Generation and characterisation of monoclonal antibodies from single RA synovial B cells. (22nd February 2012)

• Record Type: Journal Article
• Authors: Amara, Khaled; Meffre, Eric; Israelsson, Lena; Hansson, Monika; Snir, Omri; Steen, Johanna; Murray, Fiona; Wardemann, Hedda; Klareskog, Lars; Malmström, Vivianne
• Is Part Of: Annals of the rheumatic diseases. Volume 71(2012)Supplement 1
• Page Start: A40
• Page End: A41
• Publication Date: 2012-02-22
• Subjects: Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2011-201234.21
• Languages: English
• ISSNs: 0003-4967

Abstract

Abstract : Backgroundand objectives: Anticitrulline protein antibodies (ACPA) are a hallmark of HLA-associated rheumatoid arthritis (RA), but it is still not established whether these antibodies represent a cause or a consequence of arthritis. It is however clear that such antibodies can enhance murine arthritis, but a corresponding effect in patients is difficult to prove. In this study the authors aimed to assess the specificity and the immunoglobulin gene characteristics of B cells derived from RA synovial fluid of RA by using a method that allows in vitro production of monoclonal antibodies derived from single human memory B cells. Materials and methods: The authors have cloned and expressed antibodies from single flow cytometry purified CD19+IgG+ B cells from synovial fluid of two ACPA+ and two ACPA- RA patients and tested, by ELISA, the generated recombinant monoclonal antibodies for reactivity to different known citrullinated antigens. Results: On the molecular level, striking differences were found between ACPA+ and ACPA− patients taking into consideration the mutational pattern of the Ig genes. Indeed, based on DNA sequences, the authors could demonstrate that B cells from ACPA+ patients did not have more total mutations than ACPA− patients but when focusing on the CDR1, two and three regions of the Ig variable region, the ACPA+ clones displayed more replacement mutations than did the ACPA− clones. Regarding the specificity of the generated monoclonal antibodies, the authors have analysed clones from one ACPA+ and one ACPA negative patient. Our results so far show that 10 out of 23 antibodies generated from an ACPA+ RA patient, proved to bind, to variable degrees, to cyclic citrullinated peptide and different citrullinated antigens including citrullinated a-enolase, fibrinogen, vimentin and type II collagen. In contrast, no ACPA positive clones were identified amongst the antibodies derived from the ACPA− RA patient. Conclusions: Our data suggest that citrulline-reactive B cells are common in synovial fluid and the Ig molecules bear clear signs of antigen-driven maturation.