- Email: [email protected]
On the road to clinical xenotransplantation
Transplant Immunology 21 (2009) 57–59
Contents lists available at ScienceDirect
Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Editorial
On the road to clinical xenotransplantation Emanuele Cozzi ⁎ Direzione Sanitaria, Padua General Hospital, Padua, Italy Surgical Unit III, Padua General Hospital, Padua, Italy Department of Surgical and Gastroenterological Sciences “Pier Giuseppe Cevese”, University of Padua, Padua, Italy CORIT (Consortium for Research in Organ Transplantation) Padua, Italy
a r t i c l e
i n f o
a b s t r a c t
Keywords: Xenotransplantation Pig Primate review
In recent years the development of novel immunosuppressive strategies and new lines of engineered pigs have enabled improved xenograft survival in the clinically-relevant pig-to-primate models (especially in islet xenotransplantation). Furthermore, researchers have now developed appropriate biomolecular tools to address rapidly the remaining barriers and render organs from source pigs more “compatible” with man. Efficacy has been the main focus of the research conducted in the last few years. However, other fundamental issues, such as those regarding the physiology, the safety, the ethics and regulatory aspects of xenotransplantation, will need to be addressed satisfactorily prior to proceeding with clinical xenotransplantation trials. © 2009 Published by Elsevier B.V.
1. Introduction
On the other hand, it is becoming apparent that other molecular barriers may need to be the target of intervention in view of achieving long-term xenograft survival. In this respect, the natural immune response directed against pig tissues and cells may, for instance, play a greater role than originally thought. The generation of transgenic pigs for human complement regulators that effectively control primate complement activation proved to be efficient in preventing complement-mediated graft damage [6], ultimately improving the survival of porcine xenografts transplanted into primates. However, other potentially harmful components of the natural immune response directed to the xenograft exist and these may need to be counteracted adequately. In particular, recent data suggest that human anti-pig NK cells may not be appropriately controlled by the pig counterpart molecule expressed by porcine cells and this may lead to xenograft damage even when organs from αGal−/− animals are used [7]. This has been mostly associated with the incapacity of porcine MHC class I molecules to provide appropriate KIR-mediated inhibitory signals to human NK cells [8] or with the capacity of porcine ULBP-1 to activate NK cells efficiently via the NKG2D pathway [9]. Similarly, even human macrophages have been shown to induce damage to pig cells due to the incapacity of porcine CD47 to provide effective SIRPα-mediated inhibitory signals to human macrophages [10]. Taken together, published data unequivocally demonstrate the existence of a strong natural primate anti-pig immune response that is well documented both in vitro and in the context of bone marrow xenotransplantation in the primate. Nevertheless, the real impact of such findings on the long-term survival of porcine xenografts in the primates remains unclear. As a consequence, it is still unknown whether these observations will require specific engineering of source pigs.
Considerable progress has been achieved in recent years in the clinically-relevant pig-to-primate xenotransplantation models and this is comprehensively described in a series of excellent contributions in this volume of the Journal. In this light, this short editorial will exclusively discuss a limited number of selected issues with the aim of enabling the reader to better comprehend where we stand in this scientific field and which are the approaches currently being undertaken to push it further.
