Anti–CD19-CAR–transduced T cells produced cytokines in a CD19-specific manner and recognized autologous leukemia cells. (A) Staining with an anti-Fab Ab revealed expression of the anti-CD19 CAR on the surface of T cells that were administered to patient 7. Staining with an isotype control Ab is also shown. Both plots were gated on CD3⁺ lymphocytes, which made up 99% of the cells in the culture. (B) On the day of infusion, T cells of patient 7 up-regulated CD107a expression after a 4-hour culture with the CD19⁺ target cell CD19-K562 but not the negative control cell NGFR-K562 that does not express CD19. (C) On the day of infusion, anti–CD19-CAR–transduced T cells of patient 7 produced IFNγ, TNF, and IL-2 when cultured for 6 hours with the CD19⁺ target cell CD19-K562 but not the negative control cell NGFR-K562 that does not express CD19. The results shown in panels A through C are representative of the results obtained for all of the patients on the protocol. (D) Anti–CD19-CAR–transduced T cells of patient 3 were cultured with either autologous pretreatment lymphocytes or autologous remission lymphocytes overnight, and an IFNγ ELISA was performed on the supernatant. Anti–CD19-CAR–transduced T cells of patient 3 specifically recognized pretreatment lymphocytes but not remission lymphocytes obtained 7 weeks after CAR-transduced T-cell infusion. Sixty-four percent of the pretreatment lymphocytes were CD19⁺ leukemia cells. The remission lymphocytes contained only 0.1% CD19⁺ cells. (E) Anti–CD19-CAR–transduced T cells of patient 6 were cultured with either pretreatment autologous lymphocytes or autologous remission lymphocytes overnight and an IFNγ ELISA was performed on the supernatant. Anti–CD19-CAR–transduced T cells of patient 6 specifically recognized pretreatment lymphocytes but not remission lymphocytes obtained 2 weeks after CAR–transduced T-cell infusion. Seventy-six percent of the pretreatment lymphocytes were CD19⁺ leukemia cells. The remission lymphocytes contained only 0.1% CD19⁺ cells. In both panels D and E, pretreatment lymphocytes cultured alone did not produce detectable quantities of IFNγ.