Gene-knockout Pigs Research Articles

Using α1,3-galactosyltransferase gene-knockout pig organs in nonhuman primates

Purpose of review The aim of this article is to review the experience with α1,3-galactosyltransferase gene-knockout pig organ transplantation into nonhuman primates, and related in-vitro studies. Recent findings Since the first transplants of α1,3-galactosyltransferase gene-knockout pig organs into nonhuman primates were reported in 2005, there has been relatively little further in-vivo experience. This experience has demonstrated, however, the importance of pig non-Galα1,3Gal (Gal) antigens as targets for primate antipig antibodies. Several in-vitro studies have confirmed that, although the incidence and levels of anti-non-Gal antibodies in nonhuman primates and humans are significantly less when compared with total anti-pig antibodies (i.e., anti-Gal + anti-non-Gal), they can result in complement-mediated lysis of α1,3-galactosyltransferase gene-knockout pig cells. The in-vivo experience has also confirmed the importance of an immunosuppressive regimen that prevents an elicited anti-non-Gal antibody response, if acute humoral xenograft rejection is to be prevented. The antigen targets for anti-non-Gal antibodies remain uncertain. Evidence is accumulating that differences in the coagulation–anticoagulation systems between pig and primate result in coagulation dysregulation leading to the development of thrombotic microangiopathy and graft failure. Summary Although they have provided an advance over wild-type pigs as a source of organs, further genetic modification of α1,3-galactosyltransferase gene-knockout pigs is required to overcome the immune barriers of pig-to-nonhuman primate xenotransplantation.

The impact of the α1,3-galactosyltransferase gene knockout pig on xenotransplantation

Purpose of review The generation of knockout pigs was a major achievement for the field of xenotransplantation. This review will look at key findings reported within the past 12 months relevant to the role of carbohydrates in the α1,3-galactosyltransferase knockout pig lines. Recent findings The first reports of pig-to-primate solid organ transplantation show that the major barrier to clinical xenotransplantation, hyperacute rejection, has been overcome. These experiments clearly identify the next problem to be addressed by the field, the phenomenon of acute vascular rejection. The role that carbohydrates may still play in the xenograft response is highlighted by the discovery of a second glycosyltransferase that synthesizes Galα(1,3)Gal and the findings that cell surface receptors of natural killer cells recognize Galα(1,3)Gal and N-acetyllactosamine, the carbohydrate epitope exposed in GT knockout animals. Summary With reference to the recent literature, we discuss the issues that we believe need to be addressed before the field may progress towards the clinical reality of using pigs for xenotransplantation.

Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies

Pig islets have been proposed as an alternative to human islets for clinical use, but their use is limited by rejection. The availability of genetically modified pigs devoid of alpha1,3-galactosyltransferase might provide islets more suitable for xenotransplantation. To limit the costs involved in the logistics and health care of pigs for clinical xenotransplantation, we have studied whether younger, rather than older, pigs that are typically preferred can be used as islet donors. We utilized pancreases from Yorkshire and White Landrace wild-type pigs and alpha1,3-galactosyltransferase gene-knockout pigs of three main different age and size groups: (i) <6 months, (ii) 6 to 12 months, and (iii) >2 yr of age, inclusive of retired breeders. We compared isolation yield and in vitro and in vivo function of islet cells obtained from these groups. Islets from adult pigs (>2 yr) offered not only higher islet yields, but retained the ability to preserve intact morphology during the isolation process and culture, in association with high functional properties after transplantation. Following isolation, islet cells from young (<6 m) and young-adult (6 to 12 m) pigs dissociated into small aggregates and single cells, and exhibited inferior functional properties than adult islets both in vitro and in vivo. These data support the conclusion that, in view of the large number of islets needed to maintain normoglycemia after xenotransplantation, organ-source pigs need to reach adult age (>2 yr) before being considered optimal islet donors, in spite of the higher costs involved.

