Validation of the stimulation protocol.

(A) CD69 and IFN-γ expression of CD8⁺ T cells after incubation with 2 μL dimethylsulfoxide (DMSO), 1 μg HIV-peptide dissolved in 2 μL DMSO or PMA and ionomycin. Upper panels show total PBMCs stained for CD8 and IFN-γ; lower panels are gated on CD8⁺ T cells and show CD69 and IFN-γ expression of CD8⁺ T cells. (B) Response of EBV- and HIV-specific T cells to in vitro peptide stimulation. PBMCs were incubated with 0, 1, and 10 μg peptide/mL. FACS dot plots are gated on CD8⁺ T cells and demonstrate tetramer⁺ cells (y-axis), and IFN-γ production (x-axis). Percentages in the left upper corner indicate number of tetramer⁺ T cells of the CD8⁺ T-cell population. Percentages in the right upper corner indicate number of IFN-γ⁺ peptide-specific T cells (right from the dotted line) of the CD8⁺ T-cell population. (C) IFN-γ–producing antigen-specific T cells after incubation either with 1 μg peptide (“Peptide,” black bar) or with 10 μg peptide and antibodies against CD28 and CD49d (“Costimulation,” white bar). IFN-γ⁺ T cells are given as percentage relative to the total number of tetramer⁺ T cells in the nonstimulated sample.