Fig. 3

IL-1a- and IL-27-induced PD-L1 protein expression are associated with new PD-L1 mRNA transcription in tumor cells. Fourteen cultured tumor lines were treated with IL-1a (10 ng/ml) or IL-27 (50 ng/ml) for 48 h, and cell surface PD-L1 protein was detected by flow cytometry. a. IL-1a alone (left panel) or in combination with IFN-g (right panel) increased PD-L1 expression on tumor cells. ΔMFI, mean fluorescence intensity of PD-L1 staining – isotype control staining. Wilcoxon matched-pairs signed rank test, 2-sided p-values. b. IL-27 independently increased PD-L1 protein expression on tumor cells (left panel), and a further increase was observed when IL-27 was combined with IFN-g (right panel). c. Overlay of flow cytometry histograms from two representative RCC cell lines (ACHN and A498). Either IL-1a or IFN-g independently increased PD-L1 expression, and a greater increase was observed when these cytokines were combined. Note that ACHN and A498 both show constitutive PD-L1 expression in the absence of cytokine treatment. d. Overlay of flow cytometry histograms of ACHN and A498 cells treated with IL-27 or IFN-g, alone or in combination. e. Increased PD-L1 protein expression induced by IL-1a or IL-27 was associated with new PDL1 mRNA transcription in 2 RCCs tested. PD-L1 mRNA and cell surface protein were measured by qRT-PCR and flow cytometry at 16 h or 48 h after cytokine exposure, respectively