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of the immune reconstitution may represent a favorable condition for virus reactivation once lamivudine therapy is withdrawn.     

HLA class I-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen.

Knowledge of the immune effector mechanisms responsible for clearance of hepatitis B virus (HBV)-infected cells has been severely limited by the absence of reproducible systems to selectively expand and to characterize HBV-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of patients with viral hepatitis. By using a strategy involving sequential stimulation with HBV nucleocapsid synthetic peptides followed by autologous, or HLA class I-matched, HBV nucleocapsid transfectants, we now report the existence of CTLs able to lyse target cells that express endogenously synthesized HBV nucleocapsid antigen in the peripheral blood of patients with acute viral hepatitis B. The CTL response is HLA-A2 restricted, mediated by CD8-positive T cells, and specific for a single epitope, located between amino acid residues 11 and 27 of HBV core protein; these residues are shared with the secretable precore-derived hepatitis B e antigen. Equivalent lysis of target cells that express each of these proteins suggests that their intracellular trafficking pathways may intersect. The current report provides definitive evidence that HLA class I-restricted, CD8-positive CTLs that recognize endogenously synthesized HBV nucleocapsid antigen are induced during acute HBV infection in humans and establishes a strategy that should permit a detailed analysis of the role played by HBV-specific CTLs in the immunopathogenesis of viral hepatitis.     

Cytotoxic T lymphocyte responses and CTL epitope escape mutation in HBsAg, anti-HBe positive individuals

BACKGROUND/AIMS: Clearance of hepatitis B virus (HBV) is characterised by a strong cytotoxic T cell response. Persistence of HBV in chronic hepatitis B carriers may be related to failure of this response. The aim of this study was to determine whether HLA class I restricted cytotoxic T lymphocyte (CTL) responses persist in anti-hepatitis B e (HBe) positive / HBV DNA negative individuals, and to correlate the presence of viral CTL epitope mutation with clinical outcome. METHODS: An HLA/HBV dual transfectant model was used to demonstrate these CTL responses in individuals chronically infected with HBV. Subsequently, a known hepatitis B core (HBc) CTL epitope was sequenced in a family of five chronically infected individuals all sharing a HLA allele (HLA-A68.1). RESULTS: Low level HLA class I restricted cytotoxic T cell responses were detected in the peripheral blood of five of eight anti-HBe positive individuals. In the family of HLA-A68.1 positive chronically infected individuals, mutation of the HLA-A68.1 restricted hepatitis B core antigen (HBcAg) CTL epitope STLPETTVVRR was found in all four anti-HBe positive individuals but not in the sole hepatitis B e antigen (HBeAg) positive patient. CONCLUSION: These data are consistent with a continued immune selection pressure on HBV in anti-HBe positive chronically infected individuals with low replicating HBV infection and suggest that mutation of a CTL epitope may be a consequence of the immune response, as opposed to the cause of viral persistence.     

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.     

Therapeutic polypeptides based on HBV core 18-27 epitope can induce CD8^＋ CTL-mediated cytotoxicity in HLA-A2^＋ human PBMCs

AIM: To explore how to improve the immunogenicity of HBcAg CTL epitope based polypeptides and to trigger an HBV-specific HLA I-restricted CD8^+ T cell response in vitro.METHODS: A new panel of mimetic therapeutic peptides based on the immunodominant B cell epitope of HBV PreS2 18-24 region, the CTL epitope of HBcAg18-27 and the universal T helper epitope of tetanus toxoid (TT) 830-843 was designed using computerized molecular design method and synthesized by Merrifield‘s solid-phase peptide synthesis.Their immunological properties of stimulating activation and proliferation of lymphocytes, of inducing TN1 polarization,CD8^+ T cell magnification and HBV-specific CD8^+ CTL mediated cytotoxicity were investigated in vitro using HLA-A2^+ human peripheral blood mononuclear cells (PBMCs) from healthy donors and chronic hepatitis B patients.RESULTS: Results demonstrated that the therapeutic polypeptides based on immunodominant HBcAg18-27 CTL,PreS2 B- and universal TN epitopes could stimulate the activation and proliferation of lymphocytes, induce specifically and effectively CD8+ T cell expansion and vigorous HBVspecific CTL-mediated cytotoxicity in human PBMCs.CONCLUSION: It indicated that the introduction of immunodominant T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of short CTL epitopes in vitro.     

