Cytokine production by CD45RO⁺ and CD45RO⁻/RA⁺ HIV-specific CD8⁺ T cells. PBMCs were stimulated overnight with HIV peptide, and cytokine secretion (IFN-γ, TNF-α, and IL-2) was blocked after 1 hour of activation. (A) The percentage of responding CD8⁺ T cells was measured by flow cytometry after costaining with IFN-γ-FITC/multimer-PE, TNF-α-FITC/IFN-γ-PE, or IFN-γ-FITC/IL-2-PE. The total of the 3 quadrants FITC⁺ PE⁻, FITC⁺ PE⁺, and FITC⁻ PE⁺ was considered to be HIV-specific CD8⁺ T cells. (B-D) The relative percentages of CD45RO⁻/RA⁺ and CD45RO⁺ responding cells among HIV-specific cells were calculated and are shown for IFN-γ production, TNF-α production, and simultaneous IFN-γ and TNF-α production.