HLA class I-associated expansion of TRBV11-2 T cells in multisystem inflammatory syndrome in children

Abstract

Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. Superantigen specificity for different Vβ chains results in Vβ skewing, whereby T cells with specific Vβ chains and diverse antigen specificity are overrepresented in the T cell receptor (TCR) repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCRβ variable gene 11-2 (TRBV11-2), with up to 24% of clonal T cell space occupied by TRBV11-2 T cells, which correlated with MIS-C severity and serum cytokine levels. Analysis of TRBJ gene usage and complementarity-determining region 3 (CDR3) length distribution of MIS-C expanded TRBV11-2 clones revealed extensive junctional diversity. Patients with TRBV11-2 expansion shared HLA class I alleles A02, B35, and C04, indicating what we believe is a novel mechanism for CDR3-independent T cell expansion. In silico modeling indicated that polyacidic residues in the Vβ chain encoded by TRBV11-2 (Vβ21.3) strongly interact with the superantigen-like motif of SARS-CoV-2 spike glycoprotein, suggesting that unprocessed SARS-CoV-2 spike may directly mediate TRBV11-2 expansion. Overall, our data indicate that a CDR3-independent interaction between SARS-CoV-2 spike and TCR leads to T cell expansion and possibly activation, which may account for the clinical presentation of MIS-C.

Keywords: COVID-19; Immunology; T cell receptor.

Figure 1. TRBV-driven repertoire skewing in MIS-C patients.

(A) Principal component analysis (PCA) of differential TRBV, TRAV, TRGV, and TRDV gene usage in MIS-C patients (n = 20) versus febrile controls (n = 15), and between mild MIS-C (n = 4) and severe MIS-C (n = 16) patients. Statistical analysis: Pillai-Bartlett test in MANOVA of all principal components. (B) Frequencies of differentially used TRBV genes in MIS-C and febrile control patients. The boxes extend from the 25th to 75th percentiles, whiskers from min to max, and the line within the box indicates the median. Lines in dot plots indicate mean ± SEM. Individuals considered TRBV11-2 expanded are marked with a red box.

Figure 2. Clonal distribution in individual repertoires of MIS-C patients.

(A) Visualization of clonal richness and distribution in individual productive repertoires from mild (n = 4) and severe (n = 16) MIS-C patients and febrile controls (n = 15). One bubble represents one amino acid clonotype, and the area size of the bubble indicates its fraction within the repertoire. Clonotypes using TRBV11-2, TRBV24-1, or TRBV11-3 are highlighted. Individuals considered TRBV11-2 expanded are marked with a red box. (B) Correlation of serum cytokine levels with TRBV11-2 usage. Statistical analysis: Spearman’s r correlation test. (C) Distribution of nasopharyngeal SARS-CoV-2 PCR results among patients with or without TRBV11-2 expansion.

Figure 3. Junctional diversity in MIS-C patients with TRBV11-2 expansions.

(A) Overlap of CDR3 amino acid clonotypes per 200 sequences in repetitive samples of a healthy individual and between repertoires of MIS-C patients with TRBV11-2 expansion. (B) PCA and differential usage of J genes rearranged with TRBV11-2 in MIS-C patients and febrile controls. Bars indicate mean ± SD. Statistical analysis: Pillai-Bartlett test in MANOVA of all principal components. (C) Heatmap of productive CDR3 length distribution in the repertoires of MIS-C patients (n = 20) and febrile controls (n = 15). (D) CDR3 diversity in MIS-C patients with or without expanded TRBV11-2 and febrile controls displayed as positional weight matrix generated using GLAM2. (E) Unsupervised phylogenetic analysis of the amino acid sequences spanning FR2 to CDR3 versus CDR3 alone in the top 100 clones of MIS-C repertoires (n = 20), either comprising the complete TRBV sequence pool or TRBV11-2 sequences only.

Figure 4. TRAV repertoire skewing and differential gene usage in MIS-C patients.

(A) PCA of differential productive TRAV gene usage in MIS-C patients (n = 20) versus febrile controls (n = 15). MIS-C subgroups: mild MIS-C (n = 4), severe MIS-C (n = 16), MIS-C without TRBV11-2 shift (n = 9), and MIS-C with TRBV11-2 shift (n = 11). Statistical analysis: Pillai-Bartlett test in MANOVA of all principal components. (B) Frequencies of differentially used TRAV genes in MIS-C with or without TRBV11-2 shift as compared to febrile controls. The boxes extend from the 25th to 75th percentiles, whiskers from min to max, and the line within the box indicates the median. Lines in dot plots indicate mean ± SEM.

Figure 5. HLA class I alleles are associated with TRBV11-2 expansion in severe MIS-C patients.

(A) HLA genotypes of febrile controls (n = 10), severe MIS-C patients without TRBV11-2 expansion (n = 3), and severe MIS-C patients with TRBV11-2 expansion (n = 4). (B) Percentages of patient groups with HLA class I alleles. (C) Percentages of patient groups that share HLA-I A02, B35, and C04.

Figure 6. Complex formation between SARS-CoV-2 spike and TCR containing the Vβ chain encoded by TRBV11-2, and comparative analysis of TRBV sequences homologous to TRBV11-2.

(A) Binding of TCR (with Vβ chain sequentially identical to that of TRBV11-2 gene product) to the SAg-like region of SARS-CoV-2 spike (E661 to R685; colored yellow). The TCR α and β chains are shown in magenta and cyan, respectively. The spike subunits are colored dark red, beige, and gray, and the neurotoxin motif (299–356), green. (B and C) Overall and close-up view of the interactions between the SAg-like region and the TCR. Two basic residues, R682 and R683, on the SAg-like region of spike interact with 2 acidic residues, D67 and D68 in the Vβ domain, and D2 in the Vα domain. (D) Multiple sequence alignment (MSA) of the TCR Vβ chains distinguished by Vβ skewing in MIS-C (protein products of TRBV11-2, TRBV11-3, and TRBV24-1 with their corresponding UniProt IDs) and severe adult COVID-19 patients (protein products of TRBV24-1, TRBV14, and TRBV5-6). The MSA was generated by Clustal Omega (76). CDR1, CDR2, and CDR3 are indicated by orange shades. The regions enclosed in blue boxes indicate the highly conserved paratopes in Vβ chains corresponding to TRBV11-2 and TRBV14. See more extended MSA in Supplemental Figure 6.