Progress in xenotransplantation following the introduction of gene-knockout technology

The production of alpha1,3-galactosyltransferase gene-knockout (GT-KO) pigs has overcome the barrier of preformed anti-Galalpha1,3Gal (Gal) antibodies that has inhibited progress in pig-to-primate organ xenotransplantation for many years. Survival of GT-KO pig organs in nonhuman primates is currently limited by the development of a thrombotic microangiopathy that results in increasing ischemic injury of the transplanted organ over weeks or months. Potential causative factors include vascular endothelial activation from preformed anti-nonGal antibodies or cells of the innate immune system that recognize nonGal pig antigens directly, and coagulation dysregulation associated with molecular incompatibilities between pig and primate. Carefully isolated pancreatic islets from wild-type (genetically unmodified) adult pigs express minimal Gal epitopes, allowing survival sometimes for weeks or months after transplantation into nonhuman primates receiving immunosuppression directed only at T-cell function. However, there is a considerable immediate loss of islets, probably related to activation of coagulation and complement cascades. Further genetic manipulation of organ-source pigs is therefore required to overcome these problems. GT-KO pigs expressing a human complement-regulatory protein, e.g. decay-accelerating factor, and/or an 'anti-coagulant' gene, e.g. human tissue factor pathway inhibitor, might prevent the change in vascular endothelium from an anti-coagulant to a procoagulant phenotype, and protect the islets from early loss.

Elicited Antibodies in Baboons Exposed to Tissues from ??1,3-Galactosyltransferase Gene-Knockout Pigs

This study investigates anti-nonGal antibodies (Abs) in baboons after alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pig heart transplantation (Tx). Four baboons underwent pig heart Tx under chronic immunosuppression, which was discontinued after graftectomy. During follow-up, one baboon also received a pig splenocyte infusion. Hearts and splenocytes were from GalT-KO pigs (n = 3) or pigs with low Gal expression (Gal-low, n = 2), all of swine leukocyte antigen (SLA) dd haplotype. Several weeks after graftectomy, sera were tested by flow cytometry and cytotoxicity assay on porcine peripheral blood mononuclear cells (PBMC) for elicited anti-nonGal Abs. Sera were adsorbed on a Gal immunoaffinity matrix, and tested for SLA haplotype specificity using PBMC from SLA aa, cc, and dd haplotypes. Before heart Tx, no baboon had anti-nonGal Abs demonstrable by binding or cytotoxicity to GalT-KO PBMC. All four baboons developed anti-nonGal Abs after Tx, demonstrable by flow cytometry, and three sera from baboons showed cytotoxicity to GalT-KO PBMC of SLA(dd) haplotype. After adsorption of anti-Gal Abs, the elicited anti-nonGal Abs showed similar binding to PBMCs from pigs of all three haplotypes (SLA(dd), SLA(aa), SLA(cc)). Anti-nonGal Abs developed after GalT-KO pig heart Tx into baboons. The most potent of these antibodies appeared to detect antigens shared by the three pig haplotypes tested. It remains unclear whether these antibodies are directed towards shared SLA determinants or other pig antigens, and whether antibodies with specificity for allelic SLA determinants are also present, but at lower titer.

Glycobiology relating to xenotransplantation

Purpose of reviewCarbohydrates, beginning with Galα1,3Gal (Gal) antigens, have played an important part in xenotransplantation research. Now that Gal-related problems have been resolved by the breeding of α1,3-galactosyltransferase gene-knockout pigs, additional issues are becoming clear and demandi

Synergistic Effects on the Inhibition of Human CD8 + Cytotoxic T Lymphocytes-Mediated Killing Against Xenograft Cells by Coexpression of Membrane-Bound Human FasL and Decoy Fas Antigen

The principal barrier to the use of pigs as donors to humans is hyperacute rejection mediated by the interaction of α-gal abundantly expressed on pig cells and the natural anti-Gal antibody, abundantly produced in humans. This antibody-mediated hyperacute rejection may be overcome by an α1, 3 galactosyltransferase gene-knockout pig. However, xenograft cells could be rejected by T cells, especially CD8 + cytotoxic T lymphocytes (CTL)-mediated response, because these elements show great cytotoxicity against xenografted cells. We previously demonstrated that the Fas/FasL pathway is a major contributor to CD8 + CTL function. Furthermore, we sought to prevent this cytotoxicity by overexpression of membrane-bound FasL carrying the deletion at the metalloproteinase cleavage site or by decoy Fas antigen that does not contain the death domain in its cytoplasmic region. To investigate the effects of coexpression of these molecules, we cotransfected both genes into swine endothelial cells (SEC). The double-overexpression effectively prevented CD8 + CTL-mediated killing. Although cotransfectants and single transfectants of either membrane-bound FasL or decoy Fas gene showed similar inhibition of cytotoxicity, the expression levels of decoy Fas in SEC cotransfectants were much lower than those of decoy Fas single transfectants. These data suggest that beneficial effects for prevention of CTL-mediated xenocytotoxicity may be produced by the double expression of these molecules. The overexpression of both molecules on xenografted cells may decrease the innate cellular response to xenografts creating a window of opportunity to facilitate xenograft survival.

