Clinical manifestation of the patient with myocarditis after BNT162b2 vaccination. (A) Timeline for the collection of samples. (B) Cardiac magnetic resonance imaging findings. T2-weighted black blood image (left) and delayed enhancement image (right) show diffuse increased signal intensity (as compared with skeletal muscles) due to edema and abnormal subepicardial and mid-wall hyperenhancement, particularly in the anterior and antero-septal portions of the left ventricle (red and yellow arrows). (C) Endomyocardial biopsy showed active multifocal lympho-histocytic myocarditis with a few microfoci of myofiber eosinophilic changes and disrupted myocytes (marked with black arrows; H&E stain, ×200). The inflammatory infiltrates show mild extent and focal distribution of the lymphocytic type without fibrosis (left and right). (D) Left: CD3 immunostaining highlights that inflammatory infiltrates are mainly composed of T lymphocytes (in brown, black arrows) (×200). Right: CD68 immunostaining highlights that inflammatory infiltrates are mainly composed of macrophages (in brown, black arrows) (×200).|@|~(^,^)~|@|Single-cell transcriptional characteristics in the patient with myocarditis after BNT162b2 vaccination. (A) UMAP representation of cell types annotated by transcriptional profiling. cDC, classical dendritic cells; T_CM, central memory T cells; T_EM, effector memory T cells; NK, natural killer cells. (B) The violin plots were drawn with immune cell markers. (C) The fraction of cells in each cluster was counted.|@|~(^,^)~|@|Degree of changes in transcriptomic profile differs according to the cell clusters. (A) Heatmap with the gene markers from each cluster. (B) The number of DEGs satisfying the condition (minimum percentage of expressing cells > 15%, logFC > 0.15, adjusted P < 0.05) was counted. (C) Pathway analysis with DEGs of raw P value less than 0.01 in cluster 1 and 2.|@|~(^,^)~|@|Analysis of TCR repertoire in the patient with myocarditis at two time points. (A) Distribution of CDR3 length of T cell clones after the treatment (blue) and before the treatment (myocarditis; red). (B) Single cell TCR analysis was performed. The three clonotypes with the highest frequency are described with sequences. (C) The frequency of clonotypes is shown depending on the condition. (D) Pathway analysis was performed after data restoration in cluster 13 CD8 naïve T cell. DEGs with adjusted P value smaller than 0.1 were used. Dotted line shows where adjusted P value is 0.05. TCA, T cell receptor A; TCB, T cell receptor B; AP-1, activator protein 1; IL-2, interleukin-2; TGFβ, transforming growth factor-β.|@|~(^,^)~|@|Single-cell RNA seq data of BNT162b2-induced myocarditis case and vaccinated individuals are integrated. (A) UMAP representation of PBMCs from this study combined with PBMCs from GSE171964. Different colors denote different clusters. (B) Timeline indicating different time points of sample collection for two datasets. (C) The expression levels of TNFAIP3, CD69, FOS, and KLF6 are marked according to the time after vaccination in T cell subpopulations (GSE171964). Time points when PBMCs from this study were collected were marked with dashed line. (D) Violin plot shows the expression levels of TNFAIP3, CD69, FOS, and KLF6 in T cell subpopulations. *P < 0.05, **P < 0.01, ***P < 0.001; Wilcox test.|@|~(^,^)~|@|The mRNA expression level of IL7R is increased at the time of acute myocarditis. (A) T Single-cell RNA sequencing data of PBMCs of vaccinated individuals was analyzed with automatic clustering. The expression level of IL7R of each cluster is shown with different colors denoting different clusters. (B) Cluster information marked in 6A are shown on the UMAP representation. (C) The expression level of IL7R in BNT162b2-induced myocarditis case is shown in T cell clusters. **P < 0.01, ***P < 0.001; Wilcox test.

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