Figure 5. Restoration of HCV-specific IL-17A and IL-21 production by blocking multiple inhibitory pathways.

(A) Representative FACS plot demonstrating enhanced production of Th17 and Th1 cytokines by HCV NS4-specific CD4 T cells upon antibody blockade. CD25^neg PBMCs from acute HCV patients were stained with CFSE and stimulated with 1 µg/ml of HCV NS4 recombinant protein in the presence of control Igs (IgG1, Ig2a) or blocking antibodies against PD-L1, CTLA-4 and Tim-3 (10 µg/ml each). After 6 days of culture, cells were washed and re-stimulated with PMA/ionomycin to detect production of Th17 (IL17A, IL-21) and Th1 (IFN-γ and TNF-α) cytokines by ICS as described in Materials and Methods. Cells are gated on CD3+CD8^neg lymphocytes (left panels). (B, C) Enhanced cytokine expression upon antibody blockade in the SR (open symbols, n = 3–5) and CI patients (closed symbols, n = 3–5). Data is represented as percent cytokine expressing CFSE^low cells within the CD3⁺CD8^neg lymphocyte gate. P-values were calculated using Wilcoxon signed rank test.