2. Dealing with the barriers to long-term xenograft survival At this stage, the key biological events that preclude the long-term survival of porcine cardiac, renal and islet xenografts transplanted into primates appear to have been identified. These primarily consist in the anti-graft immune response [1] and in the existence of several molecular incompatibilities between pigs and primates [2]. These incompatibilities may be responsible for the thrombotic events that are central to the rejection process and the coagulopathy consistently observed in xenografted primates [3], but may also ultimately affect the immune response to the xenograft itself [4,5]. Currently, work is actively underway to counteract the antixenograft immune response and overcome the previously-identified undesirable molecular incompatibilities, some of which also affect the immune response itself. ⁎ Department of Medical and Surgical Sciences, University of Padua, Clinica Chirurgica III, Via Giustiniani, 2, 35128 Padova, Italy. Tel./fax: +39 049 8218841. E-mail address: [email protected]. 0966-3274/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.trim.2009.05.002
58
E. Cozzi / Transplant Immunology 21 (2009) 57–59
Much insight has also been gained recently on the existing molecular incompatibilities between porcine and primate proteins of the coagulation cascade. These are ultimately partly responsible for the onset of acute humoral xenograft rejection, a process usually presenting as a microvascular thrombosis, often associated with consumptive coagulopathy in xenografted primates. The current status in this field is eloquently presented by Lin et al. [3]. Of particular interest is the recent demonstration that co-incubation of porcine endothelial cells with human platelets induces the latter cells to express tissue factor with a mechanism independent of the humoral immune response. This phenomenon would therefore also occur even in a completely tolerant recipient, possibly precluding long-term xenograft survival. This new set of data may, at first consideration, be perceived as an additional obstacle on the road to clinical xenotransplantation. However, in the same article, the authors also suggest several carefully-designed pharmacological strategies that, once administered to primate recipients of appropriately engineered source pigs, could inhibit the occurrence of coagulation disorders in xenografted primates. 3. Current best results in clinically-relevant pig-to-primate models The current status of porcine solid organ and islet xenotransplantation in the clinically-relevant primate models is thoroughly described in two separate articles. As far as solid organ xenotransplantation is concerned, Ekser et al. clearly show that, using genetically engineered source animals, graft survival has considerably improved over the years, especially with regard to cardiac xenotransplantation [11]. In contrast, only a marginal improvement has apparently been achieved in the renal xenotransplantation setting. In this regard, however, it is of interest that Baldan et al. could obtain 90 day survival of a primate recipient of a renal xenograft (with 3 out of 7 renal recipients surviving for 40 days or longer) using a relatively straightforward immunosuppressive regimen that would be regarded by some as clinically applicable [12]. On the other hand, it is noteworthy that when porcine lungs or livers were transplanted into primates, only up to 8 and 5 day survival were reported, respectively. This could possibly be due to the more complex physiological discrepancies or the additional immunological barriers existing between pig and primate for these two organs. As far as islet xenotransplantation is concerned, the comprehensive review assembled by Hering et al. clearly indicates that, in contrast with solid organ xenotransplantation, long-term survival of a life-supporting islet xenograft with normoglycaemia is reproducibly achievable in the primate [13]. In this regard, it is remarkable that 6 month survival or longer was reported in two different non-human primate species by at least 4 independent investigators [14–17]. More importantly, in one case, following optimisation of the encapsulation of adult porcine islets using highly purified alginate, islet xenografts implanted subcutaneously were able to normalise glycaemia in diabetic non-human primates for up to 6 months without any immunosuppression [16]. These results are of outstanding importance as, for the first time, they unequivocally demonstrate long-term survival of a life-supporting porcine xenograft in non-immunosuppressed primates. Clearly, avoidance of any type of immunosuppression, as in this case, considerably enhances the safety profile of xenotransplantation and would represent the ideal scenario to start any clinical xenotransplantation trial. 4. Strategies to further extend survival of porcine xenografts transplanted in the primate Whilst results have considerably improved over the last few years, conventional and novel approaches may further extend survival of porcine xenografts transplanted in the primate. These approaches can be broadly divided into strategies acting directly on the xenograft recipient, enabling better control of the anti-graft immune response; and strategies rendering the source pig more “compatible” with the
primate recipient, resulting in reduced susceptibility to damage and, ultimately, improved xenograft survival. 