Fig. 4: PD-1–PD-L1 or PD-1–PD-L2 binding attenuates the phosphorylation of both TCR/CD3 complex and its downstream signaling molecules. | Communications Biology

Fig. 4: PD-1–PD-L1 or PD-1–PD-L2 binding attenuates the phosphorylation of both TCR/CD3 complex and its downstream signaling molecules.

From: PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment

Fig. 4

a 2D12 transduced with mPD-1–HaloTag were preincubated with the HaloTag ligand–TMR (cyan) and DyLight 488-labeled H57 Fab (green), plated onto the SLB as in Fig. 1a without (top) or with mPD-L1–GPI (150/μm2, middle) or mPD-L2–GPI (150/μm2, bottom), fixed at 2 min after contact, stained with Alexa Fluor 647-labeled anti-phospho (p) CD3ζ (red) and imaged by confocal microscopy. Histograms show fold fluorescent intensities of TCRβ (green), PD-1 (cyan) and pCD3ζ (red) on the diagonal yellow lines in the DIC images. b The graph shows pCD3ζ/TCRβ fluorescent intensity ratio at the T cell-bilayer interface in a in the absence (left) or presence of mPD-L1–GPI (middle) or mPD-L2–GPI (right) (n = 20). Horizontal bars, average. c Primary CD4+ T cells in Fig. 1c transduced with mPD-1–GFP (green) were prestained with the DyLight 549-labeled H57 Fab (cyan), plated onto the SLB, fixed, stained for pCD3ζ (red, left panel) or pSLP-76 (red, right panel) and imaged as in a. Histograms show fold fluorescent intensities of TCRβ (cyan), PD-1 (green) and pCD3ζ or pSLP-76 (red) on the diagonal yellow lines in the middle column. d The graphs show fluorescent intensity ratio of pCD3ζ/TCRβ (top) or pSLP-76/TCRβ (bottom) in c in the absence or presence of mPD-L1–GPI or mPD-L2–GPI (n = 20). Horizontal bars, average. e 2D12 expressing mPD-1 were stimulated with MCC88–103-prepulsed DC-1 cells not expressing (left) or expressing mPD-L1 (middle) or mPD-L2 (right) for the indicated times. The WCLs were blotted for pPLCγ1, PLCγ1, pErk1/2, or Erk1/2. The number below each line represents the intensity ratio, pPLCγ/PLCγ or pErk/Erk. f The cells in e were stimulated by 16h-aggregation culture with DC-1 cells (black) or those expressing mPD-L1 (red) or mPD-L2 (green) under the different concentrations of MCC88–103 and the concentration of IL-2 in each supernatant was measured by ELISA. g The cells in e were stimulated by 16 h-aggregation culture with MCC88–103 and DC-1 cells expressing mPD-L1 or mPD-L2 in the absence or presence of each antibody as in Fig. 2a and the concentration of IL-2 in each supernatant was measured by ELISA. h The target cell, EL-4 cell, is introduced by RLuc8 and further by mPD-L1 (red) or mPD-L2 (green). Effector primary CD8+ T cells expressing mPD-1 were cocultured with 1 nM OVA257–264-pulsed these target EL-4 cells at the indicated E:T ratios for 16 h with or without 10 μg/ml anti-PD-1 (29F.1A12). After treatment with RLuc8 substrate, the intensity of live cells was measured and the percent specific lysis was calculated. i The concentration of IFNγ in culture supernatants in h was measured by ELISA. j The cells in e were stimulated by 16 h-aggregation culture with 10 μM MCC88–103 and DC-1 cells expressing mPD-L1 and mPD-L2 in the absence or presence of anti-PD-1 (J43: brown, RMP1-14: blue, 29F.1A12: green) as in Fig. 2c at the indicated concentrations and the concentration of IL-2 in each supernatant was measured by ELISA. The right graph shows the recovery rate of IL-2 production by each anti-PD-1 at the different concentrations. All data are representatives of three independent experiments. Bars, 5 μm. Error bars, SD. Statistical analysis was by one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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