Quantitative and functional analysis of core-specific T-helper cell and CTL activities in acute and chronic hepatitis B

Abstract: Aims/Background: CD4+ T-helper cell (Th) responses to hepatitis B virus (HBV) core antigen (HBc) are increased during exacerbations in acute and chronic hepatitis B (AHB, CHB) and might influence the induction of CD8+ cytotoxic T lymphocytes (CTL) that are important for viral clearance. Methods: HBc-specific proliferative responses and cytokine release of blood mononuclear cells (PBMC) were studied in patients with AHB or CHB, as well as responders and non-responders to interferon-α treatment (IFN-R, IFN-NR), by [³H]-thymidine-uptake, enzyme-linked immunosorbent assay (ELISA) and Elispot assay and were compared to peptide HBc18–27-specific CTL precursor frequencies among CD8+ T cells derived from HLA-A2+ patients. Results: HBc-specific proliferative PBMC responses and Th frequencies were significantly increased in AHB patients compared with untreated CHB patients. PBMC derived from IFN-R showed stronger cellular responses than IFN-NR. Stimulated PBMC from all patient groups secreted significantly more IFN-γ than IL-4 indicating Th1/Th0 cell responses. Furthermore, in AHB and IFN-R patients, high peptide HBc18–27-specific CTL precursor frequencies closely correlated with strong HBc-specific Th responses, whereas in untreated CHB and IFN-NR patients lower CTL frequencies were observed without correlation to Th activities. Conclusions: HBV core-specific Th-cell responses appeared to support efficient CTL induction in patients with viral clearance, whereas in chronic HBV carriers quantitatively insufficient Th and CTL responses were observed. This observation could be important for future therapeutic strategies.     

Immunopathogenesis of hepatitis B persistent infection: implications for immunotherapeutic strategies

It has been shown that cellular immunity, especially by cytotoxic T lymphocytes (CTLs), NK cells and NK-T cells, plays a central role in the control of virus infection. In addition, CD4+ T cells facilitate both CTL and B-cell responses. Hyporesponsiveness of HBV-specific T cells in peripheral blood has been shown in patients with chronic HBV infection. Interferon and nucleos(t)ide analogs, such as lamivudine, adefovir, entecavir and tenofovir, are the currently available treatments. Unfortunately, the efficacy of nucleos(t)ide analogs is limited by viral reactivation by the emergence of escaped mutants in cases of prolonged treatment. Therefore, immunotherapy is one of the significant options to eradicate or control HBV replication without drugs. The aim of immunotherapies is to decrease the levels of viral replication and to eradicate infected hepatocytes. For this reason, new strategies for immunotherapies by vaccination target not only the induction or stimulation of CD4+ and CD8+ T cell responses, but also the induction of proinflammatory cytokines capable of controlling viral replication. We will review the immunopathogenesis of persistent HBV infection, especially focusing on the mechanisms of immune suppression. Then we will review the immunotherapy for HBV persistent infection.     

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.     

Effect of HIV infection and antiretroviral therapy on hepatitis B virus (HBV)-specific T cell responses in patients who have resolved HBV infection

Coinfection with hepatitis B virus (HBV) is a common occurrence in human immunodeficiency virus (HIV)-positive patients and an increasing cause of morbidity and mortality. The CD8(+) T cell response is critical for long-term control of HBV in patients resolving acute infection. Here, we examine the effect of HIV on HBV-specific CD8(+) T cell responses in patients who have resolved HBV infection. A cross-sectional study showed a reduction in HBV-specific CD8(+) T cell responses in HIV-positive, HBV-immune patients, compared with those in HIV-negative, HBV-immune patients. A longitudinal study of a subgroup of patients examined whether this attrition could be reversed by effective antiretroviral therapy. The introduction of highly active antiretroviral therapy (HAART) resulted in reconstitution of some HBV-specific CD4(+) and CD8(+) T cell responses, in association with restoration of CD4(+) T cell counts. These data provide a mechanism to account for the observed impairment of control of HBV infection in the setting of HIV infection and support the ability of HAART to reconstitute functionally active T cell responses.     

