Cytotoxic T cells speci�c for alpha-myosin drive immunotherapy related myocarditis

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to immune checkpoint inhibitor (ICI) utility in anti-cancer therapy1. The pathogenesis of ICI-myocarditis is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathologic features of ICI-myocarditis, including myocardial T cell in�ltration2. Single cell RNA/T cell receptor (TCR) sequencing on the cardiac immune in�ltrate of Pdcd1-/-Ctla4+/- mice identi�ed activated, clonal CD8+ T cells as the dominant cell population. Treatment with anti-CD8, but not anti-CD4, depleting antibodies rescued survival of Pdcd1-/- Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients which required CD8+ T cells. Alpha-myosin, a cardiac speci�c protein absent from the thymus3,4, was identi�ed as the cognate antigen source for three MHC-I restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from two patients with ICI-myocarditis were expanded by alpha-myosin peptides, and these alpha-myosin expanded T cells shared TCR clonotypes with diseased heart and skeletal muscles, indicating that alpha-myosin may be a clinically important autoantigen in ICI-myocarditis. These studies underscore the critical role for cytotoxic CD8+ T cells, are the �rst to identify a candidate autoantigen in ICI-myocarditis and yield new insights into ICI toxicity pathogenesis.


Main
Immune checkpoint inhibitors (ICIs) have drastically altered the treatment landscape and prognosis for many cancers.However, not all patients respond to treatment and many patients experience immunerelated adverse events (irAEs), especially when ICIs are used in combination.With increasing use of ICIs, preventing, diagnosing, and treating irAEs are urgent clinical challenges.Currently, clinically actionable biomarkers of response and toxicity are limited.Furthermore, the mechanistic basis of irAEs is poorly de ned.
Myocarditis is an uncommon irAE, affecting < 1% of ICI-treated patients, but has a mortality rate of nearly 50% 1,5 .Combination ICI therapy (with anti-PD-1 and anti-CTLA4) is the most well-established risk factor for ICI-myocarditis [6][7][8][9] .ICI-myocarditis is pathologically characterized by predominance of T lymphocytes and macrophages in the heart and often co-occurs with myositis, with early studies suggesting common clonotypes of T lymphocytes in both tissues 5 .These data suggest the possibility of shared target antigens driving T lymphocyte expansion and activation, which would be critical for pathogenesis; however, experimental data have been lacking.
Generally, mice treated with ICIs do not replicate the full spectrum of irAEs seen in patients, limiting research on mechanisms of toxicity.We have recently described a mouse model of ICI-myocarditis in which C57BL6/J mice with homozygous knockout of Pdcd1 and heterozygous deletion of Ctla4 die prematurely and speci cally due to myocarditis, recapitulating clinical and pathological features of ICImyocarditis 2 .Severe in ammation is speci c to the heart in these mice.By ow cytometry, the myocardial immune in ltrate is primarily composed of CD8+ T cells, similar to patients with ICI-myocarditis.Furthermore, treatment with abatacept, a CTLA4 fusion protein, attenuates myocarditis and increases survival in the mice, consistent with early clinical data from patients with ICI-myocarditis treated with abatacept 2,10 .Here we utilize this mouse model of ICI-myocarditis to characterize the immune in ltrates, establish CD8+ T cells as necessary for disease, and identify alpha-myosin as a cognate antigen for the most abundant TCRs in myocarditis.Furthermore, we extend some of these ndings into human disease and nd that alpha-myosin expanded TCRs are present in in amed cardiac and skeletal muscles in patients with ICI-myocarditis.
Clonal CD8+ T cells are abundant in ICI-myocarditis Fulminant myocarditis affects 50% of Pdcd1-/-Ctla4+/mice and is characterized by histologic destruction of the myocardial architecture (Fig. 1a, b) 2 .We used single cell RNA and TCR sequencing to characterize sorted CD45+ in ltrating immune cells from six healthy wild type mouse hearts and four hearts from Pdcd1-/-Ctla4+/mice affected by myocarditis.Dimensionality reduction with uniform manifold approximation and projection (UMAP) and cell type annotation assisted by SingleR reveals distinct clustering by genotype (Fig. 1c; Extended Data Fig. 1a) 11 .Activated T lymphocytes and myeloid cells comprise the majority of the immune cells in myocarditis.The largest difference is seen in the activated T cell cluster, which makes up 34% of the myocarditis immune cells, and only 2% of the control immune cells.Markers of activation such as Ccl5, Ccl4, Tigit, Nkg7, and Gzmb are upregulated in the T cell clusters in myocarditis compared to control T cell clusters (Fig. 1d).Conversely, markers of naïve status such as Ccr7, Lef1 and Sell are upregulated in control T cells.Activation markers are also upregulated in other clusters, including myeloid cell subsets, in the myocarditis samples (Extended Data Fig. 1b).Aw112010, a long noncoding RNA essential for the orchestration of mucosal immunity during infections and in colitis, is strongly upregulated in several clusters in the myocarditis samples 12,13 .In contrast, B lymphocytes make up most of the immune cells in the control heart, consistent with previous studies [14][15][16] .
