Anti–PD-L1 blocks PD-1 microcluster formation and anergic status in PD-1^hi CD8⁺ T cells. (A) OT-I-Tg Rag2^−/− mice were subcutaneously injected with 3 nmol OVA257-264 in 100 µl PBS everyday for 1 wk. Histograms show the cell surface expression of CD25, CD44, CD62L, and PD-1 on splenic CD8⁺ T cells from OT-I-Tg Rag2^−/− mice before (top) or at 7 (middle) or 14 (bottom) d after first challenge. A representative of three independent experiments is shown. (B) Naive or OVA257-264–challenged (PD-1^hi) OT-I-Tg CD8⁺ T cells were stimulated with irradiated C57/BL6 whole splenocytes for 15 h with the indicated concentrations of OVA257-264 in the absence or presence of anti-PD-1 (J43 or RMA1-30) or anti-PD-L1 (MIH5). 10 ng/ml PMA and 0.5 µM ionomycin were used for control stimulation. Concentration of IL-2 was measured by ELISA. A representative of three independent experiments is shown. Error bars represent SD. (C) Naive or OT-I-Tg PD-1^hi CD8⁺ T cells in A were stained with DyLight 649–labeled H57 Fab (red) and DyLight 488–labeled anti–PD-1 (RMA1-30) (green), plated onto an OVA257-264 prepulsed planar bilayer containing H-2K^b–, ICAM-1–, and CD80- (row 2) plus PD-L1–GPI (rows 1, 3, and 4), and real-time imaged by confocal microscopy at 2 (left) or 20 (right) min after contact in the absence (top three rows) or presence (bottom) of anti–PD-L1 (MIH5). Histograms show fold fluorescent intensities of TCR-β (red) and PD-1 (green) on the diagonal yellow lines in the merged images. Bars, 5 µm. A representative of two independent experiments is shown.