FOXA1+CD3+T lymphocytes exacerbate myocardial injury in immune checkpoint inhibitor associated myocarditis via the DLL1/Notch1/GSDMD signaling pathway

Background&Objective Immune checkpoint inhibitor-associated myocarditis (ICIAM),through occurring in only 1%-1.4% of patients, carries a mortality rate of up to 60%. Despite this high lethality, its pathogenesis remains poorly understood. While T cell-driven inflammation is a known feature of ICIAM, the key transcriptional regulators and downstream effector mechanisms responsible for cardiomyocyte death have not been identified.This study aimed to identify pivotal molecular drivers of ICIAM and elucidate the mechanisms by which they induce myocardial injury. Methods Transcriptomic profiling was performed on peripheral blood mononuclear cells (PBMCs) from ICIAM patients and matched controls to identify differentially expressed genes. In vitro, human CD3⁺ T cells were transduced with a FOXA1-overexpressing adeno-associated virus (AAV) or a control AAV, then co-cultured with AC16 cardiomyocytes. Cardiomyocyte injury and pyroptosis were assessed using Western blot, qRT-PCR, caspase-1 activity assays, LDH release assays, ELISA, and Calcein-AM/PI staining. To investigate underlying mechanisms, co-cultures were treated with disulfiram or DAPT. Chromatin immunoprecipitation and dual-luciferase reporter assays were performed to validate the transcriptional regulation of DLL1 by FOXA1. In vivo, we established an experimental autoimmune myocarditis (EAM) mouse model. FOXA1-overexpressing CD3⁺ T cells were adoptively transferred into EAM mice, which were subsequently treated with disulfiram or DAPT. Cardiac function, myocardial inflammation, pyroptosis, and structural damage were evaluated by echocardiography, Western blot, qRT-PCR, ELISA, H&E staining, immunohistochemistry, and transmission electron microscopy. Results Transcriptomic analysis identified FOXA1 as a key gene significantly upregulated in PBMCs from ICIAM patients, a finding that was recapitulated in T cells from EAM mice. In vitro, FOXA1-overexpressing CD3⁺ T cells exacerbated myocardial injury and inflammation, characterized by elevated cTnI, IL-6, TNF-α, IL-18, and IL-1β. This effect was mediated by NLRP3/caspase-1/GSDMD-dependent pyroptosis and was abrogated by treatment with disulfiram and DAPT. Mechanistically, FOXA1 promoted DLL1 transcription, leading to activation of the Notch1 signaling pathway in cardiomyocytes. Inhibition of Notch1 signaling with DAPT significantly attenuated pyroptosis and subsequent cardiomyocyte injury. In vivo, adoptive transfer of FOXA1⁺ T cells into EAM mice exacerbated cardiac dysfunction, myocardial inflammation, and pyroptosis. These pathological effects were significantly mitigated by treatment with either disulfiram or DAPT. Conclusion FOXA1⁺CD3⁺ T cells exacerbate myocardial injury by directly binding the DLL1 promoter to activate the Notch1 signaling pathway, which in turn induces NLRP3/caspase-1/GSDMD-mediated cardiomyocyte pyroptosis.Targeting this FOXA1/DLL1/Notch1 axis may provide a promising therapeutic strategy for ICIAM.