Background/Purpose: Sjögren’s disease (SjD) is a chronic rheumatic autoimmune disorder characterized by focal lymphocytic infiltration of the lacrimal and salivary glands (SG). CD4⁺ T cells dominate the foci and play a pivotal role in pathogenesis, as suggested by association of SjD with the HLA DR3/DQ2 haplotype. Here, our objectives are to conduct deep profiling of the peripheral blood (PB) TCR repertoire of SjD cases and HCs, identify disease-associated TCR motifs, and determine whether TCRs expressed on clonally expanded SG-derived CD4⁺ T cells are enriched in the peripheral blood (PB) of SjD cases compared to HCs.    

Methods: PB samples were collected from 19 SjD cases (16 DR3⁺DQ2⁺, 2 DR3^–/DQ2^–, 1 DR3⁺DQ2^–) meeting the 2016 ACR/EULAR SjD classification criteria and 19 DR3⁺DQ2⁺ HCs matched for age, sex, and race. CD3⁺CD4⁺CD45RA^– T-cells were bulk-sorted followed by RNA extraction, cDNA synthesis, and generation of TCRβ libraries for deep sequencing¹. CDR3β sequences, VDJ gene usage, and clonotype information was retrieved from sequencing data. Paired a/b SG TCR sequences were also extracted from the SjD patients (n=10 by TCR RT-PCR², n=9 by single-cell RNAseq) and included for CDR3β motif analysis. Disease-associated TCR clusters with putative shared antigen specificity were identified using the ‘GLIPH2’ algorithm³. Generation probability(pGen) scores of CDR3β amino acid sequences were calculated using OLGA software.

Results: We analyzed 1743 paired SG-CDR3 a/b sequences from cases and 2,953,511 PB-CDR3β sequences from cases and HC. For all cases with ≥50 sampled SG-T cells (n = 10), identical TCR-CDR3β expressed on clonally expanded SG-T cells were detected in the PB of the same patient. GLIPH2 TCR clusters including SG-clonal expansions (n = 31), Vb gene-enrichment and conserved CDR3 length (n = 14), were exclusively shared between the SG and PB TCR repertoires of cases and not HC. Thirteen TCR clusters shared between the two tissues were more abundant in the blood of cases vs. HC (p < 0.05, Mann Whitney U test). Such ‘shared’ clusters were detected in all 10 cases above, and the number of such clusters was inversely associated with whole unstimulated salivary flow (Spearman r = -0.7317, p = 0.02). SjD cases exhibited restricted PB-TCRβ usage, reduced TCR diversity, and higher abundance of clonotypes compared to HC (p < 0.05, Mann Whitney U test). PB-CDR3β sequences detected in cases were more ‘private’ with increasing average frequency when compared to the ‘shared’ public nature of the HC repertoire (p < 2 x 10^-16, pGen vs. average frequency generalized linear regression model).

Conclusion: This is the first study showing that SjD-associated TCRs enriched in clonally-expanded CD4⁺ T-cells of SG tissue are also abundant in the blood of the same patient. The presence of T cell clones with similar antigen specificity shared across SG and blood of multiple patients suggests that a common set of antigens may trigger pathogenic CD4⁺ T cells in SjD.

1. Mamedov, et al. Front Immunol. 2013, PMID: 24391640

2. Joachims, et al. J. Clin. Med. 2020, PMID: 32650575

3. Huang,et al. Nat Biotechnol. 2020, PMID: 32341563

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-sjogrens-disease-associated-cd4-t-cell-receptor-tcr-motifs-and-repertoire-landscape-through-tcr-deep-sequencing/