Project Summary. Myasthenia gravis (MG) is an autoimmune disorder of neuromuscular transmission. Autoantibodies to muscle specific tyrosine kinase (MuSK) can be found in patients with MG who do not have detectable antibodies to the acetylcholine receptor (AChR). Passive transfer and active immunization studies in animals have shown that MuSK serum autoantibodies are clearly pathogenic. Among the features distinguishing MuSK MG from the AChR-specific subtype is a predominantly IgG4 subclass-driven immunopathology and unique responsiveness to immunotherapy. B cells are critical in driving many aspects of MuSK MG including production of pathogenic autoantibodies. Yet their identity, specificity, function, and fate remain very poorly defined. It is well-recognized in the field of B cell-mediated autoimmunity that finding a means to isolate and study the specific autoreactive B cells is an important unsolved problem of high impact. Thus, it follows that the reason for lack of progress is not lack of interest, but rather that the problem is difficult: isolating and characterizing autoantibody-producing B cells is non-trivial. To this end, we applied novel technology for direct, ex-vivo isolation of individual MuSK autoantibody-producing B cells, which relies on flow cytometry-based separation and a fluorescent MuSK antigen. We have recently isolated a large set of memory B cells and antibody-secreting cells from patients with MuSK MG. From these cells, we have produced more than 50 unique monoclonal antibodies (mAbs) and further demonstrated their specificity to human MuSK. This important finding represents the first isolation of B lineage cells (and human mAbs) that produce autoantibodies targeting the MuSK antigen. We now wish to apply this unique approach to further describe the important biological and pathogenic characteristics of these MG autoantibodies. We propose to investigate paradigm-changing features of MuSK MG pathology that were previously not possible to study with antibodies derived from heterogeneous human serum. We will demonstrate that MuSK autoantibodies can utilize IgG subclasses other than IgG4 and that they recognize novel epitopes on MuSK. We anticipate that these findings will modify the widely accepted view of the molecular pathology. We will then explore how different MuSK autoantibody mAbs, encoding different IgG subclasses and different epitope recognition, contribute to immunopathology using both established in-vitro and in-vivo models. This study leverages an exceptionally unique approach applied to patient-derived specimens to first investigate fundamental disease mechanisms that have the potential to change the understanding of the immunopathology and second to develop clinically relevant disease models that will directly impact patient care.