THYMIC B-CELL ACTIVATION IN MYASTHENIA GRAVIS

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Thymus cell (TC) suspensions obtained from thymic tissue removed at thymectomy in myasthenia gravis (MG) patients frequently secrete immunoglobulin (Ig) and anti-acetyl-choline receptor (anti-AChR) antibodies prominently when cultured in vitro despite a paucity of B lymphocytes expressing surface IgM. Current findings in this laboratory suggest that these prominent spontaneous and pokeweed mitogen-induced humoral responses may be from B lymphocytes already differentiated with an isotype switch due to prior in vivo activation. The overall goal of this project is to extend these studies to enhance our understanding of the characteristics and underlying mechanisms in these thymic B lymphocyte humoral responses. Specific aims are to: 1) further clarify the differentiation/activation status of the B cells in these TC; 2) determine the proportion of the B cells in these TC that belong to the germinal center pool; 3) correlate the phenotype and functional capacity of MG thymic helper T cells at the mRNA level; 5) generate anti-AChR secreting hybridomas from MG thymic B cells; 6) define the variable region (V-gene) sequence of anti-AChR secreted by thymic B cells. These aims will be approached using thymic and blood cells obtained at thymectomy in MG patients. Phenotypic markers and flow cytometry will be used for analysis and separation of cell subpopulations. These cell sub-populations will than be cultured either alone or re-combined with other cell subsets. The material secreted in culture will be assayed for Ig and anti-AChR and anti-tetanus (MG irrelevant) antibodies. By analyzing these responses in cells from MG patients immunized with tetanus toxoid prior to thymectomy, the role of the auto-immune (AChR) vs. exogenous (tetanus toxoid) antigenic stimulus can be compared. Appropriate hybridoma and molecular biology technology will be applied to characterize the anti-AChR secreted by TC. These findings will be of great potential clinical importance since the anti-AChR antibody is likely pathogenic in MG and thymectomy is often beneficial in this disorder. In addition, the question of whether anti-AChR antibodies secreted by TC are encoded by: a) germ line genes; b) somatic mutation; or 3) unique gene segments can be approached.