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

Tomohiro Makino,Maki Terakawa,Satoshi Muneoka,Yuriko Nagane,Ryuichi Nakamura,Masakatsu Motomura,Kazuhiro Nagahira,Kimiaki Utsugisawa,Jyoji Yamate,Güher Saruhan-Direskeneli

doi:10.1371/journal.pone.0185976

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 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.

Highlights

Myasthenia gravis (MG) is an autoimmune disease characterized by fluctuating muscle weakness and abnormal fatigue in those affected [1,2,3]
Sex, serological results, clinical symptoms and MG Foundation of America (MGFA) clinical classification of donor patients with MG enrolled in this study are summarized in S1 Table
The population of plasmabasts ranged from 0% to 0.2% across donors (0% for MG2, 0.02% for MG1, and 0.2% for MG3 and 6) (Fig 1B), which was consistent with previous observations [45]

Summary

Introduction

Myasthenia gravis (MG) is an autoimmune disease characterized by fluctuating muscle weakness and abnormal fatigue in those affected [1,2,3]. By analyzing mAbs isolated from antigen-immunized rats via hybridoma technology, anti-nAChR Abs and their pathogenic mechanism in rodents have been extensively characterized [5,7]. A passive transfer model of experimental autoimmune MG (EAMG) mediated by monoclonal and polyclonal Abs has contributed fundamentally to our understanding of the pathogenic mechanism underlying MG [5,7,8]. Binding of these Abs to the receptors triggers a decrease in receptor density by inducing complement-dependent cytotoxicity, downmodulating the receptors on the cell surface, or even antagonizing receptor function [6,7]

Methods

Results

Discussion

Conclusion