Recently, we completed the determination and synthesis of all the molecular features on sperm-white myoglobin (Mb) that are recognized by antibodies and have found that the sites that are recognized as antigenic are independent of the immunized species and that the immune response to each antigenic site is under separate genetic control. In the present work, the molecular features recognized by T-cells will be determined with bulk cultures and specific T-cell clones that are isolated from long-term T-cell cultures. Chemical and enzymatic cleavage fragments from Mb will be used in T-cell proliferative assays to identify the most likely location to which specificity of a given T-cell clone is directed. These indicated locations will be synthesized to determine the fine specificity of the T-cells. Similar chemical approaches will be employed to determine, by binding studies, the regions recognized by a large number of monoclonal antibodies. These studies will enable us to find and investigate the physiological role of any regions that are not immunodominant in terms of antibody response and also regions that may be recognized only by T-cells and not be B-cells. Anti-idiotypic antibodies will be raised against monoclonal antibodies and T-cell clones to investigate whether antibody and T-cells, with specificity to the same region of Mb, share idiotype and the role in the antibody response of the T-helper cell sharing idiotype. Factors with activity in cell-cell communication will be isolated from supernatants of T-helper and/or T-suppressor cells. Also, we will isolate from clones T-cell receptors with binding specificity for a given region of Mb. The active sites on selected factor and receptor molecules will be localized by activity of fragments and finally by peptide synthesis. Finally, we will attempt in vivo reconstitution studies in immuno-deficient mice with T-cell clones and/or with the cell-free products.
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