Fig 2: The combinational use of anti-hPD-1 and anti-hPD-1 ligands more effectively cancels hPD-1-meidated T cell suppression.a 2D12 cells expressing hPD-1-EGFP were cocultured with MCC88-103 and DC-1 cells expressing hPD-L1 in the presence of the indicated combination of antibodies at an individual concentration calculated from EC50. The concentration of IL-2 was measured by enzyme-linked immunosorbent assay (ELISA). P, pembrolizumab; N, nivolumab; D, durvalumab; A, atezolizumab. b The ratios of the increment of IL-2 measured in (a). c The cells in a were imaged at 2 min after contact to an MCC88-103-prepulsed SLB containing I-Ek–, mICAM-1–, and hPD-L1–GPI in the absence or presence of the indicated combination of antibodies at an individual concentration calculated from EC20. d The percentages of T cells forming hPD-1 microclusters in c (n = 30). e The graph shows the ratio of the total area of microclusters to the area of the T cell-SLB interface in individual T cells (%area) in (c). Data were analyzed from the total number of cells indicated above the boxes. Box center indicates median, box edges 25th and 75th percentile, and whiskers minimum and maximum percentile. f The same coculture as in a was performed using DC-1 cells expressing both hPD-L1 and hPD-L2 in the absence or presence of the antibodies at the indicated concentrations. g The cells in a were conjugated with MCC88-103-prepulsed DC-1 cells expressing both hPD-L1-SNAP-tag (red) and hPD-L2-HaloTag (cyan) in the absence or presence of pembrolizumab and/or 29E.2A3 and/or 24 F.10C12 at a concentration of 0.1 μg/ml. The images were acquired 2 min after contacts. h The graphs show the percentages of the cells accompanied by the accumulation of hPD-1 (green), hPD-L1 (magenta), or hPD-L2 (cyan) at the T cell-DC-1 cell interface (n = 30). All data are representative of two independent experiments. ns not significant. Bars, 5 μm. Error bars, standard deviation (SD). Statistical analysis was performed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data for a, b, d–f and h are provided as a Source Data file.

Fig 3: The inhibition of hPD-1 microcluster formation is correlated with the recovery of T cell responses if blocking antibodies for hPD-1 or hPD-L1 were added in sufficient concentrations.a 2D12 cells expressing hPD-1-EGFP (green) were prestained with DyLight 650-labeled H57 Fab (red) and plated on an SLB with hPD-L1–GPI as shown in Fig. 1a. The cells were imaged by confocal microscopy 2 min after contact in the absence (top) or presence of pembrolizumab (anti-hPD-1, row 2), nivolumab (anti-hPD-1, row 3), durvalumab (anti-hPD-L1, row 4), atezolizumab (anti-hPD-L1, row 5), MIH1 (anti-hPD-L1, row 6), or 29E.2A3 (anti-hPD-L1, bottom) at a concentration of 10 µg/ml. b The graph shows the percentage of T cells forming hPD-1 microclusters in a (n = 30). c The cells in a were conjugated with MCC88-103 prepulsed (1 µM) DC-1 cells expressing hPD-L1-HaloTag (cyan) in the absence or presence of the indicated antibodies and images by confocal microscopy 2 min after T cell–APC contacts. d The cells in a were cocultured for 16 h with 1 µM MCC88-103 and DC-1 cells not expressing or expressing hPD-L1 in the absence or presence of the indicated antibodies, and the concentration of IL-2 in each supernatant was measured by enzyme-linked immunosorbent assay (ELISA) in triplicate. e Pdcd1-/- OT-I TCR-Tg T cells reconstituted by hPD-1 were cocultured with 1 nM OVA257-264-pulsed target EL-4 cells not expressing or expressing hPD-L1 at the indicated E/T ratios for 16 h without or with anti-hPD-1 or anti-hPD-L1 at a concentration of 10 µg/ml. Statistics were performed between the calculated percentage of specific lysis with and without each antibody. All data are representative of two independent experiments. ns not significant. Bars, 5 µm. Error bars, SD. Statistical analysis was performed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data for b, d and e are provided as a Source Data file.

