Our previous work explored the interaction of the CD8 alpha beta coreceptor with MHC-I, based primarily on the mouse model system. Because of the importance of CD8 function in cellular immunity, we have continued to work diligently towards obtaining biologically active recombinant human CD8alpha beta, with several goals in mind: 1) to complete the detailed characterization of the CD8 alpha beta MHC-I interaction, and 2) to understand in detail the structural aspects of the the stalk of the CD8 alpha beta molecule. Our progress has been to: 1) develop systems for the expression of the human CD8 alpha beta;2) to exploit such protein for binding and structural studies. With considerable effort, we now have engineered human CD8 alpha/beta in several mammalian cell expression systems, and are exploring the binding characteristics of these recombinant preparations. In the second major aspect of this project, we have extended our understanding of the interactions of the NK cell activating receptor and T cell coreceptor, NKG2D, with its stress induced ligands. Although the structural details of the interaction of NKG2D with human MICA and ULBP3 are known, the interaction with the mouse RAE1 is only known at low resolution, and several other murine stress induced ligands of the same family have not been studied in detail. To this end, Dr. M. Hong in the laboratory has engineered two of the previously less studied murine NKG2D ligands, MULT1, and H60, purified them, determined quantitatively their interactions with NKG2D by surface plasmon resonance, and determined several critical X-ray structures that permit us to understand the evolution of these molecules. We have determined the structure of MULT1 unliganded to 2.8 resolution, and a complex of H60 with NKG2D to a resolution of 3.3 . These structures were then confirmed by the production of a battery of mutant MULT1 and H60 molecules and measurement of their affinities for NKG2D. These studies provide additional insight into the general problem of how NKG2D can recognize a large number of distinct ligands (8 different ligands in the human and 9 in the mouse) that are structurally related but are varied in amino acid sequence idnetity (ranging from 10 to 40%). The third sub-project here is a collaboration with the laboratory of John Coligan in which we have contributed our expertise in measurement of protein/protein interactions and have begun to address the question of the specificity of the interaction of the CD300 family of NK cell/ myeloid cell proteins with a variety of molecules founds on cell surfacesmolecules that can be introduced into lipsomes. Promising preliminary data suggest that we can explore the interaction of CD300 family members with liposomes made with various compositions of phospholipids, and area that should prove valuable in understand the ligand specificity of these molecules. In the last ongoing subproject, we are collaborating with Jane Hu-Li and William Paul in efforts to engineer the cytokine IL-1beta and various mutants for targetting, via coupling to particular antibodies, to unique subsets of T lymphoctyes. Preliminary results suggest that we can prepare the needed amounts of the purified IL1 beta and its mutants to allow both biological experiments and biochemical coupling experiments.

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