4.1. Strategies acting directly on the xenograft recipient As far as obtaining better control of the anti-graft immune response, at least three different avenues should be considered. First, new immunosuppressive approaches primarily based on the use of novel pharmaceutical compounds or biologics are being developed to overcome the different forms of xenograft rejection identified to date [18]. Whilst these approaches have been instrumental in obtaining current survival data, it should be pointed out that these are usually associated with a status of generalised (non-specific) immunosuppression that makes them far less attractive than the two options described hereafter. Second, the establishment of accommodation in the primate, namely long-term graft survival notwithstanding the continuing presence of xenoreactive antibodies and complement [19], would represent a preferable alternative. However, as reported by Dehoux [20], thus far, accommodation has only been achieved in allotransplantation and in rodent xenotransplantation models. At this stage, research towards achieving accommodation in the challenging pig-to-primate model is almost non-existent and a resumption of scientific efforts in this area is eagerly awaited. Third, tolerance, or achieving recipient non-responsiveness towards the graft in the absence of continued immunosuppression, would certainly represent an ideal scenario, avoiding systemic immunosuppression and drug-related side-effects that are inevitably associated with the long-term treatment of transplanted patients. Sachs et al. summarise the current status in this area where considerable progress has recently been achieved using mixed haematopoietic chimaerism or vascularised thymus transplantation [21]. In particular, failure to raise an anti-pig immune response has recently been demonstrated in primates treated with a non-myeloablative preparative regimen and receiving large doses of αGal−/− bone marrow cells that enabled pig progenitor cell engraftment for up to 28 days. Furthermore, co-transplantation of vascularised thymic tissue along with αGal−/− kidneys led to prolonged survival of functioning kidney xenografts, without the development of anti-donor T cell responses or induced antibody production, although third-party allo-responses were maintained. In this regard, it is also very encouraging that T cell tolerance across the pig-to-human xenogeneic barrier has already been achieved in humanised mice. Continuous research in this area has, in the last year, resulted in a major breakthrough that has enabled successful clinical application of the mixed chimaerism approach to renal allograft recipients. Together these results provide optimism for the successful induction of xenograft tolerance in the primate in the near future. 4.2. Strategies rendering the source pig more “compatible” with the primate recipient As far as approaches that render the source pig more “compatible” with the primate recipient, this can be achieved either by genetically engineering the pig or by organogenesis. For genetically engineering the pig, the key modifications that may be necessary to obtain a suitable source animal are described in this issue by d'Apice et al. [22]. These modifications can be broadly divided into five categories. First, major efforts should be undertaken to reduce the immunogenicity of pig cells. In this regard, as αGal residues are widely expressed by pig cells and represent the primary target of the natural and elicited anti-graft antibody response, removal of this epitope appeared to be the first logical step to implement. Accordingly, several groups have developed the necessary technology and obtained animals from which such residues are absent [23]. Second, and complementary to this approach, as complement activation triggered by the binding of natural or elicited antibodies is central to the graft damage and it is virtually impossible to eliminate
E. Cozzi / Transplant Immunology 21 (2009) 57–59
all non-αGal-incompatibilities capable of eliciting an immune response, more “compatible” pigs will require the expression of high levels of human complement cascade inhibitors [6]. Third, as thrombosis is a central event in the rejection process of a xenograft, antithrombotic factors (such as thrombomodulin, EPCR, TFPI and CD39, or a combination of these) will need to be expressed by specifically-designed donor pigs [4]. Fourth, transgenesis conferring protection against ischaemia–reperfusion injury (targeting molecules such as CD39, A20 or VEGF) will also be useful [24]. Finally, as cellmediated immunity may constitute an additional immune obstacle, the local expression of cell specific inhibitors, such as CTLA4Ig or HLAG, will probably represent a useful adjunct to counteract locally the anti-xenograft immune responses mediated by T cells, NK cells or macrophages. As an alternative to genetically engineered donor pigs, other investigators have proposed the route of organogenesis, using precursors derived from embryonic or foetal tissues. This field is rapidly progressing and an update on this very promising subject is here presented by Hammerman [25]. In this respect, it is remarkable that it is now possible to ‘grow’ new pancreatic tissue or kidneys in situ via xenotransplantation of organ primordia from animals (organogenesis of the endocrine pancreas or kidney). Interestingly, following transplantation of embryonic pig metanephroi into rats, the majority of endothelial cells are of host origin whereas mesangial cells predominantly originate from the donor [26]. Furthermore, pig pancreatic primordia engraft in diabetic rats or rhesus macaques without immunosuppression. As a consequence, glucose intolerance can be corrected in formerly diabetic rats and ameliorated in rhesus macaques. On the other hand, engraftment of pig renal primordia transplanted directly into rats has been shown to require immunosuppression. The reason for the requirement of immunosuppression when renal, but not pancreatic, primordia are xenografted in the rat has yet to be determined.
5. Conclusions It is unquestionable that the key biological events underlying the rejection of porcine cardiac, renal and islet xenografts transplanted into primates appear to have been clarified. This has resulted, in recent years, in the development of novel immunosuppressive strategies and new lines of engineered pigs that have enabled improved xenograft survival in the clinically-relevant pig-to-primate models (especially in islet xenotransplantation) [11,13]. More importantly, researchers have now developed the appropriate biomolecular tools to address rapidly the remaining barriers and render organs from source pigs more “compatible” with man [22]. Whilst efficacy has been the main focus of the research conducted in the last few years, and was also the subject of this volume of the Journal, other fundamental issues have to be kept in mind prior to proceeding with clinical xenotransplantation. These regard the physiology, the safety, the ethics and regulatory aspects of clinical xenotransplantation. Unless each of these aspects can be satisfactorily addressed, it is the author's opinion, and that of many in this field, that we will not be ready to proceed with clinic xenotransplantation trials.
