Acquisition of N-Glycosylation Sites in Immunoglobulin Heavy Chain Genes During Local Expansion in Parotid Salivary Glands of Primary Sjögren Patients.

Annie Visser,Hendrika Bootsma,Arjan Vissink,Marieke E Doorenspleet,Frans G M Kroese,Nicolaas A Bos,Niek De Vries,Fred K L Spijkervet,Richard J Bende

doi:10.3389/fimmu.2018.00491

Abstract

Previous studies revealed high incidence of acquired N-glycosylation sites acquired N-glycosylation sites in RNA transcripts encoding immunoglobulin heavy variable region (IGHV) 3 genes from parotid glands of primary Sjögren’s syndrome (pSS) patients. In this study, next generation sequencing was used to study the extent of ac-Nglycs among clonally expanded cells from all IGVH families in the salivary glands of pSS patients. RNA was isolated from parotid gland biopsies of five pSS patients and five non-pSS sicca controls. IGHV sequences covering all functional IGHV genes were amplified, sequenced, and analyzed. Each biopsy recovered 1,800–4,000 unique IGHV sequences. No difference in IGHV gene usage was observed between pSS and non-pSS sequences. Clonally related sequences with more than 0.3% of the total number of sequences per patient were referred to as dominant clone. Overall, 70 dominant clones were found in pSS biopsies, compared to 15 in non-pSS. No difference in percentage mutation in dominant clone-derived IGHV sequences was seen between pSS and non-pSS. In pSS, no evidence for antigen-driven selection in dominant clones was found. We observed a significantly higher amount of ac-Nglycs among pSS dominant clone-derived sequences compared to non-pSS. Ac-Nglycs were, however, not restricted to dominant clones or IGHV gene. Most ac-Nglycs were detected in the framework 3 region. No stereotypic rheumatoid factor rearrangements were found in dominant clones. Lineage tree analysis showed in four pSS patients, but not in non-pSS, the presence of the germline sequence from a dominant clone. Presence of germline sequence and mutated IGHV sequences in the same dominant clone provide evidence that this clone originated from a naïve B-cell recruited into the parotid gland to expand and differentiate locally into plasma cells. The increased presence of ac-Nglycs in IGHV sequences, due to somatic hypermutation, might provide B-cells an escape mechanism to survive during immune response. We speculate that glycosylation of the B-cell receptor makes the cell sensitive to environmental lectin signals to contribute to aberrant B-cell selection in pSS parotid glands.

Highlights

Primary Sjögren’s syndrome is clinically characterized by complaints of dry mouth and dry eyes, which are associated with the presence of periductal lymphoid infiltrates in the salivary and lacrimal glands
We analyzed the presence of somatic hypermutations among the immunoglobulin heavy variable region (IGHV) sequences amplified from cDNA of parotid glands of individual Primary Sjögren’s syndrome (pSS) patients and non-pSS sicca controls
The higher percentage of germline encoded IGHV genes reflects the presence of more naïve B-cells in the parotid gland of pSS patients compared to the parotid gland of non-pSS sicca controls

Summary

Introduction

Primary Sjögren’s syndrome (pSS) is clinically characterized by complaints of dry mouth and dry eyes (sicca complaints), which are associated with the presence of periductal lymphoid infiltrates in the salivary and lacrimal glands. From a pathogenic point of view, B-cell hyperactivity is a hallmark of the disease [1] This is reflected by the presence of elevated serum levels of IgG in patients with pSS, as well as by the presence of autoantibodies, such as anti-Ro/SSA and anti-La/SSB autoantibodies, and rheumatoid factor (RF) [2, 3]. The reason for the considerable clonal expansion of B-cells in pSS is not known, but proliferation and survival mediated by enhanced signaling through the B-cell receptor (BCR) may be involved This presumption is in line with the observation that both naïve and memory B-cells in peripheral blood of pSS patients express increased levels of Bruton’s tyrosine kinase, a molecule that is critically involved in BCR signaling [12]. The elevated levels of B-cell associated cytokines, such as BAFF, APRIL, IL-6, and IL-21 present in serum and saliva of these patients, may further support the development and persistence of these clonal B-cell and plasma cell populations [13,14,15,16,17,18,19,20,21]

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Results

Conclusion