Intracellular cytokine responses measured against SIV-Env (A) and SIV-Gag (B) antigens in Experimental vaccinated macaques were shown. PBMC were unstimulated (medium control) or stimulated for 6 h with different SIV-Env and/or SIV-Gag peptide pools at 14d post immunization, day of challenge and 231d post challenge time points. Cells were gated on singlets, lymphocytes, followed by live cells and then on CD3+ T-cells and subsequently on CD3+CD4+ and CD3+CD8+ T-cell subsets. CD3+CD4+ or CD3+CD8+ T-cells were further analyzed for the presence of IFNγ, TNFα and /or IL2 positive cells by using Flowjo, and SPICE software. Increased polyfunctional responses were detected both in CD4 and CD8 T-cells, however the antigen specific CD4 responses were higher compared to CD8 specific responses. SIV-Gag specific responses were also higher compared to SIV-Env antigens. Individual animal responses are depicted by each dot and gray bars represent mean values of respective responses from all animals (n = 5). Positive symbols represent cells staining positive for a cytokine response, and minus symbols represent cells staining negative for a cytokine response. The presence of three different cytokine producing cells, two different cytokine producing cells and single cytokine producing cells are denoted under the bottom-most graphs (left to right) for each CD4 and CD8 cells as 3, 2 and 1 cytokine(s) respectively. The criterion for a positive cytokine response was a two-fold increase in frequency for that specific antigen and cytokine above the medium control culture. All values were subtracted from medium control before the analysis.