Evidence of Cardiotoxic Immune Activation by Triple Immune Checkpoint Blockade: A Translational Alert for Clinical Surveillance in Patients With Cancer

: Immune checkpoint inhibitors (ICIs) have successfully revolutionized cancer therapy, but their immune-mediated adverse events include rare, often severe myocarditis. Although dual ICI blockade is associated with increased cardiotoxic risk, little is known about the potential effects of triplet combinations currently under clinical investigation. We developed a coculture model of human cardiomyocytes and human peripheral blood mononuclear cells (hPBMCs) to evaluate immune-mediated cytotoxicity induced by ICIs. HFCs were exposed for 48h to nivolumab plus relatlimab, ipilimumab, or atezolizumab, either alone or in triplet combinations. Cell lysis was quantified by LDH release. Cytokine secretion (IL-2, granzyme B, and additional inflammatory mediators including NLRP3 activation pathway) was measured by ELISA. Digital microscopy was used for morphological assessment. Triplet combinations of nivolumab-relatlimab with ipilimumab or atezolizumab induced significantly higher cardiomyocyte lysis than single agents or doublets ( P < 0.001). This effect correlated with a robust increase in IL-2 and granzyme B secretion, and activation of proinflammatory cytokines and NLRP3 inflammasome-related mediators. Microscopic analyses confirmed immune cell activation and reduced density of HFCs exposed to triplets. Our findings demonstrate that ICI triplets elicit potent immune activation against cardiomyocytes, providing the first preclinical evidence of direct cardiotoxic potential in this setting. These results highlight the need for enhanced clinical surveillance and cardio-oncology monitoring in patients receiving triple ICI combinations, because these regimens expand in clinical practice.