Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor Kathleen Vrolix a, 1 , Judith Fraussen b,1 , Mario Losen a,1 , Jo Stevens a , Konstantinos Lazaridis c , Peter C. Molenaar a , Veerle Somers b , Maria Alma Bracho d, e, f , Rozen Le Panse g , Piet Stinissen b , Sonia Berrih-Aknin g , Jos G. Maessen h , Leen Van Garsse h , Wim A. Buurman a , Socrates J. Tzartos c , Marc H. De Baets a, b , Pilar Martinez-Martinez a, * a Department of Neuroscience, School of Mental Health and Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 50, P.O. Box 616, 6200 MD Maastricht, The Netherlands b Neuroimmunology group, Biomedical Research Institute and Transnationale Universiteit Limburg, School of Life Sciences, Hasselt University, Diepenbeek, Belgium c Department of Biochemistry, Hellenic Pasteur Institute, GR 11521 Athens, Greece d Centre Superior d’Investigació en Salut Pública (CSISP), Àrea de Genòmica i Salut, Conselleria de Sanitat, Generalitat Valenciana, València, Spain e Institut “Cavanilles” de Biodiversitat i Biologia Evolutiva (ICBiBE), Universitat de València, València, Spain f Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Ministerio de Ciencia e Innovación, Spain g UPMC UM 76/INSERM U974/CNRS UMR7215/Institute of Myology,105 Bd de l’hôpital, Paris, France h Department of Cardiothoracic Surgery, University Hospital, Maastricht, The Netherlands article info Article history: Received 1 July 2013 Accepted 12 December 2013 Keywords: Myasthenia gravis B-cell immortalization Striational autoantibodies Clonal expansion abstract Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChReMG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChReMG and patients often improve after thy- mectomy. We hypothesized that thymic B cell and antibody repertoires of AChReMG patients differ intrinsically from those of control individuals. Using immortalization with EpsteineBarr Virus and Toll- like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChReMG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChReMG versus 6% in control thymus. The IgV H gene sequence analysis showed remarkable similarities, concerning V H family gene distribution, mutation frequency and CDR3 composition, between B cells of AChReMG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR- specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The thymus plays a major role in the central immune tolerance, mainly by regulating the development and maturation of non-self- reactive T cells and the elimination of self-reactive T cells [1]. In addition, the human thymus contains small numbers of B cells [2], with the phenotype of activated B cells which are mainly present in the medulla from early after birth through adulthood [2,3]. The presence of progenitor B cells indicates that they can differentiate Abbreviations: AChR, acetylcholine receptor; AChReMG, anti-acetylcholine re- ceptor antibody positive myasthenia gravis; EBV, EpsteineBarr virus; MG, myas- thenia gravis; MS, multiple sclerosis; NMJ, neuromuscular junction; RMS, rhabdomyosarcoma; SLE, systemic lupus erythematosus; TLR9, Toll-like receptor 9. * Corresponding author. Tel.: þ31 43 3881042; fax: þ31 43 3884086. E-mail address: p.martinez@maastrichtuniversity.nl (P. Martinez-Martinez). 1 Equal contribution. Contents lists available at ScienceDirect Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm 0896-8411/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jaut.2013.12.008 Journal of Autoimmunity xxx (2014) 1e12 Please cite this article in press as: Vrolix K, et al., Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor, Journal of Autoimmunity (2014), http://dx.doi.org/10.1016/j.jaut.2013.12.008