Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs

Puga Yung GL, Li Y, Borsig L, Millard A‐L, Karpova MB, Zhou D, Seebach JD. Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock‐out pigs. Xenotransplantation 2012; 19: 196–206. © 2012 John Wiley & Sons A/S. Abstract: Background: Anti‐Galα1,3Galβ‐R natural antibodies are responsible for hyperacute rejection in pig‐to‐primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody‐mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb₃) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. Methods: The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb₃ synthase mRNA was tested by RT‐PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples. Results: Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb₃ on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H‐type sugar structures present in GalT knockout cells as compared to wild‐type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb₃ was also totally absent, whereas a fucosylated form of iGb₃ was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild‐type or GalT knockout animals. Conclusions: These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non‐Galα1,3Gal antibodies and cellular responses.     

The pathobiology of pig‐to‐primate xenotransplantation: a historical review

The immunologic barriers to successful xenotransplantation are related to the presence of natural anti‐pig antibodies in humans and non‐human primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose‐α1,3‐galactose [Gal]), and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti‐pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3‐galactosyltransferase gene‐knockout [GTKO] pigs) and express one or more human complement‐regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade‐based immunosuppressive regimen, prevents early antibody‐mediated and cellular rejection. However, low levels of anti‐non‐Gal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation‐anticoagulation systems between pigs and primates. The expression in GTKO/hCRP pigs of a human coagulation‐regulatory protein, for example, thrombomodulin, is increasingly being associated with prolonged pig graft survival in non‐human primates. Initial clinical trials of islet and corneal xenotransplantation are already underway, and trials of pig kidney or heart transplantation are anticipated within the next few years.     

The sweets standing at the borderline between allo‐ and xenotransplantation

Animal cells are densely covered with glycoconjugates, such as N‐glycan, O‐glycan, and glycosphingolipids, which are important for various biological and immunological events at the cell surface and in the extracellular matrix. Endothelial α‐Gal carbohydrate epitopes (Galα3Gal‐R) expressed on porcine tissue or cell surfaces are such glycoconjugates and directly mediate hyperacute immunological rejection in pig‐to‐human xenotransplantation. Although researchers have been able to develop α1,3‐galactosyltransferase (GalT) gene knockout (KO) pigs, there remain unclarified non‐Gal antigens that prevent xenotransplantation. Based on our expertise in the structural analysis of xenoantigenic carbohydrates, we describe the immunologically significant non‐human carbohydrate antigens, including α‐Gal antigens, analyzed as part of efforts to assess the antigens responsible for hyperacute immunological rejection in pig‐to‐human xenotransplantation. The importance of studying human, pig, and GalT‐KO pig glycoprofiles, and of developing adequate pig‐to‐human glycan databases, is also discussed.     

The effect of Gal expression on pig cells on the human T-cell xenoresponse

Wilhite T, Ezzelarab C, Hara H, Long C, Ayares D, Cooper DKC, Ezzelarab M. The effect of Gal expression on pig cells on the human T cell xenoresponse. Xenotransplantation 2012; 19: 56–63. © 2012 John Wiley & Sons A/S. Abstract: Background: Lack of Gal expression on pig cells is associated with a reduced primate humoral immune response as well as a reduction in cytokine production by human cells in vitro. We investigated whether lack of Gal expression is associated with reduced human T‐cell response in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy humans and naïve baboons. Human CD4⁺ and CD8⁺ T cells were isolated. Porcine aortic endothelial cells (pAECs) were isolated from wild‐type (WT) and α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs. WT pAECs were treated with α‐galactosidase, reducing Gal expression. Swine leukocyte antigen (SLA) class I and II expression on pAECs was measured, as was T‐cell proliferation and cytokine production in response to pAECs. Results: Reduced Gal expression on WT pAECs after α‐galactosidase treatment was associated with reduced human PBMC proliferation (P < 0.005). SLA class I and II expression on WT and GTKO pAECs was comparable. Human CD4⁺ and CD8⁺ T‐cell proliferation was less against GTKO pAECs before (P < 0.001) and after (P < 0.01 and P < 0.05, respectively) activation. Human and baboon PBMC proliferation was less against GTKO pAECs before (P < 0.05) and after (P < 0.01 and P < 0.05, respectively) activation. Human PBMCs produced a comparable cytokine/chemokine response to WT and GTKO pAECs. However, there was less production of IFN‐γ/TNF‐α by CD4⁺ and IFN‐γ/granzyme B/IP‐10 by CD8⁺ T cells in response to GTKO pAECs. Conclusions: The absence of Gal on pig cells is associated with reduced human T‐cell proliferation (and possibly selected cytokine production). Adaptive primate T‐cell responses are likely to be reduced in GTKO xenograft recipients.     

Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4⁺T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.     

Investigation of potential carbohydrate antigen targets for human and baboon antibodies

Yeh P, Ezzelarab M, Bovin N, Hara H, Long C, Tomiyama K, Sun F, Ayares D, Awwad M, Cooper DKC. Investigation of potential carbohydrate antigen targets for human and baboon antibodies. Xenotransplantation 2010; 17: 197–206. © 2010 John Wiley & Sons A/S. Abstract: Background: The continued presence of a primate antibody‐mediated response to cells and organs from α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs indicates that there may be antigens other than Galα1,3Gal (αGal) against which primates have xenoreactive antibodies. Human and baboon sera were tested for reactivity against a panel of saccharides that might be potential antigen targets for natural anti‐non‐αGal antibodies. Methods: Human sera (n = 16) and baboon sera (n = 15) of all ABO blood types were tested using an enzyme‐linked immunoadsorbent assay for binding of IgM and IgG to a panel of synthetic polyacrylamide‐linked saccharides (n = 15). Human sera were also tested after adsorption on αGal immunoaffinity beads. Sera from healthy wild‐type (WT, n = 6) and GTKO (n = 6) pigs and from baboons (n = 4) sensitized to GTKO pig organ or artery transplants (of blood type O) were also tested. Forssman antigen expression on baboon and pig tissues was investigated by immunohistochemistry. Results: Both human and baboon sera showed high IgM and IgG binding to αGal saccharides, α‐lactosamine, and Forssman disaccharide. Human sera also demonstrated modest binding to N‐glycolylneuraminic acid (Neu5Gc). When human sera were adsorbed on αGal oligosaccharides, there was a reduction in binding to αGal and α‐lactosamine, but not to Forssman. WT and GTKO pig sera showed high binding to Forssman, and GTKO pig sera showed high binding to αGal saccharides. Baboon sera sensitized to GTKO pigs showed no significant increased binding to any specific saccharide. Staining for Forssman was negative on baboon and pig tissues. Conclusions: We were unable to identify definitively any saccharides from the selected panel that may be targets for primate anti‐non‐αGal antibodies. The high level of anti‐Forssman antibodies in humans, baboons, and pigs, and the absence of Forssman expression on pig tissues, suggest that the Forssman antigen does not play a role in the primate immune response to pigs.     

40 years of heart transplantation and the DFG‐Transregio Research Group Xenotransplantation