Given the high in ltration of activated T cells, we next sought to assess the clonality of TCRs in the myocarditis samples using both bulk and single cell TCR sequencing.Cardiac tissue from affected Pdcd1-/-Ctla4+/mice had lower Shannon diversity compared to splenic tissue (whether derived from healthy wild type mice or mice with myocarditis), indicating a higher degree of clonal TCRs (Extended Data Fig. 1c).In single cell TCR data, clonal was de ned as more than two cells with the same TCR clonotype (same TCR alpha and beta CDR3 regions).No clonal cells were identi ed by single cell TCR sequencing of the healthy cardiac immune in ltrate.In contrast, 63% of cells with TCR reads in the myocarditis sample had clonal TCRs and clonal cells were identi ed in all four myocarditis samples (Extended Data Fig. 1d).Dimensionality reduction of only T cells with TCR reads showed that clonal cells overlap with the activated T cell cluster whereas not clonal cells overlap with naïve T cells (Fig. 1e).A signi cantly higher proportion of the clonal cells in myocarditis are activated T cells relative to not clonal cells in either control or myocarditis samples (Fig. 1f).Comparing gene expression by clonality shows that not clonal cells from control samples express Cd8a, Cd4, and markers associated with naïve status, but not genes associated with activation.In contrast, clonal cells from myocarditis samples express Cd8a and cytotoxicity genes such as Nkg7 and Gzmb, but do not express Cd4 or markers of naïve status (Fig. 1g).These data show that there is a large population of highly activated, clonally expanded CD8+ T cells in murine ICI-myocarditis.

CD8+ T cells are necessary for myocarditis
Using anti-CD8 and anti-CD4 depleting antibodies we tested whether depletion of these cell subsets would attenuate myocarditis and affect survival of Pdcd1-/-Ctla4+/mice. Depletion of target cells was con rmed by ow cytometry on peripheral blood (Extended Data Fig. 2a).Depletion of CD8+ cells, but not CD4+ cells, signi cantly rescued survival in these mice (Fig. 2a).Conversely, we tested whether transfer of immune cells could recapitulate disease.Adoptive transfer of whole splenocytes, but not splenocytes from which CD8+ cells were depleted, from Pdcd1-/-Ctla4+/mice with myocarditis to Rag1-/recipients was su cient to induce fatal myocarditis (Fig. 2b).CD8 depletion was con rmed by ow cytometry on transferred splenocytes (Extended Data Fig. 2b).Myocarditis was con rmed histologically on necropsy tissue (Fig. 2c).The single fatality in the CD8 depleted arm was due to a bowel obstruction and there was no evidence of myocarditis histologically.Immunohistochemistry for CD3, CD4, CD8 and F4/80 showed abundant cardiac in ltration of CD3+ and CD8+ cells in the whole splenocyte recipients but not the CD8 depleted recipients.Some CD4+ cells and limited F4/80+ cells were also found in the hearts of whole splenocyte recipients (Fig. 2d; Extended Data Fig. 3a).We performed TCR sequencing on the cardiac tissue of one donor mouse (Donor) and four whole splenocyte recipients (Rec1, Rec2, Rec3, and Rec4; Fig. 2e).High numbers (>2000) of TCR reads were seen in all sequenced hearts, indicating signi cant T cell in ltration, as expected from histology (Extended Data Fig. 3b).In all four recipient mice the single most clonal TCR beta chain occupied greater than 65% of the total cardiac TCR repertoire, indicating massive expansion of a single TCR clonotype.The most clonal TCR beta chain (CDR3: CASSLRRGEQYF) in the donor heart (which comprised 37% of the donor cardiac repertoire) was expanded in three of four recipients (Rec1, 3, 4).Interestingly, in one recipient mouse (Rec2), a low frequency TCR from the donor was expanded and occupied the majority of the TCR repertoire (CDR3: CASSLGGTVQDTQYF).This high degree of expansion from donor to recipient cardiac tissue suggests a single TCR clonotype may drive myocarditis in the recipient animals.Together, these results strongly indicated that CD8+ T lymphocytes are necessary for the development of myocarditis.
