Targeting Regnase-1 leads to durable CAR–T cell-mediated protection in an immunocompetent leukemia model. (A) Schematic of hCD19 CAR-Tg construct. (B) Kaplan-Meier survival analysis of C57BL/6 mice bearing hCD19⁺ B-ALL tumors treated with phosphate-buffered saline (PBS), or with activated control or CAR-Tg cells (5 × 10⁶ CD4⁺ and 5 × 10⁶ CD8⁺). (C-E) Naive MACS-purified CD8⁺Cas9⁺ hCD19 CAR-Tg cells were activated and transduced with nontargeting (WT CAR-T) or Regnase-1–targeting (KO CAR-T) sgRNA. Mice bearing hCD19⁺ B-ALL tumors were treated with PBS or with WT or KO CAR-T cells. Surviving mice and control naive mice were rechallenged with hCD19⁺ B-ALL cells 50 days later. (C) Rechallenge experimental design. (D) Kaplan-Meier survival analysis. (E) Tumor growth monitored by bioluminescence imaging. (F-H) CD8⁺ WT and KO CAR-T cells were cotransferred 1:1 into tumor-bearing mice. Organs were harvested at the indicated time points for analysis. (F) Cotransfer experimental design. (G) Frequency and ratio of KO/WT CAR-T cells. (H) Absolute number of WT and KO CAR-T cells shown as mean plus or minus standard error of the mean (SEM) (n = 3-5 mice per group). Significance was determined by log-rank (Mantel-Cox) test (B,D) or paired Student t test (H). Data are representative of (D-E) or pooled from (B,G-H) 2 independent experiments. *P < .05; **P < .01; ***P < .001; ****P < .0001.