PD-1^high CM9-specific CD8⁺ T cells are highly susceptible to activation-induced cell death. (A) Flow cytometry showing CM9⁺CD8⁺ T cells as well as their PD-1 expression cultured in the absence or presence of CM9 peptide for 18 to 22 hours (left panel, top row). Histograms depict the levels of annexin V positivity in Vivid⁻ CM9⁺CD8⁺ T cells. Pooled data showing the percentage of Vivid⁻annexin V⁺CM9⁺CD8⁺ T cells from 4 macaques, in the PD-1^low, PD-1^interm, and PD-1^high compartments are shown below. The spontaneous apoptosis of total, CM9-specific, and TL8-specific CD8⁺ T cells cultured for 14 hours is shown in the right panel. SIV-specific CD8⁺ T cells from 4 animals at different time points after infection were analyzed. Horizontal lines and bars depict mean values. The P values were calculated using Student t test. (B) Histograms representing the CFSE dilution in annexin V⁻CM9⁺CD8⁺ T cells cultured alone or stimulated with CM9 peptide in the absence or presence of an anti-PD-1 antibody (20 μg/mL) (left panel). The levels of annexin V positivity on CM9⁺CD8⁺ T cells, under the same treatment conditions, are also shown (left panel). The effect of treatment with the anti-PD-1 on the PD-1^high and PD-1^low CM9⁺CD8⁺ T cells in a 6-day culture is shown in the right panel. Data from 1 of 2 macaques tested are shown.