GRB2, SOS1, and IRF4 are required for ALK⁺ ALCL immunosuppression. (A) ALCL line DEL was infected with GRB2, SOSO1, IRF4, or Ctrl sgRNAs along with GFP, selected and induced to expression, after coculture with PD-1 expressing Jurkat T cells (GFP⁻; tumor: T cell ratio = 3:1) for 24 hours. T-cell activation was measured by CD69 expression. Shown are the percentage of CD69-positive T cells (GFP⁻) in each coculture condition. (B) PD-1-expressed Jurkat T cells were cocultured with ALCL cells (DEL) in the presence of indicated concentration of PD1 antibody nivolumab or with DEL infected with PD-L1, GRB2, SOSO1, IRF4, or Ctrl sgRNAs for 24 hours. Relative CD69 expression was measured by flow cytometry and normalized to the activated T-cell-only controls. Tumor: T-cell ratio = 3:1. (C-D) ALCL lines DEL and Karppas299 were infected with indicated sgRNAs, selected and induced to expression, after coculture with PD-1-expressed Jurkat T cells for 4 days. T-cell proliferation was measured by CFSE level. Shown are the CFSE staining histogram (C) and the relative CFSE MFI normalized to the activated T-cell-only controls (D). Tumor: T-cell ratio = 3:1. (E) ALCL lines DEL and Karppas299 were infected with indicated sgRNAs, selected and induced to expression, after being cocultured with PD-1 expressed Jurkat T cells. The IL-2 section was measured by ELISA and normalized to activated T-cell-only controls. Tumor: T-cell ratio = 3:1. (F) DEL line was infected with indicated sgRNAs, selected and induced to expression, after coculture with PD-1-positive NK cell line YT1 at the indicated ratio. The relative viable tumor cells remaining was shown. All error bars denote SEM of triplicates in panels B, D, and E, and SEM of duplicates in panel F.