GRB2/SOS1 are essential for PD-L1 induction downstream of ALK-NPM. (A) ALCL lines were induced to express indicated sgRNAs along with GFP. Surface PD-L1 expression in uninfected (GFP⁻) cells and sgRNA-infected (GFP⁺) cells were measured by flow cytometry. The relative PD-L1 MFI was normalized to the uninfected (GFP⁻) cells. Number of repeats for sgRNAs: GRB2, n = 8; SOS1, n = 8; KRAS, n = 3; and STAT3, n = 3. Error bars denote SEM. P < .05 for all the data. (B) ALCL lines were infected with GRB2, SOS1, or Ctrl sgRNAs, selected and expression induced. Lysates were analyzed by immunoblotting for the indicated proteins. (C) ALCL lines were infected with GRB2, SOS1, or Ctrl sgRNAs, selected and expression induced. PD-L1 gene expression was measured by real-time PCR. Error bars denote SEM of triplicates. (D) ALK IPs or total lysates from DEL, Karpas299, and FePD cells were immunoblotted for the indicated proteins. (E) ALK IPs or total lysates from DEL and Karpas299 cells treated with ALK inhibitor crizotinib (300 nM) or dimethyl sulfoxide (DMSO) for 24 hours were immunoblotted for the indicated proteins. (F) ALCL cell lines were treated with ALK inhibitor crizotinib for 24 hours at indicated concentrations; surface PD-L1 expression was measured by flow cytometry and normalized to DMSO controls. Error bars denote SEM of triplicates.