The requirement for antigen-specific stimulation mediated through the TCR for maintaining the PD-1^highCM9⁺ phenotype is independent of tissue localization. (A) Flow cytometry plots showing the PD-1 expression on total, CM9-specific, and TL8-specific CD8⁺ T cells in the same sample. Histograms depict the PD-1 expression on CM9- and TL8-specific CD8⁺ T cells from the same sample, located in mucosa and LN. (B) Pooled data showing the expression of PD-1 as percentage (left panel) or MFI (right panel) on total, CM9-specific, and TL8-specific CD8⁺ T cells from different tissues. Samples from PBMCs (n = 3), spleen (n = 3), LN (n = 11), and mucosal sites (n = 12) were analyzed. Horizontal lines depict mean values. The P values were calculated using Student t test.