This project is focused on functional studies of NK receptor and T cell receptor recognition. Our approaches in these areas revolve around: 1) the determination of the three-dimensional structure of molecules critical to immune recognition; 2) the characterization of the immunological function of animals transgenic for a single chain MHC-I molecule, expressed in a beta2 microglobulin negative background; 3) the development of model systems, using transgenic mice, for the analysis of the etiology of organ specific autoimmunity. We have determined the high resolution crystal structure of the Ly49A/H-2Dd complex, the first such MHC/NK receptor complex determined. This offers new insight into NK cell recognition. The characterization of the single chain H-2Dd transgenic mice on a beta2-m negative background has revealed several important aspects of both T cell and NK cell recognition . The single chain H-2Dd molecule, expressed on a beta2-m negative background, serves to positively select a diverse repertoire of CD8+ cells. These animals can mount a peptide specific response, indicating that they can adequately educate their T cells. In addition, antigen presenting cells from these animals clearly are capable of presenting H-2Dd-restricted peptides to specific T cells. In contrast to TCR recognition of the single chain H-2Dd molecule, NK recognition seems to be impaired. We also investigated these parameters of NK function in our single chain H-2Dd transgenic mice and observed no evidence for interaction of the single chain H-2Dd with Ly49A nor with other potential inhibitory or activating NK receptors. Because we have also engineered the Ly49A in a form that allows biotinylation and staining of cells bearing an Ly49A ligand, we were able to directly examine cells of the single chain transgenic animals for the presence of a ligand for Ly49A. None was detected, indicating in a binding assay that the single chain molecule failed to interact with the purified Ly49A. These results lead to the conclusion that the H-2Dd sites that interact with TCR and with NK receptors are distinct.With a goal of assessing the nature of the TCR recognition that is involved in autoimmune diseases, in collaboration with Dr. E. Shevach's laboratory, we have cloned the Valpha2 Vbeta2 TCR from a gastritis producing IAd restricted, H/K ATPase specific T cell clone, and expressed this as a three domain TCR. We have cloned each of these TCR V regions onto the appropriate constant region in appropriate expression vectors, and have generated several strains of transgenic mice that now express the somatically rearranged transgenic TCR alpha and beta chains of this pathogenic receptor. Founder animals in a BALB/c X BL/6 background have been identified, and they are being bred now for further analysis. To facilitate our understanding of MHC and TCR interactions in the above transgenic animals and in normal immune responses, we have recently engineered a number of MHC /peptide multivalent molecules. In addition, we have cloned and expressed a TCR from a Vbeta4 expressing T cell clone that shows specificity for a different peptide from the H/K ATPase. Transgenic animals are now being produced that will alow further analysis.
Showing the most recent 10 out of 34 publications
