Functionality of _RV19BB_CRTRBC⁻ CAR T cells and alloreactive potential of TCR⁺ T cells in vitro. (A-D) The functionality of _RV19BB_CRTRBC⁻ CAR T cells was compared to _RV19BB_TCR⁺, _RV19BB_CRTCR⁺ CARs and untransduced T cells in different functionality assays. (A) Twenty-four hours after coculturing the CAR T cells with CD19⁺ target cells in a 1:1 E:T ratio, the cells were harvested and analyzed for expression of activation markers CD137 and CD25 (n = 6). (B) Intracellular staining of IFNγ and TNFα was performed after 24 hours of coculturing effector cells with CD19⁺ cells at an E:T of 1:1 (n = 4). (C) CFSE labeled T cells were used to analyze the frequency of proliferating effector cells after contact with CD19⁺ target cells for 72 hours (n = 3). (D) CD19⁺ target-cell killing was determined by flow cytometry after target cells were cocultured for 48 hours with CAR T cells in different E:T ratios (n = 4). (E-F) Untransduced, _CRTCR⁺, _CRTRBC⁻ T cells were cocultured with allo-PBMCs pooled from 6 different donors and cocultured at an E:T ratio of 1:5. (E) After 48 hours of coculture, T cells were analyzed for surface expression of the activation markers CD69, CD25, and CD137 (n = 3). (F) Percentage of proliferating T cells after contact with allo-PBMCs was analyzed after 5 days (n = 3). A 2-tailed paired Student t test or 1-way ANOVA was performed to determine statistical significance.