Figure 3

PD-1 ligation with bead-coated PD-L1 suppressed TCR-stimulated up-regulation of p-STAT1, T-bet and production of Th1 cytokines, while anti-PD-1 blockade could reverse the suppressive effects of PD-1. Total TIL were stimulated with anti-CD3/-CD28/hIgG1 or anti-CD3/-CD28/PD-L1 coated beads (bead: cell=10:1) for 48h in the presence of 100ug/mL hIgG4 or anti-PD-1 (BMS-936558), then p-STAT1, T-bet and p-S6 were analyzed by flow cytometry. Summary data of frequency of p-STAT1 (Y701)+ (A), T-bet+ (B) and p-S6 (S235/236)+ (C) in CD8+ and CD4+ TIL with indicated conditions is shown (n=7). Supernatants of TIL stimulated with anti-CD3/-CD28/hIgG1 or anti-CD3/-CD28/PD-L1 coated beads (bead: cell=10:1) for 48h in the presence of 100ug/mL hIgG4 or anti-PD-1 (BMS-936558) were collected and stored at -80°. Th1 (IFN-γ and IL-2) and Th2 (IL-10) cytokines in the supernatants were determined by Luminex. Summary data of amount of IFN-γ (D), IL-2 (E) and IL-10 (F) in the supernatants of TIL cultured under indicated conditions is shown. The graphs present the mean ± SEM from 8 HNC patients. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p<0.05, **p<0.01.