Blocking PD-1/PD-L1 interaction reduced Treg-mediated suppression of CD8⁺ T cells in vitro. Treg suppression assay was performed as described. Proliferating CD8⁺ T cells were quantified by the dilution of CFSE resulting in a CFSE^low population and IFN-γ secretion was measured by enzyme-linked immunosorbent assay. CFSE-labeled CD8⁺ T cells were cocultured with syngeneic, anti-CD3ϵ mAb-loaded APCs with or without Tregs and/or anti–PDL1 mAb as indicated. CD8⁺ T cells, Tregs, and APCs were obtained from either WT or B6 PD-1 KO mice, as indicated. PD-1 KO CD8⁺ T cells are more resistant to WT Treg-mediated suppression of proliferation (A) and IFN-γ secretion (B) than WT CD8⁺ T cells. Compared with WT Tregs, PD-1 KO Tregs have a defective capacity to suppress the proliferation (C) and IFN-γ secretion (D) of CD8⁺ T cells. Anti–PD-L1 mAb prevents WT Treg suppression of proliferation (E) and IFN-γ secretion (F) of WT CD8⁺ T cells. Neither PD-1 KO nor WT Tregs are able to suppress proliferation of CD8⁺ T cells in the presence of PD-L1 KO APCs (G). Results from one of 3 representative experiments are shown. Bar graphs represent mean ± SD.