GSBS Dissertations and Theses

Title

Characterization of Intrahepatic T-lymphocytes in Patients with Chronic Hepatitis C Virus Infection: a Dissertation

Approval Date

November 2000

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Program in Immunology and Virology

Subjects

Liver; T-Lymphocytes; Hepatitis C; Academic Dissertations

Abstract

Hepatitis C virus (HCV) is a positive strand RNA virus that is the leading cause of chronic hepatitis. HCV infections are an important health problem because >80% of patients become chronically infected and many develop chronic hepatitis. With approximately 400 million chronic HCV infections worldwide, understanding the pathogenesis of this disease is of critical importance in order to develop appropriate therapies and/or vaccine strategies.

Strong proliferative and cytotoxic T cell responses that target multiple HCV proteins are detected in patients with self-limited infection. Conversely, HCV-specific T cell responses are minimal during acute infection in patients who become chronically infected. It is thought that the genetic diversity of HCV plays a crucial role in establishing persistence.

Chronic viral hepatitis is characterized by infiltration of T lymphocytes in the liver, which are thought to play a pivotal role in disease progression. Although virus-specific T cells can be isolated from both peripheral blood and from liver biopsy samples of chronically infected patients, there appears to be a compartmentalization of HCV-specific T cells in the liver. However, the presence of virus-specific T cells is inefficient for viral clearance. Because HCV is known to be highly variable in sequence, the detailed characterization of the interaction of individual HCV-specific CTL clones with autologous viral sequences might be important for understanding the mechanisms by which HCV is able to establish a chronic infection.

We isolated three intrahepatic CD8+ CTL clones from two individuals with chronic HCV infection and compared the recognition of prototype and autologous HCV sequences. These CTL recognized epitopes within the NS2 (amino acids 957-964) or NS3 (amino acids 1402-1410 and 1406-1415) proteins in the context of HLA B37, B8, or A2.1, respectively. The corresponding predominant autologous HCV sequences (SDWAANGL, ELAAKLVGL, ALRGMGLNAV, respectively) differed from the HCV-1 prototype sequences used for screening (RDWAHNGL, ELAAKLVAL, KLVALGINAV, respectively) at one to five residues. For each CTL clone, recognition of the autologous HCV sequence required significantly higher peptide concentrations than did recognition of the HCV-1 sequence; for two of the clones, recognition was minimal or absent at peptide concentrations as high as 25μM.

When the HLA A2.1-restricted HCV NS3-specific T cell clone was analyzed further, we found that it was cross-reactive with peptide sequences from at least three other HCV strains. The clone recognized target cells loaded with synthetic peptides derived from sequences of genotype 1b; HCVTW (KLSALGIHAV), HCVJA (KLTGLGLNAV),and HCVBK (KLSGLGINAV). This HCV-specific T cell clone was also able to recognize target cells that were loaded with a peptide derived from an autologous protein, cellular retinoic acid binding protein I (CRABP I). When we generated HLA A2.1-restricted HCV NS3-specific T cell lines from the peripheral blood of two additional patients, almost one half of the cell lines could lyse target cells loaded with the CRABP I peptide. These data show that intrahepatic HCV-specific CD8+ CTL clones can be relatively inefficient at recognizing autologous viral epitopes and that some viral-specific CTL can recognize autoantigens in vitro.

There is little information regarding the composition and stability of the liver-infiltrating T cell repertoire during chronic HCV infection. To address this issue, we used TCR complimentarity determining region 3 (CDR3) length analysis to examine the T lymphocytes in sequential biopsy samples from five individuals chronically infected with HCV. We found that although almost all TCRBV families were represented in the liver, 25-85% had skewed spectratype profiles, indicative of the presence of clonally expanded T cells. Further analysis using TCRBJ-primed run-off reactions revealed that the intrahepatic repertoires were not stable, as many expansions that existed in one biopsy sample were not detected in the other. Some expansions persisted, however, and sequencing of TCRBV-J transcripts identified CDR3 sequences that were maintained in two individuals for 10 or 45 months. Furthermore, although some expansions were found in the periphery, most were represented only in the liver. These data suggest that there is an evolution of the immune response during chronic HCV infection and that the response is largely concentrated in the liver of these individuals.

Based on our observations regarding the function of intrahepatic HCV-specific CTL and the dynamics of the intrahepatic repertoire during chronic HCV infection, we propose a model in which the co-evolution of HCV quasispecies and HCV-specific T cells contribute to both viral persistence and immunopathology.

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