SHIV-specific, mC46-expressing CD4⁺ T cells are maintained in mC46-expressing macaques. (A-B) PBMC isolated from postchallenge macaques were stimulated with SHIV peptide for 6 hours and SHIV-specific cells were identified using intracellular cytokine staining. Boolean gating was used to identify the frequencies of CD4⁺ (A) and CD8⁺ (B) cells expressing 1 or more of the following cytokines: IFN-γ, IL-2, and tumor necrosis factor (TNF)-α. To calculate percentages, all negative values after background subtracting were made equal to 0. CD4⁺ T-cell responders from control (cont.)2 data were omitted because all data failed the count >1000 filter. (C-D) The same Boolean gating data were used to observe the frequencies of SHIV-specific CD4⁺ and CD8⁺ cells within the GFP⁺ and GFP⁻ subsets. Frequencies of GFP⁺ cells are displayed as green circles; frequencies of GFP⁻ cells are displayed as red squares; data that failed positivity calls are depicted as open symbols. One to 3 samples per macaque was examined per peptide pool depending on viability and total number of gated CD4⁺ or CD8⁺ T cells. Cryopreserved samples from 2, 4, and 6 months after SHIV challenge were used for these experiments.