Regnase-1 KO CAR-T cells undergo tumor-dependent reprogramming from effector- to memory-like cells. CD8⁺ WT and KO CAR-T cells were cotransferred 1:1 into tumor-bearing or tumor-free mice. Spleens were harvested at the indicated time points. (A) Absolute number (top) and ratio of KO/WT CAR-T cells (bottom) 7 and 21 days after cotransfer. (B) Frequencies of CD44⁺CD62L⁻, CD44⁺CD62L⁺, and CD44⁺CD62L⁻ WT and KO CAR-T cells 7 days after cotransfer. Data are shown as mean plus or minus SEM and represent 3 independent experiments with 5 mice per group. Significance was determined by the paired Student t test (A, top), unpaired Student t test (A, bottom), or 2-way ANOVA with the Tukey posttest for multiple comparison (B). (C-F) Microarray analysis of transcripts in CD8⁺ WT and KO CAR-T cells sorted 7 days after cotransfer from spleens of mice with or without tumors. (C) Principal component analysis (PCA) of all samples. (D) Gene-set enrichment analysis (GSEA) comparing differential gene expression between KO and WT CAR (KO/WT) with and without tumor to the C7 immunologic signatures gene-set collection.⁵⁴  Red circles represent enrichment in KO CAR. Blue circles represent enrichment in WT CAR. The enriched subset in each gene set is highlighted in red. (E-F) WGCNA was performed on the top third of variable genes across 4 pairwise comparisons: KO vs WT without tumor, KO vs WT with tumor, KO with vs without tumor, and WT with vs without tumor; followed by filtering for differentially expressed (DE) genes at false discovery rate (FDR) < 0.05 and log₂ fold change (FC) > 0.5 in at least 1 comparison. (E) Heatmaps of genes in clusters 3 and 8, and (F) clusters 5 and 9. (G) Expression of select genes in WT and KO CAR-T cells from tumor-bearing mice 7 and 21 days after cotransfer. Data represent biological replicates of 3 to 5 mice per group (C-G). *P < .05; **P < .01; ***P < .001; ****P < .0001. DN, downregulated gene set; UP, upregulated gene set.