Myasthenia Gravis (MG) is a human paralysis due to an autoimmune response against the nicotinic acetylcholine receptor (AcChR), with thymus (T)-dependent production of antibodies. The majority of both the anti-AcChR antibodies and of the specific helper T-cells, are against determinants on one particular AcChR peptide component, the Alpha-subunit. The proposed project will involve: 1) Identification of the fine antigenic specificity of poly- and monoclonal helper T-cell lines, specific for the AcChR, obtained from myasthenic patients of known MHC haplotype. We will focus our efforts on the Alpha-subunit because it dominates the anti-AcChR response in MG. This will be accomplished by challenging the T-cells with proteolytic fragments of known sequence from the Alpha-subunit. Once the segment able to stimulate the T-cells will be identified, fine localization of the antigenic determinants will be obtained by the use of short synthetic peptides corresponding to the different parts of the stimulating fragment. 2) Establishment and propagation of B-cell hybridomas, secreting anti-AcChR monoclonal antibodies, from the same group of patients (donors of T-cells), and partial amino-terminal sequencing of the antibodies, to identify any H- or L-chain restriction of this autoimmune response. 3) Identification of the antigenic determinants (on the Alpha-subunit) recognized by the antibodies. This will be accomplished either by a competition assay using an existing, large (approximately 160) library of rat anti-AcChR monoclonals of known specificity, or by immunoprecipitation of labeled, synthetic peptides corresponding to crucial segments of the Alpha-subunit. Comparison of the sets of antigenic determinants recognized by the T- and B-cells of the same patient will give insights into the development of this autoimmune response, and will elucidate how a complex membrane antigen like AcChR is """"""""seen"""""""" by the different compartments of the human immune system. Comparison of the sets of determinants recognized by each patient with his/her MHC haplotype will give indications as to whether the MHC products, and in particular the class II molecules, determine the recognition of particular areas of the AcChR molecule. Finally, the results of the studies proposed here will indicate if the anti-AcChR response is oligo- or polyclonal, both in the T and B compartment, and how many clones are involved. To answer this question will be an important step forward both for understanding the pathogenesis of MG and for a rational approach to its treatment.
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