Figure 4. PD-1 ligation with bead-coated PD-L1 suppresses p-STAT1, T-bet, p-S6 and production of Th1 cytokines upon TCR stimulation, 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. Supernatants from each condition were collected and stored at −80°C. Th1 (IFN-γ and IL-2) and Th2 (IL-10) cytokines in the supernatants were determined by Luminex. A) Representative data showing p-STAT1 (Y701), T-bet and p-S6 (S235/236) levels in CD8⁺ TIL under the described conditions. Summary data of frequency of p-STAT1 (Y701)+ (B), T-bet+ (C) and p-S6 (S235/236)+ (D) in CD8⁺ and CD4⁺ TIL with indicated conditions is shown (n=7). E) Summary data of amount of IFN-γ, IL-2 and IL-10 in the supernatants of TIL cultured under indicated conditions is shown. The graphs present the mean ± SEM from 8 HNSCC patients. F) Immunohistochemistry analysis of PD-1 and IFN-γ in serial sections of representative HNSCC tumors. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p<0.05, **p<0.01. p>0.05 was considered to be not significant (n.s.).