title: Antibody responses to de novo identified citrullinated fibrinogen peptides in rheumatoid arthritis and visualization of the corresponding B cells.
abstract: Antibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients.
  
  
  
  An in-house ELISA was established against four cit-Fib α-subunit peptides (cit-Fib α-35; cit-Fib α-216,218; cit-Fib α-263,271 and cit-Fib α-425,426) and serum from patients with established RA (n = 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n = 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry.
  
  
  
  The frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib α-35 (RA 20%, vs PsA/AS 1%); cit-Fib α-216,218 (13% vs 0.5%); cit-Fib α-263,271 (21% vs 0.5%) and cit-Fib α-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib α-35 and cit-Fib α-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers.
  
  
  
  Our data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital Solna, 17176, Stockholm, Sweden.
authors.forename: Vijay
authors.identifier: 0000-0002-2606-8180
authors.initials: V
authors.lastname: Joshua
authors.affiliation: Department of Biomolecular Chemistry, Radboud Institute for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University, Nijmegen, Netherlands.
authors.forename: Loes
authors.initials: L
authors.lastname: Schobers
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital Solna, 17176, Stockholm, Sweden.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Titcombe
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital Solna, 17176, Stockholm, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Israelsson
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Rönnelid
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital Solna, 17176, Stockholm, Sweden.
authors.forename: Monika
authors.initials: M
authors.lastname: Hansson
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital Solna, 17176, Stockholm, Sweden.
authors.forename: Anca I
authors.initials: AI
authors.lastname: Catrina
authors.affiliation: Department of Biomolecular Chemistry, Radboud Institute for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University, Nijmegen, Netherlands.
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital Solna, 17176, Stockholm, Sweden. vivianne.malmstrom@ki.se.
authors.forename: Vivianne
authors.initials: V
authors.lastname: Malmström
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D002956:Citrulline / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005340:Fibrinogen / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000276:immunology; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D010455:Peptides; D002956:Citrulline; D005340:Fibrinogen
keywords: ACPA; Autoantibodies; Fibrinogen; PTPN22; Rheumatoid arthritis