334A AASLD ABSTRACTS HEPATOLOGY October 1995

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COMPARATIVE ANALYSIS OF THE CYTOTOXIC T CELL RESPONSE IN CHRONIC HBV AND HCV INFECTION. B.. K.M. u L G. McHurcbison. F.V. Cbisarl, The Scripps Research Institute, La Jolla, CA, USA

Cytotoxic T cells (CTL) contribute to the clearance of many viral infections, including HBV. Previous studies have demonstrated, however, that HCV per- sists in the face of a n response, raising serious questions about tbe virolo- gical and patbogenetic significance of CTL in this infection. In some of those studies, however, HCV speciftt CTL were detected not only in the peripheral blood mononuclear cells f.PBMC) of most chronically infected patients, but they could also be detected in a small fraction of uninfected normal controls. This raised the possibility that the CTL response detected in the patients might reflect in vitro priming.

To clarify this issue, we have analyzed the C’IL response to HCV in infected patients and controls using methodology that we have found ti selectively de- tect CTL that have been primed in viva. PBMC from 23 patients with chronic hepatitis C and 19 uninfected controls were stimulated in 8 replicate microwell cuitures with synthetic peptides corresp$mding to 11 previously described highly conserved HLA-A2 restricted HCV epitopes. For comparison, 5 HBV- derived CTL epitopes were used to stimulate PBMC from 9 patients with chronic HBV infection, 2 patients with acute HBV infection, and 9 normal con- trols. The cultures were restimulated with autologous irradiated feeders and pcp- tide on days 7 and 14, fed with IL-2 every 3-4 days and tested for cytolytic acti- vity using a standard slCr release assay on day 21.

Twenty-two of the 23 HCV infected patients responded to at least one of the HCV core, HCV-El, NS3, NS4 and NS5 epitopes while 18 of the 19 uninfec- ted controls did not. The core and NS3 epitopes were mbst frequently recog- nized, and the epitope response pattern in each patient was remarkably stable for up to 27 months of follow-up. In contrast, none of the chronically HBV infected patients responded to any of the HBV epitopes which were readily recognized by CTL from patients with acute HBV infection.

In conclusion, chronic HCV infection is indeed characterized by a polyclonal, multispecific, class I restricted peripheral CTL response that reflects in viva priming by this virus. This Contrasts with the lack of a peripheral CTL re- sponse to HBV-derived epitopes in most patients with chronic HBV infection, suggesting qualitatively or quantitatively different immunological mechanisms responsible for viral persistence and chronic hepatitis in these two diseases.

Accumulation of HCV-core specific T cell clonotypes in patients with chronic hepatitis C and it3 reduction following eradication of HCV-RN A by antiviral tkatment. &shi Tanaka*#, Hiroto Kitall, Masao Takatorit, Shingo lwabuchit. Kivoshi Kurokawa#, Michio ImawariP. Kusuki Nishioka*, Shim line*, and Kazuhiko Yamamoto*.*lnst. of Med. Sci. and tSecond Dept. of lnt. Med., St. Marianna Univ., School of Medicine, Kanagawa, Japan; PLiver Studv Lab.. Jichi Univ., School of Medicine, Tochiei, Japan; and #First and vhird bpt. of lnt. Med., Univ. of Tokyo, Tokyo, Jaian. . Although T cell responses to HCV play important roles in HCV infection, the behaviors of HCV-specific T cells, especially its alteration along the clinical course, are still unknown. Using a novel method for T cell clonotype analysis emp1oyin.g RT-PCR and single-strand conformation polymorphism (SSCP), we invesbgated whether HCV-specific T cells were accumulated in viva. and if so. whether this accumulation would change following the eradication of HCV-RNA by interferon (IFN) treatment. Three patients studied were HCV-RNA positive, and showed the evidence of chronic hepatitis. Two out of three could eradicate HCV-RNA from their sera by IFN administration. Under the informed consent, we got samples from liver and PBMC of patients at the same day, and 29 weeks later we obtained PBMC again. IFN was administered during this period. One part of secondly taken PBMC was directly treated to extract RNA, and the rest was stimulated with HCV-core peptide in vitro for IO days, and thereafter brought to RNA extraction. After converting the extract RNA to cDNA, we carried out PCR using a set of VI< family-specific primers and a Cfi primer. Thereafter, we denatured the amplified DNA and analyzed in non-denaturing polyacrylamide gel electrophoresis (SSCP) to discriminate the differences of nucleotides in the complementarity-determining 3 (CDR3) region of T cell receptor p chain, which is supposed to be a recognition site for an antigen. We found that oligoclonal ‘r cell clonotypes were accumulated both in PBMC and in liver of patients with chronic hepatitis C, and that some of these accumulated T cells had the same nucleotide sequence in CDR3 reaion of their T cell receptor [$ chains, and thus the same aniigen specificities, as-T cells exoanded bv in vitro stimulation of PBMC from the oatients with HCV-core peptide. Since it is assumable that stimulation with HeV-core peptide would expand T cells which are specilic for HCV-core, this finding suggests that we could directly detect HCV-core specific T cells in viva without establishing T cell clones. Furthermore, in patients who could eradicate HCV-RNA by IFN, we demonstrated that accumulation of HCV-core specific T cells was reduced following elimination of HCV-RNA. This finding clearly shows that the accumulation of antigen-specific T cell clonotypes dynamically changes following the alteration of antigenic stimulations in viva.

