Characterization of autoreactive B cells and AChR autoantibodies in myasthenia gravis

Abstract

Myasthenia gravis (MG) is a B cell mediated autoimmune disease that is characterized by muscle weakness and fatigue and associated with autoantibodies directed against muscle acetylcholine receptor (AChR) at the neuromuscular junction. Detection of these antibodies has an important role in the diagnosis, treatment and management of the disease. The antibodies are partly produced by the autoreactive B cells in germinal centres formed in the medulla of the myasthenia thymus, where there are also thymic muscle-like cells that express fetal AChRs. The antibodies circulate to the neuromuscular junction leading to complement mediated lysis of the membrane and internalization of AChRs. The patients are successfully treated by corticosteroids, plasmapheresis and immunotherapies that reduce the levels of circulating antibodies. The AChR antibodies are disease specific and thought to be pathogenic but the mechanisms involved in the failure of tolerance is not well understood. In addition, the treatments are non-specific. Better treatments, targeted at acetylcholine receptor specific B cells, or the plasma cells that make the antibodies, would be preferred. The aim of the thesis was to try to characterize AChR autoantibodies from B cells in the myasthenia gravis thymus gland. The first steps were to test sera, from patients whose thymic lymphocyte populations had been archived by Prof N Willcox, in order to identify cultures that contained cells synthesizing AChR antibodies. Radioimmunoprecipitation and cell-based assays using human embryonic kidney (HEK293T) cells transfected with adult or fetal AChR with additional rapsyn-EGFP (enhanced green fluorescent protein), were used to measure the antibodies. Thymic cultures were tested from selected patients to identify AChR synthesis. The next step was to identify the AChR-antibody specific B cells; a novel approach was to use extracellular membrane vesicles prepared from the transfected cells expressing the AChRs clustered with the intracellular protein rapsyn. Finally, the peripheral blood mononuclear cells (PBMCs) and thymic lymphocytes were characterized by fluorescence activation cell sorting (FACS), and the binding of AChR vesicles to CD19 positive B cells examined. The radioimmunoprecipitation assay was able to detect serum AChR antibodies in MG patients’ sera and to a lesser extent in thymic cell cultures. The cell-based assay also detected clustered AChR antibodies in sera of the majority but not all MG patients. Because the thymus gland contains fetal AChR rather than the adult form, fetal AChR antibodies were tested and found to be more highly represented in the patients than the antibodies to the adult form. Extracellular membrane vesicles expressed AChRs as shown by immunostaining and western blotting. FACS analysis of the AChR/rapsyn- EGFP membrane vesicle preparation showed that they could be detected by this method. To develop the methods for detecting the AChR antibody producing specific B cells, FACS analysis of lymphocyte populations was established in Raji cells and peripheral blood lymphocytes, and finally in the thymic cultures of two myasthenia patients. Analysis of CD19+ B lymphocytes from one selected thymic culture was incubated with AChR expressing vesicles which demonstrated binding of the vesicles to a population of B cells. The work done in this thesis partially achieved the aims of developing the methods for identifying AChR-specific B cells by use of AChR-expressing membrane vesicles and has provided the basis for further work. For this confirmation of the results, fresh thymic preparations from patients would clearly be desirable rather than the archived preparations available at the time of this work.