Fig. 4

p65 and STAT1 are involved in IL-1a- and IL-27-induced PD-L1 expression, respectively, in tumor cells. Cultured tumor cells were treated with IL-1a (10 ng/ml), IL-27 (50 ng/ml), or IFN-g (100 IU/ml). STAT1, STAT3, and p65 phosphorylation was detected by Western blotting 15 min after cytokine exposure. In experiments to inhibit phosphorylation, transcription factors first were knocked down by transfecting specific siRNAs; after 2 days, transfected cells were treated with cytokines and knockdown effects were assessed with Western blotting. PD-L1 cell surface protein expression was detected by flow cytometry 1 day after cytokine treatment. a. In two RCC cell lines, IL-27 exposure caused phosphorylation of both STAT1 and STAT3, while IFN-g selectively phosphorylated STAT1, and IL-1a did not phosphorylate either STAT1 or STAT3. Pos ctr, positive control; mixture of equal amounts of IFN-treated PC-3 cells as a pSTAT1 positive control, and IL-6-treated COS-7 cells as a pSTAT3 positive control. b. In 397mel, STAT1 but not STAT3 knockdown significantly reduced IL-27-induced PD-L1 expression. Results representative of 2 tumor cell lines (one MEL, one SCCHN). c. IL-1a increased p65 phosphorylation, but not STAT1 or STAT3 phosphorylation, in 14 tumor cell lines. After cytokine exposure, phosphorylation of the indicated transcription factors was detected by Western blotting. Protein bands were quantified by ImageJ and results were normalized to beta-actin expression. Because all cell lines expressed phosphorylated p65 constitutively in the absence of cytokines, values for constitutive normalized ratios have been subtracted from the data depicted for pp65. PD-L1 increased, cytokine-induced enhancement of PD-L1 cell surface expression of ≥5 MFI detected with flow cytometry (red symbols); no or lower levels of PD-L1 enhancement indicated by black symbols. Kruskal-Wallis test (Dunn’s multiple comparisons test), 2-sided p-values. d. Knocking down p65 reduced IL-1a-induced PD-L1 protein expression in 786-O. Percentage represents reduction in total PD-L1 expression with p65 knockdown compared to scrambled siRNA control; number in parentheses represents reduction in the amount of PD-L1 expression that was induced by IL-1a above the “no cytokine” baseline expression. Results in panel D are representative of 3 separate experiments with 786-O. Corresponding Western blot is provided in Additional file 2: Fig. S2. ΔMFI, mean fluorescence of specific PD-L1 staining – isotype control staining