N1 CD4⁺ CAR-T proliferate more than control CD4⁺ CAR-T in response to CD19⁺ tumor. (A) CFSE dilution by CD19-specific N1 and control CD4⁺ CAR-T after coculture with irradiated CD19⁺ Raji or K562 tumor cells. Left: flow cytometry plots are representative of 6 to 9 donors per timepoint. Percent cells divided (middle) and proliferation index (average number of divisions) at various timepoints after stimulation (right) are shown for individual donors. Paired Student 2-tailed t test. ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001. (B) Expression of IL-2R chains, STAT3 pY705, and STAT5 pY694 in CD19-specific N1 and control CD4⁺ CAR-T after 24 hours of coculture with CD19⁺ or CD19⁻/BCMA⁺ K562 tumor cells. Top: histograms depict expression following CD19⁺ K562 restimulation representative of 5 donors. Bottom: IL-2R and phosphoSTAT expression in CAR-T following coculture with CD19⁺ or CD19⁻ BCMA⁺ tumor cells. Ratio-paired Student 2-tailed t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. (C) Left: GSEA of RNAseq data generated from FACS-isolated CD19-specific N1 and control CD4⁺ CAR-T after 24 hours of coculture with CD19⁺ K562 tumor cells. Right: heatmaps depict log₂(normalized count/global average) for leading edge genes. Significance was established at P and q both <.05 after correction for multiple hypothesis testing. See also supplemental Tables 3 and 4. Ctrl, control; NES, normalized enrichment score.