SIV-specific T-cell responses in naturally SIV-infected SMs. The responses were measured by intracellular cytokine staining for IFN-γ, TNF-α, and IL-2 in response to peptide pools encompassing the entire sequence of gag, pol, env, and nef genes of SIVmac239. A positive T-cell response was defined as cytokine production greater than unstimulated negative control by at least 0.01%. (A) Representative plot of an IFN-γ response to Gag pool no. 3 and Env pool no. 3 (right) along with the unstimulated negative control (left) and positive control (PMA and ionomycin: middle). (B) Distribution of naturally SIV-infected SMs based on the presence of above-background responses to SIV by CD3⁺CD8⁺ (right) and CD3⁺CD8^- (left) T cells. Animals were divided as producing no cytokine, IFN-γ only, TNF-α only, or both cytokines in response to any pool of SIVmac239 peptides. (C) Magnitude of SIV-specific T-cell responses defined as percent of CD3⁺CD8⁺ (left) or CD3⁺CD8^- (right) T cells producing either IFN-γ or TNF-α in response to SIVmac239 peptide stimulation. Each bar represents an individual animal. The contribution of each of the tested antigens to the total SIV-specific T-cell response is shown. (D) Average magnitude of the responses to the 4 tested SIV antigens by CD3⁺CD8⁺ (left) and CD3⁺CD8^- (right) T cells. (E) Percentage of CD3⁺CD8⁺ (left) or CD3⁺CD8^- (right) T cells producing IL-2 in response to SIVmac239 peptide stimulation in 39 SIV-infected SMs. The contribution of each of the tested antigens to the total SIV-specific T-cell response is shown. (F) Magnitude of SIV-specific T-cell responses in 8 experimentally SIVsm-infected rhesus macaques (RMs) defined as percent of CD3⁺CD8⁺ (left) or CD3⁺CD8^- (right) T cells producing IFN-γ or TNF in response to SIV stimulation. The total SIV-specific 6T-cell response is broken down as response to any of the 4 tested SIV antigens.