CD5 CAR T cells recognize and kill primary T-ALL cells. (A) Production of IFN-g upon coculture with different primary T-ALL samples was assessed by intracellular cytokine staining. Numbers indicate percent or CAR⁺ T cells positive for IFN-g. (B) Production of IFN-g (left), TNFa (middle), and expression of CD107a (right) by CD5 CAR T cells upon mixing with thawed T-ALL blasts from 2 patients (T-ALL #295 and #315). Bar graphs depict frequency of cytokine-producing CD4⁺ and CD8⁺ T cells as average ± SEM from 4 donors. (C) Cytotoxicity of CD5 CAR T cells against fresh primary T-ALL blasts isolated from peripheral blood mononuclear cells of a T-ALL patient #394 was measured in a 5-hour Cr release assay. (D) Protein expression of PI-9 and (E) Bcl-2 in T-ALL blasts from donor #394 was measured by intracellular staining and flow cytometry. Expression histogram in Jurkat cells is shown with a dotted line (left). Bar graphs depict corresponding MFI compared with CD5 CAR T cells (mean ± SD from 3 donors) and Jurkat T-ALL cell line (right).