结核杆菌热休克蛋白质70作为乙型肝炎核心抗原细胞毒性T淋巴细胞表位肽载体的研究

目的探讨结核杆菌热休克蛋白质70（TB．HSP70）作为乙型肝炎病毒（HBV）核心抗原细胞毒性T淋巴细胞（CTL）表位肽载体，诱导HBV特异性免疫应答的可能性。方法体外观察重组TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物诱导慢性乙型肝炎患者外周血淋巴细胞增殖以及HBV特异性细胞毒活性；以Balb／c小鼠为体内研究对象，行流式细胞术分析免疫后小鼠外周血和脾细胞中CD4^＋与CD8^＋T淋巴细胞及自然杀伤（NK）细胞的比率，并观察能否诱导HBV特异性免疫保护作用。结果体内外研究表明TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物能有效地诱导HBV特异性细胞毒活性，体内能激活CD4^＋与CD8^＋T淋巴细胞及NK细胞增殖。在体内，TB．HSP70-CTL融合蛋白质较复合物能更有效地激活免疫应答，其杀伤率为28．9％。CD8^＋T淋巴细胞在脾细胞中比率为43．9％，NK细胞为13．6％。而单纯的TB．HSP70和CTL表位肽并不能有效地引起机体的免疫应答。结论TB．HSP70能够作为乙型肝炎核心抗原CTL表位肽的载体，提高小分子表位肽的免疫原性。     

A longitudinal analysis of cytotoxic T lymphocyte precursor frequencies to the hepatitis B virus in chronically infected patients

Individuals with acute hepatitis B virus (HBV) infection characteristically mount a strong, multispecific cytotoxic T lymphocyte (CTL) response that is effective in eradicating virus. In contrast, this response in chronic carriers is usually weak or undetectable. Since it is generally acknowledged that HBV pathogenesis is immune-mediated, the occurrence of episodes of active liver disease in many carriers suggests that these individuals can mount active CTL responses to HBV. To see whether the detection of circulating CTLs is related to these flare episodes, we have determined the CTL precursor (CTLp) frequencies to HLA-A2-restricted viral peptides in seven patients over a 12-24-month period of their disease. Limiting dilution analyses (LDA) were performed longitudinally to five epitopes comprising the viral capsid (HBc), envelope (HBs) and polymerase (pol) proteins. Assays were performed against a mixture of peptides, or against each individual peptide, to measure overall CTL activity and the multispecificity of the responses, respectively. Since two of the patients were treated with recombinant human interleukin-12 (rHuIL-12) at the time, with one individual achieving complete disease remission a year later after being treated with interferon-alpha, we were also able to examine the effects of these cytokines on HBV cytotoxicity. Our results indicate that weak but detectable CTL responses do occur in chronic carriers which are generally associated with disease flares, although CTLps were also seen occasionally during minimal disease activity. The range of specificities varied between individuals and within each individual during the course of the disease. Finally, we also provide evidence that CTL reactivity is stimulated following treatment with certain cytokines, but is dependent on the time of administration.     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

The impact of HBV-DNA fluctuations on virus-specific CD8+ T cells in HBeAg+ chronic hepatitis B patients treated with a steroid and lamivudine

Restoration of anti-viral immune response may be a requisite for sustained virological response to treatment in chronic hepatitis B patients. Over a 13-month period, we examined the dynamics of hepatitis B virus (HBV)-specific CD8+ cells in six human leucocyte antigen (HLA)-A2+ hepatitis B e antigen (HBeAg)+ 'immunotolerant' chronic hepatitis B patients treated sequentially with corticosteroid and lamivudine. Our results show that the combination treatment did not result in a sustained restoration of anti-viral specific CD8+ cells in five of the six patients studied. However, HBV-specific CD8+ cells, despite being severely compromised, were not totally deleted. Paradoxically, steroid treatment was not associated with inhibition but with a minimal increase of the HBV-specific CD8 response, and we observed that nucleocapsid-specific CD8 responses were not rescued by stable and prolonged inhibition but became detectable after rapid rebounds of HBV replication. In most patients, the transient and minimal restoration of HBV-specific immunity was not associated with clinical benefits. Our results describe a dynamic relationship between HBV-specific CD8+ cells and HBV-DNA values, that could potentially be used for a better design of HBV treatment in HBeAg+ 'immunotolerant' chronic hepatitis B patients.     

