急性乙型肝炎患者程序性细胞死亡受体1表达与记忆性T淋巴细胞形成的关系

目的 观察急性自限性乙型肝炎发病过程中患者体内病毒抗原特异性细胞毒性T淋巴细胞(CTL)上程序性细胞死亡受体1(PD-1)表达的动态变化特点及其与记忆性抗原特异性CD8+T淋巴细胞形成的关系. 方法 针对不同表位合成4种五聚体,长期随访收集11例人类白细胞抗原(HLA)-A2阳性的急性乙型肝炎患者的外周血,流式细胞仪检测病毒特异性CTL上免疫抑制性分子PD-1、记忆性分子(CCR7、CD45RA、CD127)和活化标志物CD38的表达情况,并分析其相关性.同时进行肝功能,HBsAg、抗-HBs和血清HBV DNA载量检测.结果所有急性自限性乙型肝炎患者发病早期均表现出高频度、病毒抗原多表位的特异性CTL反应,而晚期各表位CTL频率均明显下降.CTL上PD-1表达在早期明显上调;与早期比较,晚期PD-1分子的表达明显降低(t=4.314,P＜0.01).同时CTL高表达记忆性分子CCR7,CD45RA和CD127,而低表达活化标志物CD38.提示病毒清除后记忆性CD8+T淋巴细胞形成. 结论 在急性自限性乙型肝炎发病过程中,HBV特异性CTL上PD-1分子表达的动态变化与记忆性T淋巴细胞的形成密切相关.     

Virological and immunological determinants of intrahepatic virus-specific CD8+ T-cell failure in chronic hepatitis C virus infection

Virus-specific CD8+ T-cells play an important role in the outcome of acute hepatitis C virus (HCV) infection. In the chronic phase, however, HCV can persist despite the presence of virus-specific T-cell responses. Therefore, we set out to perform a full-breadth analysis of the intrahepatic virus-specific CD8+ T-cell response, its relation to the peripheral T-cell response, and the overall influence of viral escape and the genetic restriction on intrahepatic CD8+ T-cell failure. Intrahepatic and peripheral CD8+ T-cells from 20 chronically HCV infected patients (genotype 1) were comprehensively analyzed using overlapping peptides spanning the entire HCV polyprotein in concert with autologous viral sequences that were obtained for all targeted regions. HCV-specific CD8+ T-cell responses were detectable in most (90%) chronically HCV-infected patients, and two thirds of these responses targeted novel previously undescribed epitopes. Most of the responses were detectable only in the liver but not in the peripheral blood, indicating accumulation and enrichment at the site of disease. Of note, only approximately half of the responses were associated with viral sequence variations supported by functional analysis as viral escape mutations. Escape mutations were more often associated with HLA-B alleles. CONCLUSION: Our results show an unexpected high frequency of intrahepatic virus-specific CD8+ T-cells, a large part of which continue to target the present viral antigens. Thus, our results suggest that factors other than mutational escape contribute to the failure of intrahepatic virus-specific CD8+ T-cells.     

The immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function

Multiple inhibitory receptors may play a role in the weak or absent CD8+ T-cell response in chronic hepatitis B virus (HBV) infection. Yet few receptors have been characterized in detail and little is known about their complex regulation. In the present study, we investigated the role of the signaling lymphocyte activation molecule (SLAM)-related receptor CD244 and of programmed death 1 (PD-1) in HBV infection in 15 acutely and 66 chronically infected patients as well as 9 resolvers and 21 healthy controls. The expression of CD244, PD-1, and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) was analyzed in virus-specific CD8+ T-cells derived from peripheral blood or liver using major histocompatibility complex class I pentamers targeting immunodominant epitopes of HBV, Epstein-Barr-virus (EBV), or influenza virus (Flu). In chronic HBV infection, virus-specific CD8+ T-cells expressed higher levels of CD244 both in the peripheral blood and liver in comparison to the acute phase of infection or following resolution. CD244 was expressed at similarly high levels in EBV infection, but was low on Flu-specific CD8+ T-cells. In chronic HBV infection, high-level CD244 expression coincided with an increased expression of PD-1. The inhibition of the CD244 signaling pathway by antibodies directed against either CD244 or its ligand CD48 resulted in an increased virus-specific proliferation and cytotoxicity as measured by the expression of CD107a, interferon-γ, and tumor necrosis factor-α in CD8+ T-cells. Conclusion: CD244 and PD-1 are highly coexpressed on virus-specific CD8+ T-cells in chronic HBV infection and blocking CD244 or its ligand CD48 may restore T-cell function independent of the PD-1 pathway. CD244 may thus be another potential target for immunotherapy in chronic viral infections.     

