The overall objective of this proposal is to establish the nature and extent of the involvement of DP IV in the immune response and to determine the underlying molecular mechanisms. The rationale for this objective comes largely from their recent discoveries that potent synthetic inhibitors of DP IV are potent inhibitors of antigen-, but not mitogen-induced T-cell activation and that the HIV-1 Tat protein, which also exhibits an antigen-specific immunosuppressive effect, inhibits this enzyme. The applicants now propose to develop more stable variations of the potent and specific DP IV inhibitors based on the boronic acid transition-state-approach and to kinetically characterize their inhibitory behavior against DP IV isolated from a variety of sources. They will also measure their immunosuppressive activity in order to determine whether there is a correlations between the ability of these inhibitors to block enzyme activity and their ability to suppress antigen-induced T cell activation. Another aspect of the proposal is to complete a detailed kinetic analysis of Tat's inhibition of DP IV, already nearly complete for Tat 1-86, Tat 1-72, and H6-Tat, and to extend such analysis to other Tat derivatives, such as Tat 1-58, and Tat 10-58. Correlation between the ability of Tat derivatives to Inhibit DP IV and their ability to suppress antigen-induced T-cell activation will also be examined. Structural features, and the amino acid residues of Tat that are responsible for its DP IV inhibitory activity will be determined next. The approach will involve screening fragments of Tat for DP IV inhibitory activity and screening mutants of full length Tat for loss of DP IV inhibitory activity. Structural studies using 1D, 2D, and 3D NMR spectroscopy will be carried out in conjunction with the mutagenesis experiments to aid in the identification of the structural elements essential for binding to DP IV. Subsequently, the applicants will attempt to incorporate 15N labeled histidine and 13C cysteine into Tat and to conduct NMR studies directed at (i) identifying zinc ligands, (ii) determining the effect of zinc on Tat structure, and (iii) characterizing the physical-chemical properties of Tat in solution. The investigators will continue the analysis of the effects of DP IV inhibitors, both boronic acid inhibitors and Tat, on the antigen and mitogen induced response of human PBMC and murine splenic T cells; i.e., to determine the time course for the inhibitory interaction with DP IV, and to define the subset(s) of CD4+ lymphocytes that are targets for DP IV mediated inhibition. The effect of DP IV inhibition on second messenger systems in cloned human and murine CD4+ T cell lines and hybridomas will also be examined. Lastly, the applicants will use already existing anti-Tat and anti-DP IV (CD 26) mAbs and to generate a panel of new mAB to (a) define the functional domains of DP IV and the interaction of DP IV with Tat; (b) test anti-DP IV Mab for stimulatory or inhibitory activity in antigen-specific T cell activation; and (c) test anti-Tat Mab for their ability to prevent and reverse immunosuppression.
