GSI pretreatment leads to increased CAR T-cell reactivity to myeloma cell line and primary myeloma samples. (A) Percentage of cytokine⁺ of CD4⁺ (top) or CD8⁺ (bottom panels) BCMA CAR T cells after stimulation with MOLP8 myeloma cell line pretreated with 10 μM RO4929097 or 0.1 μM LY3039478 for 4 hours. (B) Proliferation of CD4⁺ or CD8⁺ BCMA CAR T cells measured by CFSE dilution after 72 hours of stimulation with primary myeloma cells (E:T 2:1) that were pretreated with 1 μM RO4929097 (blue histogram) or 1 μM LY3039478 (light green histogram) for 4 hours. No GSI control is indicated by the red line and T cells only by the lavender histogram. Fraction of cells proliferated >3 times (CD4) or >2 times (CD8) is indicated per histogram. Data shown are representative of n = 3. (C) Representative contour plot of intracellular IFN-γ staining of CD8⁺ BCMA CAR T cells stimulated for 4 hours with patient primary myeloma cells pretreated with 0.1 μM LY3039478 at an E:T ratio of 5:1. (D) Percentage of IFN-γ⁺ CD4⁺ or CD8⁺ BCMA CAR T cells after stimulation with patient primary myeloma cells pretreated with various concentrations of RO4929097 or LY3039478. Data in panel A are summarized from 2 independent experiments with BCMA CAR T cells prepared from at least 3 different donors. Error bars represent mean plus SEM. *P < .05, **P < .01, ***P < .001 as determined by 1-way ANOVA with the Tukey posttest.