Figure 4.

Assessment of Gag-specific cellular immunity elicited by Ad2 vaccine through intracellular cytokine staining (ICS) assay. The ability of polyfunctional CD4⁺ and CD8⁺ T-cell populations from immunized mice to secrete IFN-γ, TNF-α, and IL-2 cytokines in response to Gag peptide pools stimulation was assessed. The background-subtracted median responses (n = 5) in BALB/c mice following vaccination are shown. Gating strategy for flow cytometric scatter plots to analyze the frequency of the single or multiple cytokine(s)-positive CD4⁺ (A) or CD8⁺ (D) T cells. Frequencies of CD4⁺ (B) or CD8⁺ (E) T cells secrete IFN-γ, TNF-α, and IL-2 cytokines specific to various Gag antigens after adenoviral-based immunization. Further analysis was performed to show subpopulations of double cytokines-secreting CD4⁺ T cells (C) or CD8⁺ T cells (F). Data were expressed as mean ± SEM, and p-values of less than 0.05 were deemed statistically significant.*p < 0.05; **p < 0.01; ***p < 0.001; NS, no significance.