Myocarditis-derived TCRs are speci c for alpha-myosin Next, we aimed to identify the cognate antigen for clonal murine TCRs.TCR-A was derived from the single cell RNA/TCR sequencing and was associated with the activated T cell cluster (Fig. 3a).TCRs B and C were derived from the adoptive transfer of whole splenocytes.TCR-B was the most abundant TCR in the heart of the donor and three recipients (Beta CDR3: CASSLRRGEQYF).TCR-C was the most abundant TCR in the heart of recipient 2 (Beta CDR3: CASSLGGTVQDTQYF; Fig. 2e).CDR3 amino acid sequences, V genes, and J genes are shown in Table 1.These TCRs were reconstructed using Stitchr, cloned and retrovirally transduced into Jurkat nuclear factor of activated T cells (NFAT)-GFP reporter cells [17][18][19] .Syngeneic bone marrow derived dendritic cells were used as antigen presenting cells (APCs).
We used a candidate autoantigen approach for TCR screening.Analysis of published RNA sequencing data on thymic APCs (including thymic epithelial cells) showed four cardiac enriched genes (genes where expression in the heart was signi cantly enriched relative to other tissues) with no detectable expression in the thymus (Fig. 3b) 4 .Lack of thymic expression would be predicted to enable self-reactive T cells to escape negative selection, an important mechanism of tolerance.Of these four genes, MYH6 (alphamyosin) has been con rmed by other groups to not be expressed in the thymus in mice or humans and has been shown to be an MHC-II restricted autoantigen in mouse models 3,20,21 .We used a library of 130 overlapping peptides, covering all of alpha-myosin protein (Extended Data Table 1).All three TCR cell lines had NFAT activity in response to alpha-myosin peptides.TCRs A and B activated NFAT reporters in response to the same alpha-myosin peptide (MYH6 181-200 ), whereas TCR-C had NFAT activity against a distinct alpha-myosin peptide (MYH6 406-425 ; Fig. 3c).From these 20 amino acid peptides, we used TepiTool to narrow down the most likely immunogenic epitopes 22 .TCRs A and B recognize the epitope MYH6 191-198 (VIQYFASI).TCR-C recognizes the epitope MYH6 418-425 (VQQVYYSI; Fig. 3d).VIQYFASI and VQQVYYSI both have strong predicted binding to H2-Kb using TepiTool (Extended DataTable 2).The tyrosine and phenylalanine residues at position ve of the peptides are known to be key binding epitopes for H2-Kb 23 .In line with these predictions, blocking H2-Kb, but not H2-Db, with an antibody abrogates NFAT reporter activity for all three cell lines (Fig. 3e).All three clonal TCRs derived from independent murine cardiac TCR repertoires recognized alpha-myosin epitopes.Therefore, this strongly suggests that alpha-myosin is an important MHC-I restricted autoantigen in murine ICI-myocarditis.

Alpha-myosin expanded TCRs are present in fulminant myocarditis in patients
We next aimed to test the relevance of alpha-myosin as a potential autoantigen in humans, using three healthy donors and two patients with ICI-myocarditis.ICI-myocarditis patient information is summarized in Table 2. First, we tested whether it was possible to expand alpha-myosin speci c T cells from peripheral blood mononuclear cells (PBMCs).PBMCs were stimulated with alpha-myosin peptides or control cytomegalovirus, Epstein-Barr virus and in uenza (CEF) peptides (for healthy donors only) for 14 days to generate expanded PBMCs (exPBMC).Shannon diversity decreased signi cantly from preexpansion PBMC to alpha-myosin exPBMC for healthy donors and myocarditis patients, indicating clonal expansion of alpha-myosin speci c T cells.Interestingly, Shannon diversity did not signi cantly change from baseline to CEF peptide expansion, suggesting that alpha-myosin is a strong stimulus for clonal T cell expansion (Fig. 4a).For all donors, both alpha-myosin and CEF stimulation resulted in expansion of some individual TCR clonotypes (Extended Data Fig. 4 a, b).These data suggest that both healthy donors and ICI-myocarditis patients have alpha-myosin speci c T cells in the periphery that may be expanded.
