Background/Purpose: Sjögren’s syndrome (SS) is a systemic autoimmune disease characterized by lymphocytic infiltration of the salivary and lacrimal glands resulting in tissue destruction, pathological dry mouth and dry eyes and in increased risk for lymphoma. The presence of ectopic germinal centers, clonally related B cells and autoantibody production from antibody-secreting cells (ASCs) isolated from the salivary glands indicate that at least some of the ASCs arise from an antigen-driven immune response. Analyses of immunoglobulin sequences can be instrumental for determining somatic mutational patterns shaped by selective pressures, where positive selection in the Complementary Determining Regions (CDRs) and negative selection in the framework regions (FWRs) would indicate antigen-driven antibody production. An alternative would be a non-specific mode of B cell activation. Immunoglobulin variable region N-linked glycosylation acquired by somatic hypermutation (AcN-glycs) has been strongly correlated to follicular lymphoma. Bacterial lectins can bind and activate B cells via AcN-glycs – a possible non-specific mechanism for antibody production and proliferation of B cells.

Methods: To explore immunoglobulin selective pressures in our cohort, we single cell-sorted IgG+ ASCs isolated from the minor salivary glands of 4 SS patients meeting the American-European combined and ACR criteria, and 5 sicca controls. The immunoglobulin variable regions were sequenced by RT-PCR. AcN-glyc motiffs were identified using the online NetNGlyc tool. An IMGT/V-QUEST sequence analysis was performed to confirm that all identified AcN-glyc motifs were introduced by somatic mutation and not germline-encoded. To analyze for antigen-driven selection we utilized BASELINe Version 1.3, an online tool for predicting positive or negative selective pressures.

Results: We sequenced 56 variable regions from 3 SS and 21 sicca control immunoglobulins. Analysis of heavy and light chain sequences revealed SS patients had an increased frequency of AcN-glycs motifs in the FWRs and a decreased frequency in the CDRs as compared to controls (20% vs 5%; 4% vs 10%). The BASELINe analysis showed positive selection in the CDRs and negative selection in the FWRs of sequences from both SS patients and controls. When the selection strengths for immunoglobulins based on glycosylation status were compared (regardless of classification), we found that heavy chains with CDR AcN-glycs and light chains with FWR AcN-glycs had significantly less positive selection than those without AcN-glycs (p=0.05 and p=0.04, respectively).

Conclusion: Overall, immunoglobulins from ASCs infiltrating the minor salivary glands of SS patients and sicca controls in our cohort undergo positive selection in the CDRs and negative selection in the FWRs, indicating antigen-driven immune responses. Immunoglobulins with AcN-glycs have lower positive and negative selection pressures in the heavy chain CDRs and FWRs as compared to those that are non-glycosylated, indicating a potential non-specific mechanism for activation in these ASCs that could potentially give rise to autoreactive proliferations or lymphoproliferative neoplasms.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immunoglobulin-n-glycosylation-acquired-by-somatic-hypermutation-as-a-potential-mechanism-for-non-specific-b-cell-activation-in-sjogrens-syndrome/