title: Autoantibodies From Single Circulating Plasmablasts React With Citrullinated Antigens and Porphyromonas gingivalis in Rheumatoid Arthritis.
abstract: OBJECTIVE
  Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). However, the molecular basis for ACPA production is still unclear. The purpose of this study was to determine if circulating plasmablasts from RA patients produce ACPAs and whether Porphyromonas gingivalis facilitates the generation of ACPAs.
  
  
  METHODS
  Using a single-cell antibody cloning approach, we generated 217 and 110 monoclonal recombinant antibodies from circulating plasmablasts from 7 RA patients and 4 healthy controls, respectively. Antibody reactivity with citrullinated antigens was tested by a second-generation anti-cyclic citrullinated peptide (anti-CCP) kit and by enzyme-linked immunosorbent assays (ELISAs) against citrullinated human antigens. Antibody reactivity with P gingivalis was tested by ELISAs against outer membrane antigens (OMAs) and citrullinated enolase from P gingivalis.
  
  
  RESULTS
  Approximately 19.5% of plasmablast-derived antibodies from anti-CCP-positive RA patients, but none from 1 anti-CCP-negative RA patient or the healthy controls, specifically recognized citrullinated antigens. The immunoglobulin genes encoding these ACPAs were highly mutated, with increased ratios of replacement mutations to silent mutations, suggesting the involvement of active antigen selection in ACPA generation. Interestingly, 63% of the ACPAs cross-reacted with OMAs and/or citrullinated enolase from P gingivalis. The reactivity of ACPAs against citrullinated proteins from P gingivalis was confirmed by immunoblotting and mass spectrometry. Furthermore, some germline-reverted ACPAs retained their reactivity with P gingivalis antigens but completely lost their reactivity with citrullinated human antigens.
  
  
  CONCLUSIONS
  These results suggest that circulating plasmablasts in RA patients produce ACPAs and that this process may be facilitated by anti-P gingivalis immune responses.
authors.affiliation: Qilu Hospital of Shandong University, Ji'nan, Shandong, China, and University of Nebraska Medical Center, Omaha.
authors.forename: Song
authors.initials: S
authors.lastname: Li
authors.affiliation: University of Nebraska Medical Center, Omaha.
authors.forename: Yangsheng
authors.initials: Y
authors.lastname: Yu
authors.affiliation: University of Nebraska Medical Center, Omaha.
authors.forename: Yinshi
authors.initials: Y
authors.lastname: Yue
authors.affiliation: University of Nebraska Medical Center, Omaha.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Liao
authors.affiliation: University of Nebraska Medical Center, Omaha.
authors.forename: Wanqin
authors.initials: W
authors.lastname: Xie
authors.affiliation: University of Nebraska Medical Center, Omaha.
authors.forename: Jessica
authors.initials: J
authors.lastname: Thai
authors.affiliation: Veterans Affairs Nebraska-Western Iowa Health Care System, and University of Nebraska Medical Center, Omaha.
authors.forename: Ted R
authors.initials: TR
authors.lastname: Mikuls
authors.affiliation: Veterans Affairs Nebraska-Western Iowa Health Care System, and University of Nebraska Medical Center, Omaha.
authors.forename: Geoffrey M
authors.initials: GM
authors.lastname: Thiele
authors.affiliation: Veterans Affairs Nebraska-Western Iowa Health Care System, and University of Nebraska Medical Center, Omaha.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Duryee
authors.affiliation: University of Nebraska Medical Center, Omaha.
authors.forename: Harlan
authors.initials: H
authors.lastname: Sayles
authors.affiliation: University of Nebraska Medical Center, Omaha, and University of Nebraska Medical Center, Lincoln.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Payne
authors.affiliation: Veterans Affairs Nebraska-Western Iowa Health Care System, and University of Nebraska Medical Center, Omaha.
authors.forename: Lynell W
authors.initials: LW
authors.lastname: Klassen
authors.affiliation: Veterans Affairs Nebraska-Western Iowa Health Care System, and University of Nebraska Medical Center, Omaha.
authors.forename: James R
authors.initials: JR
authors.lastname: O'Dell
authors.affiliation: Sichuan University, Chengdu, China, University of Nebraska Medical Center, Omaha, and The Eppley Institute for Research in Cancer and Allied Diseases, Omaha.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: University of Nebraska Medical Center, and The Eppley Institute for Research in Cancer and Allied Diseases, Omaha.
authors.forename: Kaihong
authors.initials: K
authors.lastname: Su
mesh_terms: D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007136:Immunoglobulins / Q000235:genetics / Q000276:immunology; D013058:Mass Spectrometry; D010456:Peptides, Cyclic / Q000276:immunology*; D010950:Plasma Cells / Q000276:immunology*; D016966:Porphyromonas gingivalis / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001323:Autoantibodies; D007136:Immunoglobulins; D010456:Peptides, Cyclic; C487763:cyclic citrullinated peptide