To assess whether alpha-myosin expanded TCR clones might be involved in cardiac and skeletal muscle toxicity, we compared TCR repertoires in the heart and in amed muscle to those overrepresented in alphamyosin exPBMC relative to unexpanded PBMC.We performed bulk TCR sequencing on formalin-xed para n embedded tissues from endomyocardial biopsy (patient 1 only) and autopsy material (patients 1 and 2).Tissue samples from each myocarditis patient are summarized in Fig. 4b.High numbers of total TCR reads (>1500) were seen in each sequenced sample, consistent with high T cell in ltration (Extended Data Fig. 4c).Shannon diversity was low in heart, diaphragm, and other skeletal muscle samples, indicating clonal TCR repertoires at the sites of toxicity (Fig. 4a).Alpha-myosin expanded TCRs (shown in red; count in alpha-myosin exPBMC minus count in pre-expansion PBMC) were present in in amed tissues from both patients (Fig. 4c; Extended Data Fig. 5a, b).Some alpha-myosin expanded TCRs were present at high frequencies in the in amed heart and skeletal muscles.This overlap suggests that alphamyosin may be a relevant disease antigen for ICI-myocarditis and myositis.We performed single cell RNA/TCR sequencing on the sorted CD3+ exPBMC from patient 1 and ltered on only cells with expression of a TCR.Gene expression analysis shows expression of CD3E in all cells, presence of both CD8A and CD4 expressing cells, and a very small population of residual CD79A expressing B cells (Extended Data Fig. 5c).Clonal cells in the exPBMC are expected to be enriched for alpha-myosin speci city.We further separated cells based on whether their TCR was shared with the cardiac TCR repertoire.Shared TCR clonotypes with the heart would be expected to be enriched for disease relevant TCRs.Following dimensionality reduction with UMAP, the cells cluster distinctly by group (Fig. 4d).Of the cells with TCR clonotypes shared with the heart, a signi cantly higher proportion were clonal in the exPBMC relative to cells with TCRs not present in the heart (Fig. 4e).Clonal cells with TCRs shared with the diseased heart have high expression of CD8A relative to clonal cells with TCRs not present in the heart (Fig. 4f).These data suggest that CD8+ alpha-myosin speci c T cells are present at the site of toxicity, in line with results from our mouse model.Clonal cells with TCRs present in the heart also have high expression of markers of activation such as NKG7, GZMB, and GNLY (Fig. 4f).Expression of activation genes in the exPBMC is not expected to re ect in vivo cell state, given the two-week in vitro expansion period.Rather, higher expression of activation associated genes following stimulation and expansion with alpha-myosin peptides would be expected to further enrich for alpha-myosin speci c TCRs and suggest cytotoxic capability of progenitor cells.Taken together, these data suggest that CD8+ cytotoxic T cells speci c for alpha-myosin are present in the diseased hearts of patients with fulminant ICImyocarditis.

Discussion
Immunotherapy toxicities are important limitations to the expanding indications for ICIs.Here we present a new perspective on ICI-myocarditis as an antigen-driven, T cell mediated toxicity.We show in our mouse model that myocarditis is characterized by cytotoxic CD8+ T cells with highly clonal TCRs, and that CD8+ cells are necessary for the development of myocarditis.Three of the most clonal TCRs, derived from independent mice, recognize alpha-myosin epitopes.Two of these TCRs, though derived from independent experiments, recognize the same alpha-myosin epitope.Strikingly, alpha-myosin speci c TCRs expanded when transferred to recipient mice and occupied greater than 65% of the highly in amed cardiac TCR repertoire at the time of death from myocarditis.Lack of thymic expression increases the likelihood that alpha-myosin speci c T cells may escape central tolerance mechanisms 3 .Alpha-myosin speci c T cells can be expanded from the blood of healthy donors and patients with ICI-myocarditis.Alpha-myosin expanded TCRs overlap with TCR repertoires in the diseased hearts and skeletal muscle of two patients with fatal ICI-myocarditis.Although presence of shared clones is insu cient to assess whether alpha-myosin speci c T cells play a causal role in the initiation of myocarditis, these data suggest that alpha-myosin may be an important autoantigen in ICI-myocarditis.The presence of other high frequency TCRs in the hearts that were not enriched in the alpha-myosin expanded repertoires suggests that there are likely to be other relevant antigens, particularly by the time myocarditis has become severe.Knowledge of the most relevant disease antigens may allow for antigen-directed approaches to suppressing in ammation without sacri cing anti-tumor e cacy such as tolerogenic vaccines.Identi cation of alpha-myosin as an autoantigen may also permit identi cation of biomarkers to predict which patients are at higher risk for myocarditis.
