The nature of the antigenic determinants on human histocompatibility antigens that are recognized by antibodies and cytotoxic T lymphocytes will be investigated. Mutations in the HLA-2 molecule have been constructed by substitution of amino acids that naturally occur in other HLA-A2 variant molecules, and additional mutants containing both single and multiple substitutions will be produced. More extensive substitutions will be made by the exchange of exons and parts of exons within the 1 and 2 domains of the HLA-A2 and HLA-B7 molecules. These genes will be expressed in both murine and human cells after transfection, and analyzed for the loss or retention of antigenic determinants using both monoclonal antibodies and cytotoxic T cell clones. Such studies will allow the influence of different regions of the molecule on structure and antigenicity to be determined. The ability of such mutant molecules to induce allogeneic cytotoxic T cells will also be studied in order to determine the degree of relatedness between different mutants and whether any alterations affect the ability of the molecules to function as histocompatibility antigens. Structural alterations will be studied by the production of monoclonal antibodies against exon shuffled molecules, and the relative location of both unique determinants and those that are conserved between parental and recombinant molecules will ascertained by competitive blocking. A similar analysis of the nature of determinants that are altered on HLA antigens expressed on murine cells will also be conducted. Attempts to more directly localize determinants will be made by proteolysis of antigen -antibody complexes, followed by identification of bound peptide fragments using mass spectrometry. The studies will greatly contribute to our understanding of the nature and location of antigenic determinants on human histocompatibility antigens that are important for both T cell and antibody recognition. Such information is important in understanding the central role of these molecules as mediators of the human immune response to tissue grafts, tumors, and virally infected cells.
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