PMC10020205_01
Background and assay. Theoretical background of the T cells receptor (TCR) as a cellular fingerprint, and the process of extracting the data for evaluation. (A) Schematic representation of the V-D-J recombination of the TCR beta locus together with the creation of the CDR3 segment, which constitutes a cellular fingerprint. (B) Clonal expansion of antigen specific T cells during an immune reaction. (C) Schematic representation of the experimental assay to obtain TCR sequences for comparison and evaluation. (D) Principle of 15-mers with a 11-mer overlap. The first 50 amino acids of the SARS-CoV-2 M-protein is used as example sequence. (E) Overlapping peptides from the three proteins are pooled into a single peptide pool used for T cell stimulation.
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Initial electrocardiogram showed borderline ST elevations in I, II, AVL, AVF, V4-V6 and ST depressions in V1 and III (Supplementary Figure 1A). After application of gadolinium, there was a late midmyocardial apical enhancement with minimal pericardial effusion indicating edema and tissue injury (Supplementary Figure 1B). Histological findings showed mild interstitial edema in line with the diagnosis of acute myocarditis (Supplementary Figures 1A-D). Human herpes virus (HHV) 6a, HHV 6b, enterovirus, adenovirus, and parvovirus B19. Histology was indicative for hypoxia of the heart tissue without larger infiltrations of CD3+ T cells (Supplementary Figures 1E, F). Magnetic resonance tomography showed reduced edema but residual signs of myocardial damage (Supplementary Figure 1). All T cells are endowed with a T cell receptor (TCR) of a particular specificity which is unique to each T cell clone (Figure 1A). During an immune reaction, only T-cells with a TCR specific for the antigen are expanded (Figure 1B). By isolating antigen specific cells from one sample and comparing these to another, information about the immune cells in latter can be obtained (Figure 1C; Supplementary material). TCR sequences specific for SARS-CoV-2, peripheral blood mononuclear cells (PBMCs) were isolated from a blood sample (20 ml blood) and subjected to overnight stimulation with over lapping peptide pools from SARS-CoV-2 S, M, and N proteins. The peptide pools consist of 15-mers covering the entire SARS-CoV-2 S, M, and N proteins with an overlap of 11 amino acids (Figure 1D). All 15-mers from the S, M, and N proteins were pooled into a single stimulation pool (Figure 1E).
SARS-CoV-2 specific T cells in heart biopsy. Peripheral blood T cells were stimulated with over lapping peptide pools from SARS-CoV-2 S, M, and N proteins. Activated SARS-CoV-2-specific T cells were isolated by magnetic sorting and the T cell receptors (TCRs) were sequenced using mRNA. In parallel, TCRs from bulk mRNA from a heart biopsy were also sequenced. (A) Frequency of clonotypes identified in the heart biopsy (n = 5,473) and in the isolated SARS-CoV-2 specific T cells after activation (n = 130,447). The presence and frequency of clonotypes in either samples are compared in the dot-plot, the log-log-scale helps the linearize the distributions of clonotypes and thereby making them comparable. (B) Total frequency of clonotypes shared between the heart biopsy and SARS-CoV-2 specific T cells.
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T cells (n = 130,447); we could identify tissue infiltrating SARS-CoV-2 specific T cells and thus the possible contribution of these T cells to the myocarditis. Several clonotypes of the heart biopsy appeared SARS-CoV-2-specific as they were also present in the isolated CD154+CD137+ T cells (Figure 2A). Some were distinct to either the heart biopsy or the isolated SARS-CoV-2 specific T-cells. The cumulative frequency of SARS-CoV-2-specific clones out of the whole TCR repertoire of myocardium infiltrating T cells, that is clonotypes of the myocardium also present in the CD154+CD137+ sample, was 20.8% (Figure 2B).