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

<p>Mice were immunized and splenocytes were collected as described in the Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093143#pone-0093143-g004" target="_blank">Figure 4A</a>. 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.