To better understand the role of B cells, potential mechanisms for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's Syndrome (SS), we explored selection pressures and N-glycosylation acquired by somatic mutation (acN-glyc) in the immunoglobulin (Ig) variable regions (V-regions) of antibody secreting cells (ASCs) isolated from the minor salivary glands of SS patients and non-SS controls with sicca symptoms.
We report a novel method to produce and characterize recombinant monoclonal antibodies (mAbs) from SS patient and control labial salivary gland single-cell sorted ASC infiltrates that can be utilized to concurrently probe any other expressed genes. V-regions were amplified by RT-PCR, sequenced, and analyzed for incidence of N-glycosylation and selection pressure, then expressed as the native mAbs, or mutant mAbs lacking the acN-glyc for specificity testing. Protein modeling was used to demonstrate how even acN-glycs outside of the complementarity-determining region (CDR) could participate in, or inhibit, antigen binding.
V-region sequence analyses revealed clonal expansions and evidence for secondary light chain editing and allelic inclusion not previously reported in SS. We found increased acN-glycs in the sequences from SS patients and that acN-glycs were associated with increased replacement mutations and lowered selection pressure. We also identified a clonal set of polyreactive mAbs with differential FWR1 acN-glycs and demonstrated that removal of the acN-glyc could nearly abolish binding to the autoantigens.
Our findings support an alternative mechanism involving V-region N-glycosylation for the selection and proliferation of some autoreactive B cells in SS patients.