Fig. 2

STAT1, but not STAT3 phosphorylation is necessary for IFN-g-induced PD-L1 protein expression on tumor cells. a. IFN-g had a major effect on STAT1 phosphorylation (left panel) but only a minor effect on STAT3 phosphorylation (right panel) in 31 tumor cell lines tested, including MELs, RCCs, and SCCHNs. IL-6 had a reciprocal effect in the same cell lines. Cultured cells were treated with IFN-g 250 U/ml or IL-6 20 ng/ml. Cells were harvested after 15 min and phosphorylation of STAT1 and STAT3 was detected by Western blotting. Protein bands were quantified by ImageJ and results were normalized to beta-actin expression. Kruskal-Wallis test (Dunn’s multiple comparisons test), 2-sided p-values. b and c. Specific siRNA knockdown of STAT1, but not STAT3 mRNA expression in 397mel cells significantly reduced total and phosphorylated STAT1 proteins and reduced IFN-g-induced cell surface PD-L1 protein expression. Cultured tumor cells were transfected with 100 pmol of the indicated siRNAs and were treated 2 days later with IFN-g 250 U/ml. Total and phosphorylated STAT proteins were detected by Western blotting after 15 min of IFN-g treatment, and flow cytometry for cell surface PD-L1 was conducted 1 day later. 397mel expressed HLA-DR constitutively, and this was not affected by STAT knockdown (c). d and e. In JHU-022 cultured SCCHN cells, STAT1 knockdown reduced IFN-g-induced but not constitutive (“no cytokine”) cell surface PD-L1 protein expression. IFN-g also induced HLA-DR expression on JHU-022, which was reduced by STAT1 but not STAT3 knockdown. Percentages represent reduction in total PD-L1 or HLA-DR expression with STAT1 knockdown compared to scrambled siRNA control; numbers in parentheses represent reduction in the amount of PD-L1 or HLA-DR expression that was induced by IFN-g above “no cytokine” baseline expression. Data in panels B-E are representative of 6 tumor lines (4 MELs and 2 SCCHNs). No trans, no transfection; Pos. Ctr., positive control cell lines, mixture of equal amounts of IFN-treated PC-3 cells as pSTAT1 positive control and IL-6-treated COS-7 cells as pSTAT3 positive control; Scrambled, non-specific siRNA mixture