Generation and Characterization of Anti-Citrullinated Protein Antibody-Producing B-Cell Clones

Kristine Germar^1,2, Mark Kwakkenbos³, Sabrina Pollastro^1,2, Nathalie van Uden^1,2,4, Priscilla Kerkman⁵, Ellen I.H. van der Voort⁶, Evan Reed⁷, Karin Lundberg⁷, Niek de Vries^1,8, Lars Klareskog⁷, Hans U. Scherer⁵, René E.M. Toes⁵, Arjen Bakker³, Hergen Spits^3,9,10 and Dominique Baeten^1,2,10, ¹Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ²Amsterdam Rheumatology and immunology Center, Amsterdam, Netherlands, ³AIMM Therapeutics, Amsterdam, Netherlands, ⁴Experimental Immunology, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ⁵Rheumatology, Leiden University Medical Center, Leiden, Netherlands, ⁶Department of Rheumatology, Leiden University Medical Center, Leiden, Netherlands, ⁷Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, ⁸Clinical Immunology and Rheumatology F4-105, Amsterdam Rheumatology and immunology Center, location AMC, Amsterdam, Netherlands, ⁹Department of Cell Biology and Histology, Academic Medical Centre/University of Amsterdam, Amsterdam, Netherlands, ¹⁰These authors contributed equally to this work, Amsterdam, Netherlands

Meeting: 2016 ACR/ARHP Annual Meeting

Date of first publication: September 28, 2016

Keywords: ACPA, B cells and rheumatoid arthritis (RA)

Session Information

Date: Tuesday, November 15, 2016

Title: B Cell Biology and Targets in Autoimmune Disease - Poster II: Rheumatoid Arthritis and Other Rheumatic Diseases

Session Type: ACR Poster Session C

Session Time: 9:00AM-11:00AM

Background/Purpose: Rheumatoid arthritis (RA) is the most frequent form of autoimmune arthritis with a prevalence of almost 1% worldwide. Anti-citrullinated protein antibodies (ACPA) are the most specific biomarker for rheumatoid arthritis (RA)(1). Although therapies designed to deplete B cells, which are the precursor to antibody-secreting cells, are effective in the treatment of RA(2), they only modestly affect ACPA and having no effect on antibodies to recall antigens in serum of treated patients(3). This suggests that autoreactive B cells may contribute to RA pathophysiology by additional mechanisms independent of terminal differentiation toward ACPA-producing plasma cells. Unfortunately, the lack of tools has prevented reliable identification of citrullinated protein (CP)-reactive B cells. Our objective was to develop a novel tool to characterize and model the function of (low-frequency) autoreactive B cells in RA.

Methods: B-cell clones from either blood or synovial fluid of CCP2+ RA patients were immortalized by a technique previously described for obtaining clones for recall antigens(4). ELISA and FACS were used to identify CCP2-reactive clones and to further characterize surface marker and cytokine expression. BCR-signaling competence was tested by calcium flux, and antigen internalization was visualized on Amnis Imagestream. Global gene expression profiles were interrogated by RNA sequencing.

Results: We have identified three unique B-cell clones from two RA patients that can secrete antibodies with specific binding to CCP2 yet show no reactivity to the control arginine variant. While clones generated through this method can secrete soluble antibody, they retain surface immunoglobulin expression, mobilize calcium in response to citrullinated protein antigen and can internalize their antigen. Finally, these clones have a unique surface profile of costimulatory molecules and can secrete both pro- and anti-inflammatory cytokines.

Conclusion: We present here the application of a unique cloning method to establish autoreactive human B-cell clones in RA. We propose that this technique can also be used for other autoimmune diseases with described autoantigen reactivity and that these B cells can be in turn utilized for the identification of autoreactive T cells recognizing novel autoantigen epitopes.

References:

1. Vincent C, Nogueira L, Clavel C, Sebbag M, Serre G. Autoantibodies to citrullinated proteins: ACPA. Autoimmunity. 2005;38(1):17-24.

2. Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, Emery P, Close DR, et al. Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med. 2004;350(25):2572-81.

3. Cambridge G, Leandro MJ, Edwards JC, Ehrenstein MR, Salden M, Bodman-Smith M, et al. Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis. Arthritis Rheum. 2003;48(8):2146-54.

4. Kwakkenbos MJ, Diehl SA, Yasuda E, Bakker AQ, van Geelen CM, Lukens MV, et al. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nat Med. 2010;16(1):123-8.

Disclosure: K. Germar, None; M. Kwakkenbos, None; S. Pollastro, None; N. van Uden, None; P. Kerkman, None; E. I. H. van der Voort, None; E. Reed, None; K. Lundberg, None; N. de Vries, None; L. Klareskog, None; H. U. Scherer, None; R. E. M. Toes, None; A. Bakker, None; H. Spits, None; D. Baeten, None.

To cite this abstract in AMA style:

Germar K, Kwakkenbos M, Pollastro S, van Uden N, Kerkman P, van der Voort EIH, Reed E, Lundberg K, de Vries N, Klareskog L, Scherer HU, Toes REM, Bakker A, Spits H, Baeten D. Generation and Characterization of Anti-Citrullinated Protein Antibody-Producing B-Cell Clones [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/generation-and-characterization-of-anti-citrullinated-protein-antibody-producing-b-cell-clones/. Accessed .

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