PD-1 up-regulation on total and virus-specific CD8⁺ T cells inhibits IFN-γ production in TP patients. (A) Representative dot plots of IFN-γ intracellular staining gated on CD3⁺ T cells and CD8⁺ T cells from LTNPs and TP subjects. (B) PD-1⁻CD8⁺ but not PD-1⁺CD8⁺ T cells produced a large amount of IFN-γ in response to TCR stimulation. Dot plots show the intracellular IFN-γ staining profile of PD-1⁺CD8⁺ and PD-1⁻CD8⁺ T cells from a representative TP patient. CD8⁺ T cells that were isolated from PBMCs using the MiniMACS system were further sorted into PD-1⁺CD8⁺ and PD-1⁻CD8⁺ T cells using FACSVantage. The sorted cells were added back to CD8-deleted PBMCs at a ratio of 1:3. Cells were further stimulated with a TCR mimic for 6 hours and intracellular IFN-γ production was assessed. Values in the upper right quadrant indicate the percentage of sorted CD8⁺ T cells that express PD-1 (top) and the percentage of PD-1⁺CD8⁺ and PD-1⁻CD8⁺ T cells that express IFN-γ. (C) Blocking PD-1/PD-L1 restores the percentage of IFN-γ⁺CD8⁺ T cells. IFN-γ intracellular staining of total and HIV-specific CD8⁺ T cells is shown in dot plots from a representative TP patient. (D-E) Summary data for the percentage of IFN-γ⁺CD8⁺ T cells (D) or IFN-γ⁺pentamer⁺CD8⁺ T cells (E) from LTNPs and TPs. Horizontal bars indicate mean values.