Receptor clustering and pathogenic complement activation in myasthenia gravis depend on synergy between antibodies with multiple subunit specificities

Abstract

Myasthenia gravis is an autoimmune disorder defined by muscle weakness and fatigability associated with antibodies against proteins of the neuromuscular junction (NMJ). The most common autoantibody target is the acetylcholine receptor (AChR). Three mechanisms have been postulated by which autoantibodies might interfere with neurotransmission: direct antagonism of the receptor, complement-mediated destruction of the postsynaptic membrane, and enhanced internalization of the receptor. It is very likely that more than one of these mechanisms act in parallel. Dissecting the mechanisms of autoantibody-mediated pathology requires patient-derived, monoclonal antibodies. Using membrane antigen capture activated cell sorting (MACACS), we isolated AChR-specific B cells from patients with myasthenia gravis, and produced six recombinant antibodies. All AChR-specific antibodies were hypermutated, including isotypes IgG1, IgG3, and IgG4, and recognized different subunits of the AChR. Despite clear binding, none of the individual antibodies showed significant antagonism of the AChR measured in an in vitro neuromuscular synapse model, or AChR-dependent complement activation, and they did not induce myasthenic signs in vivo. However, combinations of antibodies induced strong complement activation in vitro, and severe weakness in a passive transfer myasthenia gravis rat model, associated with NMJ destruction and complement activation in muscle. The strongest complement activation was mediated by combinations of antibodies targeting disparate subunits of the AChR, and such combinations also induced the formation of large clusters of AChR on the surface of live cells in vitro. We propose that synergy between antibodies of different epitope specificities is a fundamental feature of this disease, and possibly a general feature of complement-mediated autoimmune diseases. The importance of synergistic interaction between antibodies targeting different subunits of the receptor can explain the well-known discrepancy between serum anti-AChR titers and clinical severity, and has implications for therapeutic strategies currently under investigation.

Serum neurofilament light chain in myasthenia gravis subgroups: An exploratory cohort and case–Control study

“…One potential cause is complete or partial atrophy of muscle fibers based on permanent local acetylcholine (ACh) deficiency because ACh could exert a trophic influence on muscle fibers in addition to transmitting the impulse at the NMJ ( 17 , 18 ). The pathophysiological mechanisms leading to ACh deficiency are well known: direct blockade of the receptors through MG auto-Abs, complement activation leading to destruction and loss of AChR content at the postsynaptic membrane, and depletion of AChR receptors by Abs-mediated crosslinking ( 29 , 50 , 51 ). These mechanisms depend on the specific IgG auto-Abs subclasses, Abs titer, and specific epitopes ( 29 , 51 ).…”

“…The pathophysiological mechanisms leading to ACh deficiency are well known: direct blockade of the receptors through MG auto-Abs, complement activation leading to destruction and loss of AChR content at the postsynaptic membrane, and depletion of AChR receptors by Abs-mediated crosslinking ( 29 , 50 , 51 ). These mechanisms depend on the specific IgG auto-Abs subclasses, Abs titer, and specific epitopes ( 29 , 51 ). Another explanation for a possible neurogenic change might be the increasing evidence for presynaptic involvement in the pathogenesis of MG ( 29 , 52 ).…”

“…These mechanisms depend on the specific IgG auto-Abs subclasses, Abs titer, and specific epitopes ( 29 , 51 ). Another explanation for a possible neurogenic change might be the increasing evidence for presynaptic involvement in the pathogenesis of MG ( 29 , 52 ). Presynaptic proteins such as synaptic vesicle glycoprotein 2A (SVP2) are enriched in patients with MG ( 29 ).…”

“…Another explanation for a possible neurogenic change might be the increasing evidence for presynaptic involvement in the pathogenesis of MG ( 29 , 52 ). Presynaptic proteins such as synaptic vesicle glycoprotein 2A (SVP2) are enriched in patients with MG ( 29 ). Notably, in the presence of high anti-AChR-Abs titers, these presynaptic proteins may be activated as a bystander effect of complement activation ( 29 ).…”

“…As the neuromuscular terminal seems to be enriched with proteins critical for NMJ structure and function, including cytoskeleton-associated proteins like Nfl ( 24 – 27 ), the different extent of neuromuscular destruction on the NMJ could have a measurable effect on Nfl release ( 28 , 29 ). Here, we investigate whether sNfl is increased in patients with MG compared to controls and whether sNfl could serve as a biomarker of disease severity.…”