CCR2+Macrophages And T-cells Infiltrate The Murine Heart Following Immune Regulator Treatment With CD40

Immune checkpoint inhibitors have revolutionized cancer treatment but have been associated with severe adverse cardiac events including myocarditis. CD40 agonist (CD40ag) antibodies are immune regulators that have emerged as promising candidates with remarkable efficacy across tumors including those thought to be resistant to established ICIs. To investigate CD40 signaling and its potential for adverse cardiac events, C57BL/6J, CCR2-GFP, and CCR2-knockout mice were injected with CD40ag or isotype antibodies for 7 days. Flow cytometry showed a specific increase in CCR2 + macrophages that was independent of monocyte recruitment. There was no change in neutrophil recruitment and only a modest increase in dendritic cells was evident. We also observed a significant expansion of CD4 and CD8 T-cells that displayed an effector memory phenotype. Bulk cardiac tissue gene expression analysis (Figure A) revealed that CD40 activation upregulates multiple inflammatory cytokines (Ifng, Tnf, Il-12β) and chemokines (Ccl3, Ccl5, Ccl7, Cxcl9, Cxcl10), which are regulated by interferon gamma and coordinate the activation of APCs and T-cells. Finally, RNA in situ hybridization (Figure B-C) demonstrated increased levels of Cxcl9 (red) expression in cells consistent with CCR2 + (white) macrophages when comparing isotype control to treatment with CD40ag. These findings reveal that CD40 activation results in a robust expansion of CCR2 + macrophages and activation of T-cells within the heart, initiating a feed forward loop of activation that is mediated by interferon gamma and generates inflammatory cytotoxic mediators that may lead to myocardial injury.