Comprehensive analysis of the HCV-specific CD8+ T cell response in individuals with chronic or resolved HCV infection

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AASLD ABSTRACTS

HEPATOLOGYOctober 2001

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COMPREHENSIVE ANALYSIS OF THE HCV-SPECIFIC C D 8 + T CELL RESPONSE IN 1 N D M D U A L S W I T H CHRONIC OR RESOLVED HCV INFECTION. Georg M Lauer, Tam N Nguyen, Cheryl L Day, Massachusetts Gen

HEPATITIS C VIRUS CORE PROTEIN INHIBITS APOPTOSIS VIA ENHANCEMENT OF BCL-XL EXPRESSION. Motoyuki Otsuka, Naoya Kato,

Hosp/Harvard Medical School, Charlestown, MA; Paul Klenerman, John Radcliffe Hospital, Oxford Uk; Ray T Chung, Massachusetts Gen Hosp/Harvard Medical School, Boston, MA; Bruce D Walker, Massachusetts Gen Hosp/Harvard Medical School, Charlestown, MA It has been shown that spontaneous resolution of acute HCV infection is associated with the presence of a vigorous and broadly directed cellular immune response. While it has been widely accepted that the CD4+ T helper cell response persists for a long time after the virus has been cleared, the fate of the CD8 + mediated response is less clear. Since most studies so far have analyzed the CD8+ T cell response to only a limited number of epitopes, we addressed this with a more comprehensive analysis for the detection of HCV-specific T cells. Methods: Twenty individuals were included in the study. Ten individuals were chronically infected with HCV, whereas ten were anti-HCV positive, but HCV RNA negative for at least 2 years. We analyzed the CD8+ cellular immune response in PBMC with an Elispot assay using peptides representing all defined HCV CTL epitopes as well as 300 20mer overlapping peptides covering the entire HCV genome. Positive responses in the Elispot were confirmed to be CD8+ or CD4+ responses by depletion experiments and by using truncated peptides. The CD8 + mediated immune response was also analyzed using class I tetramers. Results: We found HCV-specific CD8+ T cell responses in 3 out of 10 individuals with chronic HCV infection and in 8 out of 10 individuals with resolved infection. Individuals with chronic HCV infection recognized between 0 and 3 HCV epitopes (mean 0.7) and HCV RNA negative individuals between 0 and 5 epitopes (mean 1.8). The magnitude of the response against a single HCV epitope ranged between 40 to 150 SFC/106 PBMC in most individuals. Interestingly, the 2 HCV RNA negative individuals who recognized 4 and 5 epitopes respectively had much stronger responses to each of the peptides (up to 800 SFC/106 PBMC). Tetramer analysis gave concordant results in the HLA-A2 positive individuals. Conclusions: By mapping the complete HCVspecific CD8+ T cell response we found a broader and more vigorous response in individuals who cleared HCV in contrast to those with chronic HCV infection. Importantly, we show that some individuals who clear HCV infection maintain a strikingly broad and vigorous CD8+ T cell response over many years. More studies are under way to fully understand the true impact and the kinetics of the CD8+ T cell response during HCV infection.

Hiroyoshi Tanignchi, Tadashi Goto, Hideo Yoshida, Jun Kato, Yujin Hoshida, Masaru Moriyama, Yasushi Shiratori, Masao Omata, Department of Gastroenterology, Graduate Shcool of Medicine, University of Tokyo, Tokyo Japan Objectives: Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis and hepatocellular carcinoma. Previous studies suggested that core protein influences cell apoptosis, but those studies have not demonstrated the precise or direct mechanisms of the effects by core protein on apoptosis. Therefore, we investigated at which level core protein influences apoptotic pathway to clarify the underlying mechanisms. Methods: 1) To determine at which level core protein affect the apoptotic pathway, activation of caspases after anti-Fas treatment, in transiently transfected and magnetically concentrated core-producing HepG2 cells, were examined by Western blotting. 2) Using RNase protection assay, the mRNA levels of Bcl-2-related genes in core-producing HepG2 cells were investigated. 3) The effect of HCV proteins on bcl-x promoter was examined by a reporter assay. 4) By use of various inhibitors, dominant negative vectors, or deletion constructs, which signaling pathway might be involved in the effect of core protein on bcl-x promoter was investigated. Results: Core protein inhibited apoptosis at the downstream of caspase 8 and upstream of caspase 3 in the apoptotic cascade. The amount of mRNA of bcl-xl and mcll, which are known to inhibit apoptosis, increased in coreproducing cells. However, none of the other members of the bcl family tested was affected by core protein expression. Reporter assays revealed that core protein augmented the bcl-x promoter activity. This augmentation was inhibited by blocking MAPK pathway. Conclusions: Core protein enhanced the expression of Bcl-xL via MAPK pathway, and this was associated with inhibition of procaspase 3 activation. Core protein mediated Bcl-xL induction may be one of the underlying mechanisms of its inhibition of apoptosis.

