NOTCH1 agonism enhances CD4⁺ CAR-T proliferation in mice bearing lymphoma xenografts. (A) Experimental schematic for the xenograft model. (B) Frequencies of N1 and control CD4⁺ CAR-T in peripheral blood over time following injection into tumor-bearing mice. N = 10 mice per group, 2 donors. (C) Frequencies of N1 and control CD4⁺ CAR-T at peak expansion 10 days posttransfer into tumor-bearing mice. N = 10 mice per group, 2 donors. Unpaired 2-tailed Student t test. ∗∗∗∗P < .001. (D) Frequencies of N1 and control CD4⁺ CAR-T in peripheral blood, spleen, and bone marrow of tumor-bearing mice treated with 1.6 × 10⁶ N1 or control CD4⁺ CAR-T 14 days posttreatment. N = 3 mice per group, 1 donor. Unpaired 2-tailed Student t test. ∗P < .05; ∗∗∗P < .005. (E) Gene expression by RNAseq of N1 and control CD4⁺ CAR-T FACS isolated from peripheral blood 8 days posttransfer into tumor-bearing mice. N = 3 mice per group, 1 donor. Significance was established at P < .05 and fold change > 1.5. See also supplemental Table 5. (F) Left: GSEA of RNAseq generated from N1 and control CD4⁺ CAR-T FACS-isolated from peripheral blood 8 days posttransfer into tumor-bearing mice. N = 3 mice per group, 1 donor. Right: heatmaps depict log₂(normalized count/global average) for selected leading-edge genes with a cutoff at 1. Significance was established at P and q both <.05 after correction for multiple hypothesis testing. See also supplemental Table 6. (G) Tumor burden over time in mice treated with N1 or control CD4⁺ CAR-T, evaluated by intraperitoneal luciferin injection and bioluminescent imaging. N = 10 mice per group, 2 donors. Mann-Whitney U test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. Ctrl, control; GvHD, graft-versus-host disease; n.s., not significant.