Peripheral CD8 T Cell remodeling reveals proinflammatory and chemotactic endothelial interactions in immune checkpoint inhibitor associated myocarditis

Introduction Immune checkpoint inhibitor–associated myocarditis (ICI-M) is a rare but highly lethal complication characterized by myocardial infiltration of CD4⁺/CD8⁺ T cells and macrophages, which highlights a central role for T lymphocytes in the disorder. Immune checkpoint blockade induces IFN-γ– and TNF-α–dependent endothelial activation with increased CX3CL1, ICAM-1, and PD-L1 expression. T-cell receptor analyses show that myocardial T-cell clones in ICI-M are detectable among circulating CD8⁺ T cells, suggesting a pathophysiological continuum from peripheral activation to endothelial interaction and myocardial injury. Thus, circulating CD8⁺ T-cell characterization is key to understanding ICI cardiotoxicity. Objective The CD8-ImmunoMYOC study aimed to characterize phenotypic and functional alterations of circulating CD8⁺ T cells in ICI-treated patients, with or without myocarditis, before and after corticosteroid therapy, via single-cell multiparametric approaches. Methods Patients were enrolled in the prospective cohort and treated with ICIs, causing or not causing myocarditis. PBMCs were isolated from peripheral blood and CD8⁺ T cells enriched by immunomagnetic separation. CD8⁺ subsets were analyzed post-PMA-ionomycin stimulation via multiparametric flow cytometry, single-cell functional assays, secretome profiling and intracellular signaling analysis Results ICI treatment induced major remodeling of circulating CD8⁺ T cell populations. Patients with myocarditis or other cardiovascular disorders exhibited enrichment in effector and terminally differentiated (TEM/TEMRA) CD8⁺ T cells at the expense of TNAIVE/TCM populations. In contrast, patients without cardiac toxicity retained a balanced profile. This shift corresponded to reduced functional heterogeneity and emergence of a CX3CR1⁺ CCL5⁺ Ki-67⁺ signature with increased IL-8 and IL-2 production, consistent with proliferative cytotoxic subsets with vascular tropism. Reduced functionality of TEM cells suggests a dynamic transition towards highly differentiated TEMRA CD8⁺ cells. Corticosteroid treatment partially restored a TNAIVE/TCM-dominant profile, showing CD8⁺ plasticity. Co-expression of CX3CR1 and CCL5 establishes a functional link between cytotoxic subsets and activated endothelium via the CX3CR1–CX3CL1 axis, constituting a peripheral signal preceding myocardial infiltration. Conclusion During ICI myocarditis, CD8⁺ T cells specialized in a hyper-differentiated, pro-inflammatory, chemotactic TEMRA phenotype at the expense of populations responsible for peripheral tolerance. Proliferation of CX3CR1⁺ CCL5⁺ CD8⁺ T cells in an IL-2– and IL-8–enriched environment may promote endothelial adhesion and immune recruitment, contributing to myocardial injury. These mechanisms are under investigation in co-culture models combining CD8⁺ T cells and hiPSC-derived cardiac cells from patients of the prospective cohort, with encouraging preliminary results.