Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV.

Broadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard (51)Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.

Liver-derived CTL in hepatitis C virus infection: breadth and specificity of responses in a cohort of persons with chronic infection.

Hepatitis C virus (HCV)-specific CTL have been found within the inflammatory infiltrate of the liver of chronically infected individuals, but the breadth and specificity of the CTL response in relation to viral load are less well characterized. In this study, we analyzed the intrahepatic CTL response in liver biopsy specimens from 44 chronically infected subjects. Liver-infiltrating lymphocytes were expanded polyclonally in bulk cultures, and multiple clones were derived by limiting dilution. HCV-specific CTL responses directed at genotype 1a structural proteins were assessed in all subjects, and 22 subjects were tested more comprehensively using vectors expressing all structural and nonstructural HCV Ags. CTL responses were further characterized to determine the HLA restriction and optimal epitopes recognized. In those persons screened for recognition of all HCV Ags, HLA class I-restricted CTL were detected in 45%. Nineteen different CTL epitopes were identified, which were distributed throughout the genome; only one epitope was targeted by more than one person. In those persons with CTL responses, the breadth of response ranged from one to five epitopes. There was no correlation between the presence of a detectable CTL response and viral load. These results indicate considerable heterogeneity in detectable HCV-specific CTL responses in chronically infected persons. The mechanisms by which HCV persists during chronic infection remain to be clarified.