The viral clearance in interferon-treated chronic hepatitis C is associated with increased cytotoxic T cell frequencies.

BACKGROUND/AIMS: Cytotoxic T lymphocytes have been demonstrated in peripheral blood and liver tissue of patients with chronic hepatitis C virus infection, but their significance for viral clearance is unknown. Therefore, we analyzed hepatitis C virus-specific cytotoxic T lymphocyte precursor frequencies in chronic hepatitis C virus carriers during interferon-alpha treatment. METHODS: Blood mononuclear cells or CD8+ T cells from HLA-A2 positive and negative patients and controls were analyzed in chromium-release assays using a panel of 18 synthetic peptides from the HCV core, E1 and NS4 antigens bearing HLA-A2 binding motifs. Specific cytotoxic T lymphocyte precursor frequencies were studied within CD8+ T cells derived from interferon-alpha-treated patients using a TNF-alpha-based ELISPOT assay and compared to viremia levels. RESULTS: T cells from 16 of 24 HLA-A2+ but none of the six HLA-A2- patients with chronic hepatitis C and six HLA-A2+ healthy controls lysed targets pulsed with peptide cocktails. Fine specificity revealed four very immunogenic epitopes in the core (C36-44, C132-140) and the envelope regions (E332-340, E363-372). Cytotoxic T lymphocyte precursor frequencies were prospectively analyzed in 11 interferon-alpha-treated HLA-A2+ hepatitis C virus patients. Four sustained and two transient therapy responders showed lower pretreatment viremia levels and significantly higher specific cytotoxic T lymphocyte precursor frequencies during viral clearance compared to five therapy non-responders and untreated controls. CONCLUSIONS: The quantitative induction of HLA-class I restricted responses by interferon-alpha could contribute to a beneficial outcome of hepatitis C virus infections. Furthermore, it appears that the balance between viral load and specific cellular immune responses is critical for successful viral clearance.     

6 Immunopathogenesis of viral hepatitis

More than 500 million people world-wide suffer from viral hepatitis which can be caused by a variety of distinct infectious agents. The spectrum of disease, which ranges from acute self-limited hepatitis to liver cirrhosis, not only reflects the different biological properties and pathogenicity of the hepatitis viruses, but is also the result of the specific interaction between each virus and the immune system of the infected host. The immune response plays a crucial role in the elimination of the infecting virus as well as in disease pathogenesis and is described in detail for acute and chronic hepatitis B and C virus infection. Acute hepatitis B virus infection is characterized by a vigorous, polyclonal cytotoxic T lymphocyte response against HBV that is not readily detectable in patients with chronic hepatitis B, suggesting that resolution of disease is mediated by the HBV-specific CTL response in these patients. Because traces of virus as well as HBVspecific CTL can perist for decades after clinical recovery, continuous priming of new CTL by minute traces of virus is thought to protect from reactivation of disease. In contrast, the hepatitis C virus causes chronic liver disease despite a polyclonal and multispecific immune response, suggesting that distinct immunological and viral mechanisms determine the different clinical outcome of HBV and HCV infection. Their implications for the development of immunomodulatory vaccines to cure patients with chronic viral hepatitis are discussed.     

