Background/Purpose:  Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by chronic inflammation of salivary and lacrimal glands. Patients with pSS have increased clonal expansions of B-cells in glandular tissue. The driving force for this expansion is unknown. Previous Ig heavy chain (IGHV) analysis studies of both parotid and labial glands revealed that there are no indications for an antigen driven expansion. However, our sequence analysis of RNA transcripts encoding for IGHV3 (IGHV3) genes derived from parotid pSS B-cell clones revealed significantly higher incidence of acquired N-glycosylation sites due to somatic hypermutation, suggesting a role for N-glycosylation motifs (N-glycs) in the selection of Ig producing cells. The aim of this study was to assess clonal expansions with Next Generation Sequencing (NGS) technology for all IGHV family genes in parotid gland tissue derived from pSS and non-pSS sicca patients.

Methods:  mRNA was isolated from biopsies of parotid glands of 5 pSS patients diagnosed according to the AECG criteria and 5 non-pSS sicca patients. IGHV sequences of all IGHV families were amplified and sequenced using NGS. The Ig sequences were analyzed for IGHV gene usage, somatic hypermutation, CDR3 length and rearrangement by alignment with germline gene sequences of the IMGT reference directory. Sequences with the same CDR3 region (amino acids) and shared mutations within the Ig variable region were referred to as clonally related. Clones composed of more than ≥0.3% of the total amount of productive sequences were referred to as dominant clones. These clones were analyzed for expansion patterns, signs of antigen selection and acquired N-glycs.

Results:  From each patient 1800-4000 unique sequences were recovered. We observed no difference in IGHV gene usage or CDR3 length between the pSS and non-pSS IGHV sequences. A total of 70 dominant clones (mean 14.0±5.6 clones) was found in pSS biopsies, whereas in non-pSS biopsies a significantly lower (p=0.0079) number of 15 dominant clones was detected (mean 3.0±1.6 clones). No difference in percentage of mutations was seen between dominant clones from pSS and non-pSS patients. Analysis of dominant clones showed that in 4 of the 5 pSS patients, and in none of the clones of the non-pSS patients, the germline sequence was present in some of the clones. There was no evidence for antigen driven selection in the dominant clones. We observed that the IGHV sequences of dominant clones in pSS patients had acquired significantly more N-glycs compared to non-pSS sicca patients. These N-glycs were not restricted to IGHV3 genes. Remarkably, the majority of N-glycs was seen in the framework 3 region of IGHV3.

Conclusion:  The presence of germline and heavily mutated IGHV sequences in dominant clones in parotid gland tissue of Sjögren patients is direct evidence that naïve B-cells entering the parotid gland expand and differentiate locally into plasma cells. Lack of evidence for antigen-driven clonal expansion in conjunction with significantly higher incidence of N-glycs, outside the antigen-binding site, in these clones suggests an alternative selection of Ig producing B-cells in pSS. We postulate that there might be a possible role for lectins in this process.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/b-cell-clonal-expansions-in-parotid-glands-of-sjogrens-patients-are-associated-with-increased-numbers-of-n-glycosylation-motifs-in-the-immunoglobulin-heavy-chain-genes/