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

• Published in: PloS one 2017-10, Vol.12 (10), p.e0185976-e0185976
• Main Authors: Makino, Tomohiro, Nakamura, Ryuichi, Terakawa, Maki, Muneoka, Satoshi, Nagahira, Kazuhiro, Nagane, Yuriko, Yamate, Jyoji, Motomura, Masakatsu, Utsugisawa, Kimiaki
• Format: Article
• Language: English
• Subjects: Acetylcholine receptors (nicotinic); Adaptive immunity; Adoptive Transfer; Affinity; Animals; Antibodies; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - isolation & purification; Antigens; Autoantibodies; Autoantibodies - analysis; Autoantibodies - biosynthesis; B cells; B-Lymphocytes - immunology; B-Lymphocytes - pathology; Binding; Biology and Life Sciences; Cell immortalization; Disease; Engineering; Flow Cytometry; Genetic engineering; Humans; Hybridomas - chemistry; Hybridomas - immunology; Immortalization; Immunity; Immunogenicity; Immunoglobulins; Immunology; Inflammatory diseases; Laboratories; Lymphocytes B; Lymphocytes T; Medicine and health sciences; Muscles; Myasthenia; Myasthenia gravis; Myasthenia Gravis - genetics; Myasthenia Gravis - immunology; Myasthenia Gravis - pathology; Neurology; Neuromuscular junctions; Nicotinic receptors; Pathogenicity; Pathogens; Patients; Peripheral blood; Phage display; Phages; Primary Cell Culture; Protein binding; Protein Subunits - antagonists & inhibitors; Protein Subunits - genetics; Protein Subunits - immunology; Rats; Receptors, Nicotinic - genetics; Receptors, Nicotinic - immunology; Research and Analysis Methods; Rodents; Single-Cell Analysis - methods; Skeletal muscle; Torpedo californica
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0185976

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 nAChR-Abs 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 native-structured 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.