Therapeutic polypeptides based on HBcAg18-27 CTL epitope can induce antigen-specific CD8＋ CTL-mediated cytotoxicity in HLA-A2 transgenic mice

AIM: To explore how to trigger an HLAI-restricted CD8(+) T cell response to exogenously synthesized polypeptides in vivo. METHODS: Three mimetic therapeutic polypeptides based on the immunodominant CTL epitope of HBcAg, the B- epitope of HBV PreS(2) region and a common T helper sequence of tetanus toxoid were designed and synthesized with Merrifield's solid-phase peptide synthesis method. Their immunological properties of inducing T( H1) polarization, CD8(+) HBV-specific CTL expansion and CD8(+) T cell mediated cytotoxicity were investigated in HLA-A2 transgenic mice. RESULTS: Results demonstrated that the mimetic polypeptides comprised of the immunodominant CTL, B-, and T helper epitopes could trigger specifically and effectively vigorous CD8(+) HBV-specific CTL-mediated cytotoxicity and T(H1) polarization of T cells in HLA-A2 transgenic mice. CONCLUSION: A designed universal T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of CTL epitopes in vivo. And that the mimetic therapeutic peptides based on the reasonable match of the above CTL, B- and T helper epitopes could be a promising therapeutic peptide vaccine candidate against HBV infection.     

In Vivo Bioluminescence Imaging of HBV Replicating Hepatocytes Allows for the Monitoring of Anti-Viral Immunity

Immunity against hepatitis B virus (HBV) infection is complex and not entirely understood so far, including the decisive factors leading to the development of chronic hepatitis B. This lack of a mechanistic understanding of HBV-specific immunity is also caused by a limited number of suitable animal models. Here, we describe the generation of a recombinant adenovirus expressing an HBV 1.3-overlength genome linked to luciferase (Ad-HBV-Luc) allowing for precise analysis of the quantity of infected hepatocytes. This enables sensitive and close-meshed monitoring of HBV-specific CD8 T cells and the onset of anti-viral immunity in mice. A high dose of Ad-HBV-Luc developed into chronic hepatitis B accompanied by dysfunctional CD8 T cells characterized by high expression of PD1 and TOX and low expression of KLRG1 and GzmB. In contrast, a low dose of Ad-HBV-Luc infection resulted in acute hepatitis with CD8 T cell-mediated elimination of HBV-replicating hepatocytes associated with elevated sALT levels and increased numbers of cytotoxic HBV-specific CD8 T cells. Thus, the infectious dose was a critical factor to induce either acute self-limited or chronic HBV infection in mice. Taken together, the new Ad-HBV-Luc vector will allow for highly sensitive and time-resolved analysis of HBV-specific immune responses during acute and chronic infection.     

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.     

Hepatitis B virus-specific T-cell proliferation and cytokine secretion in chronic hepatitis B e antibody-positive patients treated with ribavirin and interferon alpha

Immune elimination of hepatitis B virus (HBV) during antiviral therapy depends on the activation of T-cell responses, which are generally impaired in chronic hepatitis B. HBV-specific T helper (Th)-cell reactivity has been assessed post-treatment in liver and peripheral blood of 18 anti-HBe-positive patients with chronic hepatitis B administered combined ribavirin/interferon alfa (IFN-alpha) therapy. The results showed that patients with undetectable HBV DNA by quantitative polymerase chain reaction under combination therapy were able to mount an HBV-specific CD4(+) Th-cell proliferative response and such T-cell reactivity is detectable 1 year after HBV DNA clearance. Hepatitis B virus core (HBcAg) and e (HBeAg) antigen-specific Th-cell proliferation was found more frequently in the liver and peripheral blood in those patients who sustained the alanine aminotransferase (ALT) normalization together with HBV DNA loss. However, HBV-specific IFN-gamma production in vitro in peripheral blood mononuclear cells augmented in 4 of 5 sustained responders and all 13 nonresponders, interleukin 10 (IL-10) production decreased in all 5 sustained responders but increased in 7 of 13 nonresponders. Furthermore, intrahepatic HBcAg plus HBeAg-specific Th-cell proliferation only occurred in sustained responders (2 of 3, 67%, vs. 0 of 9; P =.045) whose cells showed in vitro significantly increased productions in HBcAg/HBeAg-specific IFN-gamma and IL-12 compared with nonresponders in whom IFN-gamma and IL-12 productions decreased together with increased IL-10 secretion. In conclusion this study indicates that combined therapy with ribavirin and IFN-alpha for chronic hepatitis B not only significantly reduces viremia levels but also induces lasting CD4(+) T-cell proliferation and Th1 cytokine release at the site of infection, which may lead to sustained eradication of the HBV.