Today, organ transplantation represents a well‐established and effective therapy of terminal organ failure revealing high actuarial survival rates. Unfortunately, the enormous potential of organ transplantation cannot be tapped due to the significant gap between organ demand and organ donation. Current statistics of the International Society of Heart and Lung Transplantation prove a continuity of depressed numbers of transplantations performed per year since the late nineties. To counteract the persisting severe shortage of human organs in Germany and worldwide suboptimal donor organs and/or organs from older donors were accepted. Both the acceptance of inferior organs and the implementation of the Transplantation Law (in Germany in 1997) could not answer this problem. Increasing the donor rates emerge difficult to achieve and will ultimately result in numbers which are not sufficient. The improvement of transplant results by e.g. a less nephrotoxic immunosuppression, or the generating of hyporeactivity or even tolerance is an additional aim important to achieve. Alternative techniques to answer the tremendous organ shortage might be the differentiation of embryonic stem cells or the reprogramming of adult stem cells as a virtually unlimited source for cell replacement to treat degenerative diseases or traumatic tissue injury. Yet, disadvantages such as ethical issues and the generation of tumorigenic cells should not be underestimated. A cellular therapy by the injection of undifferentiated bone marrow (CD133⁺ stem/progenitor) cells into the myocardium in combination with or without aortocoronary surgery for chronic ischemic heart disease as well as cells from the amniotic fluid (Wharton's jelly) might also represent possible future solutions to the organ deficit but still are far from a functional substitution of the human heart. Until now there is no in‐all implantable mechanical heart assist device which is able to completely and permanently replace the human organ and provide a quality of life comparable to that after allotransplantation. In contrast, xenotransplantation, using porcine organs for human transplantation, offers a potential solution to the world‐wide lack of donor organs. The advantages of xenotransplantation are an unlimited disposability of donor organs, an elective transplantation with a subsequent reduction of ischemic time and the possibility of a pre‐operative start of the immunosuppressive therapy of the recipient. Harmful effects of the brain death of the donor to the donor organ could be excluded. Finally, genetic modifications of compatible xenografts could be made. Substantial progress of the research in the field of xenotransplantation has been possible thanks to the introduction of organs from genetically engineered pigs transgenic for human complement regulatory proteins [e.g. human decay accelerating factor (hDAF/hCD55), human membrane cofactor (hMCP/hCD46), and human membrane inhibitor of reactive lysis (hMIRL/hCD59)]. Using an effective and persistent depletion of preformed cytotoxic anti‐Galα(1,3)Gal antibodies (IgM and IgG) by a Galα(1,3)Gal therapeutic (e.g. GAS914, TPC) in combination with these transgenic pigs hyperacute rejection can be avoided successfully. During the early phase after transplant acute vascular rejection triggered by induced anti‐Galα(1,3)Gal antibodies can be controlled. Several groups developed pigs which lack the Galα(1,3)Gal xenoantigen. Studies on xenotransplantations performed with homozygous alpha(1,3)‐galactosyltransferase gene knockout pigs demonstrated that these modified pig organs offer some progress in terms of graft survival. Thus, the major xenoantigen Galα(1,3)Gal is no longer an unsurmountable immunological barrier preventing transplantation of pig organs into humans. Acute vascular rejection, however, remains as a major hurdle to clinical application of xenotransplantation due to cytotoxic anti‐pig antibodies of other specificity than Galα(1,3)Gal. Furthermore, humoral factors are not the only players in xenograft rejection. Primate anti‐pig cellular immunity is defined by multifocal lymphocytic infiltrates, with morphologic evidence of direct tissue damage. Pre‐requisites for the clinical use of xenotransplantation are PERV‐C (porcine endogenous virus C) free animals using a PERV knock down (si‐RNA) technique. Multitransgenic αGalT‐KO [alpha(1,3)‐galactosyltransferase knockout] pigs additionally expressing human complement regulator proteins, and human anticoagulants (e.g. human thrombomodulin) are necessary to reliably prevent not only hyperacute rejection as the first immunological barrier, but also acute vascular rejection at its beginning, when serum cytotoxicity to the pig heart appears to be predominantly Galα(1,3)Gal‐specific. Further co‐stimulation blockade (e.g. PD‐1L, CTLA‐4‐Ig), HLA‐E [protection against human NK (natural killer)‐cells], or haemeoxygenase‐1 (defense against disseminated intravascular coagulation) will help to suppress acute vascular and acute cellular xenograft rejection. Special pathogen free (SPF) units and breeding conditions of pig organ donors limit the risk of microbial contamination by most pathogens liable to be transmitted from a pig graft to a human recipient. Our DFG‐(German Research Council) Transregio Research Group Xenotransplantation assembles an interdisciplinary group of leading German laboratories incl. biotechnologists, immunologists, virologists, and surgeons with vast experimental expertises in the field of experimental and clinical allotransplantation and experimental xenotransplantation. The first clinical goal of xenotransplantation is xenogeneic tissue transplantation such as the transplantation of porcine islet cells (αGalT‐KO (?), CTLA‐4‐Ig expression) in diabetic patients with hypoglycemic attacks as well as porcine cornea, porcine cardiomyocytes and porcine heart valves, possibly porcine bones and teeth (?). Thereafter, xenogeneic organ transplantation starting with the more promising use of kidneys and hearts is the definitive clinical goal. In summary, clinical heart transplantation represents an accepted method of end‐stage heart failure with an outdated “standard immunosuppression” and the need of an individualized immunosuppression adjusted to the specific needs of the individual patient. The organ shortage remains the main obstacle of the heart transplantation, and other organ transplantation, respectively. In the near future, xenotransplantation will be possible!     