59
Acknowledgements The author would like to thank CORIT (Consortium for Research in Organ Transplantation, Padua, Italy) and the EU FP6 Integrated Project “Xenome” (www.xenome.eu), contract no. LSHB-CT-2006-037377 for their support, and give special thanks to Dr Michela Seveso (CORIT) and Dr Mark Pullinger for their critical review of the manuscript. References [1] Yang YG, Sykes M. Xenotransplantation: current status and a perspective on the future. Nat Rev Immunol 2007;7:519. [2] Cowan PJ, d Apice AJ. The coagulation barrier in xenotransplantation: incompatibilities and strategies to overcome them. Curr Opin Organ Transpl 2008;13:178. [3] Lin CC, Cooper DKC, Dorling A. Coagulation dysregulation as a barrier to xenotransplantation in the primate. Transpl Immunol 2009;21:75–80. [4] Le Bas-Bernadet S, Blancho G. Current cellular immunological hurdles in pig-to-primate xenotransplantation. Transpl Immunol 2009;21:60–4. [5] Billiau AD, Li S, Waer M. Xenotransplantation: role of the natural immunity. Transpl Immunol 2009;21:70–4. [6] Cozzi E, White DJ. The generation of transgenic pigs as potential organ donors for humans. Nat Med 1995;1:964. [7] Baumann BC, Schneider MK, Lilienfeld BG, et al. Endothelial cells derived from pigs lacking Gal alpha(1,3)Gal: no reduction of human leukocyte adhesion and natural killer cell cytotoxicity. Transplantation 2005;79:1067. [8] Sullivan JA, Oettinger HF, Sachs DH, Edge AS. Analysis of polymorphism in porcine MHC class I genes: alterations in signals recognized by human cytotoxic lymphocytes. J Immunol 1997;159:2318. [9] Forte P, Lilienfeld BG, Baumann BC, Seebach JD. Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D. J Immunol 2005;175:5463. [10] Ide K, Wang H, Tahara H, et al. Role for CD47-SIRPalpha signaling in xenograft rejection by macrophages. Proc Natl Acad Sci U S A 2007;104:5062. [11] Ekser B, Rigotti P, Gridelli B, Cooper DKC. Xenotransplantation of solid organ in the pig-to-primate model. Transpl Immunol 2009;21:87–92. [12] Baldan N, Rigotti P, Calabrese F, et al. Ureteral stenosis in HDAF pig-to-primate renal xenotransplantation: a phenomenon related to immunological events? Am J Transplant 2004;4:475. [13] Hering B, Walawalkar N. Pig-to-nonhuman primate islet xenotransplantation. Transpl Immunol 2009;21:81–6. [14] Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med 2006;12:301. [15] Cardona K, Korbutt GS, Milas Z, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med 2006;12:304. [16] Gianello P, Dufrane D. Encapsulation of pig islets by alginate matrix to correct streptozotocin-induced diabetes in primates without immunosuppression. Joint meeting of the International Xenotransplantation Association (IXA), the International Pancreas and Islet Transplant Association (IPITA), and the Cell Transplant Society (CTS). Xenotransplantation. Minneapolis, MN, USA: Blackwell Munksgaard; 2007. p. 441. [17] D. Cooper, 2009. In Personal Communication. [18] Pierson R. Antibody-mediated xenograft injury: mechanisms and protective strategies. Transpl Immunol 2009;21:65–9. [19] Koch CA, Khalpey ZI, Platt JL. Accommodation: preventing injury in transplantation and disease. J Immunol 2004;172:5143. [20] Dehoux JP, Gianello P. Accommodation and antibodies. Transpl Immunol 2009;21:106–10. [21] Sachs D, Sykes M, Yamada K. Achieving tolerance in pig-to-primate xenotransplantation: reality or fantasy? Transpl Immunol 2009;21:101–5. [22] D Apice AJF, Cowan PJ. Xenotransplantation: the next generation of engineered animals. Transpl Immunol 2009;21:111–5. [23] Lai L, Kolber-Simonds D, Park KW, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002;295:1089. [24] D Apice AJ, Cowan PJ. Gene-modified pigs. Xenotransplantation 2008;15:87. [25] Hammerman M. Xenotransplantation of pancreatic and kidney primordia: where do we stand? Transpl Immunol 2009;21:93–100. [26] Takeda S, Rogers SA, Hammerman MR. Differential origin for endothelial and mesangial cells after transplantation of pig fetal renal primordia into rats. Transpl Immunol 2006;15:211.
