Which B cells are pathogenic in MuSK-Myasthenia gravis?

Myasthenia gravis (MG) is a severe autoimmune disease of the neuromuscular junction (NMJ), characterized by fatigable muscle weakness that may also affect respiratory muscles. The prevalence in Austria is 15.7 in 100.000, with most patients harbouring antibodies of IgG1 subclass against the acetylcholine receptor (AChR) and a subset having antibodies of IgG4 subclass to muscle specific kinase (MuSK). Treatment consists of symptomatic therapy with acetylcholinesterase inhibitors and/or broad immunosuppression. Although the antibody-based pathology of disease is well understood, there are fundamental questions concerning MG disease mechanisms that are unanswered. The mechanisms of MG autoantibody production have not been elucidated. Specifically, the location and characteristics of the B cell subset that produces the autoantibodies have not been identified. Thus, the goal of the proposed research is to identify and characterize the cells that produce the pathogenic autoantibodies and define the B cell subtype repertoires that contribute to the B cell mediated pathology. Antigen-specific B cell populations will be isolated using flow cytometery and a novel MuSK antigen tetramer technology established at my host laboratory at the Department of Neurology at Yale University. Transcriptomes covering the variable region of the IgG of antigen specific B cells will be generated and compared to IgG proteomes produced from antigen specific IgG by mass spectrometry. This will allow us to identify which B cell subsets produce the pathogenic IgG in MG. MuSK antibodies are mainly of IgG4 subclass, which does not activate complement and have been described as partly bi-specific and thus functionally monovalent, making it an unlikely subclass to be involved with autoimmunity. Why IgG4 antibodies to MuSK are the main pathogenic entity is puzzling, and understanding the relevance of IgG4 subclass in pathogenicity may also advance our understanding of the subclass itself and other IgG4 associated diseases such as pemphigus and IgG4-related diseases. Using the transcriptomes of MuSK specific B cells, chimeric antibodies harbouring the variable region of a MuSK specific IgG4 and the constant region of MuSK IgG1 and vice versa will be produced. The chimeric antibodies will be analyzed for pathogenicity in vitro using methods established by the applicant. The project will be executed as a joint operation between Kevin OConnor (Yale) and Markus Reindl (Innsbruck),who is working on antigen-specific B cells in inflammatory demyelinating diseases. This unique project will not only identify key therapeutic targets, but also transfer novel technology that is adaptable to other diseases to Austria. Furthermore, the pathophysiology of MG is not currently investigated in Austria despite a relatively high prevalence. Our project will be the first step to establish MG research in Austria.

In the project Which B-cells are pathogenic in MuSK Myasthenia gravis, immune cells from patients with MuSK Myasthenia gravis were analyzed to determine which cell population is responsible for the production of damaging autoantibodies against the muscle protein MuSK. These autoantibodies impair the signal transmission from the nerve to the muscle, which leads to severe muscle weakness. Patients with MuSK Myasthenia gravis are severely affected, and may suffer also from a respiratory crisis with the need for artificial ventilation. The cells that produce the autoantibodies could not be found in the blood of the patients, as they might be very rare or not present in the blood but in other tissues. Furthermore, the damaging autoantibodies of IgG4 subclass in these patients were investigated. It was found that these antibodies undergo a drastic change in the body of the patients, as they split up into two halves and recombine randomly with halves of other antibodies of IgG4 subclass. The resulting antibodies are bispecific, one half still binds to the muscle protein MuSK, while the other half binds a completely different protein. This process is known as Fab-arm exchange and has consequences for the properties of the autoantibody. For example, the binding strength of the antibody is reduced when only half of the antibody still binds the target protein, and a different protein could be bound at the same time. Furthermore, cross-linking of the protein is being prevented. In fourteen other, very severe diseases, IgG4 subclass autoantibodies play an important role, and it is a new hypothesis that these autoantibodies may also undergo Fab-arm exchange. Future therapies could target the Fab-arm exchange, or perhaps new diagnostic assays could evaluate the degree of Fab-arm exchange as novel clinical parameter. In addition, a new classification system for IgG4 autoimmune diseases was developed to assess the pathogenicity of IgG4 subclass antibodies. It was found that five IgG4 autoantibodies could be classified as pathogenic, among these autoantibodies against MuSK. For these disorders, future therapies could be developed that focus directly on the IgG4 subclass. In a further project, four myasthenia gravis patients with the recently discovered autoantibodies against Lrp4 were investigated. In these patients, the thymus was surgically removed. We investigated if there are germinal centers present in the thymus, in which immune cells are developed to produce high affinity antibodies. We did not find any germinal centers, but all four patients improved after the surgery, which is why we hypothesize that the thymus may contribute by a yet unknown mechanism to the disease, and that surgical removal of the thymus could be a beneficial treatment option for these patients. Due to the small sample size, these findings need to be validated by further studies with larger patient cohorts. During the return phase an award-winning new antigen discovery project was initiated to discover new autoantibodies in MG patients and to improve diagnosis, clinical management and thus quality of life of these patients.