Background/Purpose: Sjögren’s disease (SjD) is marked by focal lymphocytic infiltration of lacrimal and salivary glands (SG) that form tertiary lymphoid structures with clonally expanding T cells that may drive disease. Here, we integrated the SG T cell receptor (TCR) repertoire of SjD cases and healthy controls (HC) with single cell transcriptomics to identify SG cellular compartments associated with clonal expansion and preferential gene usage in SjD cases vs. HCs.

Methods: Labial SG biopsies collected from SjD cases (13 Ro⁺ and 10 Ro^–) meeting the 2016 ACR EULAR criteria and 6 HCs were recovered from CryostorII, dissociated, and subjected to 5′ TCR/scRNAseq (10X Genomics). Cell Ranger pipeline was used to extract CDR3 sequences, excluding cells with >3 productive TCR chains. Group comparisons were performed using ANOVA with Tukey’s post-test or Kruskal Wallis test with Dunn’s correction as appropriate in R.

Results: We analyzed 12,232 paired TCR CDR3ab sequences from CD4⁺ (n=5,596), CD8⁺ (n=2,834), CD4⁺CD8⁺ (n=1,209), and innate T cells (n=2,593) from SjD cases and HCs. Among participants containing CD4⁺ clonal expansions (≥2 nucleotide identical TCRαβ⁺ cells), the number of expanded cells was significantly elevated in Ro⁺ cases vs. HCs (p=0.03). Although not statistically significant, CD4⁺ expansions in the Tfh/Tph, Th17, and Treg compartments were largely limited to cases and detected sparsely in one HC. In CD4⁺ clonotypes, TRAV23/DV6 and TRBJ1-3 genes were enriched in Ro⁺ (p=0.005, 0.013) and Ro^– (p=0.018, 0.007) cases, while TRAJ40 and TRBV5-5 were enriched in Ro⁺ cases (p=0.034, 0.020) vs. HCs. TRAV39 and TRBV5-5 were enriched in Ro⁺ vs. Ro^– cases (p=0.033, 0.027). In CD8⁺ clonotypes, TRAJ30 and TRBV11-3 in Ro⁺ cases (p=0.049, 0.043) and TRBV27 in Ro^– cases (p=0.018), were enriched vs. HCs. TRAV23/DV6 was enriched even across the total non-innate T cell population, in Ro⁺ (p=0.005) and Ro^– cases (p=0.039) in addition to TRBV25-1 in Ro⁺ cases (p=0.012), vs. HCs. CD4⁺ stressed/activated and Tfh/Tph cells were the largest contributors to V/J genes enriched in the total non-innate and CD4 repertoires of Ro⁺/Ro^– cases, as were FOXP3⁺ Tregs and activated T cells in the CD4 repertoire of Ro⁺ and Ro^– cases, respectively (Table 1). The V/J genes enriched in the CD8 repertoire were dominated by terminal Tmem/eff cells and Trm cells in both Ro⁺ and Ro^– cases (Table 1).

Conclusion: Early trends of SG TCR analyses in SjD indicate increased CD4⁺ clonal expansion and preferential usage of TRBV genes implicated in multiple autoimmune pathologies viz. TRBV27^1-3 and TRBV25-1^{1, 4, 5}. We identified previously unreported strong enrichment of TRAV23/DV6 in the total non-innate and CD4⁺ repertoires of both Ro⁺ and Ro^– cases. The prevalence of Tfh/Tph, Treg, and stressed/activated CD4⁺ T cell compartments in both clonal expansions and enriched V/J genes distinguish Ro⁺ cases from HCs, indicating an activated state of immune response and B cell helper profile alongside compensatory regulatory function.

Cell types constituting enriched V/J genes

ACR Meeting Abstracts - https://acrabstracts.org/abstract/ro-sjogrens-disease-patients-display-selective-enrichment-of-tcr-v-j-genes-in-the-salivary-gland-with-a-distinguishing-clonal-profile/