Contents lists available at ScienceDirect
Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Editorial
On the road to clinical xenotransplantation Emanuele Cozzi ⁎ Direzione Sanitaria, Padua General Hospital, Padua, Italy Surgical Unit III, Padua General Hospital, Padua, Italy Department of Surgical and Gastroenterological Sciences “Pier Giuseppe Cevese”, University of Padua, Padua, Italy CORIT (Consortium for Research in Organ Transplantation) Padua, Italy
a r t i c l e
i n f o
a b s t r a c t
Keywords: Xenotransplantation Pig Primate review
In recent years the development of novel immunosuppressive strategies and new lines of engineered pigs have enabled improved xenograft survival in the clinically-relevant pig-to-primate models (especially in islet xenotransplantation). Furthermore, researchers have now developed appropriate biomolecular tools to address rapidly the remaining barriers and render organs from source pigs more “compatible” with man. Efficacy has been the main focus of the research conducted in the last few years. However, other fundamental issues, such as those regarding the physiology, the safety, the ethics and regulatory aspects of xenotransplantation, will need to be addressed satisfactorily prior to proceeding with clinical xenotransplantation trials. © 2009 Published by Elsevier B.V.
1. Introduction
On the other hand, it is becoming apparent that other molecular barriers may need to be the target of intervention in view of achieving long-term xenograft survival. In this respect, the natural immune response directed against pig tissues and cells may, for instance, play a greater role than originally thought. The generation of transgenic pigs for human complement regulators that effectively control primate complement activation proved to be efficient in preventing complement-mediated graft damage [6], ultimately improving the survival of porcine xenografts transplanted into primates. However, other potentially harmful components of the natural immune response directed to the xenograft exist and these may need to be counteracted adequately. In particular, recent data suggest that human anti-pig NK cells may not be appropriately controlled by the pig counterpart molecule expressed by porcine cells and this may lead to xenograft damage even when organs from αGal−/− animals are used [7]. This has been mostly associated with the incapacity of porcine MHC class I molecules to provide appropriate KIR-mediated inhibitory signals to human NK cells [8] or with the capacity of porcine ULBP-1 to activate NK cells efficiently via the NKG2D pathway [9]. Similarly, even human macrophages have been shown to induce damage to pig cells due to the incapacity of porcine CD47 to provide effective SIRPα-mediated inhibitory signals to human macrophages [10]. Taken together, published data unequivocally demonstrate the existence of a strong natural primate anti-pig immune response that is well documented both in vitro and in the context of bone marrow xenotransplantation in the primate. Nevertheless, the real impact of such findings on the long-term survival of porcine xenografts in the primates remains unclear. As a consequence, it is still unknown whether these observations will require specific engineering of source pigs.
Considerable progress has been achieved in recent years in the clinically-relevant pig-to-primate xenotransplantation models and this is comprehensively described in a series of excellent contributions in this volume of the Journal. In this light, this short editorial will exclusively discuss a limited number of selected issues with the aim of enabling the reader to better comprehend where we stand in this scientific field and which are the approaches currently being undertaken to push it further.
