title: Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK.
authors.forename: Elisa
authors.initials: E
authors.lastname: Corsiero
authors.affiliation: Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK.
authors.forename: Michele
authors.initials: M
authors.lastname: Bombardieri
authors.affiliation: Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Carlotti
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical & Experimental Medicine, University of Pisa, Pisa, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Pratesi
authors.affiliation: Stanford University School of Medicine, Stanford, USA.
authors.forename: William
authors.initials: W
authors.lastname: Robinson
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical & Experimental Medicine, University of Pisa, Pisa, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Migliorini
authors.affiliation: Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK.
authors.forename: Costantino
authors.initials: C
authors.lastname: Pitzalis
mesh_terms: D000368:Aged; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000276:immunology / Q000378:metabolism; D015551:Autoimmunity; D001402:B-Lymphocytes / Q000276:immunology*; D002956:Citrulline / Q000276:immunology; D065206:Extracellular Traps / Q000276:immunology*; D005260:Female; D006657:Histones / Q000276:immunology*; D006801:Humans; D007596:Joints / Q000276:immunology / Q000378:metabolism; D008875:Middle Aged; D013583:Synovial Membrane / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D006657:Histones; D002956:Citrulline
keywords: Autoantibodies; B cells; Rheumatoid Arthritis