PD-1–PD-L1 binding blocks stable synapse formation. (A) AND-Tg Pdcd1^−/− Rag2^−/− CD4⁺ T cells expressing PD-1–EGFP were stained with DyLight 649–labeled H57 Fab. The cells were plated onto an MCC88-103 prepulsed planar bilayer containing I-E^k–, ICAM-1–, CD80-, and PD-1–GPI at the indicated densities, and real-time imaged by confocal microscopy at 2 (top tow rows) or 20 (bottom tow rows) min after contact. Bars, 5 µm. A representative of two independent experiments is shown. (B) The graph shows the percentage of cells in A forming a stable synapse at 20 min after contact (n = 50). (C) The cells in A were plated onto a planar bilayer containing I-E^k–, ICAM-1–, CD80-, and PD-L1–GPI prepulsed MCC88-103 at the indicated concentrations, and imaged by confocal microscopy at 2 (top tow rows) or 20 (bottom tow rows) min after contact. A representative of two independent experiments is shown. Bars, 5 µm. (D) The graph shows the percentage of cells in D forming a stable synapse at 20 min after contact (n = 50). (E) The cells in C (right column) were real-time imaged by confocal microscopy every 5 s at 10 min after contact. Bar, 5 µm. (F) The cells in A were plated onto an MCC88-103–prepulsed planar bilayer containing I-E^k–, ICAM-1–, and CD80- (row 1, 3, and 4) plus PD-L1–GPI (row 2). At 10 min after contact, the cells were further incubated with control antibodies (50 µg/ml, row 1), the Src kinase inhibitor PP2 (10 µM, row3), or anti-MHCp (14–4-4 and D4, 50 µg/ml each, row 4) for 10 min and imaged. Bars, 5 µm. A representative of two independent experiments is shown. (G) The graph shows the percentage of cells in F forming a stable synapse at 20 min after contact (n = 50).