Ibrutinib does not impair CAR gene transfer, T-cell proliferation, or cytotoxic capacity and limits Th2 activation in CTL019 cells. (A) Anti-CD19 CAR lentiviral transduction efficiency after T-cell enrichment/expansion in the presence or absence of ibrutinib (0-5 μM). (B) Proliferative capacity of T cells as demonstrated by a CFSE dilution assay in unstimulated (black peaks) and CD3/CD28 bead–stimulated (red peaks) lymphocytes in the presence or absence of short-term ibrutinib exposure (0-5 μM). Experiments were repeated at least 3 times with independent donors. Culture conditions were set up to mimic the expansion process used to produce CTL019 cells. (C) Cytotoxic capacity of CTL019 cells after overnight coculture with tumors. For cytokine analysis and the cytotoxicity assays, CAR T cells were pretreated with ibrutinib for 1 hour and washed extensively before exposure to targets. The cytotoxicity assay is representative of 4 independent experiments conducted with different healthy donors. Normalized production of IL-12 (D) and IFN-γ (E) compared with IL-4 (n = 3 independent donors) in expanded CTL019 cells exposed for 18 hours to targets expressing K562-CD19. Significant differences are depicted as *P < .05 and **P < .01 (2-tailed Student t test). DMSO, dimethylsulfoxide; Ibr., ibrutinib; n.s., not statistically significant.