Figure 4.
Figure 4. The tyrosine motifs in PD-1 are required for PD-1–mediated T cell suppression, not for PD-1 clustering. (A) AND-Tg Pdcd1−/− Rag2−/− CD4+ T cells were reconstituted with WT (top) or Y225F (row 2), Y248F (row 3), or Y225/248F (YFYF, bottom) mutant PD-1–EGFP (green), and were prestained with DyLight 649–labeled H57 Fab (red). The cells were plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, CD80-, and PD-1–GPI and real-time imaged by confocal microscopy at 2 (left) or 20 (right) min after contact. Histograms show fold fluorescent intensities of TCR-β (red) and PD-1 (green) on the two diagonal yellow lines in the merged images. Bars, 5 µm. A representative of three independent experiments is shown. (B) The graph shows the percentage of TCR microclusters colocalized by WT or mutant PD-1 at 2 min after contact in A (n = 5). Error bars represent SD. (C) AND-TCR T cell hybridomas expressing WT or Y225F, Y248F, or YFYF mutant PD-1–Flag were stimulated with 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1 for 2 min. Cell lysate immunoprecipitated by anti-Flag or WCLs were blotted for SHP2 or PD-1. A representative of three independent experiments is shown. (D) Not transduced or PD-1–transduced AND-TCR T cell hybridomas in C were stimulated with an equal number of DC-1 cells expressing PD-L1 or PD-L2 with 0.3 µM MCC88-103 for 16 h. Immobilized anti-CD3ε (2C11, 10 µg/ml) and anti-CD28 (PV-1, 2 µg/ml) were used for control stimulation. Concentration of IL-2 was measured by ELISA. A representative of two independent experiments is shown. Error bars represent SD. (E) SHP1 or SHP2 were directly attached to the cytoplasmic tail of the YFYF mutant PD-1 and transduced into AND-TCR T cell hybridomas. The inhibitory function of the PD-1 YFYF-SHP1/SHP2 chimeras was analyzed as in D. A representative of three independent experiments is shown. Error bars represent SD. (F) The graph shows the percentage of cells in A forming a stable synapse at 20 min after contact (n = 50). Error bars represent SD.

The tyrosine motifs in PD-1 are required for PD-1–mediated T cell suppression, not for PD-1 clustering. (A) AND-Tg Pdcd1−/− Rag2−/− CD4+ T cells were reconstituted with WT (top) or Y225F (row 2), Y248F (row 3), or Y225/248F (YFYF, bottom) mutant PD-1–EGFP (green), and were prestained with DyLight 649–labeled H57 Fab (red). The cells were plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, CD80-, and PD-1–GPI and real-time imaged by confocal microscopy at 2 (left) or 20 (right) min after contact. Histograms show fold fluorescent intensities of TCR-β (red) and PD-1 (green) on the two diagonal yellow lines in the merged images. Bars, 5 µm. A representative of three independent experiments is shown. (B) The graph shows the percentage of TCR microclusters colocalized by WT or mutant PD-1 at 2 min after contact in A (n = 5). Error bars represent SD. (C) AND-TCR T cell hybridomas expressing WT or Y225F, Y248F, or YFYF mutant PD-1–Flag were stimulated with 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1 for 2 min. Cell lysate immunoprecipitated by anti-Flag or WCLs were blotted for SHP2 or PD-1. A representative of three independent experiments is shown. (D) Not transduced or PD-1–transduced AND-TCR T cell hybridomas in C were stimulated with an equal number of DC-1 cells expressing PD-L1 or PD-L2 with 0.3 µM MCC88-103 for 16 h. Immobilized anti-CD3ε (2C11, 10 µg/ml) and anti-CD28 (PV-1, 2 µg/ml) were used for control stimulation. Concentration of IL-2 was measured by ELISA. A representative of two independent experiments is shown. Error bars represent SD. (E) SHP1 or SHP2 were directly attached to the cytoplasmic tail of the YFYF mutant PD-1 and transduced into AND-TCR T cell hybridomas. The inhibitory function of the PD-1 YFYF-SHP1/SHP2 chimeras was analyzed as in D. A representative of three independent experiments is shown. Error bars represent SD. (F) The graph shows the percentage of cells in A forming a stable synapse at 20 min after contact (n = 50). Error bars represent SD.

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