Extended Data Fig. 6: TCR clonotype sharing and antigen recognition screen.

a, Blood CD8T/NK UMAP highlighting circulating cells (in red) that express a TCR-β sequence found to be expanded in irMyocarditis hearts (combined scRNA-seq and bulk TCR-β sequencing data). b, A volcano plot showing the results of a logistic regression model investigating the likelihood of a cell in a given CD8T/NK cell subset from the blood containing a TCR-β CDR3 sequence that was expanded in heart tissue (n = 13). Points represent odds ratios for each cell subset. Red points denote cell subsets with statistically significant sharing (FDR < 0.05, two-sided likelihood-ratio test). Error bars represent 95% confidence intervals. c,d, Cells in blood for which the same TCR-β was expanded in paired irMyocarditis heart and blood sample from the same patient are shown in red projected on the (c) CD8T/NK blood UMAP embedding and (d) CD4T blood UMAP embedding. Each patient with paired heart and blood samples is shown. e, UMAP of heart T and NK cells highlighting cells that express expanded TCR-β CDR3 sequences that were found in CD8T/NK blood cells on a per-patient basis. f, Feature plots using colour to indicate gene expression (logCPM) levels of the indicated genes projected onto the heart T/NK UMAP embedding. Cell numbers and percentages represent gene expression across all heart T and NK cells. g, The flow cytometry gating strategy for the CD137 expression assay is shown. Lymphocytes are analyzed as either CD8⁺ or CD8^- (to assess for TCRs isolated from CD8 or CD4T cells, respectively) and then gated on intensity of Far Red, CT-Violet, or CT-CFSE staining; each combination of stains represents a unique TCR. Each TCR is then gated on mTRBC⁺ to identify cells expressing the transduced TCR construct. Each row of histograms represents a condition of Epstein-Barr virus-immortalized lymphoblastoid cell lines (EBV-LCLs), which served as antigen presenting cells, pulsed with target peptides/peptide pools, and CD137 expression is shown. PMA/Iono serves as a positive control; “DMSO,” “Ova peptide,” and “CEF” represents negative controls; RINATLETK is an α-myosin peptide known to be recognized by the positive control TCR (“Myo-TCR”)¹⁶; α-myosin pool 4 contains a 20-aa peptide with the RINATLETK sequence; and all other pools are considered test peptide pools. Irrelevant TCRs are TCRs from an unrelated donor⁷⁶ and serve as negative controls along with untransduced (UT) T cells. h, A heatmap showing the background subtracted CD137 expression of each TCR and peptide/peptide pool combination. Each column represents a unique TCR, colour coded by patient and then grouped by CD8 and CD4 TCRs. Each row represents peptides or peptide pools tested; these include the controls and pools covering the full length of the α-myosin, troponin I (TnI), and troponin T (TnT) proteins. α-myosin pool 11 could not be evaluated for TCR63 and is shown as empty space on the heatmap. In c, d, and e, red patient labels denote cases of fatal irMyocarditis. Abbreviations: CEF, Cytomegalovirus, Epstein-Barr virus, and influenza virus; EVB-LCLs, Epstein-Barr virus-immortalized lymphoblastoid cell lines; HLA, human leukocyte antigens; PMA/Iono, Phorbol 12-myristate 13-acetate/ionomycin; mTRBC, murine T-cell receptor β chain; TCR, T-cell receptor; TnI, troponin I; TnT, troponin T; UT, untransduced. Related to Fig. 3.