The majority of CM9-specific CD8⁺ T cells express high levels of PD-1. (A) PD-1 expression was measured by applying a polychromatic flow cytometry assay (left panel). The gating scheme for identification of cell populations is shown. Histograms depict the PD-1 expression on total and CM9-specific CD8⁺ T cells from the same sample and in relation to their maturation status. The combination of CD28 and CD95 was used for identification of memory populations. The control (all antibodies except aPD-1) staining is also shown. Representative flow cytometry plots depicting the PD-1^high and PD-1^low CD3⁺ T-cell populations are shown on the right panel. (B) Pooled data showing the percentage (%) of CD8⁺ T cells expressing a PD-1^high phenotype in PBMC compartment from noninfected (n = 4) and SIV-challenged or treated/SIV-challenged (n = 13) animals. (C) The percentage (%) of PD-1^high CD8⁺ T cells located in spleen, LN (axillary, n = 8; inguinal, n = 8; mesenteric, n = 4; paratracheal, n = 3), and mucosa (rectal, n = 11; jejunum, n = 9; colon, n = 4) is shown. PD-1 expression in memory populations of total and CM9⁺CD8⁺ T cells from all tissues tested is also shown. All animals were infected for at least 8 weeks. Horizontal lines depict mean values. The P values were calculated using Student t test.