Inhibition of T-cell function by GSIs is dose dependent. (A) RO4929097 or LY3039478 do not affect cell surface CD19 expression on K562/CD19. Cells were cultured with or without GSI for 16 to 18 hours and stained with anti-CD19 and isotype control antibodies. (B) Viability of CD4⁺ and CD8⁺ CD19 CAR T cells incubated with GSI for 24 hours measured by propidium iodide staining, normalized to percentage of no GSI condition. (C) Cytolytic activity of CD4⁺ and CD8⁺ CD19 CAR T cells incubated for 4 hours with K562/CD19 (circles) or K562 control (triangles) with varying concentrations of RO4929097 (filled circles) or LY3039478 (open circles) at an E:T ratio of 30:1 and 10:1 as measured by ⁵¹Cr release assay. (D) IL-2 secretion by CD4⁺ and CD8⁺ CD19 CAR T cells stimulated with K562/CD19 at an E:T ratio of 2:1 for 18 hours in the presence of various concentrations of RO4929097. (E) IL-2 secretion by CD4⁺ and CD8⁺ CD19 CAR T cells stimulated with K562/CD19 or K562/BCMA at an E:T ratio of 2:1. CAR T cells were preincubated with the indicated concentrations of RO4929097 or LY3039478. GSI was either washed out (+/−) or present during the assay (+/+). IL-2 production was measured after 18 hours coculture with K562/BCMA or K562/CD19. (F) Proliferation of CD4⁺ and CD8⁺ CD19 CAR T cells stimulated with K562/CD19 or K562/BCMA in the indicated concentrations of RO4929097 (left panels) or LY3039478 (right panels) determined by CFSE dilution after 96 hours. Data from panels A through F are representative of ≥2 independent experiments. Error bars represent mean plus SEM.