α1,3-Galactosyltransferase Gene-Knockout Pig Heart Transplantation in Baboons with Survival Approaching 6 Months

The recent generation of alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pigs has allowed investigation of the survival of GalT-KO pig organs in nonhuman primates. Heterotopic heart transplantation from GalT-KO pigs was carried out in baboons (n=8) using a human antihuman CD154 monoclonal antibody-based immunosuppressive regimen. In six of the eight cases, graft survival extended to between approximately 2 and 6 months. All grafts developed thrombotic microangiopathy (TM). In particular, the clinical course of one baboon in which the graft functioned for 179 days is summarized. This baboon received aspirin (40 mg on alternate days) from day 4 in addition to heparin, which may have been a factor in the delay of onset and progression of TM and in prolonged graft survival. Maintenance therapy with anti-CD154 mAb, mycophenolate mofetil, and methylprednisolone was associated with persistently low numbers of CD3CD4 and CD3CD8 cells. Despite persisting depletion of these cells, no infectious complications occurred. It remains to be established whether TM is related to a very low level of natural preformed or T-cell-induced antibody deposition on the graft, inducing endothelial activation and injury, or to molecular incompatibilities in the coagulation mechanisms between pig and baboon, or to both. However, function of a pig organ in a baboon for a period approaching six months, which has not been reported previously, lends encouragement that the barriers to xenotransplantation will eventually be overcome.

Preformed Antibodies to α1,3-Galactosyltransferase Gene-Knockout (GT-KO) Pig Cells in Humans, Baboons, and Monkeys: Implications for Xenotransplantation

Objective The aim of our study was to determine the prevalence and cytotoxicity of primate antibodies directed to antigens other than Galα1,3Gal (Gal), termed nonGal antigens. Methods Sera from human, baboon, and cynomolgus monkeys were tested by flow cytometry for IgM and IgG binding to both wild-type (WT) and GT-KO pig peripheral mononuclear cells (PBMC). Also, complement-dependent cytotoxicity assays were performed. Results All species demonstrated significantly higher antibody binding and cytotoxicity to WT cells compared to GT-KO cells ( P < .01). Cynomolgus monkeys had significantly higher IgM binding to WT and GT-KO cells than did baboons or humans ( P < .01). Furthermore, approximately 50% of both human and baboon sera proved to be lytic to GT-KO cells, compared to 76% of monkey sera ( P < .01). Conclusions We confirm the advantage of using GT-KO pig grafts over WT pig grafts. However, our results suggest that, compared to the cynomolgus monkey, the baboon may be a more suitable model to study antibody-mediated rejection of GT-KO pig grafts.

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The low rate of homologous recombination in somatic cells is considered to be an urgent issue. Therefore, we molecularly cloned three genes that relate to efficient homologous recombination.Polymerase chain reaction (PCR) was performed to isolate candidate cDNA fragments from a pig spleen cDNA library with the corresponding primer sets deduced from multiple alignment analysis of other mammalian genes. A 5′- and 3′-RACE PCR experiment was performed to determine the complete cDNA sequences.The complete cDNA sequences of the pig RAD51, RAD52, and RAD54 genes, which are closely related to homologous recombination events, were identified using molecular cloning technique. The cDNA sequences of three genes were successfully isolated by PCR-based methods. As a result, we determined the sequences of pig RAD51 (1663 bp, 339 aa), RAD52 (1884 bp, 406 aa), and RAD54 (2884 bp, 747 aa). The nucleic acid sequence homologies of the pig RAD51, RAD52, and RAD54 genes compared with the corresponding human genes were 92.9%, 77.3%, and 90.0%; the corresponding amino acid sequence homologies were 98.8%, 71.1%, and 95.0%, respectively.The knockout of α-1,3-galactosyltransferase in pigs resulted in a drastic reduction in xenoantigenicity. However, other xenoantigens, in particular, the non-Gal antigens, also need to be down-regulated. Gene transfer to alter expression levels of these recombination-related molecules and/or ex ante evaluation of expression profiles of these genes in primary cultures of somatic cells constitute a new approach to enhancing homologous recombination events during the production of gene knockout pigs.

