Xenotransplantation: Where are we in 2008?

Xenotransplantation holds promise to solve the ever increasing shortage of donor organs for allotransplantation. In the last 2 decades, major progress has been made in understanding the immunobiology of pig-into-(non)human primate transplantation and today we are on the threshold of the first clinical trials. Hyperacute rejection, which is mediated by pre-existing anti-αGal xenoreactive antibodies, can in non-human primates be overcome by complement- and/or antibody-modifying interventions. A major step forward was the development of genetically engineered pigs, either transgenic for human complement regulatory proteins or deficient in the α1,3-galactosyltranferase enzyme. However, several other immunologic and nonimmunologic hurdles remain. Acute vascular xenograft rejection is mediated by humoral and cellular mechanisms. Elicited xenoreactive antibodies play a key role. In addition to providing B cell help, xenoreactive T cells may directly contribute to xenograft rejection. Long-term survival of porcine kidney- and heart xenografts in non-human primates has been obtained but required severe T and B cell immunosuppression. Induction of xenotolerance, e.g. through mixed hematopoietic chimerism, may represent the preferred approach, but although proof of principle has been delivered in rodents, induction of pig-to-non-human primate chimerism remains problematic. Finally, it is now clear that innate immune cells, in particular macrophages and natural killer cells, can mediate xenograft destruction, the determinants of which are being elucidated. Chronic xenograft rejection is not well understood, but recent studies indicate that non-immunological problems, such as incompatibilities between human procoagulant and pig anticoagulant components may play an important role. Here, we give a comprehensive overview of the currently known obstacles to xenografting: immune and non-immune problems are discussed, as well as the possible strategies that are under development to overcome these hurdles. Xenotransplantation holds promise to solve the ever increasing shortage of donor organs for allotransplantation. In the last 2 decades, major progress has been made in understanding the immunobiology of pig-into-(non)human primate transplantation and today we are on the threshold of the first clinical trials. Hyperacute rejection, which is mediated by pre-existing anti-αGal xenoreactive antibodies, can in non-human primates be overcome by complement- and/or antibody-modifying interventions. A major step forward was the development of genetically engineered pigs, either transgenic for human complement regulatory proteins or deficient in the α1,3-galactosyltranferase enzyme. However, several other immunologic and nonimmunologic hurdles remain. Acute vascular xenograft rejection is mediated by humoral and cellular mechanisms. Elicited xenoreactive antibodies play a key role. In addition to providing B cell help, xenoreactive T cells may directly contribute to xenograft rejection. Long-term survival of porcine kidney- and heart xenografts in non-human primates has been obtained but required severe T and B cell immunosuppression. Induction of xenotolerance, e.g. through mixed hematopoietic chimerism, may represent the preferred approach, but although proof of principle has been delivered in rodents, induction of pig-to-non-human primate chimerism remains problematic. Finally, it is now clear that innate immune cells, in particular macrophages and natural killer cells, can mediate xenograft destruction, the determinants of which are being elucidated. Chronic xenograft rejection is not well understood, but recent studies indicate that non-immunological problems, such as incompatibilities between human procoagulant and pig anticoagulant components may play an important role. Here, we give a comprehensive overview of the currently known obstacles to xenografting: immune and non-immune problems are discussed, as well as the possible strategies that are under development to overcome these hurdles. The development of organ allotransplantation in the second half of the twentieth century can be considered a landmark in the history of medicine. Major advances in understanding transplantation immunology, development of immunosuppressives, and surgical techniques have made allografting the treatment of choice for end-stage organ failure such as renal insufficiency. As a result of its own success, however, allotransplantation faces, since several decades, an acute shortage in donated human organs. Currently, there are approximately 75 000 and 11 300 patients on the kidney transplant waiting list in the United States and Europe, respectively.1.Andreoni K.A. Brayman K.L. Guidinger M.K. et al.Kidney and pancreas transplantation in the United States, 1996–2005.Am J Transplant. 