FIG 2.

Frequency and functional characterization of antigen-specific cytokine-producing T cells after vaccination with HCV rNS3 and NS3 pepmix. CB6F1 mice were vaccinated three times at 2-week intervals with rNS3 or NS3 pepmix formulated in CAF09, as indicated. Splenocytes isolated from individual mice were restimulated with a pool of 62 NS3 peptides and analyzed by multiparameter flow cytometry at various time points. The bar charts show frequencies of NS3-specific IFN-γ-, TNF-α-, and IL-2-producing CD44⁺ CD4⁺ T cells (A, C, and E) or CD44⁺ CD8⁺ T cells (B, D, and F) out of the total CD4⁺ or CD8⁺ T cell population at week 6 (A and B) (n = 4) and week 12 (C and D) (n = 4) after the priming vaccination. (E and F) In a parallel experiment, a third boost was administered to NS3 pepmix-vaccinated mice at week 12, and T cell responses were assessed 14 weeks after the priming vaccination (rNS3 was not done [N.D.]; n = 8). The data are shown as means and standard errors of the mean (SEM). The pie charts show cytokine-producing CD44⁺ CD4⁺ T cells or CD44⁺ CD8⁺ T cells divided into seven distinct subpopulations based on their ability to coproduce IFN-γ, TNF-α, and IL-2 in any combination. The black arcs indicate IFN-γ-producing T cell subsets, and the gray arcs indicate TNF-α-producing T cell subsets. The data shown are representative of two independent experiments. *, P < 0.05; **, P, < 0.01; ***, P < 0.001; and ****, P < 0.0001.