CD19-CAR^CD7− T cells maintain antitumor activity and secrete proinflammatory cytokines after repeated antigen exposure. (A-B) Serial stimulation analysis of effector cells in the presence of BV173 at a 1:1 E:T ratio. (A) Tumor cell lysis was evaluated every 72 hours by a luciferase-based assay, and in cases where lysis was >50%, fresh leukemia cells were added to the coculture (N = 4; stimulation 1, P < .001 between NT and CD19-CAR groups, ns between Bulk and CD7 at stimulation 1 and stimulation 5; 1-way ANOVA and nonpaired t test). Double-gradient heat map (teal, 100% target cell lysis; white, 0% target cell lysis). (B) Supernatant from odd-numbered stimulations starting at stimulation 1 was collected 24 hours after the addition of leukemia cells. Multiplex analysis of cytokine production of CAR T cells after 24 hours of restimulation (N = 4). Double-gradient heat map (blue, 10° pg/mL; white, 10² pg/mL; yellow, 10⁶ pg/mL). CD19-CAR^Bulk vs CD19-CAR^CD7− at stimulation 1 and 5, data ns by 2-way ANOVA. (C) Clonality as measured by next-generation sequencing of NT^Bulk, NT^CD7−, CD19-CAR^Bulk, and CD19-CAR^CD7− TCR repertoires throughout repeated exposure to antigen; samples were collected every 72 hours. Double-gradient heat map showing the Top10 index values (see “Methods”). Values closer to 1.0 (teal) indicate higher clonality, whereas values closer to 0.0 indicate a more even repertoire. ns, not significant.