Loss of CD7 does not alter phenotype or effector function of CAR T cells. (A) T cells were electroporated with Cas9 complexed with CD7-specific or control (CD19 specific) gRNA and transduced with CD19 CAR. Representative dot plots show expression of CD7 and CD19 CAR in T cells electroporated with Cas9+gRNA 7 days posttransduction. Nontreated activated T cells were used as control. Numbers denote frequency of cells in corresponding quadrants. (B) Frequency of naïve-like cells (naïve; CCR7⁺CD45RA⁻), central memory (CM; CCR7⁺CD45RA⁻), effector memory (EM; CCR7⁻CD45RA⁻), and effector memory RA (EMRA; CCR7⁻CD45RA⁺) in CD19 CAR T cells assessed by flow cytometry on day 7 posttransduction. (C) Frequency of CD4⁺ and CD8⁺ CD19 CAR T cells 7 days posttransduction. (D) CD19 CAR T cells were incubated with CD19⁺ NALM6 cells, and production of TNFα and IFNγ in CD4⁺ cells was assessed by intracellular cytokine staining. Dot plots represent cytokine production in CD19 CAR T cells in the presence of NALM6 or in media alone. Summarized data from 3 donors are shown on the right. (E) Control or CD7^KO CD19 CAR T cells or control nontransduced T cells were cocultured with GFP⁺ Raji cells at the effector-to-target ratio 1:1 for 72 hours. Dot plots show representative frequency of gated CAR T cells and GFP⁺ tumor cells at the end of coculture. Total numbers of live tumor cells (F) and CD19 CAR T cells (G) were counted by flow cytometry at 72 hours using counting beads. Lines denote individual donors. Data represent 2 independent experiments with n = 3 donors in each. *P < .05; **P < .01; ****P < .0001.