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Generation and characterisation of monoclonal antibodies from single RA synovial B cells
  1. Khaled Amara1,
  2. Eric Meffre2,
  3. Lena Israelsson1,
  4. Monika Hansson1,
  5. Omri Snir1,
  6. Johanna Steen1,
  7. Fiona Murray1,
  8. Hedda Wardemann3,
  9. Lars Klareskog1,
  10. Vivianne Malmström1
  1. 1Rheumatology Unit, Karolinska Institutet, Center for Molecular Medicine, Stockholm, Sweden
  2. 2Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA
  3. 3Max Planck Research Group, Molecular Immunology, Max-Planck-Institute for Infection Biology, Berlin, Germany

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.

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