2. Dealing with the barriers to long-term xenograft survival At this stage, the key biological events that preclude the long-term survival of porcine cardiac, renal and islet xenografts transplanted into primates appear to have been identified. These primarily consist in the anti-graft immune response [1] and in the existence of several molecular incompatibilities between pigs and primates [2]. These incompatibilities may be responsible for the thrombotic events that are central to the rejection process and the coagulopathy consistently observed in xenografted primates [3], but may also ultimately affect the immune response to the xenograft itself [4,5]. Currently, work is actively underway to counteract the antixenograft immune response and overcome the previously-identified undesirable molecular incompatibilities, some of which also affect the immune response itself. ⁎ Department of Medical and Surgical Sciences, University of Padua, Clinica Chirurgica III, Via Giustiniani, 2, 35128 Padova, Italy. Tel./fax: +39 049 8218841. E-mail address: [email protected]. 0966-3274/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.trim.2009.05.002
58
E. Cozzi / Transplant Immunology 21 (2009) 57–59
Much insight has also been gained recently on the existing molecular incompatibilities between porcine and primate proteins of the coagulation cascade. These are ultimately partly responsible for the onset of acute humoral xenograft rejection, a process usually presenting as a microvascular thrombosis, often associated with consumptive coagulopathy in xenografted primates. The current status in this field is eloquently presented by Lin et al. [3]. Of particular interest is the recent demonstration that co-incubation of porcine endothelial cells with human platelets induces the latter cells to express tissue factor with a mechanism independent of the humoral immune response. This phenomenon would therefore also occur even in a completely tolerant recipient, possibly precluding long-term xenograft survival. This new set of data may, at first consideration, be perceived as an additional obstacle on the road to clinical xenotransplantation. However, in the same article, the authors also suggest several carefully-designed pharmacological strategies that, once administered to primate recipients of appropriately engineered source pigs, could inhibit the occurrence of coagulation disorders in xenografted primates. 3. Current best results in clinically-relevant pig-to-primate models The current status of porcine solid organ and islet xenotransplantation in the clinically-relevant primate models is thoroughly described in two separate articles. As far as solid organ xenotransplantation is concerned, Ekser et al. clearly show that, using genetically engineered source animals, graft survival has considerably improved over the years, especially with regard to cardiac xenotransplantation [11]. In contrast, only a marginal improvement has apparently been achieved in the renal xenotransplantation setting. In this regard, however, it is of interest that Baldan et al. could obtain 90 day survival of a primate recipient of a renal xenograft (with 3 out of 7 renal recipients surviving for 40 days or longer) using a relatively straightforward immunosuppressive regimen that would be regarded by some as clinically applicable [12]. On the other hand, it is noteworthy that when porcine lungs or livers were transplanted into primates, only up to 8 and 5 day survival were reported, respectively. This could possibly be due to the more complex physiological discrepancies or the additional immunological barriers existing between pig and primate for these two organs. As far as islet xenotransplantation is concerned, the comprehensive review assembled by Hering et al. clearly indicates that, in contrast with solid organ xenotransplantation, long-term survival of a life-supporting islet xenograft with normoglycaemia is reproducibly achievable in the primate [13]. In this regard, it is remarkable that 6 month survival or longer was reported in two different non-human primate species by at least 4 independent investigators [14–17]. More importantly, in one case, following optimisation of the encapsulation of adult porcine islets using highly purified alginate, islet xenografts implanted subcutaneously were able to normalise glycaemia in diabetic non-human primates for up to 6 months without any immunosuppression [16]. These results are of outstanding importance as, for the first time, they unequivocally demonstrate long-term survival of a life-supporting porcine xenograft in non-immunosuppressed primates. Clearly, avoidance of any type of immunosuppression, as in this case, considerably enhances the safety profile of xenotransplantation and would represent the ideal scenario to start any clinical xenotransplantation trial. 4. Strategies to further extend survival of porcine xenografts transplanted in the primate Whilst results have considerably improved over the last few years, conventional and novel approaches may further extend survival of porcine xenografts transplanted in the primate. These approaches can be broadly divided into strategies acting directly on the xenograft recipient, enabling better control of the anti-graft immune response; and strategies rendering the source pig more “compatible” with the
primate recipient, resulting in reduced susceptibility to damage and, ultimately, improved xenograft survival. 