H and L Chain Affinity Maturation and/or Fab N-Glycosylation Influence Immunoreactivity toward Neutrophil Extracellular Trap Antigens in Rheumatoid Arthritis Synovial B Cell Clones

J Immunol. 2020 May 1;204(9):2374-2379. doi: 10.4049/jimmunol.1901457. Epub 2020 Mar 27.


We previously showed that anti-neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19+ B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab-N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19+ B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (N→Q) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab-N-linked-glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibody Formation / immunology
  • Antigens / immunology*
  • Arthritis, Rheumatoid / immunology*
  • B-Lymphocytes / immunology*
  • Citrulline / immunology
  • Extracellular Traps / immunology*
  • Glycosylation
  • Histones / immunology
  • Immunoglobulin Variable Region / immunology*
  • Mice
  • Mice, SCID
  • Mutation / immunology
  • Neutrophils / immunology*


  • Antibodies, Monoclonal
  • Antigens
  • Histones
  • Immunoglobulin Variable Region
  • Citrulline