Direct Ex Vivo Functional Analysis of HCV-Specific T Cells

CD8⁺ and CD4⁺ T cells play a critical role in viral clearance during self-limited HCV infection. Moreover, induction of robust and polyfunctional memory T cells is a primary goal in the development of T-cell-based HCV prophylactic vaccines. Direct ex vivo function of HCV-specific T cells without in vitro expansion can be assessed by measuring cytokine secretion or cell proliferation upon in vitro stimulation with HCV antigens using enzyme-linked immunospot (ELISpot) assays, intracellular cytokine staining (ICS) assays, and flow cytometry-based T-cell proliferation assays. In particular, polyfunctionality of T cells can be assessed by ICS assays using multicolor flow cytometry. Here, we provide protocols to measure direct ex vivo functions of HCV-specific T cells.