4.1. Strategies acting directly on the xenograft recipient As far as obtaining better control of the anti-graft immune response, at least three different avenues should be considered. First, new immunosuppressive approaches primarily based on the use of novel pharmaceutical compounds or biologics are being developed to overcome the different forms of xenograft rejection identified to date [18]. Whilst these approaches have been instrumental in obtaining current survival data, it should be pointed out that these are usually associated with a status of generalised (non-specific) immunosuppression that makes them far less attractive than the two options described hereafter. Second, the establishment of accommodation in the primate, namely long-term graft survival notwithstanding the continuing presence of xenoreactive antibodies and complement [19], would represent a preferable alternative. However, as reported by Dehoux [20], thus far, accommodation has only been achieved in allotransplantation and in rodent xenotransplantation models. At this stage, research towards achieving accommodation in the challenging pig-to-primate model is almost non-existent and a resumption of scientific efforts in this area is eagerly awaited. Third, tolerance, or achieving recipient non-responsiveness towards the graft in the absence of continued immunosuppression, would certainly represent an ideal scenario, avoiding systemic immunosuppression and drug-related side-effects that are inevitably associated with the long-term treatment of transplanted patients. Sachs et al. summarise the current status in this area where considerable progress has recently been achieved using mixed haematopoietic chimaerism or vascularised thymus transplantation [21]. In particular, failure to raise an anti-pig immune response has recently been demonstrated in primates treated with a non-myeloablative preparative regimen and receiving large doses of αGal−/− bone marrow cells that enabled pig progenitor cell engraftment for up to 28 days. Furthermore, co-transplantation of vascularised thymic tissue along with αGal−/− kidneys led to prolonged survival of functioning kidney xenografts, without the development of anti-donor T cell responses or induced antibody production, although third-party allo-responses were maintained. In this regard, it is also very encouraging that T cell tolerance across the pig-to-human xenogeneic barrier has already been achieved in humanised mice. Continuous research in this area has, in the last year, resulted in a major breakthrough that has enabled successful clinical application of the mixed chimaerism approach to renal allograft recipients. Together these results provide optimism for the successful induction of xenograft tolerance in the primate in the near future. 4.2. Strategies rendering the source pig more “compatible” with the primate recipient As far as approaches that render the source pig more “compatible” with the primate recipient, this can be achieved either by genetically engineering the pig or by organogenesis. For genetically engineering the pig, the key modifications that may be necessary to obtain a suitable source animal are described in this issue by d'Apice et al. [22]. These modifications can be broadly divided into five categories. First, major efforts should be undertaken to reduce the immunogenicity of pig cells. In this regard, as αGal residues are widely expressed by pig cells and represent the primary target of the natural and elicited anti-graft antibody response, removal of this epitope appeared to be the first logical step to implement. Accordingly, several groups have developed the necessary technology and obtained animals from which such residues are absent [23]. Second, and complementary to this approach, as complement activation triggered by the binding of natural or elicited antibodies is central to the graft damage and it is virtually impossible to eliminate
E. Cozzi / Transplant Immunology 21 (2009) 57–59
all non-αGal-incompatibilities capable of eliciting an immune response, more “compatible” pigs will require the expression of high levels of human complement cascade inhibitors [6]. Third, as thrombosis is a central event in the rejection process of a xenograft, antithrombotic factors (such as thrombomodulin, EPCR, TFPI and CD39, or a combination of these) will need to be expressed by specifically-designed donor pigs [4]. Fourth, transgenesis conferring protection against ischaemia–reperfusion injury (targeting molecules such as CD39, A20 or VEGF) will also be useful [24]. Finally, as cellmediated immunity may constitute an additional immune obstacle, the local expression of cell specific inhibitors, such as CTLA4Ig or HLAG, will probably represent a useful adjunct to counteract locally the anti-xenograft immune responses mediated by T cells, NK cells or macrophages. As an alternative to genetically engineered donor pigs, other investigators have proposed the route of organogenesis, using precursors derived from embryonic or foetal tissues. This field is rapidly progressing and an update on this very promising subject is here presented by Hammerman [25]. In this respect, it is remarkable that it is now possible to ‘grow’ new pancreatic tissue or kidneys in situ via xenotransplantation of organ primordia from animals (organogenesis of the endocrine pancreas or kidney). Interestingly, following transplantation of embryonic pig metanephroi into rats, the majority of endothelial cells are of host origin whereas mesangial cells predominantly originate from the donor [26]. Furthermore, pig pancreatic primordia engraft in diabetic rats or rhesus macaques without immunosuppression. As a consequence, glucose intolerance can be corrected in formerly diabetic rats and ameliorated in rhesus macaques. On the other hand, engraftment of pig renal primordia transplanted directly into rats has been shown to require immunosuppression. The reason for the requirement of immunosuppression when renal, but not pancreatic, primordia are xenografted in the rat has yet to be determined.
5. Conclusions It is unquestionable that the key biological events underlying the rejection of porcine cardiac, renal and islet xenografts transplanted into primates appear to have been clarified. This has resulted, in recent years, in the development of novel immunosuppressive strategies and new lines of engineered pigs that have enabled improved xenograft survival in the clinically-relevant pig-to-primate models (especially in islet xenotransplantation) [11,13]. More importantly, researchers have now developed the appropriate biomolecular tools to address rapidly the remaining barriers and render organs from source pigs more “compatible” with man [22]. Whilst efficacy has been the main focus of the research conducted in the last few years, and was also the subject of this volume of the Journal, other fundamental issues have to be kept in mind prior to proceeding with clinical xenotransplantation. These regard the physiology, the safety, the ethics and regulatory aspects of clinical xenotransplantation. Unless each of these aspects can be satisfactorily addressed, it is the author's opinion, and that of many in this field, that we will not be ready to proceed with clinic xenotransplantation trials.
