T cell antigen recognition involves a contact between the T cell receptor (TcR) and the foreign antigenic peptide bound to the restricting major histocompatibility complex (MHC) molecule. MHC restriction is learned in the thymus. The T cell receptor (TCR) on a developing thymocyte must bind self MHC molecules on thymic cortical epithelium, in order for the thymocyte to be positively selected and allowed to complete maturation. Positive selection in the thymus occurs in the absence of foreign peptides. Self peptides, bound to self MHC molecules, play an important role in this process. However, the relative importance of TCR:self peptide versus the TCR:MHC contact during positive selection remains controversial. Elucidation of the molecular details of this process is of central importance for our understanding of fundamental principles of TCR recognition, selection of a diverse TCR repertoire, tolerance and autoimmunity. To investigate the TCR:peptide:MHC contact during positive selection, two unique systems will be used. A new MHC class I molecule was engineered to evaluate whether, under physiological conditions, all TCRs require a contact with peptides during positive selection, or whether some may rely on the MHC contact alone. In the second system, functional differences in positive selection by two class 1 MHC variants correlate to a TCR utilization pattern. We shall analyze functional reactivities and TCR sequences of these cells, to position the TCR over the selecting MHC molecule, with the help of molecular modeling and crystal structures of both MHC variants. Site-directed mutagenesis and single-chain TCR transgenic animals will then be used to determine which contacts result in restricted selection patterns. Above studies will be significantly enhanced by testing of the fine TCR repertoire using a recently developed method of peptide priming of class 1 restricted T cells.