NOTCH1 agonism programs cytokine production by inducing AhR and c-MAF. (A) Ahr, Tbx21, Gata3 and genes enriched in N1 compared with control CD4⁺ CAR-T at day 3 of culture. Heatmaps depict log₂(normalized count/global average) with a cutoff at 1.5-fold change. N = 3 donors per group. Significance was established as a minimum fold change of 1.5 and P < .05. See also supplemental Table 2. (B) Intracellular staining for AhR, c-MAF, T-bet, and GATA-3 in N1 and control CD4⁺ CAR-T at day 3 of culture. Histograms are representative of 5 donors. (C) Expression of AhR- and c-MAF–driven genes in N1 and control CD4⁺ CAR-T quantified by RNAseq on day 3 of culture. Heatmaps depict log₂(normalized count/global average) with a cutoff at 1.5-fold change. N = 3 donors per group. (D) Luminex quantification of cytokine production by CD19-specific N1 CD4⁺ CAR-T grown in DMSO or 5 μM CH-223191 after 24 hours of coculture with irradiated CD19⁺ Raji or K562 tumor in absence of drug. Graph shows mean and standard deviation of relative sample values from 8 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001. (E) Intracellular staining for IL-22 in N1 CD4⁺ CAR-T grown in DMSO or 5 μM CH-223191 after restimulation for 5 to 8 hours with PMA/ionomycin or CD19⁺ Raji tumor. N = 6 donors. Ratio-paired 2-tailed Student t test. ∗∗∗P < .005; ∗∗∗∗P < .001. (F) Luminex quantification of cytokine production by N1 CD4⁺ T cells transduced with a scrambled or Maf-targeting shRNA after 24 hours of stimulation with PMA and ionomycin. Graph shows mean and standard deviation of relative sample values from 6 donors. One-way ANOVA of log-transformed pg/ml values with Dunnett’s multiple comparison testing. ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001. (G) Intracellular staining for IL-22 in N1 CD4⁺ T cells transduced with a scrambled or Maf-targeting shRNA after restimulation for 5 to 8 hours with PMA/ionomycin. N = 6 donors. One-way ANOVA of log-transformed pg/ml data with Dunnett’s multiple comparison testing. Ctrl, control; DMSO, dimethyl sulfoxide; n.s., not significant.