Double knockout pigs deficient in N‐glycolylneuraminic acid and Galactose α‐1,3‐Galactose reduce the humoral barrier to xenotransplantation

Background

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα‐1,3‐Gal (Gal) and N‐glycolylneuraminic acid (Neu5Gc) are two known xenoantigens.

Methods

We report the homozygous disruption of the α1, 3‐galactosyltransferase (GGTA1) and the cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase (CMAH) genes in liver‐derived female pig cells using zinc‐finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody‐binding and antibody‐mediated complement‐dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal.

Results

This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1.

Conclusions

This technology will accelerate the development of pigs for xenotransplantation research.     

An in vitro model of pig liver xenotransplantation--pig complement is associated with reduced lysis of wild-type and genetically modified pig cells

Hara H, Campanile N, Tai H‐C, Long C, Ekser B, Yeh P, Welchons D, Ezzelarab M, Ayares D, Cooper DKC. An in vitro model of pig liver xenotransplantation—pig complement is associated with reduced lysis of wild‐type and genetically modified pig cells. Xenotransplantation 2010; 17: 370–378. © 2010 John Wiley & Sons A/S. Abstract: Background: After pig liver transplantation in humans, the graft will produce pig complement (C). We investigated in vitro the lysis of wild‐type (WT), α1,3‐galactosyltransferase gene‐knockout (GTKO), and CD46 transgenic (CD46) pig peripheral blood mononuclear cells (PBMC) caused by human anti‐pig antibodies (Abs) + pig C. Methods: Human serum IgM/IgG binding to WT and GTKO PBMC was determined by flow cytometry, and lysis of pig PBMC by a C‐dependent cytotoxicity assay using (i) human serum (human Abs + C), (ii) GTKO pig serum (anti‐Gal Abs + pig C), (iii) heat‐inactivated human serum (human Abs) + rabbit C, or (iv) human Abs + pig C (serum). Results: Binding of human IgM and IgG to GTKO PBMC was less than to WT PBMC (P < 0.05). In the presence of human Abs, lysis of WT and GTKO PBMC by rabbit C was 87 and 13%, respectively (WT vs. GTKO, P < 0.01), but was only 37 and 0.4% in the presence of pig C (WT vs. GTKO, P < 0.05). Human/rabbit C‐induced lysis was greater than pig C‐induced lysis for both WT and GTKO PBMC. CD46 pig PBMC reduced rabbit/human C‐ and pig C‐mediated lysis (P < 0.05). Conclusions: Pig livers, particularly from GTKO and CD46 pigs, are likely to have an immunologic advantage over other organs after transplantation into humans. In the absence of pig antibodies directed to human tissues, pig complement is unlikely to cause problems after liver xenotransplantation, especially if GTKO/CD46 pigs are used as the source of the livers.     

Intravenous synthetic αgal saccharides delay hyperacute rejection following pig‐to‐baboon heart transplantation

