Recombinant Acetylcholine Receptor Immunization Induces a Robust Model of Experimental Autoimmune Myasthenia Gravis in Mice
, Niklas Huntemann 1, Saskia Räuber 1, Vera Dobelmann 1, Derya Cengiz 1, Alexander Herrmann 1, Kathrin Koch-Hölsken 1, Norbert Gerdes 2, Hao Hu 2, Philipp Mourikis 2, Amin Polzin 2, Malte Kelm 2, Hans-Peter Hartung 1,3,4, Sven G. Meuth 1, Christopher Nelke 1,*,†and Tobias Ruck 1,*,†
Linda L. Kusner
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
In this manuscript, Theissen and coauthors established and characterized a model of EAMG using a recombinant protein containing the immunogenic sequence of AChR α subunit in C57BL/6 mice. Aside from confirming the development of fatigable weakness and anti-AChR Ab production, they demonstrated complement and IgG deposition at the neuromuscular junction (NMJ). The model exhibits dysregulated T helper compartment, B cell activation with higher levels of CXCR5. Using recombinant AChR subunits or its extracellular domains to induce EAMG, while not a new concept, has been performed mostly in rats. The authors demonstrated the feasibility of utilizing the same approach in mice. Overall, the manuscript is well written with clear data presentation. Methodology is standard and straightforward. Interpretations are logical. Here are some questions/comments.
- This model requires two immunizations. Was CFA used at both immunizations?
- The authors showed examples of immunofluorescence staining for IgG and C3 deposits at the NMJs. The manuscript will be strengthened further if the model also exhibits decreased AChR content or simplification of post-synaptic folds.
- A brief discussion on the distinct findings of dysregulated T cells and B cells in the spleen vs lymph node in this model will be helpful.
- Minor comments:
a. For Figure 2E and 3E, it is better to show examples of scatterplots with values reflecting their bar graphs rather than just gating strategy.
b. Consistency in the abbreviation for interferon-γ (IFN-γ).
Author Response
Please see the attachment.
Sincerely,
Lukas Theissen, Christopher Nelke and Tobias Ruck
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsRecombinant acetylcholine receptor immunization induces a robust model of experimental autoimmune myasthenia gravis in mice.
The authors have described a study which uses a peptide of the immunogenic region of the acetylcholine receptor to produce an experimental autoimmune myasthenia gravis model. The EAMG animals demonstrate weakness starting at 14 weeks post injection and the experiment is terminated at 17 weeks. The neuromuscular junctions show antibody binding and C3 deposition. Further assessment of the splenocyte and lymphocyte population demonstrate changes in the B cells, T follicular helper cells, and T regulatory cells. The authors postulate that the peptide induced EAMG mouse model can be used for future studies in MG.
The manuscript can be improved by providing additional information.
Lack of detail in the methods:
How many animals were used per group (please state in the methods section)?
Did the control animals receive the CFA (please state in the methods section)?
What areas were injected with the CFA?
Was CFA used for both injections?
Was additional Mycobacterium tuberculosis used in the CFA?
Were the animals assessed in a blind fashion?
How was the grip meter used? How many pulls to determine grip and was the value provided the maximum pull or an average?
What was the care of the animals? Food? Light/Dark cycle? How many were housed together? Where the control animals kept in a different cage?
What was the rationale for terminating the study at week 17?
The ELISA information (Hölzel-Biotech, BOS-PA1203) in the methods is incorrect since the website has it as a CHRNA1 Antibody.
No data for the ELISA information is contained in the manuscript. The ability for the EAMG model to produce antibodies should be a figure in the manuscript with the ability of the antibodies to demonstrate isotype switching.
Figure 2 and 3 can be combined.
Figures 5 and 6 can be combined.
The results on the weakness requires additional experiments that demonstrate the weakness is due to reduction of AChR due to complement destruction. The deposition of C3 does not indicate that the NMJ has reduced AChR, especially given the expression of complement regulators at the junction.
From the premise of the study “To improve the availability and reproducibility of this model”, the discussion requires the data on the B and T cells are supported by the literature. The use of Mantegazza et al (2016) and Gilhus et al (2019) as a citation for the information is incorrect. These articles do not refer to the dysregulation of B cells and do not have an in-depth description of the T helper cells in EAMG or MG patients. The results of changes in splenic and lymphocyte populations (figures 2-6) should be discussed with citation that have noted these alterations due to EAMG in the mouse.
Author Response
Please see the attachment.
Sincerely,
Lukas Theissen, Christopher Nelke and Tobias Ruck
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have addressed the concerns of the reviewer.
