Figure 5.
NOTCH1 agonism enhances CD4+ CAR-T proliferation and cytokine production in response to antigen. (A) CTV dilution by CD19-specific N1 and control CD4+ CAR-T 72 hours after restimulation on plates coated with various concentrations of rhCD19. Left: histograms of 1 donor representative of 6. Right: percent CAR-T divided and proliferation index at 72 hours. Paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01. (B) Luminex quantification of cytokine production by CD19-specific N1 or control CD4+ CAR-T after 24 hours restimulation on plate-coated rhCD19. N = 6 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01. (C) Flow cytometry analysis of IL-2R chain, STAT3 pY705, and STAT5 pY694 expression by CD19-specific N1 or control CD4+ CAR-T after 24 hours restimulation on plate-coated rhCD19. N = 5 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. (D) Schematic depicting sorting of EGFRtINT N1 and control CD4+ CAR-T (left) and the CAR expression levels of the sorted cells (right). Representative histograms of 6 donors. (E) Luminex quantification of cytokine production by CD19-specific EGFRtINT N1 and control CD4+ CAR-T after 24 hours restimulation on plate-coated rhCD19. N = 6 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. (F) CTV dilution by untransduced N1 and control CD4+ T cells after 24 hours restimulation on plate-coated agonistic αCD3 mAb (OKT3) followed by 48 hours of culture on uncoated plates. Representative plots of 5 donors. Paired 2-tailed Student t test. ∗P < .05. (G) Luminex quantification of cytokine production by untransduced N1 and control CD4+ T cells after 24 hours restimulation on plate-coated OKT3. N = 5 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01. (H) Flow cytometry analysis of IL-2R chain, STAT3 pY705, and STAT5 pY694 expression by untransduced N1 and control CD4+ T cells after 24 hours restimulation on plate-coated OKT3. N = 6 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. FSC-A, forward scatter area; n.s., not significant.

NOTCH1 agonism enhances CD4+ CAR-T proliferation and cytokine production in response to antigen. (A) CTV dilution by CD19-specific N1 and control CD4+ CAR-T 72 hours after restimulation on plates coated with various concentrations of rhCD19. Left: histograms of 1 donor representative of 6. Right: percent CAR-T divided and proliferation index at 72 hours. Paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01. (B) Luminex quantification of cytokine production by CD19-specific N1 or control CD4+ CAR-T after 24 hours restimulation on plate-coated rhCD19. N = 6 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01. (C) Flow cytometry analysis of IL-2R chain, STAT3 pY705, and STAT5 pY694 expression by CD19-specific N1 or control CD4+ CAR-T after 24 hours restimulation on plate-coated rhCD19. N = 5 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. (D) Schematic depicting sorting of EGFRtINT N1 and control CD4+ CAR-T (left) and the CAR expression levels of the sorted cells (right). Representative histograms of 6 donors. (E) Luminex quantification of cytokine production by CD19-specific EGFRtINT N1 and control CD4+ CAR-T after 24 hours restimulation on plate-coated rhCD19. N = 6 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. (F) CTV dilution by untransduced N1 and control CD4+ T cells after 24 hours restimulation on plate-coated agonistic αCD3 mAb (OKT3) followed by 48 hours of culture on uncoated plates. Representative plots of 5 donors. Paired 2-tailed Student t test. ∗P < .05. (G) Luminex quantification of cytokine production by untransduced N1 and control CD4+ T cells after 24 hours restimulation on plate-coated OKT3. N = 5 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01. (H) Flow cytometry analysis of IL-2R chain, STAT3 pY705, and STAT5 pY694 expression by untransduced N1 and control CD4+ T cells after 24 hours restimulation on plate-coated OKT3. N = 6 donors. Ratio-paired 2-tailed Student t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. FSC-A, forward scatter area; n.s., not significant.

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