Romano E, Neethling FA, Nilsson K, Kosanke S, Shimizu A, Magnusson S, Svensson L, Samuelsson B, Cooper DKC Intravenous synthetic αgal saccharides delay hyperacure rejection following pig-to-baboon heart transplantation. Xenotransplantation 1999; 6: 00-00 Several oligosaccharides containing the terminal structure Galα1–3Gal (αGal) and different side chains were tested in vitro for their ability to block natural antiαGal antibodies. A di-and a trisaccharide (diαGal and triαGal) were selected. A blood group B baboon, having IgG and IgM natural antipig titers of 1 : 256 and 1 : 1024 and a hemolytic titer (to pig red blood cells, RBCs) of 1 : 8, was chosen to measure pharmacokinetic parameters of the saccharides and to assess the extent of in vivo neutralization of the antibodies. Three grams each of the diαGal and the triαGal dissolved in saline were administered by bolus intravenous (i.v.) injection. Blood samples were collected at various times and urine was collected at 8 and 24 h. Plasma and urine concentrations of the αGal saccharides were estimated by an ELISA specially developed for this study. A fast distribution phase followed by equilibrium and excretion phases were observed, indicating a T1/2 in the order of 1 h. Fifty-eight per cent of the saccharides were recovered in the urine within 24 h. Determination of antipig antibody binding by FACS analysis and of serum cytotoxicity titers for pig endothelial cells demonstrated that a 70% reduction in binding and cytotoxicity could be achieved with plasma saccharide levels of 300–400 µg/ml. Six months later, a pig heart was transplanted heterotopically into the baboon. A 3-g bolus of the saccharide mixture (1.5 g of each saccharide) was given i.v. before allowing blood reperfusion of the transplanted heart, followed by an i.v. infusion of 1 g/hr for 1 hr and 0.5 g/hr for the 3 succeeding hours. Blood concentrations of the saccharides, CH50, hematology and cytotoxicity for PK15 cells were estimated in blood samples taken at various times. Heart function was observed to be satisfactory for 8 h, but was found to have ceased at 18 h. Myocardial biopsies taken at 3 and 5 h showed congestion only, suggestive of minimal vascular rejection, but by 18 h demonstrated severe vascular rejection. In conclusion, αGal saccharide therapy given for a period of 4 h delayed, but did not totally prevent, the development of vascular rejection in the pig-to-baboon heart transplant model. αGal saccharide therapy may be one of several useful approaches for the prevention of hyperacute rejection in pig-to-primate organ transplantation.     

Effect of αGal on corneal xenotransplantation in a mouse model

Choi HJ, Kim MK, Lee HJ, Jeong SH, Kang HJ, Park C‐S, Park C‐G, Kim SJ, Wee WR. Effect of αGal on corneal xenotransplantation in a mouse model. Xenotransplantation 2011; 18: 176–182. © 2011 John Wiley & Sons A/S. Abstract: Background: It has been reported that hyperacute rejection (HAR) does not occur after pig‐to‐nonhuman corneal xenotransplantation. However, considering that immune privilege is already disrupted in most human corneal recipients, and the expression of αGal can be gradually reduced after pig‐to‐rat corneal transplantation, the long‐term survival of corneal grafts from wild‐type pigs cannot be guaranteed. Accordingly, we aimed to investigate the effect of αGal on the change in anti‐Gal antibodies, using sensitized α1,3‐galactosyltransferase gene‐knockout (GTKO) mice recipients. Methods: C57BL/6 (B6) and GTKO mice were divided into 5 groups and underwent orthotopically full thickness cormeal transplantation as follows (n=4 for each group): (1) group 1: B6 to B6; (2) group 2: fresh porcine posterior corneal lamella to B6; (3) fresh porcine posterior corneal lamella to GTKO; (4) group 4: decellularized porcine posterior corneal lamella to GTKO, and (5) group 5: B6 to GTKO. Before transplantation, all GTKO recipients were sensitized using intraperitoneal injections of rabbit blood cells. Median survival times (MST) for the corneal grafts of the different groups were compared and plasma concentrations of IgG/IgM anti‐Gal antibodies were evaluated at 1 week, 2 weeks and 3 weeks post‐transplantation. Results: There were no differences in MSTs between groups. Although there was no HAR of fresh porcine posterior corneal grafts even in sensitized GTKO recipients, αGal expression was induced in the transplanted fresh porcine corneal grafts and plasma concentration of IgG anti‐Gal antibody was gradually increased in fresh porcine cornea‐grafted GTKO recipients. On the contrary, αGal expression did not increase in the grafts and plasma concentration of anti‐Gal antibodies did not change after transplantation using decellularized porcine corneas. Conclusions: Our findings suggest that αGal may affect the long‐term survival of porcine corneal xenografts via antibody‐mediated rejection, although αGal does not have an effect on acute rejection and decellularized porcine corneas may enable the long‐term survival of porcine corneal xenografts.     

