Overcoming the Immunological Barriers
The biggest hurdle to xenotransplantation has been that of hyperacute xenograft rejection (HAR). HAR is characterised by diffuse interstitial haemorrhage, oedema and thrombosis of small vessels and capillaries. These changes are secondary to endothelial activation and damage. At early time points following xenotransplantation, natural killer (NK) cell and neutrophil infiltrates are observed in the graft as is the extensive deposition of fibrin, immunoglobulins and complement (C3b and C5b-9). Clarification of the mechanisms responsible for the onset of HAR have demonstrated the central role of complement and antibodies in its pathogenesis. As a consequence, approaches have been undertaken which interfere with either the interaction of xenoreactive natural antibodies with their primary target, Gal α1-3Gal α1-4GlcNAc-R structures (known as αGal epitopes) or, alternatively, with the complement cascade.
In this context, most research emphasis has focused on the production of genetically engineered pigs expressing inhibitors of the human complement cascade. The validity of this approach has been convincingly demonstrated by several groups. Most importantly, Yamada et al. and Kuwaki et al. have recently demonstrated that organs (heart, kidney) from genetically engineered pigs lacking functional _ α 1,3-galactosyltransferase and thus lacking expression of Gal epitopes (GalT-KO pigs), do not undergo HAR once transplanted into primates.
Whilst HAR has been overcome in the pig-to-primate model by using α GalT-KO pigs, xenografts still eventually fail as a consequence of acute humoural (AHXR), otherwise known as delayed xenograft rejection (DXR), or acute vascular rejection (AVR). Several elements have been found to be involved in the pathogenesis of AVR and its pathology is primarily characterised by vascular thrombosis, blood extravasation and oedema. Deposits of fibrin, immunoglobulins and complement in the graft do not differ substantially from those observed in HAR. Cellular infiltrates include neutrophils, macrophages, CD8+ T cells and a few NK cells.
AVR is characterised by the progressive deposition of antibodies and complement and is associated with the apoptosis and necrosis of endothelial cells, contributing to platelet aggregation and thrombosis in the graft. Taken together, the existing data support the current view that elicited xenoreactive antibodies (specific for both α Gal and non-αGal epitopes) bind to porcine endothelial cells, leading to type II activation and the up-regulation of genes promoting inflammation and thrombosis, overwhelming the effects of potentially protective molecules and ultimately resulting in pro-inflammatory and pro-coagulant changes. On the other hand, the contribution of ischaemia reperfusion injury (IRI) to the pathogenesis of AVR has yet to be defined. Indeed, early apoptotic events, such as those observed following IRI, may result in changes in the endothelium resulting in the development of a pro-coagulant and pro-adhesive phenotype, similar to the events observed in AVR.
Taken together, studies performed to date have demonstrated that, with the introduction of genetically engineered pigs such as those currently available expressing human complement inhibitors or lacking expression of _ α Gal epitopes, the HAR barrier can be overcome. However, it should be noted that none of the engineered organs currently available are able to fully protect against the binding of anti-non- α Gal xenoreactive natural antibodies or avoid the dysregulation of the coagulation cascade. Indeed, xenografts from α GalT-KO pigs often present devastating thrombotic microangiopathy.
Thus, controlling AVR and microangiopathy will require a multifaceted transversal approach able to confront immunological, inflammatory/apoptotic and coagulation events, which eventually contribute to the onset of this irreversible form of rejection. Therefore, in order to obtain a donor animal that is compatible for xenotransplantation into primates (using an immunosuppression regimen compatible with current clinical standards), several new modifications of the pig genome will be required.
Recent refinements in genetic engineering of farm animals, including pigs, has enabled highly efficient, multiple genetic manipulations within a single animal, including the insertion of transgenes on a heterozygous _ α GalT knockout background (personal communication, D. Ayares, IXA congress 2005). These previous successes demonstrate a proof of concept that multiple manipulations can be achieved and without compromising the health of the animals.
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