Chronic antigen-specific stimulation is indispensable for persistent high expression of PD-1 on CM9⁺CD8⁺ T cells. (A) Representative flow cytometry showing the simultaneous detection of CM9- and TL8-specific CD8⁺ T-cell populations from the same sample as well as their memory status. Histogram depicts the PD-1 expression on total, CM9-specific, and TL8-specific CD8⁺ T cells from the same sample. (B) Representative plots showing the CM9- and TL8-specific CD8⁺ T-cell populations from one animal at different time points before and after infection. The animal was challenged with SIVmac251 on day 0. (C) The expression of PD-1 (as % or MFI) before and after infection on total, CM9-specific, and TL8-specific CD8⁺ T cells is shown. The plasma viral load for all animals is also shown (top left panel). PD-1 expression was analyzed in total (top panel), CD28^highCD95⁺ (middle panel), and CD28⁻CD95⁺ (bottom panel) CD8⁺ T-cell memory populations for each animal individually and pooled data from 4 animals are shown. Bars depict means (± SD). The P values were calculated using Student t test.