Pig-to-nonhuman primate organ xenotransplantation

Purpose of review The availability of α1,3-galactosyltransferase gene-knockout pigs in which the major target for primate anti-pig antibodies has been deleted is considered a major step toward clinical xenotransplantation. A review of progress in pig-to-nonhuman primate transplantation is therefore timely. Recent findings Several groups have continued their studies using pigs transgenic for one or more human complement regulatory proteins, with graft survival reaching more than 3 months in some cases. Significant complications have resulted from the intensity of immunosuppression required to achieve these results, however. The use of organs from α1,3-galactosyltransferase gene-knockout pigs has eliminated hyperacute rejection in baboons. Heterotopic heart α1,3-galactosyltransferase gene-knockout xenografts maintained with chronic immunosuppression survived up to 6 months, with graft failure associated with thrombotic microangiopathy. Life-supporting renal xenografts maintained with a tolerance-inducing regimen survived over 80 days, with the longest survivors dying with functioning grafts. Summary The availability of α1,3-galactosyltransferase gene-knockout donor pigs has eliminated the need for inhibition of antibodies to α1,3 galactose (Gal) or complement to prevent hyperacute rejection. It has also had a positive impact on the survival of heterotopic xenogeneic hearts transplanted under chronic immunosuppression and on the survival of orthotopic kidneys transplanted with donor thymus tissue in an attempt to induce tolerance. Problems remain to be resolved, including the effects of the variable presence of antibodies to non-Gal antigens, the high incidence of thrombotic microangiopathy in heart xenotransplants, and loss of animals to other causes in the recipients of orthotopic renal transplants. Nevertheless, the results with α1,3-galactosyltransferase gene-knockout donors are encouraging for the future of this field.

Production of α1,3‐galactosyltransferase gene knockout pigs expressing both human decay‐accelerating factor and N‐acetylglucosaminyltransferase III

Heterozygous alpha 1,3-galactosyltransferase (GT) gene knockout pigs were produced with transgenic pig fetal cells expressing both human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GnT-III). In this study, we assessed the gene targeting efficiency in the transgenic pig fetal cells derived from different fetal tissues such as brain, skin, heart, and liver, or fetal carcass. Targeted cell colonies were selected by hygromycin B. The GT-knockout colonies (KO colonies) were obtained equally from the cells derived from all tissues except liver. Staining with five antibodies against intermediate filaments, all examined KO cell lines stained positive for vimentin with the exception of a colony that stained positive for both vimentin and glial fibrillary acidic protein simultaneously. This is the first study to produce KO cells from the astrocytes. Some of these KO cell lines were used for nuclear transfer (NT) to obtain KO pig fetuses. Fourteen fetuses were obtained from two recipients of the embryo transfer and eight of them had normal ploidy. The cells from the KO pig fetuses were also used for NT to produce cloned KO pigs. Two healthy clone pigs were born. These pigs were determined to have a heterozygous knockout GT gene and the two transgenes. The cells collected from the KO pigs were shown to have similar expression levels of hDAF and GnT-III compared to their original transgenic pigs and less than a half levels of the alphaGal epitopes existed in wild-type pig cells.

Potential of aspirin to inhibit thrombotic microangiopathy in α1,3-galactosyltransferase gene-knockout pig hearts after transplantation in baboons

Hearts from α1,3-Galactosyltransferase gene-knockout (GaIT-KO) pigs were transplanted heterotopically into 8 baboons that received an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen and heparin. Three baboons died or were euthanized with beating grafts on 16, 23, and 56 days, respectively, and the remaining 5 grafts functioned for 59–179 days. Hyperacute rejection did not occur, and classical features of acute humoral xenograft or acute cellular rejection were rare. However, thrombotic microangiopathy (TM) developed in all cases; its onset was delayed in 2 baboons that received aspirin. Function of a pig organ in a baboon for a period approaching 6 months has not been reported previously and lends encouragement that the barriers to xenotransplantation will be overcome, but TM requires investigation.

