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T cells specific for α-myosin drive immunotherapy-related myocarditis


Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1–/–Ctla4+/– mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1–/–Ctla4+/– mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1–/–Ctla4+/– mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

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Fig. 1: scRNA–TCR-seq reveals abundant clonal effector CD8+ T cells in ICI-MC.
Fig. 2: CD8T cells are necessary for myocarditis.
Fig. 3: α-myosin is a MHC-I-restricted autoantigen in mouse myocarditis.
Fig. 4: α-myosin-expanded TCRs are present in cardiac and skeletal muscle of patients with ICI-MC.

Data availability

Sequencing data have been deposited in the Gene Expression Omnibus under accession number GSE213486Source data are provided with this paper.


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We thank the patients and families who donated tissue to this study; M. Madden and M. Korrer for assistance with sample acquisition; members of the J.M.B. laboratory and B. I. Reinfeld for their constructive input; and E. J. Philips, S. Mallal, P. Steinberger and J. Sidney for cell lines and reagents. This work was supported by F30CA236157, T32GM007347 (to M.L.A.), T32DK007061 (to J.J.W.), R01HL141466 (to J.J.M.), R01HL155990 (to J.J.M.), R01CA227481 (to D.B.J. and J.M.B.), R01HL156021 (to J.M.B. and J.J.M.), P30 AI110527 (to S.M.), Susan and Luke Simons Directorship in Melanoma, Van Stephenson Melanoma Research Fund, and James C. Bradford Melanoma Fund (to D.B.J). K.A. is supported by an American Heart Association Career Development Award (929347). W.C.M. is supported by the Mandema-Stipendium of the Junior Scientific Masterclass 2020-10 of the University Medical Center Groningen and by the Dutch Heart Foundation (Dekker grant 03-005-2021-T005). S.Z. is supported by National Natural Science Foundation of China (82100677). E.J.P. receives funding from the National Institutes of Health R01HG010863, R01AI152183 U01AI154659) and from the National Health and Medical Research Council of Australia. J.P.A. is a CPRIT Distinguished Scholar in Cancer Research. We acknowledge the Translational Pathology Shared Resource supported by NCI/NIH Cancer Center Support grant P30CA068485 and the Shared Instrumentation grant S10 OD023475-01A1 for the Leica Bond RX. Staff at The Vanderbilt VANTAGE Core, including A. Jones and L. Raju, provided technical assistance for this work. VANTAGE is supported in part by a CTSA grant (UL1TR002243), the Vanderbilt Ingram Cancer Center (P30 CA68485), the Vanderbilt Vision Center (P30 EY08126), the NIH/NCRR (G20 RR030956), and an NIH High-End Equipment Grant (S10OD025092). Figs. 1a and 4b were created with

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Authors and Affiliations



M.L.A., J.J.M. and J.M.B. conceived and designed the study and drafted the manuscript. W.C.M., E.M.S., E.T., J.Q., S.Z. and Y.Z. managed the mouse colony and identified appropriate mice for each experiment. M.L.A., W.C.M., E.M.S., M.G.C., X.S., J.J.W., E.T., J.Q., A.H. and A.L.T. performed mouse necropsies, tissue dissociation and cryopreservation. M.L.A. performed scRNA–TCR-seq and bulk TCR-seq data analyses and figure generation. M.L.A., A.L.T. and X.S. prepared samples for single-cell sequencing. W.C.M. and E.M.S. performed antibody-mediated depletion studies. A.S. provided expertise and performed tail vein injections for adoptive transfer studies. M.L.A. and M.G.C. performed antigen discovery, epitope identification and MHC blocking experiments. B.C.T. performed quantitative PCR. W.T. performed the dexamethasone treatment study. S.C.W. and J.P.A. provided expertise regarding the mouse model and myocarditis phenotype. S.R.O. provided technical expertise in experimental design and manuscript editing. J.C.R. provided immunology expertise. P.B.F. provided expertise regarding single-cell studies. E.J.P. and S.M. provided expertise in experimental design regarding LCLs, antigen discovery and MHC blockade. K.A. provided expertise in cardiac pathology from non-myocarditis phenotypes. D.B.J. and J.J.M. provided clinical expertise, assistance in acquiring human samples for this study and assistance with manuscript writing. M.L.A. analysed all data generated in this study. J.J.M. and J.M.B. obtained funding for this study.

Corresponding authors

Correspondence to Javid J. Moslehi or Justin M. Balko.

