Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools

Abstract

The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChRAbs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to nativestructured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases.

Autoimmune Pathology in Myasthenia Gravis Disease Subtypes Is Governed by Divergent Mechanisms of Immunopathology

“…Using in vitro cell culture approaches, it has been demonstrated that B cells expressing AChR autoantibodies exist within the circulation, lymph nodes, and in the bone marrow (44-48). Other studies have identified autoantibody-producing B cells in the circulation through production of recombinant human monoclonal AChR autoantibodies (mAb) from these cells (49).…”

“…The first recombinant AChR-specific autoantibodies were cloned from phage display libraries isolated from thymocyte-associated immunoglobulin sequences (31, 43, 50). Several additional AChR-specific human-derived mAbs have been produced using a number of different approaches, including single-cell technology (49). These recombinant human-derived mAbs emulate the properties of AChR autoantibodies found in the serum: They compete for binding to regions of the AChR recognized by serum-derived autoantibodies (50), and they possess pathogenic properties demonstrated through passive transfer of MG (49).…”

“…Several additional AChR-specific human-derived mAbs have been produced using a number of different approaches, including single-cell technology (49). These recombinant human-derived mAbs emulate the properties of AChR autoantibodies found in the serum: They compete for binding to regions of the AChR recognized by serum-derived autoantibodies (50), and they possess pathogenic properties demonstrated through passive transfer of MG (49). These mAbs, coupled with investigations using human serumderived AChR autoantibodies, have provided a clear illustration of the three AChR autoantibody pathogenic mechanisms.…”

Antigen specific B cells in myasthenia gravis patients

“…Eight of their Abs were positive for a cell based assay and they showed pathogenic effects both in vivo and in vitro using the Ab with the highest AChR binding capacity. Intriguingly, more than 50% (between 60% and 100%) of the binding activity of anti-AChR antibodies in the sera of patients with MG was blocked by this monoclonal antibody [18]. These findings suggested the existence of a MIR and could lead to the development of new therapies involving these antibodies [19].…”

Features of self-tolerance loss in patients with different clinical phenotypes of myasthenia

“…According to Sprent (2002), normal expression of nAChR by thymus myoid cells plays an important role in inducing central immune tolerance to muscle proteins of the body (Sprent & Kishimoto, 2002). The certain subunits of AChR were expressed by thymus epithelial cells (Levinson, 2013;Makino et al, 2017;Nakamura et al, 2018). This mechanism is partially controlled by the autoimmune regulator AIRE (Berrih-Aknin, 2014).…”