Preventing human CD8 + cytotoxic T lymphocyte–mediated cytotoxicity against swine endothelial cells by overexpression of human decoy Fas antigen

Although the birth of homozygous α1, 3 galactosyltransferase gene-knockout pigs raised hopes for an imminent breakthrough in the prevention in the antibody-mediated rejection of pig to human discordant xenotransplants, human CD8 + cytotoxic T lymphocyte (CTL)–mediated killing may represent a new immunological barrier to long-term survival in xenograft recipients. In this study, we demonstrated that the cytotoxicity of human CD8 + CTL against swine endothelial cells (SEC) is highly detrimental and mediated at least in part by the Fas/FasL pathway. To prevent this CTL-mediated xenocytotoxicity, we overexpressed the human decoy Fas antigen, which does not contain a death domain in its cytoplasmic region, by means of binding competition with endogenous pig Fas antigen on SEC for the common ligand, human FasL. Furthermore, we generated a membrane-bound form of human FasL that cannot be cleaved by a putative metalloproteinase to produce a soluble form, which was assessed as an inhibitor of CTL cytotoxicity. Both human decoy Fas and membrane-bound FasL were effective to prevent CTL-mediated killing, suggesting that these novel molecules may represent a step forward toward preventing CD8 + CTL-mediated xenograft rejection. The combined expression of both molecules may be more beneficial to protect xenograft cells.

Heart transplantation in baboons using α1,3-galactosyltransferase gene-knockout pigs as donors: initial experience

Hearts from alpha1,3-galactosyltransferase knockout pigs (GalT-KO, n = 8) were transplanted heterotopically into baboons using an anti-CD154 monoclonal antibody-based regimen. The elimination of the galactose-alpha1,3-galactose epitope prevented hyperacute rejection and extended survival of pig hearts in baboons for 2-6 months (median, 78 d); the predominant lesion associated with graft failure was a thrombotic microangiopathy, with resulting ischemic injury. There were no infectious complications directly related to the immunosuppressive regimen. The transplantation of hearts from GalT-KO pigs increased graft survival over previous studies.

An Efficient Method for Producing α(1,3)-Galactosyltransferase Gene Knockout Pigs

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the α(1,3)-galactosyltransferase (α1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of α1,3-GT gene knockout pigs using these procedures. Seven α1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neoR) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro–matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. α1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.

Investigation of red blood cells from alpha1,3-galactosyltransferase-knockout pigs for human blood transfusion.

Pigs are a potential source of red blood cells (RBCs) for transfusion into humans, but the presence of galactose-alpha1,3-galactose (Gal) epitopes on their surface, against which humans have anti-Gal, has been perceived as a major barrier. alpha1,3-Galactosyltransferase gene-knockout pigs, which do not express Gal epitopes on RBCs (Gal-/-), have recently become available. In vitro, RBCs from Gal-/- pigs were exposed to sera from naïve humans or baboons or from baboons previously sensitized to pig antigens; immunoglobulin binding was measured by flow cytometry, and cytotoxicity, by a hemolytic assay. In vivo, relatively small numbers of Gal-/- RBCs were transfused into two nonsensitized untreated baboons. The survival of pig RBCs was detected by flow cytometry. In vitro, binding of immunoglobulin (Ig) M from naïve human or baboon sera was detected to Gal-/- RBCs but was significantly less than to Gal+/+ RBCs; IgG binding to Gal-/- RBCs was absent or minimal. Sera had minimal cytotoxicity to Gal-/- RBCs compared to Gal+/+ RBCs. Sensitized baboon sera demonstrated much higher IgG binding to Gal-/- RBCs and increased cytotoxicity, but again these were less than to Gal+/+ RBCs. In vivo, the transfusion of relatively small volumes of Gal-/- RBCs was followed by detection of the cells in the baboon's blood for only 5 minutes. Pig RBCs are rapidly phagocytosed from the primate circulation by a mechanism not involving anti-Gal.

Clinical xenotransplantion—how close are we?

