STAT3 inhibition suppresses PD-L1 expression in ATLL cells. (A) Cell surface expression of PD-L1 in KK1 and ST1 ATLL cells transduced with the indicated sgRNA by flow cytometry. PD-L1 mean fluorescent intensity (MFI) was normalized by that of control sgRNA (sgAAVS1)-transduced cells. (B) Cell surface expression of PD-L1 in ATLL (KK1, Su9T01, and ST1) and ALK+ ALCL (DEL, Karpas299, and SUDHL1) cells treated with ruxolitinib for 24 hours by flow cytometry. MFI was normalized by that of dimethyl sulfoxide (DMSO)-treated cells. (C) Immunoblot analysis of p-STAT3, STAT3 and α-tubulin in ruxolitinib-treated ATLL and ALK⁺ ALCL cells. (D) Schema for the genomic locations targeted by sgRNAs for CRISPR/Cas9-mediated gene editing in the PD-L1 3′-UTR region. (E-F) Cell surface expression of PD-L1 in KK1 PD-L1 SV#7 cells and KK1 PD-L1 SV#15-12 cells by flow cytometry. The cells were treated with the indicated concentrations of ruxolitinib for 24 hours, and MFI was normalized by that of DMSO-treated cells in panel F. (G) Immunoblot analysis of PD-L1, p-STAT3, STAT3, and α-tubulin in KK1 PD-L1 SV#7 cells and KK1 PD-L1 SV#15-12 cells treated with or without ruxolitinib (312.5 nM) for 24 hours. Error bars represent the mean with standard error of the mean (SEM) of replicates. ∗P < .05; ∗∗P < .01, Welch 2-sample t test. All experiments were repeated at least 2 times.