The long-term goal of the proposed studies is to achieve a comprehensive picture of the molecular interactions involved in the recognition of the acetylcholine receptor (AcChR) in the autoimmune disease Myasthenia Gravis (MG), in different functional compartments of the immune system, i.e. the class II restriction elements, the antigen specific CD4+ helper T-cells, CD8+ """"""""suppressor"""""""" cells, which may be specific for the antigen itself or for the T-helper idiotypes, and the autoreactive B cells. Polyclonal Cd4+ T-helper (THE) cell lines and libraries of corresponding T- clones, specific for the human AcChR, will be established from the blood of several MG patients of different HLA haplotype by cycles of stimulation with pools of synthetic peptides, corresponding to the complete sequence of different human AcChR subunits. The sequence segments containing epitopes recognized by the different THE cells will be identified by challenging the polyclonal and monoclonal CD4+ cell lines with the individual synthetic peptides corresponding to the different parts of the AcChR subunit sequences. The ability of synthetic peptides to bind to the HLA class II (DR) molecules expressed by the patients will be studied, and correlated with the presence, along their sequence, of THE epitopes. Possible preferential use of Valpha and Vbeta gene subfamilies for the T-cell receptor expressed by the anti-AcChR THE cells in MG will be investigated. The actual structure of T-epitopes will be investigated by the use of panels of peptide analogous, carrying single residue substitutions of the sequences segment known to contain T-epitopes. These peptides will be used both to challenge anti-AcChR THE clones and for binding studies with different DR molecules. These studies will indicate the aminoacid residues involved in the interaction with the T cell receptor (TCR) and those interacting with the restricting DR molecule. CD8+ cells present in MG patients, which inhibit the Cd4+ T-cell response to AcChR and may have suppressive function, will be propagated, and their function and antigen specificity will be investigated. In particular, we will study whether they are specific for the AcChR of the idiotype expressed by the AcChR-specific THE cells, and whether they exert their function by direct interaction with the TH cells or by secretion of soluble factors. Finally, the phenotype of B-cells able to bind purified AcChR, which are present in the blood of MG patients, will be investigated, and they will be used to attempt to establish B-cell lines or hybridomas secreting anti- AcChR monoclonal antibodies.
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