Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients
Kazushiro Takata, Panos Stathopoulos, Michelangelo Cao, Marina Mané-Damas, Miriam L. Fichtner, Erik S. Benotti, Leslie Jacobson, Patrick Waters, Sarosh R. Irani, Pilar Martinez-Martinez, David Beeson, Mario Losen, Angela Vincent, Richard J. Nowak, Kevin C. O’Connor
Kazushiro Takata, Panos Stathopoulos, Michelangelo Cao, Marina Mané-Damas, Miriam L. Fichtner, Erik S. Benotti, Leslie Jacobson, Patrick Waters, Sarosh R. Irani, Pilar Martinez-Martinez, David Beeson, Mario Losen, Angela Vincent, Richard J. Nowak, Kevin C. O’Connor
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Research Article Immunology

Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients

Abstract

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction. Most patients have autoantibodies against the acetylcholine receptor (AChR), but a subset of patients have autoantibodies against muscle-specific tyrosine kinase (MuSK) instead. MuSK is an essential component of the pathway responsible for synaptic differentiation, which is activated by nerve-released agrin. Through binding MuSK, serum-derived autoantibodies inhibit agrin-induced MuSK autophosphorylation, impair clustering of AChRs, and block neuromuscular transmission. We sought to establish individual MuSK autoantibody clones so that the autoimmune mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from 6 MuSK MG patients using a fluorescently tagged MuSK antigen multimer, then generated a panel of human monoclonal autoantibodies (mAbs) from these cells. Here we focused on 3 highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells, and at mouse neuromuscular junctions, where they colocalized with AChRs. These 3 IgG isotype mAbs (2 IgG4 and 1 IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The mAbs inhibited AChR clustering, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation, which suggests an alternative mechanism for inhibiting AChR clustering.

Authors

Kazushiro Takata, Panos Stathopoulos, Michelangelo Cao, Marina Mané-Damas, Miriam L. Fichtner, Erik S. Benotti, Leslie Jacobson, Patrick Waters, Sarosh R. Irani, Pilar Martinez-Martinez, David Beeson, Mario Losen, Angela Vincent, Richard J. Nowak, Kevin C. O’Connor

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Figure 4

AChR-clustering assay in C2C12 mouse myotubes demonstrates pathogenic capacity of MuSK mAbs.

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AChR-clustering assay in C2C12 mouse myotubes demonstrates pathogenic ca...
The presence of agrin in C2C12 myotube cultures leads to dense clustering of AChRs that can be readily visualized with fluorescent α-bungarotoxin and quantified. Pathogenic MuSK autoantibodies disrupt this clustering. Three different human MuSK-specific mAbs, the humanized murine control MuSK mAb 4A3, and 3 human non–MuSK-specific mAbs derived from AChR MG patient plasmablasts (plasmablasts 64-2, 64-7, and 64-8) were tested for their ability to disrupt the AChR clustering. Each mAb was added to the cultures at 1 μg/mL. (A–D) Representative images (original magnification, ×100) from the clustering experiments are shown. (A) Cultured myotubes do not show AChR clustering until (B) agrin is added (bright spots reveal AChR clusters). (C) The mAb MuSK1A added at 1 μg/mL inhibits clustering (D), whereas a control mAb does not inhibit the formation of AChR clusters. (E) Clustering of AChR was quantified relative to the measured effect of agrin. Quantitative results are normalized to clustering induced by only agrin. Each data point represents the mean value from an independent experiment. Bars represent the mean of means and error bars the SDs. Multiple-comparisons ANOVA (against the pooled results for the 3 human non–MuSK-specific mAbs), Dunnett’s test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, shown only when significant.