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. 2009 Jul 8;83(18):9122–9130. doi: 10.1128/JVI.00639-09

FIG. 7.

FIG. 7.

Blocking Tim-3 enhances HCV-specific proliferative responses for both CD4+ and CD8+ T cells. Four chronic HCV infection patients were chosen on the basis of their previously demonstrated ability to produce IFN-γ in response to HCV-specific peptide pools as assessed by ELISPOT assays (both CD4+ and CD8+ T cells). PBMCs were CFSE stained and cultured for 7 days with DMSO or appropriate HCV-specific peptide pools, and the effect of 10 μg/ml of IgG isotype control was compared to the effect of 10 μg/ml of anti-Tim-3 MAb (clone 1G5). A decrease in CFSE (% CFSELow cells) is indicative of proliferated cells. The HCV peptide pools used were as follows: for patient 1, p7, NS5B5, and NS5B4; for patient 2, E1B and NS2A; for patient 3, core (A and B), E1A, E1B, and NS5B5; for patient 4, NS37H, NS4A, and NS5B6 (the composition of the peptide pools is described fully in reference 20). (A) Representative flow plots of CFSE staining are shown for one individual patient, although cells do not appear to have undergone multiple rounds of proliferation. (B) The combined data from all four patients tested comparing the effect of anti-Tim-3 to that of the IgG control in the presence of antigen-specific stimulation is shown. The black dotted lines (with circles) represent CD4+ T cells, and the grey solid lines represent CD8+ T cells. (C) Proliferative responses are shown minus the DMSO control for four individual patients. (D) Supernatants from the proliferation assay were tested for levels of IFN-γ and IL-10. As shown, culture in the presence of the anti-Tim-3 antibody induced IFN-γ. A concomitant decrease in IL-10 was observed. (E) The high level of proliferated cells detected in the assays using peptide pools suggests that there may also be proliferation of non-antigen-specific cells in this assay, although the staining suggests that the cells have not undergone multiple rounds of division; therefore, we took a more conventional approach and tested the ability of the blocking antibody to induce proliferation of pentamer-positive cells.