Expansion of Pathogenic Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

Abstract

Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte-associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages. We used an established murine ICI myocarditis model (<i>Ctla4</i>^<i>+/-</i><i>Pdcd1</i>^<i>-/-</i> mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies. We observed marked increases in CCR2 (C-C chemokine receptor type 2)⁺ monocyte-derived macrophages and CD8⁺ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2⁺ subpopulation highly expressing <i>Cxcl9</i> (chemokine [C-X-C motif] ligand 9), <i>Cxcl10</i> (chemokine [C-X-C motif] ligand 10), <i>Gbp2b</i> (interferon-induced guanylate-binding protein 2b), and <i>Fcgr4</i> (Fc receptor, IgG, low affinity IV) that originated from CCR2⁺ monocytes. It is important that a similar macrophage population expressing <i>CXCL9</i>, <i>CXCL10</i>, and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and <i>Cxcl9</i>^<i>+</i><i>Cxcl10</i>^<i>+</i> macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8⁺ T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of <i>Cxcl9</i>^<i>+</i><i>Cxcl10</i>^<i>+</i> macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis. These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ-induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.