Intrahepatic CD8+ T-cell failure during chronic hepatitis C virus infection

The precise mechanisms responsible for the failure of intrahepatic hepatitis C virus (HCV)-specific CD8+ T cells to control the virus during persistent infection have not been fully defined. We therefore studied the CD8+ T-cell response in 27 HLA-A2-positive patients using four previously well-defined HLA-A2-restricted HCV epitopes. The corresponding HCV sequences were determined in several patients and compared with the intrahepatic HCV-specific CD8+ T-cell response. The results of the study indicate: (1) intrahepatic HCV-specific CD8+ T cells are present in the majority of patients with chronic HCV infection and overlap significantly with the response present in the peripheral blood. (2) A large fraction of intrahepatic HCV-specific CD8+ T cells are impaired in their ability to secrete interferon gamma (IFN-gamma). This dysfunction is specific for HCV-specific CD8+ T cells, since intrahepatic Flu-specific CD8+ T cells readily secrete this cytokine. (3) T-cell selection of epitope variants may have occurred in some patients. However, it is not an inevitable consequence of a functional virus-specific CD8+ T-cell response, since several patients with IFN-gamma-producing CD8+ T-cell responses harbored HCV sequences identical or cross-reactive with the prototype sequence. (4) The failure of intrahepatic virus-specific CD8+ T cells to sufficiently control the virus occurs despite the presence of virus-specific CD4+ T cells at the site of disease. In conclusion, different mechanisms contribute to the failure of intrahepatic CD8+ T cells to eliminate HCV infection, despite their persistence and accumulation in the liver.     

PD-1表达对急性乙型肝炎患者HBV特异性CD8＋T细胞功能的影响

目的 观察急性乙型肝炎发病过程中患者体内病毒特异性CD8^＋细胞（CTL）上免疫抑制性分子程序性细胞死亡受体1（PD-1）的表达及临床意义。方法合成4种识别HBV不同抗原表位的五聚体，同时采集16例HLA-A2阳性急性乙型肝炎患者外周血，用五聚体染色后，通过流式细胞仪分析病毒特异性CTL细胞的频率及其表面PD-1分子的表达特点。同时进行肝功能、乙型肝炎病毒（HBV）血清标志物和血清HBVDNA检测。结果所有急性乙型肝炎患者发病早期均表现出多特异性的CTL反应，且PD-1在这些细胞上表达明显上调，显著高于来自同一患者的巨细胞病毒（CMV）和Flu特异性CTL上PD-1分子的表达水平。体外阻断PD-1／PD-L1间的相互作用能够恢复HBV特异性CTL分泌细胞因子的能力。相关性分析显示PD-1分子的上调表达与转氨酶水平正相关。结论在HBV急性感染过程中，尤其是在HBV基本被清除后，HBV特异性CTL细胞PD-1分子表达明显升高，有效抑制病毒特异性CTL分泌IFN-γ的能力，后者可能有利于避免急性乙型肝炎患者肝脏遭受更为严重的免疫损伤。     

Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection

BACKGROUND & AIMS: Cytotoxic T cells have been suggested to be responsible for lysis of hepatitis B virus (HBV)-infected hepatocytes and control of virus infection. The frequency, kinetics, phenotype, and capacity for clonal expansion of circulating HBV-specific CD8 cells were analyzed directly in patients with acute HBV infection to clarify their pathogenetic role. METHODS: Three HLA-A2 peptide tetramers able to visualize HBV core, envelope, and polymerase epitope-specific cytotoxic T lymphocytes were synthesized and used for flow cytometric analysis of antigen-specific populations. RESULTS: Tetramer-positive cells specific for the core 18-27 epitope were found at a higher frequency than those specific for polymerase 575-583 and envelope 335-343 epitopes in most patients with acute HBV. The number of HBV-specific CD8 cells was highest during the clinically acute stage of infection and decreased after recovery. These cells expressed an activated phenotype and had an impaired capacity to expand in vitro and to display cytolytic activity in response to peptide stimulation. Recovery of these functions was observed when the frequency of specific CD8 cells decreased, coincident with a progressive decrease in their expression of activation markers. CONCLUSIONS: This study provides the first ex vivo evidence that the highest frequency of circulating HBV-specific CD8 cells coincides with the clinically acute phase of hepatitis B. These cells exhibit an activated phenotype with limited further proliferative capacity that is restored during recovery.     

Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms

After hepatitis B virus (HBV) infection, liver injury and viral control have been thought to result from lysis of infected hepatocytes by virus-specific cytotoxic T cells. Patients are usually studied only after developing significant liver injury, and so the viral and immune events during the incubation phase of disease have not been defined. During a single-source outbreak of HBV infection, we identified patients before the onset of symptomatic hepatitis. The dynamics of HBV replication, liver injury, and HBV-specific CD8+ and CD4+ cell responses were investigated from incubation to recovery. Although a rise in alanine transaminase (ALT) levels was present at the time of the initial fall in HBV-DNA levels, maximal reduction in virus level occurred before significant liver injury. Direct ex vivo quantification of HBV-specific CD4+ and CD8+ cells, by using human leukocyte antigen (HLA) class I tetramers and intracellular cytokine staining, showed that adaptive immune mechanisms are present during the incubation phase, at least 4 weeks before symptoms. The results suggest that the pattern of reduction in HBV replication is not directly proportional to tissue injury during acute hepatitis B in humans. Furthermore, because virus-specific immune responses and significant reductions in viral replication are seen during the incubation phase, it is likely that the immune events central to viral control occur before symptomatic disease.     

Programmed death 1 expression during antiviral treatment of chronic hepatitis B: Impact of hepatitis B e-antigen seroconversion

Hyperexpression of the programmed death 1 (PD-1) molecule is a hallmark of exhausted T-cells, having a negative impact on T-cell activation and function. We studied longitudinally 18 hepatitis B e antigen (HBeAg)-positive patients undergoing treatment with direct antivirals (telbivudine or lamivudine) to determine the relationship between treatment-induced viremia reduction and HBeAg seroconversion with respect to PD-1 levels and T-cell reactivity. PD-1 expression was assessed by (1) flow cytometry and (2) quantitative real-time polymerase chain reaction; hepatitis B virus (HBV)-specific CD8+ T-cells were quantitated by pentamer staining; T-cell reactivity to HBV antigens was determined by interferon gamma (IFNgamma) and interleukin 10 (IL-10) enzyme-linked immunosorbent spot (ELISPOT) assays; and central/effector memory phenotypes were defined by phenotypic markers. PD-1 expression correlated closely with viremia levels. On therapy, PD-1 decreased significantly on total CD8+ T-cells, HBV-specific CD8+ T-cells, and CD3+/CD8- T-cells both as the percentage of positive cells (P < 0.01) and as the mean fluorescent intensity (P < 0.05), and this was paralleled by a marked reduction of PD-1 messenger RNA levels (P = 0.001). HBeAg serocoversion (in 6/18 patients) resulted in a further PD-1 decrease with a 50% reduction in the frequency of PD-1+/CD8+ T-cells, which was not observed in patients remaining HBeAg-positive. The decrease in PD-1 expression was associated with increased frequencies of IFNgamma-producing T-cells and decreased frequencies of IL-10 producing T-cells. At baseline, PD-1 expression correlated directly with the frequency of hepatitis B core antigen (HBcAg) central and effector memory phenotypes, whereas an inverse correlation was observed between PD-1 expression and HBcAg-specific effector phenotypes. CONCLUSION: These results demonstrate that in chronic HBV infection, both viremia levels and HBeAg drive PD-1 expression and resulting T-cell impairment. Treatment-induced suppression of HBV replication reduces PD-1 expression; however, additional immunotherapeutic interventions are needed for restoration of T-cell functions.     

Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen

BACKGROUND & AIMS: Impaired T-cell reactivity is believed to be the dominant cause of chronic hepatitis B virus (HBV) infection. We characterized HBV-specific T-cell responses in chronic hepatitis B surface antigen carriers who received bone marrow from HLA-identical donors with natural immunity to HBV and seroconverted to antibody to hepatitis B surface antigen. METHODS: T-cell reactivity to HBV antigens and peptides was assessed in a proliferation assay, the frequency of HBV core- and surface-specific T cells was quantified directly by ELISPOT assays, and T-cell subsets were analyzed by flow cytometry. RESULTS: CD4+ T-cell reactivity to HBV core was common in bone marrow donors and the corresponding recipients after hepatitis B surface antigen clearance, whereas none reacted to surface, pre-S1, or pre-S2 antigens. Furthermore, CD4+ T cells from donor/recipient pairs recognized similar epitopes on hepatitis B core antigen; using polymerase chain reaction for the Y chromosome, the recipients' CD4+ T lymphocytes were confirmed to be of donor origin. The frequency of core-specific CD4+ and CD8+ T cells was several-fold higher than those specific for surface antigen. CONCLUSIONS: This study provides the first evidence in humans that transfer of hepatitis B core antigen-reactive T cells is associated with resolution of chronic HBV infection. Therapeutic immunization with HBV core gene or protein deserves further investigation in patients with chronic hepatitis B.     

乙型肝炎相关性肝功能衰竭患者外周血PD-1表达的临床意义

目的 观察乙型肝炎相关性肝功能衰竭患者细胞程序性死亡因子-1 (PD-1)表达特点及临床意义.方法 针对不同表位合成3种pentamer,采集16例急性和慢加急性肝功能衰竭患者外周血,并收集15例急性乙型肝炎(AHB)患者外周血作为对照,流式细胞仪分析各组病毒特异性CTL细胞的频率及其表面PD-1分子表达水平,同时观察与肝功能和血清HBV DNA载量的相关性.组间比较采用Mann-whitneyU检验.结果 与AHB患者比较,肝功能衰竭患者发病早期病毒特异性CD8 T淋巴细胞PD-1表达水平显著降低,该阶段PD-1表达水平与转氨酶呈显著正相关,但与病毒载量无明显相关性.随访发现,AHB患者早期,PD-1高水平表达,随着疾病康复,PD-1表达逐渐降低；而肝功能衰竭患者早期PD-1表达较低,随后显著增加,而后随着疾病恢复,PD-1进一步降低.结论 肝功能衰竭患者发病早期外周血PD-1表达水平较AHB患者显著降低,而PD-1延迟高表达则与急性肝功能衰竭的发生密切相关.     

Lamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy

The hepatitis B virus (HBV) cytotoxic T lymphocyte (CTL) response in patients with chronic HBV infection is generally weak or totally undetectable. This inability to mount protective CTL responses is believed to be a crucial determinant of viral persistence, and its correction represents an important objective of immune therapies for chronic hepatitis B. However, amplification of CTL responses in vivo may be ineffective if HBV-specific CD8 cells are either absent or nonresponsive to exogenous stimulation. In this study, we asked whether antiviral treatments able to inhibit viral replication and to reduce viral and antigen load can successfully reconstitute CTL responses creating the appropriate conditions for their therapeutic stimulation. For this purpose, the HBV-specific CTL response before and during lamivudine therapy was studied longitudinally in 6 HLA-A2-positive patients with HBeAg+ chronic hepatitis B. Both HBV-specific cytotoxic T cell activity measured by chromium release assay on peptide stimulation in vitro and CD8+ T cell frequency measured ex vivo by HLA-A2/peptide tetramer staining were significantly augmented by lamivudine therapy. This enhancement followed the reconstitution of CD4 reactivity and the decline of viral load induced by therapy. Our study shows that lamivudine treatment in chronic hepatitis B can restore CTL reactivity, making CTL susceptible to exogenous stimulation. This effect may enhance the probability that T cell-based immune therapies delivered after lamivudine treatment can successfully reconstitute a protective CTL response able to cure chronic HBV infection.     

Flow cytometry of fine-needle-aspiration biopsies: a new method to monitor the intrahepatic immunological environment in chronic viral hepatitis

