Fig. 4: IL-20 induced PD-L1 expression, and 7E neutralized IL-20-induced PD-L1 expression.

a Pancreatic tissue from an orthotopic PDAC model mouse was analyzed by immunofluorescence labeling of IL-20 (green) and PD-L1 (red). Nuclei (blue) were visualized by DAPI staining. Scale bar, 50 μm. b KPC cells were treated with IL-20 or IL-20 plus 7E, and the expression of PD-L1 in the KPC cells was analyzed using RT-PCR with specific primers. c KPC cells were treated with mIL-20 for the indicated time, and the fold increase in the expression of IFN-α was analyzed using RT-qPCR with specific primers (n = 1 strain of cell culturing; one-way ANOVA, p = 0.0488). d IFN-α mRNA expression (n = 1 strain of cell culturing; one-way ANOVA, p < 0.0001) and e PD-L1 mRNA expression in KPC cells were analyzed after IL-20 treatment using RT-qPCR (n = 1 strain of cell culturing; one-way ANOVA, p = 0.0042). *p < 0.05, **p < 0.01, and ***p < 0.001. KPC mice and orthotopic PDAC model mice were treated with 7E, and pancreatic tissue samples were analyzed for f PD-L1 (one-way ANOVA, p < 0.0001), g, h CD8, and PD-1 staining quantification. The rightmost panel shows the IgG-stained negative control. Scale bar, 50 μm. g CD8 and PD-1: one-way ANOVA, p < 0.0001. h CD8 and PD-1: one-way ANOVA, p < 0.0001. ****p < 0.001 compared with the mIgG-treated controls. KPC/Luc cells were injected into the pancreas of WT mice. PBS, mIgG, an anti-PD-1 antibody, 7E, or 7E + the anti-PD-1 antibody (n = 5) were injected (i.p.) into the mice twice per week for 28 days. i Analysis of tumor growth by using the in vivo luminescence activity of KPC/Luc cells on day 21. j, k Tumors were collected and weighed (n = 5; one-way ANOVA, p = 0.005). l Luciferase activity was measured on day 21 posttreatment and used to quantify tumor growth. m, n Tumors were harvested and analyzed (n = 5; one-way ANOVA, p < 0.0001). *p < 0.05, **p < 0.01, and ****p < 0.0001. Values represent the mean ± SD. Statistical significance was determined by one-way ANOVA Sidak’s multiple comparisons test (c–h, k, n). The experiments in c–e were repeated three times independently with similar results, and the data of one representative experiment are shown.