Measuring HIV Gag-specific immune responses: intracellular cytokine staining. (A) Flow cytometric gating strategy for RMCs. Doublet and dead cell discrimination is used to reduce background noise (second and third graphs). For CD8⁺ T cells, positive responses for each individual function (CD107a, IFN-γ, IL-2, MIP-1β, and TNF-α) are selected. Boolean gates are created from these 5 individual gates to divide responding cells into 31 distinct populations corresponding to all possible combinations of these functions. These are graphed using SPICE software to better visualize polyfunctional responses. (B) CD8⁺ T-cell responses in both peripheral blood and rectal mucosa from an elite controller using standard bivariate plots.