A comparative study of 3 different approaches for generating 7CAR T cells. (A) Schematic diagram of CD7CAR constructs and schemes of the optimized protocol for generating Neg7CAR, NS7CAR, and KO7CAR T cells. (B) A comparison of the fold expansion of bulk T and 3 different CAR T cells, including Neg7CAR, NS7CAR, and KO7CAR T cells. (C) In vitro cell culture viability of Neg7CAR, NS7CAR, KO7CAR T cells, and bulk T cells at days 5, 7, and 14. (D) CAR expression was determined by FCM using tagged truncated epidermal growth factor receptor. (E) The percentages of CD7⁺ T cells in bulk T, and Neg7CAR, NS7CAR, and KO7CAR T cells are shown. Data are presented as the mean ± SD. A 2-tailed, unpaired 2-sample t test was used for statistical analysis (n = 4 for Neg7CAR and KO7CAR, n = 5 for NS7CAR, and n = 3 for bulk T cells). (F) CD7 protein (upper panel) and mRNA (lower panel) expression in Neg7CAR, NS7CAR, KO7CAR T, and control T cells were determined by RT-PCR and WB. The photographs of the gels were edited to show only the lanes relevant to this experiment. (G-I) Significant levels of CD7 mRNA and protein were detected by RT-PCR and WB from NS7CAR and control T-cell samples but not from Neg7CAR or KO7CAR samples. CAR T cells were cocultured with CFSE-labeled CCRF-CEM at the indicated E:T ratio. Cytolytic activities (G) and the cytokines concentrations (H-I) of Neg7CAR, NS7CAR, and KO7CAR are indicated. Data are presented as the mean ± SD. (J) B-NDG mice (n = 6 per group) were IV-injected with 1 × 10⁶ CCRF-CEM-Luc cells, followed by a single IV injection of 1 × 10⁷ control samples, NS7CAR, or KO7CAR cells 3 days later. Total photon flux after CAR-T injection was plotted. Data are presented as mean ± SEM (n = 6). 7CAR T, anti-CD7 CAR T; Ctl, control; E:T, effector cells: target cells; FACS, fluorescence-activated cell sorting; KO7CAR, CD7 knocked-out CAR T cells; Neg7CAR, CD7-negative T cells derived 7CAR T cells; NS7CAR, naturally selected anti-CD7 CAR T cells; PBS, phosphate-buffered saline.