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Hepatitis C virus (HCV)-specific CTL responses were evaluated in two chimpanzees (Pan troglodytes) during the acute and chronic phases of HCV infection. CD8+ T lymphocytes were isolated from liver tissue homogenates using anti-CD8 antibody-coated magnetic beads and then stimulated with anti-CD3 antibodies, IL-2, and irradiated human PBMC using limiting dilution culture conditions. HCV-specific cytotoxic activity of expanding CD8+ cell lines was assessed against autologous lymphoblastoid cell lines infected with recombinant vaccinia virus vectors encoding HCV Ag. CD8+ T cell lines specific for structural and nonstructural proteins of HCV were established from both animals. Cytolytic activity was blocked with anti-CD8 or anti-class I MHC antibodies, indicating that class I MHC molecules were involved in presentation of viral Ag to the CTL. Overlapping synthetic peptides were used to define a 12 …