Tables Table 1.Summary of TCR CDR3, V and J genes for murine TCRs used in antigen discovery experiments.

Methods
Mice.Pdcd1-/-Ctla4+/mice were maintained as previously described 2 .Female mice were used in these studies due to their higher incidence of myocarditis.Rag1-/mice were purchased from The Jackson Laboratory (#002216) 24 .For the generation of survival curves, events were de ned as either death (i.e., mice found dead) or identi cation of mice requiring euthanasia (e.g., due to lethargy, moribund, dyspnea, weight loss).All mice were housed at Vanderbilt University Medical Center vivarium, an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited, speci c pathogen-free (SPF) animal facility.All experiments were performed in accordance with Vanderbilt University Medical Center Institutional Animal Care and Use Committee (IACUC) guidelines.
Preparation of cardiac dissociates for single-cell RNA/TCR sequencing.Single-cell suspensions were obtained from murine hearts by mincing followed by enzymatic digestion with 125 U/mL DNase I (Worthington; cat no.LS002138) and 250 U/mL Collagenase 3 (Worthington; cat no.LS004182).Dissociated hearts were ltered through a 30μm lter.Red blood cells were lysed using ACK lysing buffer (KD Medical/MediaTech; cat no.NC0274127).Single-cell suspensions were either used fresh or cryopreserved in 10% DMSO 90% FBS.Prior to sorting, cells were stained with Alex Flour 488 anti-mouse CD45 (BioLegend; clone 30-F11; cat no.103122) for 20 minutes at 4°C.Following staining and washing with PBS, cells were resuspended in PBS with DAPI.Live CD45+ immune cells were sorted by uorescence-activated cell sorting on AF488-positive DAPI-negative events.The wildtype control sample consisted of pooled, without hashing, cardiac immune in ltrates from six female animals, in order to obtain su cient cells as the healthy heart has a low frequency of cardiac immune cells.The myocarditis sample consisted of four in amed hearts from female Pdcd1-/-Ctla4+/mice.In ammation was con rmed by ow cytometry for CD45.Only mice with CD45+ cells comprising greater than ten percent of the total single cells were included.Mice ranged from three to six weeks in age.One in amed heart was run as an individual sample on the 10X Genomics chromium platform.The additional three in amed hearts were hashed together using Total Seq C reagents according to the manufacturer's instructions (BioLegend: TotalSeq™-C0301 cat# 155861, TotalSeq™-C0302 cat# 155863, TotalSeq™-C0303 cat# 155865).
Single-cell RNA/TCR sequencing.Each sample (targeting 5,000 -15,000 cells/sample) was processed for single cell 5' RNA and TCR sequencing utilizing the 10X Chromium system.Libraries were prepared following the manufacturer's protocol.The libraries were sequenced using the NovaSeq 6000 with 150 bp paired end reads.RTA (version 2.4.11;Illumina) was used for base calling and analysis was completed using 10X Genomics Cell Ranger software.Data were analyzed in R using the ltered h5 gene matrices in the Seurat package [25][26][27] .Brie y, samples were subset to include cells with greater than 200 but less than 3000 unique transcripts to exclude likely non-cellular RNA reads and doublets.Cells with greater than 15% of reads coming from mitochondrial transcripts were also excluded as likely dying cells.For murine hearts, hash tag oligos were deconvoluted using HTODemux with positive quantile set at 0.85.Samples were downsized so that equivalent numbers of cells originating from healthy wild type or myocarditis Pdcd1-/-Ctla4+/cardiac in ltrating immune cells were included (2509 cells per genotype of origin).Ten clusters were identi ed using a resolution of 0.4.UMAP was used for dimensionality reduction with 15 nearest neighbors and minimum distance of 0.5.Clonal is de ned as more than two cells with the same TCR clonotype (de ned by unique combinations of CDR3 regions).