Comparison of hematologic,biochemical, and coagulation parameters in α1,3‐galactosyltransferase gene‐knockout pigs,wild‐type pigs,and four primate species

Ekser B, Bianchi J, Ball S, Iwase H, Walters A, Ezzelarab M, Veroux M, Gridelli B, Wagner R, Ayares D, Cooper DKC. Comparison of hematologic, biochemical, and coagulation parameters in α1,3‐galactosyltransferase gene‐knockout pigs, wild‐type pigs, and four primate species. Xenotransplantation 2012; 19: 342–354. © 2012 John Wiley & Sons A/S. Abstract: Background: The increasing availability of genetically engineered pigs is steadily improving the results of pig organ and cell transplantation in non‐human primates (NHPs). Current techniques offer knockout of pig genes and/or knockin of human genes. Knowledge of normal values of hematologic, biochemical, coagulation, and other parameters in healthy genetically engineered pigs and NHPs is important, particularly following pig organ transplantation in NHPs. Furthermore, information on parameters in various NHP species may prove important in selecting the optimal NHP model for specific studies. Methods: We have collected hematologic, biochemical, and coagulation data on 71 α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs, 18 GTKO pigs additionally transgenic for human CD46 (GTKO.hCD46), four GTKO.hCD46 pigs additionally transgenic for human CD55 (GTKO.hCD46.hCD55), and two GTKO.hCD46 pigs additionally transgenic for human thrombomodulin (GTKO.hCD46.hTBM). Results: We report these data and compare them with similar data from wild‐type pigs and the three major NHP species commonly used in biomedical research (baboons, cynomolgus, and rhesus monkeys) and humans, largely from previously published reports. Conclusions: Genetic modification of the pig (e.g., deletion of the Gal antigen and/or the addition of a human transgene) (i) does not result in abnormalities in hematologic, biochemical, or coagulation parameters that might impact animal welfare, (ii) seems not to alter metabolic function of vital organs, although this needs to be confirmed after their xenotransplantation, and (iii) possibly (though, by no means certainly) modifies the hematologic, biochemical, and coagulation parameters closer to human values. This study may provide a good reference for those working with genetically engineered pigs in xenotransplantation research and eventually in clinical xenotransplantation.     

alpha1,3-Galactosyltransferase gene-knockout miniature swine produce natural cytotoxic anti-Gal antibodies

BACKGROUND: The expression of galactose alpha 1,3 galactose (Gal) in pigs has proved a barrier to xenotransplantation. Miniature swine lacking Gal (Gal pigs) have been produced by nuclear transfer/embryo transfer. METHODS: The tissues of five Gal pigs of SLA dd haplotype (SLA) were tested for the presence of Gal epitopes by staining with the Griffonia simplicifolia IB4 lectin. Their sera were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (Gal) SLA-matched pigs; serum cytotoxicity was also assessed. The cellular responses of PBMC from Gal swine toward Gal SLA-matched PBMC were tested by mixed leukocyte reaction and cell-mediated lympholysis assays. RESULTS: None of the tissues tested showed Gal expression. Sera from all five Gal pigs manifested IgM binding to Gal pig PBMC, and sera from three showed IgG binding. In all five cases, cytotoxicity to Gal cells could be demonstrated, which was lost after treatment of the sera with dithiothreitol, indicating IgM antibody-mediated cytotoxicity. PBMC from Gal swine had no proliferative or cytolytic T-cell response toward Gal SLA-matched PBMC. CONCLUSIONS: Gal pigs do not express Gal epitopes and develop anti-Gal antibodies that are cytotoxic to Gal pig cells. The absence of an in vitro cellular immune response between Gal and Gal pigs is related to their identical SLA haplotype and indicates the absence of immunogenicity of Gal in T-cell responses. The model of Gal organ transplantation into a Gal SLA-matched recipient would be a valuable large animal model in the study of accommodation or B-cell tolerance.     

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs

A combination of genetic manipulations of donor organs and target‐specific immunosuppression is instrumental in achieving long‐term cardiac xenograft survival. Recently, results from our preclinical pig‐to‐baboon heterotopic cardiac xenotransplantation model suggest that a three‐pronged approach is successful in extending xenograft survival: (a) α‐1,3‐galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal‐specific antibody‐mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co‐stimulation blockade of CD40‐CD40L pathways with anti‐CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non‐Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co‐stimulation blockade‐based immunomodulatory regimen.     

Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate

Akasaka E, Watanabe S, Himaki T, Ohtsuka M, Yoshida M, Miyoshi K, Sato M. Enrichment of xenograft‐competent genetically modified pig cells using a targeted toxin, isolectin BS‐I‐B4 conjugate. Xenotransplantation 2010; 17: 81–89. © 2010 John Wiley & Sons A/S. Abstract: Background: The recent availability of α‐1,3‐galatosyltransferase knockout pigs has eliminated anti‐Gal antibodies to the galα1‐3gal (αgal epitope) as the major barrier to xenotransplantation. These αgal epitope‐negative animals can also be produced by somatic cell nuclear transfer of cells overexpressing endo‐β‐galactosidase (EndoGalC), an enzyme capable of digesting the αgal epitope. For this, selection of cells with highly reduced synthesis of αgal epitope is a prerequisite. In this study, we developed a novel method of selection using isolectin BS‐I‐B₄‐conjugated saporin (IB4‐SAP), a targeted cytotoxin, that is specific for the terminal αgal epitope. Methods: A mixture of αgal epitope‐expressing and non‐expressing pig cells was obtained by transfection with an EndoGalC expression vector. These cells were incubated with a solution containing IB4‐SAP for 2 h at 37 °C, and subsequently cultivated for over 2 months under general conditions. Results: Almost all (98%) of surviving cells were completely negative for expression of αgal epitope, as confirmed by cytochemical staining using fluorescence‐labeled IB4. FACS analysis also confirmed that the IB4‐SAP‐treated cells exhibited a staining pattern similar to that of the IB4‐negative human cells. Extended cultivation (more than 6 months) of these IB4‐SAP‐treated cells did not alter the above staining pattern. RT‐PCR analysis revealed the presence of EndoGalC mRNA in these cells. Conclusions: This IB4‐SAP‐mediated method of selection of αgal epitope‐negative cells will provide an alternative to the present method of cytotoxicity‐based selection using specific antibody and complement.     

Recipient Tissue Factor Expression Is Associated With Consumptive Coagulopathy in Pig‐to‐Primate Kidney Xenotransplantation

Consumptive coagulopathy (CC) remains a challenge in pig‐to‐primate organ xenotransplantation (Tx). This study investigated the role of tissue factor (TF) expression on circulating platelets and peripheral blood mononuclear cells (PBMCs). Baboons (n = 9) received a kidney graft from pigs that were either wild‐type (n = 2), α1,3‐galactosyltransferase gene‐knockout (GT‐KO; n = 1) or GT‐KO and transgenic for the complement‐regulatory protein, CD46 (GT‐KO/CD46, n = 6). In the baboon where the graft developed hyperacute rejection (n = 1), the platelets and PBMCs expressed TF within 4 h of Tx. In the remaining baboons, TF was detected on platelets on post‐Tx day 1. Subsequently, platelet‐leukocyte aggregation developed with formation of thrombin. In the six baboons with CC, TF was not detected on baboon PBMCs until CC was beginning to develop. Graft histopathology showed fibrin deposition and platelet aggregation (n = 6), but with only minor or no features indicating a humoral immune response (n = 3), and no macrophage, B or T cell infiltration (n = 6). Activation of platelets to express TF was associated with the initiation of CC, whereas TF expression on PBMCs was concomitant with the onset of CC, often in the relative absence of features of acute humoral xenograft rejection. Prevention of recipient platelet activation may be crucial for successful pig‐to‐primate kidney Tx.     

In vitro investigation of pig cells for resistance to human antibody‐mediated rejection

Although human complement‐dependent cytotoxicity (CDC) of α1,3‐galactosyltransferase gene‐knockout (GTKO) pig cells is significantly weaker than that of wild‐type (WT) cells, successful xenotransplantation will require pigs with multiple genetic modifications. Sera from healthy humans were tested by (i) flow cytometry for binding of IgM/IgG, and (ii) CDC assay against peripheral blood mononuclear cells and porcine aortic endothelial cells from five types of pig – WT, GTKO, GTKO transgenic for H‐transferase (GTKO/HT), WT transgenic for human complement regulatory protein CD46 (CD46) and GTKO/CD46. There was significantly higher mean IgM/IgG binding to WT and CD46 cells than to GTKO, GTKO/HT, and GTKO/CD46, but no difference between GTKO, GTKO/HT, and GTKO/CD46 cells. There was significantly higher mean CDC to WT than to GTKO, GTKO/HT, CD46, and GTKO/CD46 cells, but no difference between GTKO and GTKO/HT. Lysis of GTKO/CD46 cells was significantly lower than that of GTKO or CD46 cells. CD46 expression provided partial protection against serum from a baboon sensitized to a GTKO pig heart. GTKO/CD46 cells were significantly resistant to lysis by human serum and sensitized baboon serum. In conclusion, the greatest protection from CDC was obtained by the combination of an absence of Gal expression and the presence of CD46 expression, but the expression of HT appeared to offer no advantage over GTKO. Organs from GTKO/CD46 pigs are likely to be significantly less susceptible to CDC.     