2007; 7: 1359-1375Crossref PubMed Scopus (165) Google Scholar,2.UNOS. United Network for Organ Sharing: Richmond, VA. Available at http://www.unos.orgGoogle Scholar,3.Eurotransplant International Foundation. Leiden, The Netherlands. Available at http://www.transplant.orgGoogle Scholar In Europe, only 3 540 kidney transplants were performed in 2005, with an average waiting time of 3.2 years. The overall mortality on the waiting list is approximately 6% per year.2.UNOS. United Network for Organ Sharing: Richmond, VA. Available at http://www.unos.orgGoogle Scholar,3.Eurotransplant International Foundation. Leiden, The Netherlands. Available at http://www.transplant.orgGoogle Scholar The use of xenogeneic cells or tissues is one of the alternative solutions that could alleviate the gap between organ supply and demand. Although nonhuman primates are phylogenetically closely related to humans, the pig is considered as the most suitable xenograft donor. Pig organs are physically and physiologically suited for transplantation in humans, they breed easily without posing major ethical problems, and they can be genetically manipulated to improve suitability of donor organs. Here, we review the current progress toward the development of clinical xenotransplantation, and focus on recent findings in the pig-to-nonhuman model, as this is considered most preclinically relevant. Early attempts at xenografting, which date from the 1960s, revealed that immunological barriers to xenografts far exceeded those to allografts. In the past two decades, intensive research into the immunobiology of xenografts has resulted in significant insights into the principles of xenograft immune rejection and possible strategies to circumvent it. A major step forward was the observation that hyperacute rejection (HAR), which is the first immune barrier to pig-to-(non)human primate organ transplantation and is mediated by natural anti-αGal xenoreactive antibodies, could be controlled by removing these antibodies4.Lin S.S. Weidner B.C. Byrne G.W. et al.The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.J Clin Invest. 1998; 101: 1745-1756Crossref PubMed Scopus (232) Google Scholar and by depleting or inhibiting complement.5.Kobayashi T. Taniguchi S. Neethling F.A. et al.Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy.Transplantation. 1997; 64: 1255-1261Crossref PubMed Scopus (147) Google Scholar This has led to the development of pigs expressing transgenes for human complement regulatory proteins6.Schuurman H.J. Pino-Chavez G. Phillips M.J. et al.Incidence of hyperacute rejection in pig-to-primate transplantation using organs from hDAF-transgenic donors.Transplantation. 2002; 73: 1146-1151Crossref PubMed Scopus (93) Google Scholar,7.Diamond L.E. Quinn C.M. Martin M.J. et al.A human CD46 transgenic pig model system for the study of discordant xenotransplantation.Transplantation. 2001; 71: 132-142Crossref PubMed Scopus (162) Google Scholar,8.Niemann H. Verhoeyen E. Wonigeit K. et al.Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.Transplantation. 2001; 72: 1898-1906Crossref PubMed Scopus (53) Google Scholar and α1,3-galactosyltransferase(αGalT)-deficient pigs that do not express the αGal xenoepitope.9.Lai L. Kolber-Simonds D. Park K.W. et al.Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.Science. 2002; 295: 1089-1092Crossref PubMed Scopus (1072) Google Scholar However, successful control of HAR has equally revealed the importance of other or previously unknown immunological and nonimmunological obstacles to xenograft survival. Rejection of xenografts entails participation of the innate immune system with natural antibody-producing B cells, natural killer (NK) cells, macrophages, and complement, as well as adaptive immune responses from T and B cells (Figure 1). In addition, it is becoming increasingly clear that physiological incompatibilities, such as incompatibilities between human and pig coagulation pathways, lead to xenograft injury and contribute significantly to rejection. The established nomenclature of the different types of xenograft rejection includes HAR, AVR (acute vascular rejection) and its major component acute humoral xenograft rejection (AHXR), and chronic rejection, and refers to the time course and predominant mechanism of rejection. HAR develops within 24 h. It results from the binding of preexisting xenoreactive antibodies to endothelial cells and activation of the complement system, resulting in vascular deposition of immunoglobulins and complement, endothelial swelling, and microvascular thrombosis.10.Schuurman H.J. Cheng J. Lam T. Pathology of xenograft rejection: a commentary.Xenotransplantation. 