T cell receptor sequencing.TCR sequencing and clonality quanti cation was assessed in formalin-xed para n embedded (FFPE) or snap frozen samples of murine hearts or spleens.All human samples were derived from FFPE or isolated PBMC.For FFPE, RNA was extracted from 10µm sections using the Promega Maxwell 16 FFPE RNA kits and the manufacturer's protocol.TCRs were sequenced using the TCR Immunoverse all chain assay following the manufacturer's protocol (Invitae/ArcherDX).Sequencing results were evaluated using the Archer Immunoverse analyzer.CDR3 sequences and frequency tables were extracted from the manufacturers' analysis platform and imported into R for analysis using the Immunarch package (https://immunarch.com) in R.
Antibody-mediated depletion.Female Pdcd1-/-Ctla4+/mice were randomly assigned to control, anti-CD8a, or anti-CD4 injections at 21 days of age.Mice were injected intraperitoneally three times a week with 200µg of anti-CD4 (BioXCell, Cat# BE0003-1, clone GK1.5) or anti-CD8 (BioXCell, Cat# BE0061, clone 2.43) depleting antibodies or vehicle, all in a maximum volume of 100µL.Treatment lasted until 90 days of age.Peripheral blood was sampled via tail prick for assessment of depletion e ciency at week 3.
Adoptive transfer.Splenocytes were isolated from Pdcd1-/-Ctla4+/mice with myocarditis by manual dissociation, ltering, and red blood cell lysis.Myocarditis of the donor mice was con rmed by either H&E or dissociation of the heart and ow cytometry for CD45+ immune cells.A portion of each spleen underwent CD8 depletion using magnetic bead isolation (Miltenyi CD8 (TIL) MicroBeads, Mouse, Cat# 130-116-478).One million whole or CD8 depleted splenocytes were injected into each Rag1-/recipient mouse in 100μL PBS via tail vein injection.Mice were monitored for death or signs of distress.At death or euthanasia, hearts, spleens, livers, lungs, and kidneys were stained by H&E and evaluated microscopically.
TCR sequences and cloning.TCR sequences were generated from CDR3 regions, V genes and J genes using Stitchr 17 .Alpha genes and beta genes were separated using a T2A sequence.Restriction digest sites were added to either end.Full TCR gene blocks were synthesized as custom orders from Genewiz.Full TCR block sequences can be found in supplemental material.TCR sequences were cloned into MSCV-IRES-Thy1.1DEST vector.MSCV-IRES-Thy1.1 DEST was a gift from Anjana Rao (Addgene plasmid # 17442; http://n2t.net/addgene:17442 ; RRID:Addgene_17442) 28 .Retrovirus was made using the platA retroviral packaging cell line (Cell BioLabs RV-102).Jurkat-TCR-ko-CD8+-NFAT-GFP reporter cells were a gift from Dr. Peter Steinberger.Reporter cells were retrovirally transduced with TCRs of interest.TCR expression was con rmed via ow cytometry for Thy1.1 and TCR-beta chain.Retrovirally transduced cells were sorted on the WOLF cell sorter (NanoCellect) for Thy1.1-AF488.Cells were con rmed to be >90% Thy1.1 positive post-sort prior to use in downstream assays.
