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Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs
  1. Elisa Corsiero1,
  2. Michele Bombardieri1,
  3. Emanuela Carlotti1,
  4. Federico Pratesi2,
  5. William Robinson3,
  6. Paola Migliorini2,
  7. Costantino Pitzalis1†
  1. 1Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK
  2. 2Clinical Immunology and Allergy Unit, Department of Clinical & Experimental Medicine, University of Pisa, Pisa, Italy
  3. 3Stanford University School of Medicine, Stanford, USA
  1. Correspondence to Dr Michele Bombardieri and Professor Costantino Pitzalis, Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK; m.bombardieri{at}qmul.ac.uk, c.pitzalis{at}qmul.ac.uk

Abstract

Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium.

Methods Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA.

Results RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a ‘citrullinome’ multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS−) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS.

Conclusion We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.

  • Rheumatoid Arthritis
  • B cells
  • Autoantibodies

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