Background Immune checkpoint inhibitor(ICI)-related Myocarditis (irMyocarditis) is a potentially lethal complication of ICI therapy.¹ Human translational studies have provided insight into disease pathogenesis.^2–5 However, the urgent initiation of high-dose corticosteroids to treat irMyocarditis^{6 7}often precedes biospecimen collection and has an unknown impact on analytes.

Methods Peripheral blood mononuclear cells (PBMCs) from 25 irMyocarditis patients were collected across multiple timepoints, including at the time of irMyocarditis diagnosis (‘pre-steroid’; n=17) and shortly after the initiation of corticosteroids (‘post-steroid’; n=19; range 1–34, median 4 days). Control PBMCs were collected from ICI-treated cancer patients without irAEs (n=28). Single-cell RNA-sequencing (scRNA-seq) with simultaneous measurement of 197 surface proteins was performed.^8–11 Surface protein data was used to validate scRNA-seq abundance analyses. Abundance, differential gene expression, and gene set enrichment analysis results were considered significant at a false-discovery rate<0.1. Heart tissue collected during endomyocardial biopsies or autopsies in patients with suspected irMyocarditis (irMyocarditis n=15, control n=2) underwent scRNA-seq with T-cell receptor (TCR) sequencing.

Results Clustering 366,066 blood cells using scRNA-seq data identified expected lineages and 37 cell subsets. Compared to controls, pre-steroid irMyocarditis PBMC samples demonstrated decreased abundance of plasmacytoid dendritic cells (pDCs), classical dendritic cells (cDCs), B/plasma cells, and CD4T cell lineages; other mononuclear phagocyte (MNP) lineage cells were more frequent in irMyocarditis cases. Among our cell subsets, 13 lymphocyte subsets, cDC1, and cDC2 were decreased in irMyocardits; two MNP subsets and cDC3 were more abundant in irMyocarditis. Populations defined by surface protein data supported our scRNA-seq findings. The expression of gene sets associated with interferon responses and cell adhesion were increased in irMyocarditis samples compared to controls.

Pre-steroid versus post-steroid analysis identified only three differentially abundant cell subsets but marked transcriptional changes (4,613 significant lineage-level genes). The expression of gene sets associated with irMyocarditis onset were decreased after steroid administration.

9,134 intracardiac T/NK cells were recovered. The evaluation of T-cell clones from a donor contributing both pre-steroid biopsy and post-steroid autopsy specimens demonstrated that the 13 most expanded TCR clones were found at both timepoints. However, post-steroid clones had different transcriptional profiles, most notably lower expression of cell cycle genes (e.g., STMN1).

Conclusions Decreased circulating cDCs and pDCs may represent novel biomarkers of irMyocarditis onset. Transcriptional profiles of tissue and blood immune cells underwent significant changes soon after corticosteroid initiation for the treatment of irMyocarditis. The collection and analysis of pre-steroid biospecimens are crucial for distinguishing features of disease pathobiology from steroid effects.

Acknowledgements We are deeply grateful to all donors and their families. We also thank the Mass General Cancer Center, Ellison 16 staff, the cardiac catheterization laboratory, and the Severe Immunotherapy Complications Service for their collaboration and support. S.M.B was supported by a National Institutes of Health T32 Award (2T32CA071345-21A1) and a SITC-Mallinckrodt Pharmaceuticals Adverse Events in Cancer Immunotherapy Clinical Fellowship. D.A.Z. was supported by a National Institutes of Health T32 Award T32HL007208 and K24HL150238-02. This work was made possible by the generous support from the National Institute of Health Director’s New Innovator Award (DP2CA247831; to A.C.V.), the Massachusetts General Hospital Transformative Scholar in Medicine Award (to A.C.V.), the Damon Runyon-Rachleff Innovation Award (to A.C.V.), The Melanoma Research Alliance Young Investigator Award, the MGH Howard M. Goodman Fellowship (to A.C.V.), the Arthur, Sandra, and Sarah Irving Fund for Gastrointestinal Immuno-Oncology (to A.C.V.), the Kraft Foundation Award (to. K.L.R. and A.C.V.), and by the generous support of an anonymous donor (to. K.L.R. and A.C.V.).

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Ethics Approval Informed consent was obtained from all patients or their appropriate representatives. All research protocols were approved by the Dana-Farber/Harvard Cancer Center Institutional Review Boards (#11-181 and 13-416).