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Acknowledgements The author would like to thank CORIT (Consortium for Research in Organ Transplantation, Padua, Italy) and the EU FP6 Integrated Project “Xenome” (www.xenome.eu), contract no. LSHB-CT-2006-037377 for their support, and give special thanks to Dr Michela Seveso (CORIT) and Dr Mark Pullinger for their critical review of the manuscript. References [1] Yang YG, Sykes M. Xenotransplantation: current status and a perspective on the future. Nat Rev Immunol 2007;7:519. [2] Cowan PJ, d Apice AJ. The coagulation barrier in xenotransplantation: incompatibilities and strategies to overcome them. Curr Opin Organ Transpl 2008;13:178. [3] Lin CC, Cooper DKC, Dorling A. Coagulation dysregulation as a barrier to xenotransplantation in the primate. Transpl Immunol 2009;21:75–80. [4] Le Bas-Bernadet S, Blancho G. Current cellular immunological hurdles in pig-to-primate xenotransplantation. Transpl Immunol 2009;21:60–4. [5] Billiau AD, Li S, Waer M. Xenotransplantation: role of the natural immunity. Transpl Immunol 2009;21:70–4. [6] Cozzi E, White DJ. The generation of transgenic pigs as potential organ donors for humans. Nat Med 1995;1:964. [7] Baumann BC, Schneider MK, Lilienfeld BG, et al. Endothelial cells derived from pigs lacking Gal alpha(1,3)Gal: no reduction of human leukocyte adhesion and natural killer cell cytotoxicity. Transplantation 2005;79:1067. [8] Sullivan JA, Oettinger HF, Sachs DH, Edge AS. Analysis of polymorphism in porcine MHC class I genes: alterations in signals recognized by human cytotoxic lymphocytes. J Immunol 1997;159:2318. [9] Forte P, Lilienfeld BG, Baumann BC, Seebach JD. Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D. J Immunol 2005;175:5463. [10] Ide K, Wang H, Tahara H, et al. Role for CD47-SIRPalpha signaling in xenograft rejection by macrophages. Proc Natl Acad Sci U S A 2007;104:5062. [11] Ekser B, Rigotti P, Gridelli B, Cooper DKC. Xenotransplantation of solid organ in the pig-to-primate model. Transpl Immunol 2009;21:87–92. [12] Baldan N, Rigotti P, Calabrese F, et al. Ureteral stenosis in HDAF pig-to-primate renal xenotransplantation: a phenomenon related to immunological events? Am J Transplant 2004;4:475. [13] Hering B, Walawalkar N. Pig-to-nonhuman primate islet xenotransplantation. Transpl Immunol 2009;21:81–6. [14] Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med 2006;12:301. [15] Cardona K, Korbutt GS, Milas Z, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med 2006;12:304. [16] Gianello P, Dufrane D. Encapsulation of pig islets by alginate matrix to correct streptozotocin-induced diabetes in primates without immunosuppression. Joint meeting of the International Xenotransplantation Association (IXA), the International Pancreas and Islet Transplant Association (IPITA), and the Cell Transplant Society (CTS). Xenotransplantation. Minneapolis, MN, USA: Blackwell Munksgaard; 2007. p. 441. [17] D. Cooper, 2009. In Personal Communication. [18] Pierson R. Antibody-mediated xenograft injury: mechanisms and protective strategies. Transpl Immunol 2009;21:65–9. [19] Koch CA, Khalpey ZI, Platt JL. Accommodation: preventing injury in transplantation and disease. J Immunol 2004;172:5143. [20] Dehoux JP, Gianello P. Accommodation and antibodies. Transpl Immunol 2009;21:106–10. [21] Sachs D, Sykes M, Yamada K. Achieving tolerance in pig-to-primate xenotransplantation: reality or fantasy? Transpl Immunol 2009;21:101–5. [22] D Apice AJF, Cowan PJ. Xenotransplantation: the next generation of engineered animals. Transpl Immunol 2009;21:111–5. [23] Lai L, Kolber-Simonds D, Park KW, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002;295:1089. [24] D Apice AJ, Cowan PJ. Gene-modified pigs. Xenotransplantation 2008;15:87. [25] Hammerman M. Xenotransplantation of pancreatic and kidney primordia: where do we stand? Transpl Immunol 2009;21:93–100. [26] Takeda S, Rogers SA, Hammerman MR. Differential origin for endothelial and mesangial cells after transplantation of pig fetal renal primordia into rats. Transpl Immunol 2006;15:211.