Gal Knockout Pigs: Any More Carbohydrates?
The key event that has revived interest and heightened the confidence of the potential success of xenotransplantation was the production of α1,3galactaosyltransferase knockout pigs (GT−/−) (4–6). It was widely anticipated that the production of these knockout pigs would overcome this perceived modest increase provided by expression of CRP and lead to indefinite survival of pig organs. However, similar survival times were observed in the first reports of GT−/− pig-to-primate vascularized organ transplants (7–9). The significant finding however is that hyperacute rejection has indeed been overcome, and thus allows the dissection of the subsequent immunological barriers that need to be addressed and overcome.
The well-timed overview by Cooper et al. in this issue of Transplantation focuses on the recent progress in the xenotransplantation using GT−/− pigs (10). They identify thrombotic microangiopathy, seen in rejected GT−/− organs in primates with reduced chronic immunosuppression, as the next phase to be addressed. Seeing that it is not clear if this is dependent on the immune system or due molecular incompatibilities between the pig and primate coagulation system, the authors highlight the novel immunosuppression or approaches to overcome the coagulation dysregulation they consider are required to improve survival outcomes. Above all, the authors clearly express the view that identification of novel pig antigens that are targets for human natural antibodies is a priority for the field. Both the quantity (number) and quality (protein versus carbohydrate) of these antigens need to be elucidated.
N-glycolylneuraminic acid is correctly identified as a non-Galα(1,3)Gal antigen that may require attention. However, Galα(1,3)Gal may still be an important epitope in pig-to-human xenotransplantation (11). Galα(1,3)Gal is also synthesized by a second α1,3galactaosyltransferase, iGb3 synthase (12, 13), and low levels of Galα(1,3)Gal has been reported in Gal−/− mice and pigs (13, 14). Furthermore iGb3 has been shown to activate human natural killer T (NKT) cells (15). While it is clear that iGb3 is not involved in hyperacute rejection, as GT−/− pig organs transplanted into either immunosuppressed or nonimmunosuppressed baboons do not undergo hyperacute rejection, the role this lipid plays in xenograft rejection is uncertain. It may be speculated that anti-iGb3 antibodies, either naturally occurring or induced and of higher affinity, may bind to iGb3 on endothelial cells leading to activated, more procoagulant endothelial cells. Alternatively human NKT cells may be activated by iGb3 and mediated xenograft rejection. Another carbohydrate to be considered is N-acetyllactosamine (NAcLac), which at least in Gal−/− mice is expressed at higher levels than wild type mice (16). Human NKRP1A molecules have been shown bind to NAcLAc as well as Galα(1,3)Gal (17); however, it is unclear whether this is of significance to xenograft rejection. Further studies are required to address these possibilities.
In summary, the authors have drawn attention to the fact further studies are required to identify non-Gal antigens that may require future gene targeting.