IRF4 and BATF3 directly regulate PD-L1 transcription in ALK⁺ ALCL. (A) DEL cells transduced with WT or DNA binding mutant 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. IRF4 expression (right) was measured by intracellular flow cytometry. Error bars denote SEM of triplicates. **P < .01. (B) Diagrammatic representation of the AICE motif in PD-L1 predicted enhancer region (up). Chromatin immunoprecipitation from indicated antibodies was subjected to real-time PCR analysis for the PD-L1 enhancer region in DEL and Karpas299 lines (low). Error bars denote SEM of triplicates. (C) ALCL lines were infected with BATF3 or Ctrl sgRNAs, along with GFP. Surface PD-L1 expression in uninfected (GFP⁻) cells and sgRNA infected (GFP⁺) cells was measured by flow cytometry. The relative PD-L1 MFI was normalized to the uninfected (GFP⁻) cells. Error bars denote SEM of 4 repeats. P < .05 for all the data. (D) ALCL lines were infected with BATF3 or Ctrl sgRNAs, selected and induced to expression. Lysates were analyzed by immunoblotting for the indicated proteins. (E) ALCL lines were infected with BATF3 or Ctrl sgRNAs, selected and induced to expression; PD-L1 gene expression was measured by real-time PCR. Error bars denote SEM of triplicates. (F) ALCL lines were infected with BATF3 or Ctrl shRNAs along with GFP. Surface PD-L1 expression in uninfected (GFP⁻) cells and shRNA-infected (GFP⁺) cells was measured by flow cytometry. The relative PD-L1 MFI was normalized to the uninfected (GFP⁻) cells. Error bars denote SEM of triplicates. P < .05 for all the data. One of the represented histogram is shown (right). (G) ALCL lines were infected with BATF3 or Ctrl shRNAs, selected and induced to expression. Lysates were analyzed by immunoblotting for the indicated proteins.