In the thymus, immature T-cells undergo a positive and negative selection process which is mediated in part by the functional interaction of T-cell receptors (TCR) on developing T-cells with self-peptides in the context of self major histocompatibility complex (MHC) molecules on the surface o thymic epithelial cells. The high reactivity of TCR with MHC molecules has been known for decades, yet the basis of the specificity has been mysterious. Recent experiments have demonstrated that the resulting MHC class II activity of the TCR repertoire is not only enforced by positive selection in the thymus, but is inherent in germline TCR sequences. The goal of this proposal is to understand the contribution of germline-dependent and -independent TCR residues to the interactions of preselection TCR with MHC molecules, and in particular to their discrimination between MH class I and class II molecules. The first proposal aim will determine if the preselection repertoire is as MHC class I reactive as a normal MHC-selected repertoire and if there are differences in the reactivity of TCR repertoires with class I versus class II MIHC molecule. The second aim will investigate whether specific TCR V-alpha and V-beta regions confer preferential MHC class I or class II reactivity and examine the role of terminal deoxynucleotidyl transferase-mediated, template-independent "N-region" nucleotide addition in diminishing or enhancing MHC-reactivity. In the third aim, an H-2k-specific preselection TCR repertoire will be generated and used to examine potential MHC class II contact residues that underlie the MHC reactivity of germline encoded TCR. The proposed studies will address a key series of unresolved issues concerning the fundamental reactivity of TCR with MHC molecules. Results from the proposed studies may lead to a better understanding of how T-cells recognize and respond to specific antigen-MHC complexes during immune responses against infections and cancer.