Fig. 7: The combinational use of anti-hPD-1 and anti-hPD-1 ligands more effectively cancels hPD-1-meidated T cell suppression. | Nature Communications

Fig. 7: The combinational use of anti-hPD-1 and anti-hPD-1 ligands more effectively cancels hPD-1-meidated T cell suppression.

From: Evaluation of therapeutic PD-1 antibodies by an advanced single-molecule imaging system detecting human PD-1 microclusters

Fig. 7

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.

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