In vivo frequency of infection of splenic CD4⁺IL-21⁺ T cells in SIV-infected RMs. CD4⁺ T cells that produce IL-21, TNF-α, or IFN-γ after in vitro stimulation were sorted by flow cytometry from the spleen of 4 SIV-infected RMs. Cells were initially gated based on light scatter, followed by positive staining for CD3 without binding to the dead cell dye, and then for CD4 (without CD8 staining). Memory CD4⁺ T cells, gated based on characteristic expression patterns of CD28 and CD95, were then divided based on the production of IL-21, TNF-α, or IFN-γ (as shown in supplemental Figure 3). The infection frequencies of the highly purified splenic CD4⁺IL-21⁺, CD4⁺TNF-α⁺, and CD4⁺IFN-γ⁺ T cells were then determined by quantitative PCR for SIVgag DNA.