Associations Between Circulating Proteins and Intracardiac Immune Populations in Immune Checkpoint Inhibitor-Associated Myocarditis

Introduction: Myocarditis due to immune checkpoint inhibitors (ICIs), a type of cancer immunotherapy, is associated with high morbidity and mortality. The cellular and molecular pathogenesis of ICI myocarditis remains largely unknown. Identification of circulating factors associated with intracardiac pathology may aid new clinical approaches. Hypothesis: We hypothesized that ICI myocarditis is associated with increased abundance of intracardiac immune cells and upregulated inflammatory genes in the heart and serum. Methods: Heart tissue from 13 patients with ICI myocarditis was acquired by endomyocardial biopsy or autopsy; peripheral blood mononuclear cells (PBMCs) were also collected from ten of these patients and eight additional ICI myocarditis patients. Control heart tissue was derived from six hearts declined for transplantation (non-ICI-exposed) and from the biopsy and autopsy of one ICI-treated patient without myocarditis. The 10x Genomics Chromium system was used to generate single-cell RNA sequencing (scRNAseq) data from heart and PBMC specimens. Serum proteins were measured by core lab assay (for troponin T) or by multiplexed Luminex immunoassay. Results: Unbiased clustering of scRNAseq data from 77,071 cells recovered from heart samples revealed 37 cell subsets across 10 cell lineages. Myocarditis heart tissue demonstrated enrichment of T/NK cells (odds ratio 8.3, p=0.0006), B/plasma cells (OR 5.1, p=0.01), and dendritic cells (DCs) (OR 9.2, p=0.006) relative to controls. Circulating DC abundance was decreased in fatal (n=3) versus nonfatal (n=15) myocarditis cases (p=0.008), while intracardiac DC abundance was directly associated with serum troponin T values (p=0.02). The levels of five immunomodulatory factors - IL-15, CXCL9, CCL3, TNFα, and CCL21 - were found to be significantly upregulated transcriptionally in at least one intracardiac cell subset and at the protein level in the serum of myocarditis cases. Conclusions: Fatality status and troponin level associated with intracardiac DC abundance and multiple immunomodulatory genes were upregulated in both the heart and serum in ICI myocarditis. These observations may guide novel diagnostic and therapeutic strategies.