2003; 10: 293-299Crossref PubMed Scopus (63) Google Scholar Hyperacute rejection affects xenografts transplanted between discordant species, such as pig and (non)human primates, and represents the first barrier to porcine xenograft survival in (non)human primates. The putative xenoantibodies are predominantly directed against Galα1,3Galβ1,4GlcNAc (αGal) carbohydrate residues (Figure 1). These are abundantly expressed on endothelial cells of non-primate mammals and New World monkeys, but are absent in humans, apes, and Old World monkeys as these species lack the αGalT gene to synthesize αGal. As a result, they produce anti-αGal antibodies early in life upon encounter of αGal-expressing microorganisms in the gastrointestinal tract, and it is estimated that these ‘natural anti-αGal antibodies’ constitute 1% of immunoglobulins in the circulation. Strategies to prevent HAR target the anti-αGal natural antibody response and/or the participation of complement (Table 1). Removal of anti-αGal antibodies and depletion or inhibition of complement in nonhuman primate recipients were found to protect pig xenografts from HAR but not from AHXR, which is mediated by returning anti-αGal or newly elicited anti-non-αGal xenoreactive antibodies.23.Yang Y.G. Sykes M. Xenotransplantation: current status and a perspective on the future.Nat Rev Immunol. 2007; 7: 519-531Crossref PubMed Scopus (228) Google Scholar Transgenic pigs were developed expressing human complement regulatory proteins, for example, hDAF (human decay-accelerating factor, CD55),6.Schuurman H.J. Pino-Chavez G. Phillips M.J. et al.Incidence of hyperacute rejection in pig-to-primate transplantation using organs from hDAF-transgenic donors.Transplantation. 2002; 73: 1146-1151Crossref PubMed Scopus (93) Google Scholar MCP (membrane cofactor protein, CD46),7.Diamond L.E. Quinn C.M. Martin M.J. et al.A human CD46 transgenic pig model system for the study of discordant xenotransplantation.Transplantation. 2001; 71: 132-142Crossref PubMed Scopus (162) Google Scholar or protectin (CD59),8.Niemann H. Verhoeyen E. Wonigeit K. et al.Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.Transplantation. 2001; 72: 1898-1906Crossref PubMed Scopus (53) Google Scholar but although grafts from these donor pigs were protected from HAR, they are not completely protected from AHXR.24.Dwyer K.M. Cowan P.J. d'Apice A.J. Xenotransplantation: past achievements and future promise.Heart Lung Circ. 2002; 11: 32-41Abstract Full Text PDF PubMed Scopus (12) Google Scholar Using a combination of CD46 transgene pig heart grafts, the α-galactosyl-polyethylene glycol conjugate TPC, and conventional immunosuppression in baboons, McGregor et al.25.McGregor C.G. Teotia S.S. Byrne G.W. et al.Cardiac xenotransplantation: progress toward the clinic.Transplantation. 2004; 78: 1569-1575Crossref PubMed Scopus (80) Google Scholar,26.McGregor C.G. Davies W.R. Oi K. et al.Cardiac xenotransplantation: recent preclinical progress with 3-month median survival.J Thorac Cardiovasc Surg. 2005; 130: 844-851Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar have obtained a median heart graft survival time of 75–90 days. More recently, αGalT−/− pigs were developed9.Lai L. Kolber-Simonds D. Park K.W. et al.Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.Science. 2002; 295: 1089-1092Crossref PubMed Scopus (1072) Google Scholar and prolonged survival of αGalT−/− porcine kidney11.Yamada K. Yazawa K. Shimizu A. et al.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.Nat Med. 2005; 11: 32-34Crossref PubMed Scopus (480) Google Scholar and heart12.Kuwaki K. Tseng Y.L. Dor F.J. et al.Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience.Nat Med. 2005; 11: 29-31Crossref PubMed Scopus (526) Google Scholar grafts has equally been obtained. Yamada et al.11.Yamada K. Yazawa K. Shimizu A. et al.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.Nat Med. 2005; 11: 32-34Crossref PubMed Scopus (480) Google Scholar reported xenokidney graft survival of up to 80 days, whereas αGal-specific and non-αGal-specific xenoantibody responses were absent and kidney grafts did not show histological signs of rejection. This study involved the induction of T-cell xenotolerance through vascularized xenothymus transplantation and intense immunosuppression with recipient thymectomy, splenectomy, T-cell depletion, CD154 blockade, and treatment with MMF (mycophenolate mofetil).11.Yamada K. Yazawa K. Shimizu A. et al.