PD-L1⁺lymphoma cells secrete IL-10 cytokine stimulating STAT3 activation. (A) Cytokine protein array was analyzed in 7 PD-L1⁺ cell lines and 5 PD-L1⁻ cell lines. Arrows point to dot intensity of secreted IL-10. The table shows the cytokines included in this array. (B) Supernatants from the 12 cell lines in (A) were analyzed for IL-10 secretion using ELISA. (C) The Spearman correlation method was used to compare the protein expression levels of pSTAT3 and PD-L1 vs IL-10 secretion in 12 cell lines in (A). (D) TMD-8 and HBL-1 cells were treated with recombinant IL-10 (10 ng/mL) or IL-10 with IL-10R antibodies for 24 hours. Protein extracts were analyzed for pSTAT3, STAT3, PD-L1, and actin. (E) TMD-8 and HBL-1 cells were transiently transfected with control or predesigned and validated STAT3 siRNAs. At 48 hours after transfection, proteins purified from transfected cells were subjected to western blotting for STAT3, PD-L1, and actin (loading control). (F) PD-L1⁻ DLBCL cell lines (RC and MZ) and PD-L1⁺ DLBCL cell lines (OCI-LY10 and HBL-1) were transiently transfected with a control siRNA or STAT3 siRNA. At 48 hours posttransfection, ChIP assays, followed by qPCR, were performed. Bar graphs show the results of qPCR of PD-L1 promoter region pulled down by the STAT3 antibody as fold enrichment relative to the background signals from the isotype-control immunoglobulin G antibody. NS, not statistically significant.