Figure 2.
Secreted IL-10, CD25+ and TGF-β-bound cells were involved in the inhibition by HCV variants. A-B: PBMCs were cultured with peptide NS3358-375 (5 μM), variant-peptide pool VP1, or VP2 (1 μM/each peptide) for three hours, then 15 μg/ml of each antibody to human IL-10, CD25 and TGF-β were added. Parallel cultures with either NS3358-375 or VP1 and VP2 without antibody, and PBMC with medium alone were used as controls. At day four, cells were pulsed overnight with 1.0 μCi/well tritiated thymidine. Radioactive label incorporation (cpm) was measured at day five. C-D. CD25+ or TGF-β-bound cells were deleted from PBMCs using antibodies to CD25+ or TGF-β and magnetic beads. The remaining cells were cultured with either NS3358-375 or VP1 and VP2. Proliferation measurement and controls were the same as described in A-B. E: 50 μl of a supernatant pool alone or the pool with 15 μg/ml TGF-β antibody was added into each culture of PBMC with VP1 or VP2. The supernatant pool consisted of each culture of VP1 and VP2 described in A. F. PBMCs without TGF-β-bound cells were cultured with VP1 or VP2 and 50 μl of the supernatant pool. All experiments were repeated twice, a single representative experiment is shown G. Levels of IFN-γ, IL-10 and TGF-β induced by peptide NS3358-375, VP1 and VP2 were determined by cytokine-specific ELISA. Each 100 μl of supernatant from the cultures described in A was used for ELISA analysis of IFN-γ, IL-10 and TGF-β. Experiments were performed in duplicate. A single representative of five experiments is shown.