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Competing interests

M.L.A. is listed as a co-inventor on a provisional patent application for methods to predict therapeutic outcomes using blood-based gene expression patterns, which is owned by Vanderbilt University Medical Center, and is currently unlicensed. S.C.W. is currently an employee of, and has ownership interest in, Spotlight Therapeutics. S.C.W. has received personal fees from BioEntre and AlphaSights. S.C.W. is an inventor on a patent related to a genetic mouse model of immune checkpoint blockade induced immune-related adverse events (PCT/US2019/050551) pending to the Board of Regents, The University of Texas System and patent applications submitted by Spotlight Therapeutics related to in vivo gene editing. K.A. serves on the Data Safety Monitoring Board for ACI Clinical. J.C.R. is a founder, scientific advisory board member and stockholder of Sitryx Therapeutics, a scientific advisory board member and stockholder of Caribou Biosciences, a member of the scientific advisory board of Nirogy Therapeutics, has consulted for Merck, Pfizer and Mitobridge within the past 3 years, and has received research support from Incyte, Calithera Biosciences and Tempest Therapeutics. P.B.F. receives research support from Incyte. D.B.J. has served on advisory boards or as a consultant for BMS, Catalyst Biopharma, Iovance, Jansen, Mallinckrodt, Merck, Mosaic ImmunoEngineering, Novartis, Oncosec, Pfizer and Targovax, has received research funding from BMS and Incyte, and has patents pending for use of MHC-II as a biomarker for ICI response, and abatacept as treatment for irAEs. J.P.A. reports personal fees from Achelois, Adaptive Biotechnologies, Apricity Health, BioAtla, Candel Therapeutics, Codiak BioSciences, Dragonfly Therapeutics, Earli, Enable Medicine, Hummingbird, ImaginAb, Jounce Therapeutics, Lava Therapeutics, Lytix Biopharma, Marker Therapeutics, PBM Capital, Phenomic AI, BioNTech, Polaris Pharma, Time Bioventures, Trained Therapeutics, Two Bear Capital, and Venn Biosciences outside the submitted work. In addition, J.P.A. has a patent for a genetic mouse model of immune-checkpoint-blockade-induced irAEs pending to The University of Texas MD Anderson Cancer Center and has received royalties from intellectual property licensed to BMS and Merck. J.J.M. has served on advisory boards for Bristol Myers Squibb, Takeda, Audentes, Deciphera, Janssen, Immuno-Core, Boston Biomedical, Amgen, Myovant, Kurome Therapeutics, Star Therapeutics, ProtinQure, Pharmacyclics, Pfizer, Mallinckrodt Pharmaceuticals, Silverback Therapeutics, Cytokinetics, and AstraZeneca. J.M.B. receives research support from Genentech/Roche, and Incyte, and is an inventor on provisional patents regarding immunotherapy targets and biomarkers in cancer. No disclosures are reported by the other authors.

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Extended data figures and tables

Extended Data Fig. 1 Myocardial immune infiltrate does not differ by sex.

a) Quantification of immunohistochemistry (IHC) for CD8 and CD4 in male and female Pdcd1−/−Ctla4+/− mice with MC. Cells are counted as number of positive cells per high power (40x) field (HPF). Each data point represents an average of three high power fields per mouse. n = 4 female mice, n = 4 male mice. Box plots show the median, first and third quartiles. The whiskers extend to the maxima and minima but no further than 1.5 times the inter-quartile range. b) Representative IHC for IgG and B220 (CD45R) in hearts of mice with MC and positive control staining in spleen. Images are representative of n = 8 independent Pdcd1−/−Ctla4+/− mice with MC (n = 4 male; n = 4 female). Scale bars represent 50 µm

Source data

Extended Data Fig. 2 MC is characterized by activated immune cells and clonal T cells.

a) Gene expression of key identity genes, showing cell types of clusters. b) Differential gene expression for T, c) myeloid, B and NK cells in MC compared to control cardiac CD45+ cells. Red indicates FDR-corrected p-value (q-value) < 0.05. Black indicates not significant.

Extended Data Fig. 3 T cells in MC are effector or proliferating, tissue-resident and clonal.

a) Expression of key T cell genes by cluster in single cell data. b) Differential gene expression for Cluster 0 vs. Cluster 3 T cells. Red indicates FDR-corrected p-value (q-value) < 0.05. Black indicates not significant. c) Violin plots show expression of key tissue residency associated genes by cluster and MC vs. control. d) Shannon diversity on bulk TCR sequencing beta chain repertoires. Color indicates sex. Shape indicates whether the tissue was derived from a control wild type mouse (open circle) or a Pdcd1−/−Ctla4+/− mouse with MC (filled circle). P = 0.0002, two-sided Wilcoxon test. Box plots show the median, first and third quartiles. The whiskers extend to the maxima and minima but no further than 1.5 times the inter-quartile range. e) TCR counts in single cell data. MC sample is divided by mouse of origin. Clonal TCRs are found in all 4 sequenced hearts

Source data

Extended Data Fig. 4 Confirmation of cell type depletion.

a) Female Pdcd1−/−Ctla4+/− mice were treated with dexamethasone (1mg/kg/day; n = 18) or vehicle (n = 17) starting at 21 days of life. Time is measured since birth. P = 0.49, two-sided cox proportional hazard test. b) Representative flow cytometry gated on live CD45+ cells isolated from blood of different treatment groups, at week 3 of treatment. c) Representative flow cytometry on CD8 depleted (via magnetic beads) compared to whole splenocytes used for adoptive transfer. d) Representative immunohistochemistry on hearts of a CD8 depleted splenocyte recipient compared to a whole splenocyte recipient. Only cardiac sections are shown. Scale bars show 50 μm. Representative of n = 10 animals per group. e) Total TCR reads for cardiac TCR beta chain sequencing on donor and recipient hearts