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.Nat Med. 2005; 11: 32-34Crossref PubMed Scopus (480) Google Scholar Chen et al.13.Chen G. Qian H. Starzl T. et al.Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys.Nat Med. 2005; 11: 1295-1298Crossref PubMed Scopus (267) Google Scholar subsequently showed that in this combination, a preclinical regimen of immunosuppression including ATG, tacrolimus, and MMF equally could prevent HAR of αGalT−/− kidneys, but xenografts survived for 8–16 days only, and invariably showed signs of AHXR.Table 1Different approaches to overcome xenograft rejectionInterventionEffectRemoval of anti-αGal antibodiesPrevention of HAR4.Lin S.S. Weidner B.C. Byrne G.W. et al.The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.J Clin Invest. 1998; 101: 1745-1756Crossref PubMed Scopus (232) Google ScholarInhibition of complement systemPrevention of HAR5.Kobayashi T. Taniguchi S. Neethling F.A. et al.Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy.Transplantation. 1997; 64: 1255-1261Crossref PubMed Scopus (147) Google ScholarCD55, CD46, and CD59 transgenic pigsPrevention of HAR6.Schuurman H.J. Pino-Chavez G. Phillips M.J. et al.Incidence of hyperacute rejection in pig-to-primate transplantation using organs from hDAF-transgenic donors.Transplantation. 2002; 73: 1146-1151Crossref PubMed Scopus (93) Google Scholar,7.Diamond L.E. Quinn C.M. Martin M.J. et al.A human CD46 transgenic pig model system for the study of discordant xenotransplantation.Transplantation. 2001; 71: 132-142Crossref PubMed Scopus (162) Google Scholar,8.Niemann H. Verhoeyen E. Wonigeit K. et al.Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.Transplantation. 2001; 72: 1898-1906Crossref PubMed Scopus (53) Google ScholarαGalT−/− pigsPrevention of HAR 11.Yamada K. Yazawa K. Shimizu A. et al.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.Nat Med. 2005; 11: 32-34Crossref PubMed Scopus (480) Google Scholar,12.Kuwaki K. Tseng Y.L. Dor F.J. et al.Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience.Nat Med. 2005; 11: 29-31Crossref PubMed Scopus (526) Google ScholarPrevention of AHXR mediated by newly elicited αGal antibodies13.Chen G. Qian H. Starzl T. et al.Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys.Nat Med. 2005; 11: 1295-1298Crossref PubMed Scopus (267) Google ScholarAttenuated T-cell responsesaLin YJ, Hara H, Tai H-C et al. Xenotransplantation 2007; 14: 460.Attenuated macrophage responses14.Jin R. Greenwald A. Peterson M.D. et al.Human monocytes recognize porcine endothelium via the interaction of galectin 3 and alpha-GAL.J Immunol. 2006; 177: 1289-1295Crossref PubMed Scopus (56) Google ScholarHLA-E transgenic pigsPrevention of NK-mediated xenorejection15.Lilienfeld B.G. Crew M.D. Forte P. et al.Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity.Xenotransplantation. 2007; 14: 126-134Crossref PubMed Scopus (47) Google ScholarULBP1 transgenic pigsPrevention of NK-mediated xenorejection16.Lilienfeld B.G. Garcia-Borges C. Crew M.D. et al.Porcine UL16-binding protein 1 expressed on the surface of endothelial cells triggers human NK cytotoxicity through NKG2D.J Immunol. 2006; 177: 2146-2152Crossref PubMed Scopus (42) Google ScholarhuCD47 transgenic pigsAttenuation of macrophage responses17.Wang H. VerHalen J. Madariaga M.L. et al.Attenuation of phagocytosis of xenogeneic cells by manipulating CD47.Blood. 2007; 109: 836-842Crossref PubMed Scopus (82) Google ScholarhuCD39 transgenic pigsPrevention of thrombosis18.Dwyer K.M. Robson S.C. Nandurkar H.H. et al.Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation.J Clin Invest. 2004; 113: 1440-1446Crossref PubMed Scopus (146) Google ScholarT/B-cell suppressionAttenuation of T- and/or B-cell responsesMixed chimerismT-cell tolerance19.Sykes M. Shimizu I. Kawahara T. Mixed hematopoietic chimerism for the simultaneous induction of T and B cell tolerance.Transplantation. 2005; 79: S28-S29Crossref PubMed Scopus (29) Google ScholarB-cell tolerance19.Sykes M. Shimizu I. Kawahara T. Mixed hematopoietic chimerism for the simultaneous induction of T and B cell tolerance.Transplantation. 2005; 79: S28-S29Crossref PubMed Scopus (29) Google ScholarNK hyporesponsiveness20.Kawahara T. Rodriguez-Barbosa J.I. Zhao Y. et al.Global unresponsiveness as a mechanism of natural killer cell tolerance in mixed xenogeneic chimeras.Am J Transplant. 