Improved persistence and expanded T_PEX formation in Regnase-1 KO CAR-T cells is TCF-1 dependent. (A) Luciferase activity of HEK293T cells after transfection with Tcf7 mRNA 3′ UTR reporter, together with control (mock), Regnase-1 wild-type, or Regnase-1 D141N-expressing plasmid. (B-E) Naive MACS-purified CD8⁺Cas9⁺ hCD19 CAR-Tg cells were activated and transduced with sgRNA targeting Regnase-1 (KO CAR) or Regnase-1 and TCF-1 (DKO CAR). Cells were sorted based on fluorescent reporter expression and cotransferred 5:1 (KO:DKO) into tumor-bearing mice. Organs were harvested at the indicated time points. (B) Frequency and numbers of KO and DKO CAR-T cells (normalized to input ratio). (C) Relative expression of select genes from microarray analysis of KO and DKO CAR-T cells harvested from spleens of tumor-bearing mice 7 days after cotransfer (n = 5 mice per group). (D) Frequencies of CD127⁺KLRG1⁻ and CD127⁻KLRG1⁺ KO and DKO CAR-T cells 7 days after cotransfer. (E) Representative histograms (spleen only) and frequencies of IFNγ⁺, IL-2⁺, TNFα⁺, and granzyme B⁺ KO and DKO CAR-T cells from the spleens and bone marrow of tumor-bearing mice 7 days after cotransfer. Endogenous host CD8⁺ T cells are shown as a gating control. Significance was determined by 1-way ANOVA with Tukey posttest for multiple comparisons (A) or paired Student t test (B,D-E). Data are shown as mean plus or minus SEM (A-B,D-E) and represent 3 (A) or 2 independent experiments with 5 mice per group (B,D-E). *P < .05; **P < .01; ***P < .001; ****P < .0001.