SUMMARY: Information about the character and grade of the intrahepatic immune response in viral hepatitis is important for the evaluation of disease stage and effect of therapy. Complications like haemorrhage limit the frequent performance of tissue-needle biopsies (TB), and the cells of peripheral blood have to be used as surrogate markers instead. Fine-needle-aspiration biopsy (FNAB) of the liver represents a safe and atraumatic method that allows frequent cytological sampling. Our aim was to investigate whether flow cytometry of FNAB specimens allows co-analysis of phenotype, function and specificity of key populations of liver-infiltrating lymphocytes (LIL). In 20 consecutive patients with chronic viral hepatitis [10 hepatitis B virus (HBV), 10 hepatitis C virus (HCV)], flow cytometry was performed on FNAB cytology, and simultaneously on lymphocytes isolated from a TB and peripheral blood mononuclear cells (PBMC). The ratio of CD8+/CD4+ lymphocytes in FNAB correlated well with LIL from TB (r =0.78, P < 0.05) but differed from PBMC (mean ratio: 2.6, 2.1 and 0.7, respectively). Similarly, a correlation was observed for percentage CD56+ natural killer (NK) cells (mean %: 29.9, 32.3 and 14.5, respectively; r = 0.69, P < 0.05). The percentage of interferon (IFN)-gamma-producing CD3+ lymphocytes in both FNAB and TB was higher than in PBMC (mean %: 41, 44 and 22, respectively; P < 0.05). Furthermore, tetrameric complexes allowed analysis of HBV-specific T cells in FNAB specimens. In conclusion, flow cytometry of FNAB allows easy, atraumatic and reliable analysis of lymphocytes obtained from the intrahepatic compartment. Therefore, the FNAB is a valuable tool in the study of the immunopathology of viral hepatitis, and it may contribute to the improved clinical evaluation of chronic viral liver disease.     

Peripheral T-lymphocyte subpopulations in different clinical stages of chronic HBV infection correlate with HBV load

AIM： To characterize the peripheral T-cell subpopulation profiles and their correlation with hepatitis B virus （HBV） replication in different dinical stages of chronic HBV infection.
METHODS： A total of 422 patients with chronic HBV infection were enrolled in this study. The patients were divided into three stages： immune-tolerant stage, immune active stage, and immune-inactive carrier stage. Composition of peripheral T-cell subpopulations was determined by flow cytometry. HBV markers were detected by enzyme-linked immunosorbent assay. Serum HBV DNA load was assessed by quantitative real-time poiymerase chain reaction.
RESULTS： CD8^＋ T-cells were significantly higher in patients at the immune-tolerant stage than in patients at the immune-active and -inactive carrier stages （36.87 ± 7.58 vs 34.37 ± 9.07, 36.87 ± 7.58 vs 28.09 ± 5.64, P 〈 0.001）. The peripheral blood in patients at the immune-tolerant and immune active stages contained more CD8^＋ T-cells than CD4^＋ T-cells （36.87 ± 7.58 vs 30.23 ± 6.35, 34.37 ± 9.07 vs 30.92 ± 7.40, P 〈 0.01）, whereas the peripheral blood in patients at the immune- inactive carrier stage and in normal controls contained less CD8^＋ T-cells than CD4^＋ T-cells （28.09 ± 5.64 vs 36.85 ±6.06, 24.02 ± 4.35 vs 38.94 ± 3.39, P 〈 0.01）. ANOVA linear trend test showed that CD8^＋ T-cells were significantly increased in patients with a high viral load （39.41 ± 7.36, 33.83 ± 7.50, 31.81 ± 5.95 and 26.89 ± 5.71, P 〈 0.001）, while CD4^＋ T-cells were significantly increased in patients with a low HBV DNA load （37.45 ± 6.24, 33.33 ± 5.61, 31.58 ± 6.99 and 27.56 ± 5.49, P 〈 0.001）. Nultiple regression analysis displayed that log copies of HBV DNA still maintained its highly significant coefficients for T-cell subpopulations, and was the strongest predictors for variations in CD3^＋, CD4^＋ and CD8^＋ cells and CD4^＋/CD8^＋ ratio after adjustment for age at HBV-infection, maternal HBV-infection status, presence of hepatitis B e antigen and HBV mutation.
CONCLUSION： Differences in peripheral T-cell subpopulation profiles can be found in different clinical stages of chronic HBV infection. T-cell impairment is significantly associated with HBV load.     

The role of the virus specific T-cell response in acute and chronic HBV and HCV infection

Infections with hepatitis B (HBV) and hepatitis C virus (HCV) are worldwide one of the most frequent causes for chronic liver disease, liver cirrhosis and hepatocellular carcinoma. The mechanisms responsible for the elimination or the persistence of the virus are not well understood. The immunopathogenesis of HBV and HCV infection is primarily mediated by virus specific CD4+- and CD8+-T-cells. During acute infection a strong and multispecific T-cell response against different viral epitopes can be detected and is associated with the clearance of the virus. In case of viral persistence virus specific T-cells contribute to liver inflammation. In this article we summarize the current concepts about the role of the virus specific T-cell response in acute and chronic HBV and HCV infection.     