2007; 7: 2090-2097Crossref PubMed Scopus (20) Google ScholarXenothymus transplantationT-cell tolerance21.Yamada K. Shimizu A. Ierino F.L. et al.Thymic transplantation in miniature swine. I. Development and function of the ‘thymokidney’.Transplantation. 1999; 68: 1684-1692Crossref PubMed Scopus (68) Google Scholar,22.Yamada K. Shimizu A. Utsugi R. et al.Thymic transplantation in miniature swine. II. Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients.J Immunol. 2000; 164: 3079-3086Crossref PubMed Scopus (95) Google ScholarAHXR, acute humoral xenograft rejection; αGalT, α1,3-galactosyltransferase; HAR, hyperacute rejection; HLA, human leukocyte antigen; NK, natural killer.a Lin YJ, Hara H, Tai H-C et al. Xenotransplantation 2007; 14: 460. Open table in a new tab AHXR, acute humoral xenograft rejection; αGalT, α1,3-galactosyltransferase; HAR, hyperacute rejection; HLA, human leukocyte antigen; NK, natural killer. In a similar model studying αGalT−/− xenoheart grafts in baboons, Kuwaki et al.12.Kuwaki K. Tseng Y.L. Dor F.J. et al.Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience.Nat Med. 2005; 11: 29-31Crossref PubMed Scopus (526) Google Scholar reported survival of xenografts of 2–6 months under a regimen including T-cell depletion, thymic irradiation, complement depletion, anti-CD154 mAb, and MMF. HAR was prevented but grafts ultimately failed with signs of focal AHXR and prominent intragraft presence of thrombotic microangiopathy.12.Kuwaki K. Tseng Y.L. Dor F.J. et al.Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience.Nat Med. 2005; 11: 29-31Crossref PubMed Scopus (526) Google Scholar These observations suggest that residual xenoimmune reactivity or a dysregulated coagulation pathway as a result of incompatibilities between xenogeneic coagulation systems, may contribute to xenograft failure on the longer term. Whereas prevention of HAR of αGalT−/− pig organs was a major step forward, these key studies also indicate that application in humans would require the development of clinically applicable regimens to prevent AHXR, and additional adjustments to prevent other types of xenograft injury. Porcine xenografts that are protected from HAR by anti-αGal natural antibodies are subject to a second phase of rejection that typically occurs within days, referred to as AVR (Figure 1). Elicited xenoreactive antibodies, directed at non-αGal xenoantigens (and αGal antigens when αGalT+/+ organs are used) play a major role, in concert with activated complement and coagulation systems (AHXR).27.Cascalho M. Platt J.L. The immunological barrier to xenotransplantation.Immunity. 2001; 14: 437-446Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar The pathological signature of AVR resembles that of HAR, but usually includes infiltration by innate immune cells such as NK cells, macrophages, and neutrophils,10.Schuurman H.J. Cheng J. Lam T. Pathology of xenograft rejection: a commentary.Xenotransplantation. 2003; 10: 293-299Crossref PubMed Scopus (63) Google Scholar and their role is discussed below. Although xenoreactive T cells are well known to contribute to solid organ xenograft rejection by participating in the T-cell-dependent elicited B-cell antibody response to αGal and non-αGal xenoantigens, the direct contribution of T cells to solid organ xenograft rejection is not well understood. Attempts to overcome HAR and AHXR in nonhuman primate studies so far have included profound T-cell suppression and/or T-cell tolerance induction. Under these regimens, not only T-cell help for B-cell responses is impaired, but also infiltration of T cells in rejected grafts is not seen (Table 1).11.Yamada K. Yazawa K. Shimizu A. et al.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.Nat Med. 2005; 11: 32-34Crossref PubMed Scopus (480) Google Scholar,12.Kuwaki K. Tseng Y.L. Dor F.J. et al.Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience.Nat Med. 2005; 11: 29-31Crossref PubMed Scopus (526) Google Scholar,13.Chen G. Qian H. Starzl T. et al.Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys.Nat Med. 2005; 11: 1295-1298Crossref PubMed Scopus (267) Google Scholar Davila et al.28.Davila E. Byrne G.W. LaBreche P.T. et al.T-cell responses during pig-to-primate xenotransplantation.Xenotransplantation. 