The specific activation of T lymphocytes results from a ternary interaction between the T cell receptor, an antigen ligand, and a major histocompatibility complex molecule expressed on antigen presenting cells. This application addresses two issues with respect to this interaction. The first issue relates to the mechanism of ligand interactions that occur. Using cloned genes encoding both chains of the antigen-specific receptor polypeptide chains, the structural basis of specificity will be determined by gene transfection and a functional analysis of the resulting extragenic T cells. Genes from phenotypically related T cell clones will be transfected in different combinations and, in addition, a fine mapping of the primary structure of the receptor will be accomplished by in vitro site-directed mutagenesis. Transfections will be accomplished by selection for an antibiotic resistance gene present in pSV2neo. These studies will show the sequence basis for functional antigen recognition and MHC molecule recognition. The second issue relates to the selection of the T cell population during the course of stem cell maturation to antigen-inducible T lymphocytes. Cloned receptor genes from a T cell line characterized for specificity will be transfected into bone marrow stem cells and used to repopulate lethally irradiated mice. Transfections will be accomplished via recombinant retrovirus vectors capable of infecting a large percentage of cells. Under such conditions, a significant percentage of maturing T cells will express a particular receptor specificity. Investigations will focus on the selection of the extragenic T cells that mature under several conditions. In particular, experiments will determine the characteristics of the maintenance of tolerance to self determinants, and the selection of the T cell repertoire in thymus for self MHC molecule recognition. Receptor genes will be transfected into mice such that the extragenic T cells would react against self MHC molecules, respond to antigen in association with self MHC molecules, or have no specificity for self MHC molecules at all.
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