Source data

Extended Data Fig. 5 Thymic expression of Myh6 and flow cytometry gating for murine α-myosin tetramers.

a) Gene expression for Myh6, Nppa, Nppb, and Sbk2 in the heart and thymus of n = 3 each male and female Pdcd1−/−Ctla4+/− mice. Gene expression is normalized to beta-actin. Gene expression is plotted as 2^-(Ct gene of interest minus Ct of beta-actin). Box plots show the median, first and third quartiles. The whiskers extend to the maxima and minima but no further than 1.5 times the inter-quartile range. b) Gating strategy for H2-Kb tetramers on murine heart samples. Debris, doublets and dead cells (Zombie Violet positive) are excluded. CD3+CD8+ cells are used for tetramer analysis. Staining for Control (SIINFEKL) H2-Kb, and VQQVYYSI H2-Kb tetramers are shown. c) Quantification of spleen tetramer positive CD3+CD8+ cells, by sex of the mouse. The spleens used in this experiment correspond to the mice show in Fig. 3f, which all have α-myosin tetramer positive MC

Source data

Extended Data Fig. 6 TCR sequencing on exPBMC shows expansion of α-myosin and CEF specific TCRs.

a) Comparison of TCR beta chain abundance in α-myosin exPBMC and pre-expansion PBMC for all patients. Each plot is within the same patient only. Color represents change from PBMC to exPBMC. Minimal change is less than a 50 read count change. b) Comparison of TCR beta chain abundance in CEF exPBMC and pre-expansion PBMC for all healthy donors. Color represents change from PBMC to CEF exPBMC. Minimal change is less than a 50 read count change. c) Total TCR reads for biopsy (heart), autopsy, and PBMC samples from patients 1, 2 and 3.

Extended Data Fig. 7 α-myosin expanded TCRs are found in the hearts and skeletal muscles of patients with ICI-MC.

a) Change in TCR counts from PBMC to α-myosin exPBMC plotted by abundance of the same TCR beta chain in the autologous inflamed cardiac or skeletal muscle tissue of each patient. Minimal change is less than a 50 read count change. Not present means not found in either PBMC or exPBMC, but present in indicated tissue. b) Comparison of TCR beta chain abundance in α-myosin exPBMC and heart (biopsy for patient 1 and 3 or right ventricle for patient 2). Color represents change from PBMC to exPBMC. Minimal change is less than a 50 read count change. Not present means not found in either PBMC or exPBMC, but present in heart.

Extended Data Fig. 8 Purity analysis for single cell sequencing on exPBMCs from patient 1.

a) Gene expression is shown on single cell sequencing of CD3 sorted exPBMCs from patient 1. b) Violin plots of key gene expression by presence or absence in cardiac TCR repertoire and clonality in exPBMC. Identity genes are shown in light blue. Genes associated with naïve T cells are shown in dark blue. Genes associated with T cell activation are shown in red.

Extended Data Fig. 9 TCR from Pt 1 exPBMC recognizes α-myosin epitope.

a) TCR-Pt1, which was cloned and transduced into Jurkat NFAT-GFP reporter cells, is shown in red on the same plot show in Fig. 4c. This shows the expansion of this TCR in the exPBMC and abundance in the heart. b) Representative flow cytometry scatter plots are shown for the TCR-pt1 Jurkat cell line is stained with A*24:02 tetramer with RINATLETK or A*03:01 tetramer with RINATLETK. c) Full flow cytometry gating strategy for human PBMC and exPBMC tetramer staining. Debris, doublets and dead cells (Zombie Violet positive) are excluded. CD3+CD8+ cells are used for tetramer analysis. Tetramer staining for all samples is shown.

Extended Data Fig. 10 Tumor-specific MYH6 expression.

a) MYH6 transcripts per million are shown for n = 91 pre-treatment RNA-sequencing melanoma samples. Bars are colored by what ICI treatment the patient received. b) MYH6 expression is shown for n = 363 melanoma samples accessed from TCGA. Samples to the right of the dotted lines have detectable MYH6 expression.

Extended Data Table 1 Amino acid sequences for α-myosin, ANP, BNP and SBK2 peptides included in the peptide library
Extended Data Table 2 Prediction scores for binding of α-myosin peptides to MHC-I molecules in C57BL/6 mice generated by TepiTool
Extended Data Table 3 HLA types of healthy donors and patients with ICI-MC

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Full nucleotide sequences for reconstructed TCRs.

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Axelrod, M.L., Meijers, W.C., Screever, E.M. et al. T cells specific for α-myosin drive immunotherapy-related myocarditis. Nature 611, 818–826 (2022).

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