Project Summary. Myasthenia gravis (MG) is an autoimmune disorder affecting neuromuscular transmission. MG patients suffer from severe muscle weakness and increased muscle fatigability due to diminished neuromuscular signaling. MG is caused by autoantibodies that target components of the neuromuscular junction (NMJ); in most patients this is the nicotinic acetylcholine receptor (AChR). These AChR autoantibodies mediate pathology through three mechanisms: (i) interfering with acetylcholine docking, (ii) internalization of the AChR, and (iii) activation of the classical complement cascade leading to tissue damage. There is heterogeneity within the AChR disease subtype that includes early-onset and late-onset MG (EOMG and LOMG), which is based on age of onset, sex, and HLA-association. Although MG patients with different subtypes share similar disease presentations, the underlying immunopathology may be distinct. This is well Illustrated by the AChR EOMG and LOMG subtypes. Notably, EOMG is characterized by a thymic lymphocytic infiltrate, which includes AChR-specific IgG, T cells, and B cells (including AChR autoantibody-producers) that organize as tertiary lymphoid organs, resembling germinal centers. In contrast, LOMG rarely includes thymic abnormalities. While details of the heterogenous immunopathology are lacking, a deeper understanding of the mechanisms underlying the differences is highly important for both the patient and clinician. This is because treatments that are anticipated to work well in one subtype may not have a biological basis for use in the other subtype(s). Non-responding AChR MG patients in a number of recent clinical trials, including B cell depletion, and complement inhibition therapies, clearly highlight this gap in our knowledge. Thus, in this renewal period we will investigate details of the divergent immunomechanisms underlying the EOMG and LOMG MG subtypes. Our study is sharply focused on the B cells that directly contribute to disease pathology, those which produce AChR autoantibodies. Using new approaches, we will isolate these rare AChR autoantibody producing B cells from thymus tissue and blood and define their repertoire characteristics. Human recombinant monoclonal antibodies (mAb) will then be produced from these B cells so that molecular mechanisms of pathology can be defined using a suite of novel assays to identify their ability to affect pathology through complement-directed tissue damage, blocking, or modulation. Finally, we will define the phenotypes of autoantibody-producing B cell subsets and T helper cell subsets including those specific for the AChR antigen. This study is designed to provide detailed insights into both the role of autoantigen-specific immune cell subsets and molecular mechanisms used by autoantibodies to facilitate the pathology of the EOMG and LOMG subtypes. These clearly defined immunomechanisms are expected to impact treatment outcomes through informing application of biological therapeutics used to treat specific MG disease subsets.

Project Narrative (relevance to public health) This study is aimed at further understanding immunological events that underlie the pathology of human neurologic autoimmune disease. The investigation will use novel molecular approaches to dissect the mechanisms of heterogeneous autoantibody-mediated pathology, and cellular approaches to further understand the differentiation and function of B cells and T cells that contribute to the production of autoantibodies. Specimens derived from patients with myasthenia gravis (MG), a chronic, disabling neurological autoimmune disease, will be studied; yet it is anticipated that the findings will define both clinically relevant biomarkers and highly specific treatment targets for similar autoimmune diseases.