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Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses, and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1, attenuate differentiation, and enhance proliferation and antitumor efficacy in vivo. Here, we used a cell-free culture system to provide an agonistic NOTCH1 signal during naïve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation, transcription factor expression, cytokine production, and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from naïve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF, driving heightened production of interleukin-22 (IL-22), IL-10, and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T, driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors. Overall design: 18 samples (9 controls)ENARNA polymerase II core promoter proximal region sequence-specific binding, T cell activation, T-cell leukemia, biological signaling, xnotch, T-cell surface antigen T4/Leu-3, hN1, T-cell surface antigen T4|Leu-3, T-lymphocyte activation, AGL4, 9930111A19Rik, zinc ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, homeobox 1, Factor, lin-12, SEPALLATA 2, metal ion regulated sequence-specific DNA binding RNA polymerase II transcription factor activity, Transcription Factor, transcription factor activity, N1, L3T4, Motch, zinc ion regulated core promoter proximal region sequence-specific DNA binding, NOTCH, T-cell differentiation antigen L3T4, RNA polymerase II distal enhancer sequence-specific DNA binding transcription factor activity, T-cell activation, Ly-4, RNA polymerase II transcription factor activity, sequence-specific distal enhancer binding RNA polymerase II transcription factor activity, notch, metal ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, KNOTTED1-like homeobox gene 5, F14P3.4, xnotch1, Transcription, Factors, T lymphocyte activation, T-cell surface glycoprotein CD4, F14P3_4, F10N7_150, CD4mut, metal ion regulated core promoter proximal region sequence-specific binding, copper ion regulated core promoter proximal region sequence-specific binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding transcription factor activity, Transcription factor, F10N7.150, signalling, Antigen., notch1-a, signalling process, RNA polymerase II proximal promoter sequence-specific DNA binding, AOVD1, signaling process, copper ion regulated proximal promoter sequence-specific DNA binding, Xotch, AOS5, copper ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, AGAMOUS-like 4, CD4, notch-1, zinc ion regulated proximal promoter sequence-specific DNA binding, sequence-specific transcription regulatory region DNA binding RNA polymerase II transcription factor recruiting transcription factor activity, T14, TAN1, Tan1, metal ion regulated sequence-specific DNA binding, Mis6, metal ion regulated proximal promoter sequence-specific DNA binding, TRANSCRIPTION FACTOR, single organism signaling, RNA polymerase II distal enhancer sequence-specific bindingRNA polymerase II core promoter proximal region sequence-specific binding, T cell activation, T-cell leukemia, biological signaling, xnotch, T-cell surface antigen T4/Leu-3, hN1, T-cell surface antigen T4|Leu-3, T-lymphocyte activation, AGL4, 9930111A19Rik, zinc ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, homeobox 1, Factor, lin-12, SEPALLATA 2, metal ion regulated sequence-specific DNA binding RNA polymerase II transcription factor activity, Transcription Factor, transcription factor activity, N1, L3T4, Motch, zinc ion regulated core promoter proximal region sequence-specific DNA binding, NOTCH, T-cell differentiation antigen L3T4, RNA polymerase II distal enhancer sequence-specific DNA binding transcription factor activity, T-cell activation, Ly-4, RNA polymerase II transcription factor activity, sequence-specific distal enhancer binding RNA polymerase II transcription factor activity, notch, metal ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, KNOTTED1-like homeobox gene 5, F14P3.4, xnotch1, Transcription, Factors, T lymphocyte activation, T-cell surface glycoprotein CD4, F14P3_4, F10N7_150, CD4mut, metal ion regulated core promoter proximal region sequence-specific binding, copper ion regulated core promoter proximal region sequence-specific binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding transcription factor activity, Transcription factor, F10N7.150, signalling, Antigen., notch1-a, signalling process, RNA polymerase II proximal promoter sequence-specific DNA binding, AOVD1, signaling process, copper ion regulated proximal promoter sequence-specific DNA binding, Xotch, AOS5, copper ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, AGAMOUS-like 4, CD4, notch-1, zinc ion regulated proximal promoter sequence-specific DNA binding, sequence-specific transcription regulatory region DNA binding RNA polymerase II transcription factor recruiting transcription factor activity, T14, TAN1, Tan1, metal ion regulated sequence-specific DNA binding, Mis6, metal ion regulated proximal promoter sequence-specific DNA binding, TRANSCRIPTION FACTOR, single organism signaling, RNA polymerase II distal enhancer sequence-specific bindingfalseNOTCH1 signaling during CD4+ T-cell activation alters transcription factor networks and enhances antigen responsiveness [CULTURED_NCT07]NOTCH1 signaling during CD4+ T-cell activation alters transcription factor networks and enhances antigen responsiveness [CULTURED_NCT07]2025-09-242022-07-06PRJNA854669GSE2073149606