PROJECT SUMMARY Xenotransplantation (i.e. cross-species transplantation using pig organ in human) offers a potential solution to address persistent organ shortage. Organ shortage more severely impacts minorities, such as African Americans, Asians, and Hispanics. However, pig-human incompatibility results in destructive human immune response and ultimate rejection of pig organs. The long-term goal of this proposal is to develop an effective and safe method to prevent the human immune response to transplanted pig cells, tissues, and organs. Human NK cell mediated-direct killing of porcine endothelial cells is one of the barriers in xenotransplantation. A balance of signals from porcine cell ligands and human NK cell receptors interaction determines human NK cell function. This proposal focuses on manipulation of porcine cell surface proteins to inhibit/block NK cells recognition and activation. In our preliminary data, we have demonstrated that expression of HLA-G on porcine cell inhibits NK cell activation and reduces pro-inflammatory cytokine production. An antibody mediated- blocking of NKG2D receptor can diminish NK cell activation. We hypothesize that manipulation of porcine ligands may abolish human NK cell-mediated cytotoxicity. Expression of inhibitory ligands and removal of activating ligands on porcine cells may induce NK cell tolerance and inhibition. The hypothesis will be addressed in the experiments of the following Specific Aims: 1) to determine the inhibition of human NK cell by co-expression of HLA class I molecules HLA-C, HLA-E, and HLA-G on porcine cells; and 2) to determine whether elimination of NKG2D porcine ligands blocks human NK cell recognition and activation. This exploratory study will expand our understanding of the mechanism of cross-species immune recognition and response, develop a novel approach to induce local immune tolerance/acceptance, and guide engineering of pigs to meet xenotransplantation needs.