Kinetics of peripheral hepatitis B virus-specific CD8+ T cells in patients with onset of viral reactivation

Background

Patients with resolved hepatitis B virus (HBV) infection undergoing chemotherapy or immunosuppressive therapy are potentially at risk of HBV reactivation. However, it remains unclear how liver disease develops after HBV reactivation. To compare the host immune response against HBV, we performed immunological analyses of six HBV reactivation patients.

Methods

The numbers of peripheral HBV-specific CD8⁺ T cells were investigated longitudinally in six HLA-A2- and/or A24-positive patients with HBV reactivation. In addition, 34 patients with resolved HBV, 17 patients with inactive chronic hepatitis B (ICHB), 17 patients with chronic hepatitis B (CHB) and 12 healthy controls were analyzed. The number and function of HBV-specific CD8⁺ T cells were assessed by flow cytometry using tetramer staining and intracellular IFN-γ production. Furthermore, the numbers of CD4⁺CD25⁺ or CD4⁺Foxp3⁺ T cells and serum inflammatory cytokine levels were analyzed.

Results

The frequency of HBV-specific CD8⁺ T cells was significantly increased in HBV reactivation patients compared with ICHB and CHB patients. In addition, the number of HBV-specific CD8⁺ T cells was increased in resolved HBV patients compared with ICHB patients. PD-1 expression was decreased in HBV reactivation patients compared with ICHB and CHB patients. The numbers of HBV-specific CD8⁺ T cells and CD4⁺CD25⁺ or CD4⁺Foxp3⁺ T cells were negatively correlated following onset of HBV reactivation.

Conclusions

During HBV reactivation, the frequency of HBV-specific CD8⁺ T cells increased even though the administration of immunosuppressive drugs and interactions with CD4⁺ regulatory T cells may be important for the onset of liver disease.     

Tracking the source of the hepatitis B virus-specific CD8 T cells during lamivudine treatment

Lamivudine treatment in chronic hepatitis B leads to the reconstitution of virus-specific T cells in the circulation, but it is not clear whether this is the preferential result of T cell efflux from the liver or lymph nodes. To address this question, the frequency and function of liver-, lymph node-, and blood-derived hepatitis B virus (HBV)-specific CD8 T cells were analyzed in patients treated with lamivudine and undergoing liver transplantation. HBV-specific CD8 T cells, identified in portal lymph nodes, were able to expand in vitro after antigen-specific stimulation and displayed a heterogeneous profile of cytokine production. These findings suggest that the peripherally reconstituted HBV-specific CD8 T cells can originate from precursor cells within lymph nodes.     

Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B

BACKGROUND/AIMS: Lamivudine therapy in patients with chronic hepatitis B can induce the recovery of antiviral T cell responses. It is unknown whether the recovery of T cell responsiveness is long-lasting and persists throughout the treatment and whether the elevation of viremia which follows therapy withdrawal can restore a condition of T cell unresponsiveness. METHODS: Frequency and function of circulating hepatitis B virus (HBV)-specific CD4 and CD8 cells from 12 hepatitis e surface antigen + patients with chronic hepatitis B were studied longitudinally before, during and after lamivudine therapy by intracellular cytokine staining, proliferation and cytotoxicity assays against HBV proteins and peptides. CD4-mediated responses were analyzed in all patients, whereas CD8 cells were studied in 6 HLA-A2+ patients. RESULTS: HBV-specific CD4 and CD8 reactivity showed a bi-phasic behavior under lamivudine therapy with an early enhancement of T cell frequency and intensity of responses followed by a persistent decline starting from the 5th to 6th month of treatment. CONCLUSIONS: Since restoration of HBV-specific T cell reactivity is only transient, our study indicates that therapeutic stimulation of HBV-specific T cell responses to complement lamivudine treatment should be done early after the initiation of lamivudine. Moreover, the transient nature of the immune reconstitution may represent a favorable condition for virus reactivation once lamivudine therapy is withdrawn.     

