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. 2017 Feb 6;17:30–44. doi: 10.1016/j.ebiom.2017.02.004

Fig. 3.

Fig. 3

High-affinity A132L PD-1 Ig increases T cell proliferation and cytokine production in vitro after allogeneic stimulation. (A) A132L PD-1 Ig shows increased binding to human mature dendritic cells expressing PD-L1 and PD-L2, compared to wild type PD-1. Non-binding (NB) PD-1 Ig shows negligible binding only at high concentration (50 μg/ml). Data are representative of five independent experiments. (B) Kd values calculated from binding data for wild type and HA PD-1 Ig, respectively; the horizontal lines indicate the median values. Kd-s were obtained by one-site fitting of the binding curves (Y = Bmax ∗ X/(Kd + X), where X is the concentration in nM, Y is specific binding expressed as MFI; Bmax: maximum binding). Data from five independent experiments are shown, analyzed by non-parametric Wilcoxon rank test, *p < 0.05. (C) Increased CD4+ T cell proliferation measured in the presence of HA PD-1 Ig, but not wild type PD-1 Ig, relative to the untreated group (no Ig). Blocking antibody to PD-L1 was used as positive control, K78A (NB) mutant PD-1 Ig or human IgG were used as negative controls. Groups were compared by ANOVA, followed by Bonferroni's post-test, ***p < 0.0001. (D) T cell proliferation is enhanced by anti-PD-L1, and HA PD-1 Ig, but not wild type PD-1 Ig, in allogeneic MLR reactions. Proliferation was normalized to the untreated control (100%) within each experiment. Data from 4 independent experiments are shown, each symbol representing average normalized value (of 4 replicates) from one individual experiment. Horizontal bars represent median values. Treatments were compared to wild type PD-1 Ig, (Kruskall-Wallis test, *p < 0.05). Secretion of T cell cytokines IFN-γ (E), IL-12 (F), IL-2 (G), TNF-α (H), TNF-β (I), IL-5 (J), IL-13 (K), and IL-10 (L) is enhanced after PD-1 blockade in the presence of either HA PD-1 Ig, or PD-L1 blocking antibody. Treatment reagents were used at 5 μg/ml, unless noted otherwise on the graphs. Groups were compared by ANOVA, followed by Dunnett's post-test, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.