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. 2020 Aug 20;217(12):e20200513. doi: 10.1084/jem.20200513

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© 2020 Fichtner et al.

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Figure 3. — UCAs of MuSK autoantibodies have pathogenic capacity. AChR-clustering assay in C2C12 mouse myotubes demonstrates pathogenic capacity of MuSK mAbs. 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. The three different human MuSK-specific mAbs and their UCAs were tested for their ability to disrupt the AChR clustering using conditions that were previously validated (Takata et al., 2019). (A) Representative images (original magnification, ×100) from the clustering experiments are shown. Cultured myotubes (a and f) do not show AChR clustering until agrin is added (b and g; bright spots reveal AChR clusters). The mAbs MuSK1A (d), MuSK1A UCA (e), and MuSK1A Fab (i) added at 1 µg/ml inhibit clustering. A control mAb (c and h) and the MuSK1A UCA Fab (j) do not inhibit the formation of AChR clusters. The scale bar corresponds to 100 µm. (B) The effect of the mAb on agrin-dependent clustering was tested. Quantitative measurements of C2C12 clustering were normalized to the agrin-only effect of each individual experiment. Each data point represents the mean value of the normalized agrin effect (%) from at least three independent experiments. Bars represent the mean of means and error bars represent SDs. Multiple-comparison ANOVA with Dunnett’s correction of the independent experimental data points was used to compare the effect on clustering by each mAb or Fab against the pooled results for the three human non-MuSK-specific mAbs. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (only shown when significant).