NOTCH1 agonism during CD4⁺ T_N activation results in a less differentiated phenotype. (A) Left: flow cytometry of NOTCH receptor expression compared with isotype in human CD4⁺ T_N at rest (top) and 3 days after activation with αCD3/CD28 mAb-coated beads and IL-2 (bottom). Histograms show 1 representative donor. Right: percent of CD4⁺ T_N expressing each NOTCH receptor at rest and over time after activation. N = 6 donors. (B) Schematic of the N1 culture method using αNOTCH1 mAb- and RetroNectin-coated plates. (C) Reverse transcription quantitative polymerase chain reaction evaluation of Hes1 (left) and Dtx1 (right) expression relative to B2m in activated CD4⁺ T_N after 48 hours of culture on plates coated with different combinations of 2.5 μg/mL αNOTCH1 mAb, 2.5 μg/mL M IgG1κ, and 5 μg/mL RN. N = 3 donors. One-way ANOVA with Dunnett’s multiple comparisons test. ∗∗P < .01; ∗∗∗P < .005. (D) Expression of Hes1 relative to B2m in activated CD4⁺ T_N after 48 hours of N1 culture in the presence of various doses of RO4929097 γ-secretase inhibitor (γSI). N = 3 donors. One-way ANOVA with Dunnett’s multiple comparisons test. ∗P < .05; ∗∗P < .01. (E) Fold expansion of N1 and control CD4⁺ T cells (left), viability assessed by propidium iodide exclusion (middle) and CAR lentivirus transduction rate (right) measured after 11 days of culture. N = 8 donors. Ratio-paired 2-tailed Student t test. ∗∗P < .01. (F) CD45RA and CD62L expression on N1 and control CD4⁺ CAR-T at day 11. Left: representative flow cytometry plots. Right: frequencies of N1 and control CD4⁺ CAR-T with T_SCM, T_CM, effector memory and terminal effector phenotypes across 11 donors. Ratio-paired 2-tailed Student t test. ∗∗P < .01; ∗∗∗∗P < .001. (G) Left: GSEA of RNAseq data from day 11 N1 and control CD4⁺ CAR-T. Right: heatmaps depict log₂(normalized count/global average) for master regulator TFs and selected leading edge genes with a cutoff at 1.5-fold change. Significance was established at P and q both <.05 after correction for multiple hypothesis testing. See also supplemental Table 1. Ctrl, control; DMSO, dimethyl sulfoxide; FDR, false discovery rate; NES, normalized enrichment score; n.s., not significant; RN, RetroNectin.