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. 2014 Mar 26;9(3):e93143. doi: 10.1371/journal.pone.0093143

Figure 6. Assessment of polyfunctional SIVgag-specific CD8+ T cellular immunity elicited by the SIVgag-LC3b fusion antigen.

Figure 6

Mice were immunized and splenocytes were collected as described in the Materials and Methods and Figure 4A. Splenocytes from four mice in each group were mixed together and 500,000 cells were acquired and analyzed by the FACSAria instrument using the FlowJo software. The ability of functional CD8+ T cell populations from immunized mice to secrete IFN-γ, TNF-α, and IL-2 cytokines in response to the SIVgag peptide pool stimulation was assessed. (A) Gating strategy for flow cytometric scatter plots to analyze the frequency of cytokine(s)-positive CD8+ T cells positive in this study. Column graphs depicting subpopulations of single-, double-, or triple-positive CD8+ T cells secreting the cytokines IFN-γ, TNF-α, and IL-2 induced by DNA-based immunization at week 4 (B) or adenoviral -based immunization at week 8 (C). Pie chart analysis was performed to represent subpopulations of cytokine-secreting CD8+ T cells positive for combinations of IFN-γ, TNF-α, and IL-2 induced by DNA-based immunization at week 4 (D) or adenoviral -based immunization at week 8(E). The representative data shown here were obtained from two independent experiments from 8–10 mice for each group.