Our aim is to cloe genes encoding proteins that regulate the expression of the human T lymphocyte antigen CD8 during development of the lymphocyte. With the gene, we can study how expression of lymphocyte specific molecules is regulated in normal cells and in lymphomas and leukemias. Abnormal regulation of genes can lead to carcinogenesis. If abnormal regulation of lymphocyte specific gene products was detected, we would study this in detail. We will be using a novel approach for cloning such genes. CD8 loss mutants of CD8+ T cell lines will be isolated and analyzed to identify mutants in genes encoding regulatory proteins. Such mutants will serve as recipients for transfection with a cDNA direct expression library in order to complement the defect. The library is made with a new Epstein Barr Virus shuttle vector that allows stable episomal replication of cDNA clones. Recovery of intact cDNA clones from transformants can be readily achieved by Hirt isolation followed by transformation of E.coli. The other advantage of this vector is that high efficiences of stable transformation of human cells can be achieved which allows the introduction of entire high complexity expression libraries into recipient cells. Therefore, after transfection variants re- expressing CD8 will be selected using anti-CD8 monoclonal antibody and a fluorescence-activated cell sorter (FACS). The cDNA plasmid responsible for complementation will be isolated by Hirt extraction and transformation of E. coli. We will try to determine the role of CD8 in thymic education of the T lymphocyte whereby T cells learn to distinguish self major histocompatibility antigens (MHC) from non-self. CD8 is an important molecule for interaction of T lymphocytes with MHC molecules in what is known as MHC restriction. CD8 is usually required for interaction with MHC class I molecules by mature T cells. However, it is expressed on thymocytes in a different molecular form--it is covalently associated with the differentiation antigen CD1. Does this association enhance interaction with MHC class I molecules or diminish interaction. We will transfect the CD1 gene into a human T cytotoxic cell line and determine the effect on interaction of the T cell with its target cell.
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