Research Project
3445920
A major limitation to wider use of bone marrow and organ transplantation in the treatment of hematologic malignancies, immunodeficiencies, and endsage kidney and heart disease, are the body's cellular immune mechanisms involved in both allograft rejection and graft vs host response. These cellular mechanisms involve the recognition of different HLA molecules, the so-called "transplantation antigens", by cytotoxic T lymphocyte (CTL) and result in lysis of cells bearing these "foreign" antigens. A better understanding of how these foreign antigens trigger CTL lysis may be helpful in developing new therapeutic approaches to control or eliminate graft rejection and graft vs host disease. The specific problem addressed by this proposal is to investigate the structure and expression of normal and mutated HLA genes to identify the number and organization of various immunological epitopes and further, to define the amino acid residues involved in recognition by 1) allospecific and framework antibodies; 2) allospecific cytotoxic T lymphocytes (CTL); and 3) virus-immune, HLA-restricted CTL. The correlation of protein structure with function will be accomplished by direct manipulation of their genes using the recombinant DNA strategies of small DNA segment exchanges between diferend HLA genes more specific alterations at predetermined nucleotide positions using point mutagenesis with synthetic oligonucleotides. These altered class I genes will be transfected into various tissue culture cell lines by somatic cell gene transfer techniques and these cells carrying the altered genes will be analyzed for expression and function by a variety of immunological methods. The approach of mutagenesis initially involves specific alterations directed at transferring the recognition of one HLA molecule to another, and thus not just random disruption of functional recognition. Subsequently, more random approaches such as directed alteration of charged residues for nonpolar residues and visa-versa will follow. Once constructed and characterized, the library of cell lines expressing HLA-A2 and -B7 mutant gene products will be a valuable reagent to further generate monoclonal antibodies and CTL populations recognizing less immunodominant or novel epitopes on the HLA proteins. Because these HLA molecules are also important for restricting the body's detection and killing of virally-infected or cancerous cells, understanding the mechanism of these components of the body's immune system may provide a more effective approach to the treatment of human disease and malignancy, as well as prevent the rejection of transplanted organs.