Role of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-Prone CD8 T cells in persistent hepatitis B virus infection

An excess of coinhibitory signals has been proposed to drive the T-cell exhaustion characteristic of persistent viral infections. In this study we examined the contribution of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) to CD8 T cell tolerance in chronic hepatitis B virus (HBV) infection (CHB). CD8 T cells in patients with CHB have an increased propensity to express the coinhibitory receptor CTLA-4 and this correlates with viral load. CTLA-4 is up-regulated on those HBV-specific CD8 T cells with the highest levels of the proapoptotic protein Bim, which we have previously shown mediates their premature attrition; abrogation of CTLA-4-mediated coinhibition can reduce Bim expression. Longitudinal study of CHB patients beginning antiviral therapy reveals that HBV DNA suppression induces transient reconstitution of HBV-specific CD8 T cells but does not reprogram their CTLA-4(hi) Bim(hi) tolerogenic phenotype. Blocking CTLA-4 is able to increase the expansion of interferon gamma (IFN-γ)-producing HBV-specific CD8 T cells in both the peripheral and intrahepatic compartments. The rescue of anti-HBV responses by either CTLA-4 or PD-L1 blockade is nonredundant. CONCLUSION: CTLA-4 is expressed by HBV-specific CD8 T cells with high levels of Bim and helps to drive this proapoptotic phenotype. CTLA-4 blockade could form one arm of a therapeutic approach to modulate the diverse patterns of coregulation of T-cell exhaustion in this heterogeneous disease.     

Antiplatelet therapy prevents hepatocellular carcinoma and improves survival in a mouse model of chronic hepatitis B

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis of HBV-associated HCC involves both viral and host factors. The latter include a functionally inefficient CD8(+) T-cell response that fails to clear the infection from the liver but sustains a chronic necroinflammatory process that contributes to the development of HCC. According to this scenario, amelioration of immune-mediated chronic liver injury may prevent HCC. Because platelets facilitate immune-mediated liver injury by promoting the hepatic accumulation of virus-specific CD8(+) T cells, we evaluated the long-term consequences of antiplatelet therapy in an HBV transgenic mouse model of chronic immune-mediated necroinflammatory liver disease that progresses to HCC. Treatment with aspirin and clopidogrel during the chronic phase of the disease diminished the number of intrahepatic HBV-specific CD8(+) T cells and HBV-nonspecific inflammatory cells, the severity of liver fibrosis, and the development of HCC. Antiplatelet therapy improved overall survival without causing significant side effects. In contrast, the same antiplatelet regimen had no antitumor effect when HCC was induced nonimmunologically by chronic exposure to a hepatotoxic chemical. The unprecedented observation that antiplatelet therapy inhibits or delays immune-mediated hepatocarcinogenesis suggests that platelets may be key players in the pathogenesis of HBV-associated liver cancer and supports the notion that immune-mediated necroinflammatory reactions are an important cause of hepatocellular transformation during chronic hepatitis.     

乙型肝炎病毒感染临床转归与宿主细胞免疫的相关性研究

目的 比较慢性乙型肝炎患者、HBV携带者、HBV既往无症状感染者之间T细胞亚群和HBV特异性CD4+ T细胞应答强度的差异,分析宿主的细胞免疫状态对HBV感染后临床转归的影响,探讨乙型肝炎的发病机制,为慢性乙型肝炎的治疗提供新的线索.方法 选取2004年2-10月在北京协和医院肝炎门诊就诊的慢性乙型肝炎患者30例、HBV携带者22例、HBV既往无症状感染者9例以及正常对照11例,使用流式细胞仪检测其T细胞亚群,使用酶联免疫斑点法检测病毒特异性CD4+T细胞应答强度,分析其差异及临床意义.结果 慢性乙型肝炎组CD4+T细胞计数显著低于HBV既往无症状感染组和正常对照组;慢性乙型肝炎组、HBV携带者组、HBV既往无症状感染组病毒特异性CD4+ T细胞应答强度分别为(156±105)、(56±68)、(229±114)SFC/106PBMC(每106个外周血单个核细胞中斑点形成细胞的个数);慢性乙型肝炎组明显高于HBV携带者组(P＜0.01),而低于HBV既往无症状感染组(P＜0.05).结论 慢性乙型肝炎患者、HBV携带者、HBV既往无症状感染者病毒特异性T细胞应答强度存在差异,这种差异可能是造成HBV感染后不同临床转归的主要因素之一.