2006; 13: 31-40Crossref PubMed Scopus (52) Google Scholar recently showed, however, that pig hearts, protected from HAR in B-cell-depleted baboons were subject to direct injury by porcine aortic endothelial cell-specific CD4 and CD8 T cells. Porcine islets—in contrast to solid organ xenografts—are rejected through predominantly cellular mechanisms involving T cells and macrophages; αGal is poorly expressed on pig islet endocrine cells, and pig endothelial cell survival in culture is very low, rendering islet xenografts poorly susceptible to HAR and AHXR. Rather, rejected pig islets are characterized by marked infiltration with CD4 and CD8 T cells.29.Cardona K. Korbutt G.S. Milas Z. et al.Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways.Nat Med. 2006; 12: 304-306Crossref PubMed Scopus (361) Google Scholar,30.Hering B.J. Wijkstrom M. Graham M.L. et al.Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates.Nat Med. 2006; 12: 301-303Crossref PubMed Scopus (406) Google Scholar Ohdan et al.31.Ohdan H. Swenson K.G. Kruger Gray H.S. et al.Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice.J Immunol. 2000; 165: 5518-5529Crossref PubMed Scopus (101) Google Scholar demonstrated that B1b-like B cells are responsible for natural antibody production against the well-characterized xenoantigen αGal. The nature of non-αGal antigens, and the characteristics of non-αGal xenoreactive T- and B-cell responses are less well understood and are under study. Studies on sera from baboons that rejected xenografts indicate that anti-non-αGal xenoreactive antibodies comprise a limited repertoire of antigen specificity. Chen et al.13.Chen G. Qian H. Starzl T. et al.Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys.Nat Med. 2005; 11: 1295-1298Crossref PubMed Scopus (267) Google Scholar demonstrated a predominant antibody response against a 47-kDa protein, and Byrne et al. found that antibody responses were mainly directed against extracellular stress response and inflammation-related proteins, as well as a number of intracellular antigens (Byrne G et al. Xenotransplantation 2007; 14: 432). Li et al.32.Li S. Yan Y. Lin Y. et al.Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells.Blood. 2007; 110: 3926-3935Crossref PubMed Scopus (30) Google Scholar recently demonstrated that the T-cell-independent xenoantibody response in a concordant xenogeneic combination is exclusively mediated by marginal zone B cells. This B-cell population is well known for producing antibody responses against blood-borne T-independent type II antigens such as capsular polysaccharide antigens, and xenoantigens involved in this model are thought to have a related carbohydrate structure. However, the data also indicated that other types of xenoantigens may involve other B-cell populations, in particular in T-cell-sufficient recipients where T-cell-dependent help in antibody responses becomes important.32.Li S. Yan Y. Lin Y. et al.Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells.Blood. 2007; 110: 3926-3935Crossref PubMed Scopus (30) Google Scholar In vitro studies have shown that activation of human T cells by porcine endothelial cells is more vigorous than that elicited by allogeneic cells.33.Murray A.G. Khodadoust M.M. Pober J.S. et al.Porcine aortic endothelial cells activate human T cells: direct presentation of MHC antigens and costimulation by ligands for human CD2 and CD28.Immunity. 1994; 1: 57-63Abstract Full Text PDF PubMed Scopus (241) Google Scholar,34.Yamada K. Sachs D.H. DerSimonian H. Human anti-porcine xenogeneic T cell response. Evidence for allelic specificity of mixed leukocyte reaction and for both direct and indirect pathways of recognition.J Immunol. 1995; 155: 5249-5256PubMed Google Scholar A very recent finding is that the human T-cell responses toward αGalT−/− cells may be less pronounced than toward wild-type cells (Lin YJ et al. Xenotransplantation 2007; 14: 460), suggesting that αGalT−/− pig organs may provide additional advantages with regard to cellular xenoimmune response. Interestingly, Porter et al.35.Porter C.M. Bloom E.T. Human CD4+CD25+ regulatory T cells suppress anti-porcine xenogeneic responses.Am J Transplant. 2005; 5: 2052-2057Crossref PubMed Scopus (32) Google Scholar recently showed that human CD4+CD25+ regulatory T cells can suppress in vitro CD4+ T-cell-mediated antiporcine xenogeneic responses. From the three key nonhuman primate studies, it is clear that AVR can be prevented on the condition that severe T- and B-cell depletion and/or suppression is provided.11.Yamada K. Yazawa K. Shimizu A. et al.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.Nat Med. 2005; 11: 32-34Crossref PubMed Scopus (480) Google Scholar,12.Kuwaki K. Tseng Y.L. Dor F.J. et al.Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience.Nat Med. 2005; 11: 29-31Crossr