GalT-KO Pigs: Is the Cup Half Empty or Half Full?

In their timely overview on the importance of the α1,3-galactosyltransferase gene-knockout (GalT-KO) pig to the future of organ xenotransplantation, Cooper et al. have painted a picture of the field as a cup “half empty.” While they have appropriately identified tolerance as the ultimate goal for these studies, there appears to be a logical flaw in their conclusion that major problems, particularly of the coagulation system, will need to be overcome before tolerance can be achieved. In our eyes, the current assessment should rather be that of a cup “half full,” with the problems they have described occurring when tolerance was not achieved. Because the organ xenotransplant data on which their commentary was based were generated largely in this research center, we would like to take this opportunity to revise some of their interpretations. Cooper et al. suggest that the microangiopathy observed in the cited studies of xenografted hearts and kidneys is likely to be the main barrier to successful organ xenotransplantation. However, in the majority of these studies, xenograft recipients were treated with prolonged immunosuppression rather than with regimens directed toward tolerance induction. In the single study cited in which the goal was tolerance induction (1), the xenografts showed little if any evidence of the microangiopathy that was so prevalent in hearts and kidneys prolonged by immunosuppressive drugs alone. Furthermore, in the setting of tolerance induction strategies, life-supporting kidneys continued to function without evidence of rejection for prolonged periods when compared to previously reported xenograft kidneys from other strains of pigs or even to GalT-KO pigs transplanted without a regimen directed toward tolerance induction. What is the implication of these observations? Cooper et al. claim that “cellular infiltrates in GalT-KO grafts have been sparse or nonexistent, suggesting that the T-cell effector responses have been adequately suppressed” using an immunosuppressive regimen containing co-stimulatory blockade by anti-CD154 monoclonal antibodies. This conclusion allows them to treat other hurdles to xenotransplantion, such as coagulation and thrombotic disorders, as independent from T-cell responses. We disagree with this conclusion and raise expression of CD39 as an example of the integration of innate and adaptive responses by a set of extracellular, regulatory ectoenzymes (2, 3). Consistent with this view, the histopathologic studies on the GalT-KO heart xenotransplants in question (which were performed in this laboratory [4]) demonstrated small but definite T cell infiltrates in every heart examined (see Fig. 1). These infiltrates correlated with the development of vascular changes and the deposition of induced anti-pig IgG in these heart grafts (Shimizu et al., manuscript submitted). Since even minimal T cell activation can result in antibody isotype switching and the production of IgG against other, non-Gal epitopes expressed by xenografts, these T cells may well have been the initiators of the subsequent microangiopathic lesions. Thus, while strategies to inhibit coagulation may be useful adjuncts, elimination of injurious immune response through tolerance may avoid this problem altogether.FIGURE 1.: Immunohistochemical staining for CD3 of day 7 (left) and 110 (right) heart biopsies from baboon B223. This animal subsequently developed severe microangiopathy (4) as a prominent feature of rejection. CD3+ infiltrates of varying intensity as well as IgG deposits were seen in every heart reported in this study.When GalT-KO kidney xenografts have been transplanted using immunosuppression not directed toward tolerance induction, early rejection has likewise ensued, with the rejected kidneys showing T-cell infiltrates, anti-pig immunoglobulin G, and microangiopathy (1, 5). These results suggest that kidneys may show more florid changes in response to activation of residual T cells than do hearts, but that both are undoubtedly susceptible to the same kind of rejection. Figure 1 of the Cooper overview is misleading because it compares animal (not graft) survivals for life-supporting kidneys prolonged by a tolerance-induction regimen to graft survivals for heterotopic, nonfunctioning hearts, prolonged with chronic immunosuppression. In fact, most of the animals receiving the tolerance regimen died with functioning, nonrejecting kidney xenografts. A more reasonable comparison would be to life-supporting kidneys with chronic immunosuppression, which did reject (see Fig. 2).FIGURE 2.: Comparison of the survivals of baboons receiving life-supporting GaIT-KO kidneys with either chronic immunosuppression or with the simultaneous transplantation of vascularized donor thymic tissue, in an approach directed toward tolerance induction. Most of the latter baboons expired with functioning renal xenografts showing no rejection and minimal, if any, microangiopathy.In contrast, cotransplantation of vascularized thymic tissue along with GalT-KO kidneys, in an attempt to induce tolerance, has led to prolonged survival of functioning kidney xenografts, without the development of antidonor T cell responses or of induced antibody, and with maintenance of third-party alloresponses (1, 6). This success is reflective of previously reported tolerance induction with allogeneic thymus transplantation (7–10) and of tolerance approaches in other xenogeneic models, including pig to mouse (11) and pig to humanized mice (12). Although the initial regimen utilized for GalT-KO porcine thymus-kidney transplantation in baboons led to frequent infectious complications, the longest survivor in our series, which maintained its life-supporting transplant for 83 days, was treated with a steroid-free regimen and died not from infection but from a thromboembolic event related to flushing of its arterial catheter (1). We are therefore encouraged that successful induction of T cell tolerance across this xenogeneic barrier is not only achievable, but has probably already been achieved. Cooper et al. have treated both natural killer (NK) cell immunity and dysregulation of coagulation as issues unrelated to T-cell immunity. In our thymus-induced tolerance studies, however, minimal if any NK cell infiltrates were observed (Shimizu, manuscript in preparation). Furthermore, in vitro assays in the long-term survivors of kidney-thymus transplants demonstrated anti-pig unresponsiveness in bulk CML cultures containing recipient NK cells (1), suggesting that controlling T-cell responses led also to NK cell unresponsiveness. In contrast to previous pig kidney xenotransplant models using normal pig or hDAF transgenic pig donors (13–15), we did not observe lethal coagulation disorders in these baboons. We did, however, observe proteinuria and glomerulopathy in most of these animals, consistent with endothelial cell damage due to preformed anti-non-Gal antibodues (IgM). With regard to potential human recipients with high levels of anti-non-Gal antibodies, studies by Sykes and colleagues have demonstrated that nonmyeloablative mixed chimerism induction achieves B-cell tolerance both for Gal (16, 17) and for non-Gal (18) specificities, even in intentionally presensitized animals (19). We therefore consider the next important step for xenotransplantation to be induction of B cell tolerance along with T-cell tolerance, perhaps through a combined thymus and mixed chimerism approach. Introduction of additional transgenes to the GalT-KO pig should help us deal with the downstream effects of such antibodies in patients with high levels of preformed antibodies or may avoid the need for B-cell tolerance. Indeed, we are actively pursuing the addition of such transgenes to our GalT-KO line in collaboration with d'Apice and colleagues, in the hope that this approach may extend the range of recipients in whom tolerance may be achievable. Finally, it is likely that infection, due to the intensive immunosuppression currently utilized to prevent xenograft rejection (20), has contributed to many of the adverse events described by Cooper et al. including endothelial activation, coagulopathy and NK cell activation (21). The strategic use of prophylactic antiviral agents and minimization of exogenous immune suppression will undoubtedly reduce these complications. Hopefully such integrated strategies for xenotransplantation will take the cup from “half full” to “close to the brim” in the not-too-distant future. ACKNOWLEDGMENTS The authors thank Annette Sugrue and Mary Piggott for their secretarial assistance. Dr. Sachs serves as a Board member of ICON Development, Inc., Vancouver, B.C., Canada.

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