910 HEPATITIS C VIRUS-SPECIFIC CYTOTOXIC T LYMPHOCYTES FROM LIVER AND PERIPHERAL tiLO6D OF A PATIENT WITH + SUSTAITD RESPO/‘lSE TO mC)N THEFY. DKH Wpna MJ Koaiel , D DudIev , JT Wona, N Afdahl , C Rice , M Houghton: d Bb Walker Infect~oy Dtsease Umt, Massachusetts General Kosospltal an hton Cltv Hosoital . Bostc In, MA; Washington University’, St. Louis,

Emeryville, CA. MO; and dhiron ‘Co ‘ration4 Cytotmic T lymp ocytes P

oathoohvsioloav of chronic hepatitis C virus IHCV) infection. We have ICTLs) are thought to be important in the

brevi&Iy ch&cterized HCV-sdecific CTL frond the hver biopsy of subject 94F. In this study, we have established analogous HCV-specific CTL clones from peripheral blood. Methods: Subject 94F was treated with rlFNRb 3 MU S.C. TIW x 5 months. Peripheral blood mononuclear cells (PBMC) were obtained at the time of liver biopsy before treatment (Pl: ALT 139, HCV RNA-positive), 16 weeks a&x starting treatment (P2: ALT 12, HCV RNA-negative), and 8 weeks after completing treatment (P3: ALT 30, HCV RNA-negative). PBMC and liver biopsy were stimulated with the bispecfic monoclonal antibody CD3,4b, which leads to polyclonal expansion of CD8+ cells. PBMC were. also stimulated with autologous B lymphoblastoid cells (B-LCL) infected with recombinant HCV-vaccinia vimses expressing aa l-966 and aa 827-3011, which together span the entire translated polypeptide of HCV-H. After 2 weeks, bulk stimulated cells were tested for their ability to lyse autologous B-LCL targets infected with HCV-vaccinia constructs in a standard (5 1)Cr release assay. Bulk stimulated cells were cloned at limiting dilution. CTL epitopes were mapped using overlapping 20 aa synthetic HCV peptides. Results: CD3,4b stimulated cells from liver (Ll) but not PBMC (Pl-P3) had detectable HLA class I- restricted CTL activity. HCV-H stimuIated PBMC had CTL activity against aa l-966 and aa 827-3011 expressing B-LCL targets at least 20% greater than negative controls. No CTL activity was generated in 3 HCV- seronegative controls. CTL clones from 94F were established from liver (Ll) and PBMC (PI-3) and mapped to the followinn epitopes: Region

62 f-:28 HLA Pl

E21?iS2 All g* 11 P2 P3 111

E2iNS2 621-640 217 ;: NS3 1265-1274 :I31 71 103 11.5 NS3 1399-1407 B8

2 11.8

NS4 1590-2050 All 21 50 42 *Refers to individual clone, ie clone Ll-35. Conclusions: HCV-specific, HLA class I-restricted CTL are present in peripheral blood but only detectable with virus-specific in vitro stimulation.

912 VIRAL CLEARANCE DURING ACUTE HEPATITIS C IS ASSOCIATED WITH A NS3-SPECIFIC CD4+ T CELL RESPONSE: FUNCTIONAL AND MOLECULAR ANALYSIS AT THE CLONAL LEVEL. H&nut M. Dien older.

“! Wierenea. .w” Elcheniaub. Institute for Immunology and Department of Medicine 11, Klinikum GroBhadem, University of Munich, Munich, Germany

More than 50% of acute hepatitis C virus (HCV) infections lead on to chronic disease and spontarieous recovery horn chronic infection is excentional. Thus. the first few months of interaction between the patiebt’s immune response and the viral population seem to determine the outcome of infection.

We studied the response of peripheral blood mononuclear cells (PBMC) to recombinant HCV proteins core, NS3, NS4, and NS5 in 13 patients with acute hepatitis C and 65 patients with chronic hepatitis C. Ninr. of I3 acutely infected patients showed a PBMC response to NS3 as compared to 5165 with chronic hepatitis C (C* p<O.OOOl, t-test p<O.OOOl). Seven of those nine patients had a sustained PBMC response to NS3 and all 7 were negative for HCV-RNA with normal aminotransferases at the end of follow-up (mean 18.4 months, range 4- 41 months). In contrast, all four patients lacking a PBMC response to NS3 and two who lost the response to NS3 early in the course of acute infection developed chronic hepatitis C. NS3-specific Cb4+ T cell clones from five patients were isolated and characterized. Bv the use of truncated NS3 orotein fraements individual T cell-epitopes were mapped to aa 12b7-1278 an2 to aa 1271-1488, both within the region with suspected helicase activity. All clones analyzed so far were restricted either by DRBl*llOl or by DRBl*1501 and at least one of these alleles was present in all patients with self-limited hepatitis C. The majority of T cell clones produced large amounts of y-interferon and variable amounts of IL-4 and IL-5. Our study provides the first data on the cellular immune response during viral elimination in acute hepatitis C and it suggests that a THO/THl-like CD4+ T lymphocyte response to NS3 contributes to successful viral clearance.