Xenotransplantation with pig organs offers a medium-term solution to the shortage of organs available for clinical transplantation. The immunological barriers to xenotransplantation have been, and remain, formidable. In the early 1990s, the identification of Galalpha1,3Gal (Gal) as the main target for human xenoreactive (anti-pig) antibodies and the development of pigs transgenic for a human complement regulatory protein, decay-accelerating factor (hDAF), were major advances. The presence of hDAF on the vascular endothelium of pig organs provided some protection against complement-mediated hyperacute rejection. This protection, however, was short-lived, and, until recently, the longest median time for organ survival that had been achieved (with combinations of biological and pharmacological immunosuppressants) in a series of pig-to-primate organ transplants was under a month. Christopher McGregor and colleagues recently reported to the International Society of Heart and Lung Transplantation (J Heart Lung Transplant 2003; 22: S89) that, by combining the use of organs which express hDAF with the administration of a soluble Gal glycoconjugate and other immunosuppressive agents, the survival of pig hearts in baboons can be extended to a median of 76 days. McGregor's work suggests that immunological barriers to xenotransplantation are not insurmountable. WHERE NEXT? The recent generation of pigs that do not express Gal epitopes (alpha1,3-galactosyltransferase gene-knockout pigs) might remove the need both for the expression of hDAF and the administration of a soluble Gal glycoconjugate. The absence of a natural antibody response will allow investigation of the cellular immune response and of any molecular incompatibilities between pig and primate that may be detrimental to graft survival. Furthermore, the absence of a humoral response may open the way for the induction of immunological tolerance (or unresponsiveness in the absence of exogenous immunosuppression) to a transplanted pig organ.

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Solid organ xenotransplantation is a potential solution to current organ shortages in allotransplantation. We performed four heart transplantations from alpha1, 3-galactosyltransferase gene-knockout (GT-KO) pigs to cynomolgus monkeys and monitored immunological parameters before and after transplantation.After blood typing of the cynomolgus monkeys, we assessed the binding activity of immunoglobulin G (IgG) and IgM of monkey serum and serum toxicity toward porcine peripheral blood mononuclear cells (PBMCs) using flow cytometry. Immunosuppressive protocols consisted of anti-thymocyte globulin (25 mg/kg), rituximab (20 mg/kg), anti-CD154mAb (20 mg/kg), cobra venom factor (0.05 mg/kg), tacrolimus, and steroid. Cynomolgus monkeys with A or AB blood type with the lowest antibody binding and serum toxicity activity on porcine PBMCs were selected as recipients.Absolute numbers of CD3+ T cells, CD20+ B cells, and CD3+CD95+ memory T cells in the peripheral blood were suppressed upto 24 days after transplantation. Interferon gamma production of T cells in response to porcine antigens were also significantly suppressed. Heart xenografts from GT-KO pigs survived for upto 24 days without pathologic evidence of rejection.We successfully performed 4 heart xenotransplantations using GT-KO pigs. We overcame hyperacute rejection by using GT-KO pigs, and all of the heart xenografts from the GT-KO pigs survived between 11 and 24 days without pathologic evidence of rejection, disseminated intravascular coagulation, or consumptive coagulopathy; however, we need to optimize protocols for immune modulation and postoperative care to attain long-term survival of solid organ xenografts.

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Solid organ xenotransplantation is a potential solution to current organ shortages in allotransplantation. We performed four heart transplantations from alpha1, 3-galactosyltransferase gene-knockout (GT-KO) pigs to cynomolgus monkeys and monitored immunological parameters before and after transplantation.After blood typing of the cynomolgus monkeys, we assessed the binding activity of immunoglobulin G (IgG) and IgM of monkey serum and serum toxicity toward porcine peripheral blood mononuclear cells (PBMCs) using flow cytometry. Immunosuppressive protocols consisted of anti-thymocyte globulin (25 mg/kg), rituximab (20 mg/kg), anti-CD154mAb (20 mg/kg), cobra venom factor (0.05 mg/kg), tacrolimus, and steroid. Cynomolgus monkeys with A or AB blood type with the lowest antibody binding and serum toxicity activity on porcine PBMCs were selected as recipients.Absolute numbers of CD3+ T cells, CD20+ B cells, and CD3+CD95+ memory T cells in the peripheral blood were suppressed upto 24 days after transplantation. Interferon gamma production of T cells in response to porcine antigens were also significantly suppressed. Heart xenografts from GT-KO pigs survived for upto 24 days without pathologic evidence of rejection.We successfully performed 4 heart xenotransplantations using GT-KO pigs. We overcame hyperacute rejection by using GT-KO pigs, and all of the heart xenografts from the GT-KO pigs survived between 11 and 24 days without pathologic evidence of rejection, disseminated intravascular coagulation, or consumptive coagulopathy; however, we need to optimize protocols for immune modulation and postoperative care to attain long-term survival of solid organ xenografts.