Distorted TCR repertoires define multisystem inflammatory syndrome in children

Abstract

While the majority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) display mild or no symptoms, rare individuals develop severe disease presenting with multisystem inflammatory syndrome (MIS-C). The reason for variable clinical manifestations is not understood. Here, we carried out TCR sequencing and conducted comparative analyses of TCR repertoires between children with MIS-C (n = 12) and mild (n = 8) COVID-19. We compared these repertoires with unexposed individuals (samples collected pre-COVID-19 pandemic: n = 8) and with the Adaptive Biotechnologies MIRA dataset, which includes over 135,000 high-confidence SARS-CoV-2-specific TCRs. We show that the repertoires of children with MIS-C are characterised by the expansion of TRBV11-2 chains with high junctional and CDR3 diversity. Moreover, the CDR3 sequences of TRBV11-2 clones shift away from SARS-CoV-2 specific T cell clones, resulting in distorted TCR repertoires. In conclusion, our study reports that CDR3-independent expansion of TRBV11-2+ cells, lacking SARS-CoV-2 specificity, defines MIS-C in children.

Fig 1. TRBV11-2 is expanded in the repertoire of children with MIS-C.

(a) Overview of study design. (b) Principal component analysis (PCA) of differential TRBV usage in children with MIS-C (n = 12), children with mild symptoms (n = 9), and pre-COVID-19 children (n = 8). Values in brackets show the percentage of variation explained by each principal component. (c) Frequencies of TRBV genes in the patient cohorts. Bars indicate mean + SEM. Insert: TRBV11-2 usage in patient cohorts. Differential gene expression analysis of TRBV genes was conducted using the EdgeR package. TRBV11-2 is expanded in MIS-C compared to children with mild symptom (p-val < 0.0001). (d) Differential usage of J genes rearranged with TRBV11-2. Bars indicate mean + SEM. TRBV11-2 rearrangement frequencies were analysed using the EdgeR package. (e) CDR3 diversity of TRBV11-2 in children with mild disease and MIS-C displayed as positional weight matrix. CDR3 sequences containing 13 amino acids are shown as examples.

Fig 2. The expansion of TRBV11-2 in children with MIS-C doesn’t alter the TCR repertoire’s overall architecture.

(a, b) TCR repertoire metrics of patients. Significance determined by unpaired Wilcoxon test between each pediatric group, with adjustment for multiple comparisons using Benjamini-Hochberg correction, indicated by: * p<0.05, ** p<0.01, and *** p<0.001. Lack of notation for specified comparisons indicates no statistical significance. (c) Overview of sequence similarity network analysis: Each repertoire was downsampled to 1,000 clones. Pairwise amino acid sequence similarity of these 1,000 clones was calculated by constructing Levenshtein distance (LD) matrices of their CDR3 sequences. Each node in the TCR similarity network represents a unique amino acid clone, and edges between nodes are constructed by connecting nodes that differ by no more than 2 amino acids (LD1-2) in their CDR3 sequences. Classic graph metrics, such as node and edge number, node degree and the size of the largest cluster were calculated. The random down sampling and network construction were repeated 10 times and the mean value of the network metrics was used for the downstream analysis. (d) TCR sequence similarity network metrics of patient cohorts. Significance determined by unpaired Wilcoxon test between each paediatric group, with adjustment for multiple comparisons using Benjamini-Hochberg correction, indicated by: * p<0.05, ** p<0.01, and *** p<0.001. Lack of notation for specified comparisons indicates no statistical significance.

Fig 3. Distance to MIRA analysis.

(a) Overview of distance to MIRA analysis: A distance matrix was constructed between all clones in a repertoire and those that mapped to the Adaptive MIRA database, and the distance to the closest MIRA clone was identified. (b) Distance to class I MIRA distribution of TRBV11-2 clones in the repertoires of children with mild COVID-19 or MIS-C. (c) Distance to class II MIRA distribution of TRBV11-2 clones in the repertoires of children with mild COVID-19 or MIS-C.