IRF4 regulates PD-L1 expression in ALK⁺ ALCL. (A) ALCL lines were transduced with IRF4 or Ctrl sgRNAs along with the green fluorescent protein (GFP). Surface PD-L1 expression in transduced (GFP⁺) cells and sgRNA un-transduced (GFP⁻) cells were measured by flow cytometry. The relative PD-L1 MFI was normalized to the un-transduced (GFP⁻) cells. Number of repeats: DEL, n = 10; Karpas299, n = 10; SR786, n = 5; L-82, n = 4; and SUDHL1, n = 4). Error bars denote standard error of the mean (SEM). P < .05 for all the data. (B) DEL and Karpas 299 cells transduced with WT IRF4 or with a control vector, were induced to express control or IRF4 shRNAs along with GFP. Surface PD-L1 expression in uninfected (GFP⁻) cells and shRNA-infected (GFP⁺) cells were measured by flow cytometry. The relative PD-L1 MFI was normalized to the uninfected (GFP⁻) cells. Error bars denote SEM of 4 repeats. **P < .01; *P < .05. (C) ALCL lines were infected with IRF4 or Ctrl sgRNAs, selected and expression induced. Lysates were analyzed by immunoblotting for the indicated proteins. (D) ALCL lines were infected with IRF4 or Ctrl sgRNAs, selected and expression induced, PD-L1 gene expression was measured by real-time PCR. Error bars denote SEM of triplicates. (E) Expression of PD-L1, IRF4, and CD30 in ALK⁺ ALCL primary cases by IHC. Immunohistochemical PD-L1, IRF4, and CD30 staining are shown in 2 cases. Section of lymph nodes were examined microscopically, using a 200× magnification. The depicted images are representative for the 14 ALK⁺ ALCL cases examined. (F) Correlation between PD-L1 IHC scores with IRF4 IHC scores, calculated by Spearman's rank correlation methods, in 14 ALK⁺ ALCL cases.