Antibodies directed at various targets (especially Gal al,3Gal) and complement are pivotal mediators of hyperacute rejection of the heart, lung, and other organs. However in pig-to-human and pig-to-non-human primate models, we have consistently found that potent complement regulation coupled with efficient removal of anti-pig antibody is associated with rapid dysfunction of lung xenografts. Thus hyperacute lung rejection (HALR) is mediated by mechanisms in addition to those that cause hyperacute rejection of other organs. This paradigm is reinforced by the preliminary studies, where three GalT-KO swine lungs perfused ex vivo retained their function for an average of 2 hours (60, 134, and 170 minutes), far longer than controls (<10 minutes). Failure was due to sudden, rapid increase in pulmonary vascular resistance. Although biopsies obtained at 10 and 30 minutes of perfusion were histologically unremarkable, intravascular thrombi and capillary congestion were observed at graft failure, despite anticoagulation with high-dose heparin. Coagulation pathway activation (Fl+2) was delayed but not prevented, platelet activation (thrombospondin release) was not attenuated, and over 70% of platelets in the perfusate were sequestered in the lung within minutes of initiating perfusion. Complement activation and deposition in the lung, while reduced, were not prevented. Based on these observations and our previous work demonstrating pivotal roles for thrombin, platelets, complement, and pulmonary intravascular macrophages in hyperacute rejection of Gal+ lungs, we hypothesize that dysregulated intravascular coagulation and residual complement activation are the principle cause of acute injury of GalT KO pig lung xenograft. To test this hypothesis, a combination of genetic (GalTKO lungs expressing human tissue factor pathway inhibitor or decay accelerating factor) and pharmacologic approaches (specific platelet receptor, thrombin, or complement inhibitors) will be used in an established ex vivo perfusion model. Approaches that yield optimal lung function ex vivo will then be validated in vivo in a life-supporting pig-to-baboon lung xenograft model. As a result of the studies proposed, we anticipate that hyperacute lung rejection will be successfully prevented for the first time using primarily donor-directed, mechanism-based strategies to consistently achieve life-supporting function of a pig lung in a baboon, allowing subsequent immunologic barriers to be addressed in this organ system.
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