]. Funding This study was funded by the National Natural Science Foundation of China (Grant 81071372 to L.T.), and by National Key Technologies R&D Program for the 12th Five-year Plan (Grant 2012ZX10001003-001 to L.T.). Conflict of interest None declared. Ethical Approval Not required. Appendix A. Sequences and purities of the HBV core protein pools: HBV core protein (183 amino acids, genotype C (serotype adw2), Entrez Protein Locus: AAP06598): 6 mdidpykefg asvellsflp sdffpsirdl ldtasalyre alespehcsp hhtalrqail 7 cwgelmnlat wvgsnledpa srelvvsyvn vnmglkirql lwfhiscltf gretvleylv 8 121 sfgvwirtpp ayrppnapil stlpettvvr rrgrsprrrt psprrrrsqs prrrrsqsre sqc
46
W. Cao et al. / Journal of Clinical Virology 61 (2014) 40–46 Peptide pool C1–C12
Peptide pool C13–C24
Peptide pool C25–C35
No.
Sequence
Purity (%)
No.
Sequence
Purity(%)
No.
Sequence
Purity (%)
1 2 3 4 5 6 7 8 9 10 11 12
mdidpykefgasvel ykefgasvellsflp asvellsflpsdffp lsflpsdffpsirdl sdffpsirdlldtas sirdlldtasalyre ldtasalyrealesp alyrealespehcsp alespehcsphhtal ehcsphhtalrqail hhtalrqailcwgel rqailcwgelmnlat
82.3 81.9 85.9 85.9 88.4 95.7 86.5 80.8 82.4 80.7 89.4 93.9
13 14 15 16 17 18 19 20 21 22 23 24
cwgelmnlatwvgsn mnlatwvgsnledpa wvgsnledpasrelv ledpasrelvvsyvn srelvvsyvnvnmgl vsyvnvnmglkirql vnmglkirqllwfhi kirqllwfhiscltf lwfhiscltf gretv scltfgretvleylv gretvleylvsfgvw leylvsfgvwirtpp
84.6 80.1 81.3 81.9 87.4 80.4 94.9 80.4 91.3 82.8 83.9 88.5
25 26 27 28 29 30 31 32 33 34 35
sfgvwirtppayrpp irtppayrppnapil ayrppnapilstlpe napilstlpettvvr stlpettvvrrrgrs ttvvrrrgrsprrrt rrgrsprrrtpsprr prrrtpsprrrrsqs psprrrrsqsprrrr rrsqsprrrrsqsre prrrrsqsresqc
81.0 97.5 89.8 91.6 83.4 87.7 94.7 92.2 89.9 82.8 95.2
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