Antigen discovery.Jurkat-NFAT-GFP cell lines with reconstructed TCRs were used for antigen screening.Syngeneic (derived from C57BL6 mice) bone marrow derived dendritic cells (BMDCs) were used as APCs.BMDCs were generated by ushing femurs and tibias from mice with PBS, ltering the cells though a 70μM lter, lysing RBCs, and plating in RMPI + 10% FBS + 1% HEPES + 20ng/mL GM-CSF (ProSpec Cat# CYT-222).BMDCs were polarized in GM-CSF containing media for 9 days (replacing the media at days 3 and 6) prior to harvesting the adherent fraction via mechanical dissociation using a cell scrapper and cryopreservation for future experiments.For antigen discovery, BMDCs were thawed into GM-CSF containing media in at bottom plates the day before adding TCR cell lines and peptides.Cells were plated at a ratio of 1 TCR cell to 3 BMDCs.The alpha myosin peptide library was generated as 20aa peptides with 5aa overlaps from GenScript.Due to insolubility in aqueous solution, two 20aa peptides were replaced by three 10aa peptides each.Sequences of all 130 alpha-myosin peptides are shown in Extended Data Table 1.Peptides were added at a concentration of 10μg/mL and co-cultures were incubated overnight.TCR cell lines were stained with DAPI to assess viability and analyzed via ow cytometry for NFAT-GFP reporter activity.MHC blocking.Jurkat TCR cell lines were co-cultured with EL-4 cells as APCs with or without 10μg/mL cognate peptide overnight.EL-4 cells were a gift from Dr. Simon Mallal.Blocking antibodies (anti-Db clone 28-14-8S or anti-Kb clone B8-24-3) were added to cells at a concentration of 10μg/mL for 1 hour prior to adding peptides.Blocking antibodies were generously provided by Dr. John Sidney.TCR cell lines were stained with Thy1.1-APC/Cy7 (BioLegend, cat# 202506, Clone OX-7) to differentiate from EL-4s and with DAPI to assess viability and analyzed via ow cytometry for NFAT-GFP reporter activity.
Patients.Healthy donors provided informed consent under an institutionally approved protocol (IRB# 030062).Myocarditis patients and families provided informed consent for research use of biospecimens and clinical data (IRB# 191213).
PBMC expansion.PBMCs were isolated from EDTA collection tubes and processed using a Ficoll gradient.Antigen-speci c PBMC expansion was adapted from previously described protocols 31,32 .Fresh or cryopreserved PBMCs were stimulated with 130 pooled alpha-myosin peptides at a nal concentration of 400ng/mL of each peptide or a pool of control CMV, EBV, and u (CEF) peptides (AnaSpec, AS-61036-003).PBMCs were cultured in CTS OpTmizer medium (CTS OpTmizer T Cell Expansion SFM with CTS supplement A1048501, substituted with 2mM L-glutamine, and 2% human serum, Sigma-Aldrich, H3667) with cytokine supplementation (25ng/mL each of rhIL-2, rhIL-7 and rhIL-15, Peprotech).For myocarditis patients 1 and 2, expansion cultures were also supplemented with autologous LCLs to serve as antigen presenting cells at a ratio of 1 APC per 10 PBMC.For healthy donors, expansion was done directly from fresh, not cryopreserved, blood.Peptides were only added on the rst day of culture.On day 3, additional media with cytokines was added.On day 7, cells were transferred to a new culture dish with fresh media with cytokines.Cells were analyzed or cryopreserved on day 14.
Single cell sequencing of exPBMC.Expanded PBMCs (exPBMC) from patient 1 were prepared for single cell sequencing as follows.exPBMC were incubated with Human TruStain FcX™ (Fc Receptor Blocking Solution; BioLegend cat# 422302) for 5 minutes on ice, then washed and incubated with human anti-CD3-AF488 (BioLegend, cat# 300319, clone HIT3a) for 30 minutes on ice, and then washed and resuspended to a concentration of 5x10 5 cells/mL.SYTOX AADvanced™ Ready Flow™ Reagent (Invitrogen, cat# R37173) was used following the manufacturer's instructions to exclude dead cells.CD3+ live cells were sorted on the WOLF cell sorter (Nanocellect).Cells were sequenced and data were analyzed as described above.Data were analyzed in R using the ltered h5 gene matrices in the Seurat package 25- 27 .Brie y, samples were subset to include cells with greater than 200 but less than 4000 unique transcripts to exclude likely non-cellular RNA reads and doublets.Cells with greater than 15% of reads coming from mitochondrial transcripts were also excluded as likely dying cells.Clonal is de ned as more than two cells with the same TCR clonotype (de ned by unique combinations of CDR3 regions).For exPBMC, 5,816 cells with TCR reads were analyzed.To identify TCRs overlapping with the cardiac repertoire, beta CDR3 sequences were used.
Statistical analysis.All statistical analyses were performed in R. All single-cell statistical analyses were calculated in R using the Seurat package [25][26][27] .Visualization and graph generation was performed in R.
Data Availability.All data will be made available upon reasonable request to J.M.B.

Declarations
Figures

Figure 1 Single
Figure 1

Figure 3 Alpha
Figure 3

Figure 4 Alpha
Figure 4

Table 2 .
Summary of myocarditis patient information.