Triple immune checkpoint blockade induces immune-mediated cardiomyocyte injury: translational implications for cardio-oncology surveillance

Background Immune checkpoint inhibitors (ICIs) have transformed cancer care, yet immune-related adverse events involving the cardiovascular system—particularly myocarditis—remain among the most severe and life-threatening complications. While dual ICI regimens are known to increase cardiotoxic risk, the cardiovascular safety profile of emerging triple ICI combinations remains largely undefined. As multi-agent immune checkpoint blockade expands in clinical development, understanding its potential to trigger immune-mediated cardiac injury has become a critical unmet need in cardio-oncology. Methods A translational co-culture model combining human induced pluripotent stem cell–derived cardiomyocytes and human peripheral blood mononuclear cells was established to evaluate immune-driven cardiotoxicity. Cultures were exposed for 48 hours to immune checkpoint blockade with nivolumab plus relatlimab, ipilimumab, or atezolizumab, administered as monotherapies, doublets, or triplet combinations. Cardiomyocyte injury was quantified by lactate dehydrogenase release. Immune activation was assessed by measurement of IL-2, granzyme B, and pro-inflammatory cytokines, including components of the NLRP3 inflammasome pathway. Digital microscopy was used to evaluate immune–cardiomyocyte interactions and structural integrity. Results Triple immune checkpoint blockade induced a marked increase in cardiomyocyte injury compared with single-agent or dual ICI exposure (p < 0.001). This cytotoxic effect was associated with robust immune activation, characterised by elevated IL-2 and granzyme B secretion and amplification of pro-inflammatory and inflammasome-related signalling. Morphological analyses confirmed pronounced immune cell activation and a significant reduction in cardiomyocyte density following triplet exposure. In contrast, single and dual ICI regimens elicited comparatively limited cardiomyocyte damage. Conclusions These data provide the first preclinical evidence that triple immune checkpoint blockade can directly promote immune-mediated cardiomyocyte injury through exaggerated inflammatory and cytotoxic immune responses. The findings raise a translational alert for cardio-oncology, underscoring the need for intensified cardiovascular monitoring and immune-related cardiotoxicity surveillance in patients receiving multi-agent ICI regimens. As triplet immune checkpoint strategies advance toward clinical application, proactive integration of cardio-oncology expertise will be essential to mitigate cardiovascular risk without compromising oncological efficacy.