Gal knockout pigs: any more carbohydrates?

The need and potential benefit of xenotransplantation has been the driving force to identify approaches to eliminate the first immunological barrier, hyperacute rejection. In the early 1990s, Gal (1,3)Gal was identified as the key epitope that had to be removed to prevent natural antibody binding. In view of the fact that the technology was not available to inactivate genes in any animal larger than mice, the hallmark of that time was the transgenic modification of the germ-line of pigs to express human complement regulators (CRP). The challenge for the field was to identify which combination of human CRP would provide optimal xenograft protection. Some of the human complement regulators prevented hyperacute rejection and extended the survival of pig organs in primates to days in unmodified recipients, or up to 5 months if the recipient was heavily immunosuppressed (1–3). The key event that has revived interest and heightened the confidence of the potential success of xenotransplantation was the production of 1,3galactaosyltransferase 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-Gal (1,3)Gal antigen that may require attention. However, Gal (1,3)Gal may still be an important epitope in pigto-human xenotransplantation (11). Gal (1,3)Gal is also synthesized by a second 1,3galactaosyltransferase, iGb3 synthase (12, 13), and low levels of Gal (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 Gal (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. These must include exposed or altered carbohydrate. Furthermore modification of pigs for successful xenotransplantation is likely to include not only targeting the coagulation pathway, but judicious breeding of the existing human CRP transgenic pigs onto the Gal / background.