Fig. 5: hPD-L2 forms hPD-1 microclusters to suppress T cell response in a similar fashion as hPD-L1.
a 2D12 cells expressing hPD-1-EGFP (green) were imaged as shown in Fig. 1c on an MCC88-103-prepulsed SLB containing I-Ek– and mICAM-1–GPI without (top) or with hPD-L2–GPI (bottom). TCRβ, red. Histograms show fold fluorescent intensities of TCRβ (magenta) and hPD-1 (green) on the diagonal yellow lines in the DIC images. b The graph shows the percentages of TCR microclusters colocalized with hPD-1 2 min after contact in a (n = 30). c The cells in a were imaged in the absence (top) or presence of pembrolizumab (anti-hPD-1, row 2), nivolumab (anti-hPD-1, row 3), or 24 F.10C12 (anti-hPD-L2, bottom) at a concentration of 10 μg/ml. d The graph shows the percentages of T cells forming hPD-1 microclusters in c (n = 30). e The cells in a were conjugated with MCC88-103 prepulsed (1 μM) DC-1 cells expressing hPD-L2-HaloTag (cyan) in the absence or presence of the indicated antibodies at a concentration of 10 μg/ml and imaged 2 min after contacts. f The cells in a were cocultured for 16 h with 1 μM MCC88-103 and DC-1 cells not expressing or expressing hPD-L2 in the absence or presence of each antibody at a concentration of 10 μg/ml. Concentration of IL-2 in each supernatant was measured by enzyme-linked immunosorbent assay (ELISA). g Data in f were normalized among three antibodies and T cell response curves were depicted by a 4-parameter logistic function, as shown in Fig. 4e. Results are the summarized data from three independent experiments. Each plot is the average of these experiments. 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 Student’s t-test and one-way ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data for a, b, d, f and g are provided as a Source Data file.
