These investigators have previously shown effective strategies to achieve cytoprotection of human umbilical vein endothelial cells (HUVEC) from human cytotoxic T lymphocytes (huCTL) by genetic transduction of the anti-apoptotic caspase-resistant Bcl-2 gene. By contrast, porcine aortic endothelial cells (PAEC) transduced with Bcl-2 are resistant to some inducers of apoptosis but sensitive to xenogeneic huCTL. Comparison of the sensitivity of these transduced lines suggests that the sensitivity of porcine EC represents a gain of function when compared to human EC. The differences in sensitivity will be characterized with regard to factors regulating the Fas mediated signaling pathway and the extent of caspase activation will be characterized biochemically. Based on this characterization additional genes will be examined to further achieve cytoprotection of PAEC in vitro. The activity of both CD4 and CD8 T cells can be inhibited by cells known as regulatory T cells, which may play important roles in regulating development of autoimmunity and transplantation. The prototypic CD4+CD25+ regulatory T cells will be isolated and characterized functionally and biochemically. The mechanisms these regulatory cells utilize to inhibit allo- and xeno- interactions with CD4 and CD8 T cells will be compared. These cell types offer an additional strategy to reduce graft rejection. Finally, in vivo models using SCID/beige mice will be utilized to evaluate cytoprotective strategies and the function of regulatory T cells. A protocol has been established that very effectively shows cytoprotection of human microvessels by Bcl-2 transduction. We will utilize this model to study cytoprotective strategies using porcine microvessels.