Fig 4: hPD-1 gathers with TCR to form microclusters at a T cell-bilayer interface in a ligand-binding fashion.a AND-TCR-Tg CD4+ T cells expressing hPD-1–EGFP (green) were plated onto an MCC88-103-prepulsed SLB containing I-Ek–, mICAM-1–, and without (top) or with hPD-L1–GPI (bottom) and real-time imaged by TIRF microscopy (times are above images; Supplementary Movie 1). b Clustering and centripetal movement of hPD-1 on the diagonal yellow lines in a are presented as horizontal elements in kymographs. c The cells in a were prestained with DyLight 650-labeled anti-TCRß (H57) Fab (red), plated onto an SLB as in a, and real-time imaged by confocal microscopy 2 (left) or 20 min (right) after contact. Histograms show fold fluorescent intensities of TCRß (magenta) and hPD-1 (green) on the diagonal yellow lines in the DIC images. d The graph shows the percentage of TCR microclusters colocalized with hPD-1 2 min after contact in T cells in c (n = 10). e AND-TCR T cell hybridomas, 2D12 cells, expressing hPD-1–EGFP (green) were prestained with DyLight 650-labeled H57 Fab (red), conjugated with an MCC88-103 prepulsed (1 µM) I-Ek-expressing APC line, DC-1 cell, not expressing (top) or expressing (bottom) hPD-L1-HaloTag (cyan), and real-time imaged by confocal microscopy 2 min after T cell–APC contact. All data are representative of two independent experiments. Bars, 5 µm. Data are presented as mean values ± SD. Statistical analysis was performed by an unpaired two-sided t-test. ****p < 0.0001. Source data for c and d are provided as a Source Data file.

Fig 5: hPD-1–hPD-L1 binding induces the recruitment of SHP2 to the hPD-1 microclusters to suppress T cell signaling by dephosphorylation of receptors and their downstream signaling molecules.a 2D12 cells expressing both hPD-1–HaloTag (red) and EGFP–SHP1 (left, green) or –SHP2 (right, green) were plated onto the SLB without (top) or with hPD-L1–GPI (bottom). Histograms show fold fluorescent intensities of hPD-1 (magenta) and SHP1 (green) or SHP2 (green) on the diagonal yellow lines in the DIC images. b 2D12 cells expressing hPD-1–EGFP were conjugated by MCC88–103-prepulsed DC-1 cells not expressing (left) or expressing hPD-L1 (right). Cells were lysed, immunoprecipitated for hPD-1 by anti-GFP, and blotted for SHP1, SHP2, or hPD-1. The WCLs were blotted for SHP1, SHP2, or GFP. c 2D12 cells expressing hPD-1–EGFP (green) were prestained with DyLight 549-labeled H57 Fab (cyan), plated onto an SLB without (top) or with hPD-L1–GPI (bottom), fixed at 2 min after contact, stained with anti-phospho (p) CD3? (red). Histograms show fold fluorescent intensities of TCRß (cyan), hPD-1(green), and pCD3? (magenta) on the diagonal yellow lines in the DIC images. d The scatter plot summarizing the Pearson’s colocalization coefficients (PCC) values in (c). PCC was calculated between pCD3?/TCRß in the absence (left, 0.6232 ± 0.05) or presence of hPD-L1–GPI (right, 0.1198 ± 0.05) by 20 randomly plotted profiles on 20 cells. e The cells in b were stimulated with MCC88-103-prepulsed DC-1 cells not expressing (left) or expressing hPD-L1 (right). The WCLs were blotted for pPLC?1, PLC?1, pAkt, Akt, pErk1/2 or Erk1/2. f The graphs show the intensity ratio of pPLC?/PLC? (left), pAkt/Akt (middle) or pErk/Erk (right) in (e). g The cells in b were cocultured with DC-1 cells (black) or those expressing hPD-L1 (gray) plus indicated concentrations of MCC88-103. The concentration of IL-2 was measured by ELISA. n.d. not detected. All data are representative from two independent experiments. Bars, 5 µm. Data are presented as mean values ± SD. Statistical analysis was performed by unpaired two-sided t-test and one-way ANOVA. ****p < 0.0001. Source data for a–g are provided as a Source Data file.