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HIGH AND LONG-LASTING FREQUENCY OF CIRCULATING C D 8 + T LYMPHOCYTES IS ASSOCIATED W I T H CONTROL OF HCV INFECTION. Simona Urbani, Carolina Boni, Azienda Hosp di Parma, Parma Italy;

HCV CORE PROTEIN EXPRESSION IN HUMAN B CELL LINES DOES NOT SIGNIFICANTLY MODIFY PROLIFERATIVE AND APOPTOSIS PATHWAYS. Carlo Giannini, INSERM U-370 Pasteur-Necker Institutes and

Jacopo Uggeri, Azienda Hosp di Parma, Parma Itlay; Monica Malpeli, Gabriele Missale, Simona Cerioni, Carlo Ferrari, Azienda Hosp di Parma, Parma Italy The study of the immune response during the acute phase of hepatitis C virus infection is essential to define the early events that are likely to influence the subsequent course and outcome of the associated liver disease. Still limited is the information about the specificity and the kinetics of the immune response during this critical stage of the disease since acute hepatitis C is generally asymptomatic. In this study, we analyzed the frequency of HCV-specific CD8 + T cells longitudinally from the onset of the disease to one year of follow-up in eleven acute patients; three HLA-A2 tetramers containing the HLA-A2 restricted cytotoxic T lymphocyte (CTL) epitopes NS3 1073-1081, NS3 14061415, NS5 1992-2000 were used. Seven patients developed chronic HCV infection; three patients had a self-limited acute hepatitis with persistently normal ALT and negative HCV-RNA; an additional patient was treated with IFN-a for six months with complete and persistent resolution of the hepatitis. While cytotoxic T cells specific for the three HCV non-structural epitopes were detectable in the peripheral blood of all the patients who resolved the disease, only in three of the patients who developed a chronic infection tetramer positive cells were measurable. The frequency of tetramer+ CD8 cells was extremely high during the acute phase of hepatitis in patient who resolved the infection (frequencies ranging from 1.3% to 4.7% of total CD8+ lymphocytes) compared to the frequency of those who developed chronic hepatitis (frequencies from 0.1% to 0,5%). Patients with a self-limited hepatitis conserved an high frequency (ranging from 0.1% to 0,3%) of tetramer positive cells up to 120 weeks from the onset and displayed persistent cytolytic activity in vitro, while in patients with a chronic evolution of the disease HCV-specific cytotoxic T cells became undetectable few weeks after the acute phase of infection. The present study indicate that vigorous and long-lasting CTL responses focused on immunodominant epitopes are associated with an efficient and persistent control of HCV infection. In contrast, weak and transient CTL responses are associated with chronic evolution of hepatitis.

Department Internal Medicine, University of Florence, Paris France; Patrizio Caini, Francesca Giannelli, Department of Internal Medicine, University of Florence, Florence Italy; Francesca Fontana, Nadira Delhem, Christian Brechot, INSERM U-370 Pasteur-Necker Insts, Paris France; Anna Linda Zignego, Department of Internal Medicine, University of Florence, Florence Italy Hepatitis C virus has been shown to infect lymphoid cell, preferentially B-lymphocytes. In fact, HCV chronic infection has been associated with lymphoproliferative disorders (mixed cryoglobulinaemia and non-Hodking's lymphoma). Several studies performed on hepatoma and fibroblast cell lines suggest a role of HCV core protein in the activation of cellular transduction pathways leading to cell proliferation and inhibition of programmed cell death. No data are however available in literature concerning the effects of HCV core expression on B-lymphocyte proliferation and apoptosis. Methods and Results: We established B-lymphocyte cell lines permanently expressing HCV core protein using B-ceUlines with different degrees of differentiation: WIL2-ns, a well differentiated B-lymphoblastoid cell line, Ramos and BL-2, two transformed, Burkitt's lymphoma derived, EBV negative cell lines. We cloned different HCV lb core full-length sequences (CJ-NT, CJ-T, C191) isolated from chronicany infected patients into mammalian expression vectors. B-cell clones and pool of clones permanent expressing HCV core were selected and characterized for protein expression by Western blot and immunofluorescence. Expression levels were constant all along the study and resulted higher in WIL-core cell lines in respect to Ramos-core and BL-2-core cell lines. Expression of HCV core proteins did not show to significantly enhance cell proliferation rate in normal culture condition. Under mitogenic sfimulation (PMA, PWM, LPS, SAC), B-cells stably expressing core protein showed a slight increase of cell proliferation when compared to neo-clones. Analysis of SRE, TRE, CRE, NF-kB pathways by luciferase reporter genes did not show a significant influence of HCV core expression on these signal transduction cascades in B-lymphocytes. Since lymphoproliferation could be originated, not only by a sustained proliferation, but also by a reduced clearance of B-cells populations, we analysed the effects of HCV core on anti-lgM and FAS induced apoptosis in B-cell lines. Due to a more differentiated phenotype, W1L- derived cell lines were less sensitive to anti-IgM induced apoptosis when compared to Ramos and BL-2. By contrast, agonist anti-FAS antibody treatment induced a massive apoptosis in all cell lines tested. In our experimental condition, HCV core expression did not significantly modify the apoptotic profile of B-lymphocyte cell lines tested. In conclusion, our data suggest a cell type specific effect of core protein expression. In fact, by transiently or stably expressing three different HCV lb core sequences (the most prevalent genotype in our regions) in three different B-cell lines we were not able to show a significant contribution of HCV core expression in the activation of major B-cell signal transduction pathways involved in regulation of proliferation and programmed cell death, in contrast with the results reported in hepatoma cell lines.