Figure 6.
Figure 6. PD-1–PD-L1 binding attenuates phosphorylation of TCR downstream molecules and CD28–PKC-θ association. (A) AND-TCR T cell hybridomas expressing PD-1-EGFP (green) were stained with DyLight 549–labeled H57 Fab (red), plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, and CD80- (top) plus PD-L1–GPI (bottom), fixed at 2 min after contact, stained with Alexa Fluor 647–labeled anti–phospho-CD3ζ (cyan), and imaged by confocal microscopy. Histograms show fold fluorescent intensities of TCR-β (red), PD-1 (green), and phosho-CD3ζ (cyan) on the diagonal yellow lines in a DIC image. Bars, 5 µm. A representative of two independent experiments is shown. (B) The point graphs show pCD3ζ/TCR-β fluorescent intensity ratio at the T cell–bilayer interface in the absence or presence of PD-L1–GPI (left, n = 33) and the inverse correlation between PD-1 intensity and pCD3ζ/TCR-β ratio at the interface in the presence of PD-L1–GPI (right, y = 0.578 − 0.0173 x, R = 0.37294) in A. *, P < 0.001 with Student’s t test. Horizontal bars represent the mean of the intensity ratio. (C) AND-TCR T cell hybridomas expressing PD-1 and PKC-θ–EYFP were stimulated with unpulsed or MCC88-103 prepulsed DC-1 cells expressing PD-L1 or PD-L2 in the absence or presence of 50 ng/ml PMA. The cells were lysed, immunoprecipitated with anti-CD28, and blotted for PKC-θ or CD28. WCLs were blotted for PKC-θ, CD28, phospho-Vav1, Vav1, phospho-PLCγ1, PLCγ1, phospho-Erk1/2, or Erk1/2. A representative of two independent experiments is shown. (D) AND-Tg Rag2−/− CD4+ T cells were transduced with CD28-CyPet (cyan) and PD-1-YPet (yellow), stained with DyLight 649–labeled H57 Fab (red), plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, and PD-L1–GPI (top two rows) plus CD80-GPI (bottom two rows), and real-time imaged by confocal microscopy at 2 or 20 min after contact. Histograms show fold fluorescent intensities of TCR-β (red), CD28 (cyan), and PD-1 (yellow) on the diagonal yellow lines in the DIC images. Bars, 5 µm. A representative of four independent experiments is shown.

PD-1–PD-L1 binding attenuates phosphorylation of TCR downstream molecules and CD28–PKC-θ association. (A) AND-TCR T cell hybridomas expressing PD-1-EGFP (green) were stained with DyLight 549–labeled H57 Fab (red), plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, and CD80- (top) plus PD-L1–GPI (bottom), fixed at 2 min after contact, stained with Alexa Fluor 647–labeled anti–phospho-CD3ζ (cyan), and imaged by confocal microscopy. Histograms show fold fluorescent intensities of TCR-β (red), PD-1 (green), and phosho-CD3ζ (cyan) on the diagonal yellow lines in a DIC image. Bars, 5 µm. A representative of two independent experiments is shown. (B) The point graphs show pCD3ζ/TCR-β fluorescent intensity ratio at the T cell–bilayer interface in the absence or presence of PD-L1–GPI (left, n = 33) and the inverse correlation between PD-1 intensity and pCD3ζ/TCR-β ratio at the interface in the presence of PD-L1–GPI (right, y = 0.578 − 0.0173 x, R = 0.37294) in A. *, P < 0.001 with Student’s t test. Horizontal bars represent the mean of the intensity ratio. (C) AND-TCR T cell hybridomas expressing PD-1 and PKC-θ–EYFP were stimulated with unpulsed or MCC88-103 prepulsed DC-1 cells expressing PD-L1 or PD-L2 in the absence or presence of 50 ng/ml PMA. The cells were lysed, immunoprecipitated with anti-CD28, and blotted for PKC-θ or CD28. WCLs were blotted for PKC-θ, CD28, phospho-Vav1, Vav1, phospho-PLCγ1, PLCγ1, phospho-Erk1/2, or Erk1/2. A representative of two independent experiments is shown. (D) AND-Tg Rag2−/− CD4+ T cells were transduced with CD28-CyPet (cyan) and PD-1-YPet (yellow), stained with DyLight 649–labeled H57 Fab (red), plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, and PD-L1–GPI (top two rows) plus CD80-GPI (bottom two rows), and real-time imaged by confocal microscopy at 2 or 20 min after contact. Histograms show fold fluorescent intensities of TCR-β (red), CD28 (cyan), and PD-1 (yellow) on the diagonal yellow lines in the DIC images. Bars, 5 µm. A representative of four independent experiments is shown.

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