Cellular immunological barriers in xenotransplantation: current status and prospects for avoidance of graft destruction

Genetic modification of pigs (e.g. transgenic expression of human complement regulatory molecules or inactivation of α1,3galactosyltransferase) enabled the development of promising strategies to overcome hyperacute rejection after pig‐to‐primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Cellular rejection of porcine cells in xenotransplantation models is mediated by macrophages, T cells and NK cells. Activation of human monocytes by pig cells is partly due to the incapacity of porcine ligands to bind the inhibitory receptor SIRPα (signal regulatory protein α). Thus, one approach to impair the ability of human macrophages to phagocyte porcine cells is the overexpression of the human ligand for SIRPα in porcine cells. To inhibit human NK cell reactivity after xenotranslantation transgenic expression of HLA‐E in pigs has been shown to be a promising concept. Cells from these pigs were partially protected from lysis by human NK cells. Our group focuses on manipulation of human anti‐pig T cell responses by negative costimulatory signals. Thus, we asked whether overexpression of PD‐L1 on porcine cells can (i) downregulate human anti‐pig cellular responses in vitro, and (ii) inhibit rat anti‐pig cellular immune responses in vivo. Pig cells overexpressing PD‐L1 triggered reduced proliferation and low amounts of IL‐2, IFNγ, TNF‐alpha, IL‐4, and IL‐5 in human CD4⁺ T cells compared to control pig cells. The concentration of IL‐10, however, was increased. In long‐term cultures of human CD4⁺ T cells and PD‐L1 transfectants a high frequency of CD4⁺ CD25^high FoxP3⁺ cells showed up which had the capacity to suppress the activation of conventional CD4⁺ T cells. Cytotoxic CD8⁺ T cells and NK cells lysed pig control cells very efficiently. In contrast, PD‐L1 transfected pig cells were partially protected from lysis by human effector cells. Overexpression of PD‐L1 on porcine cells was not sufficient to prevent rejection after transplantation under the rat kidney capsule. However, in rats that had been grafted with PD‐L1 expressing cells we observed reduced cellular infiltrates in the kidneys and lower antibody responses compared to rats grafted with control cells. Together these observations support the assumption that PD‐1/PD‐Ligand pathways are interesting targets to prevent cellular immune responses after xenotransplantation. PD‐L overexpression might not only impede the initiation of an anti‐pig T cell response by suppressing CD4⁺ T cells but may also protect pig cells from destruction by cytotoxic effectors. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forschergruppe “Xenotransplantation”, FOR 535).     

Antigen-binding specificity of anti-αGal reagents determined by solid-phase glycolipid-binding assays. A complete lack of αGal glycolipid reactivity in α1,3GalT-KO pig small intestine

Diswall M, Gustafsson A, Holgersson J, Sandrin MS, Breimer ME. Antigen‐binding specificity of anti‐αGal reagents determined by solid‐phase glycolipid‐binding assays. A complete lack of αGal glycolipid reactivity in α1,3GalT‐KO pig small intestine. Xenotransplantation 2011; 18: 28–39. © 2011 John Wiley & Sons A/S. Abstract: Background: αGal‐specific lectins, monoclonal and polyclonal antibodies (Abs) are widely used in xenotransplantation research. Immunological assays such as immunohistochemistry, flow cytometry, Western blot and thin layer chromatography are often the only applicable characterization procedures when limited amount of tissue is available and biochemical characterization is impossible. Hence, detailed knowledge of the Ab/lectin carbohydrate‐binding specificity is essential. Methods: The binding specificity of human blood group AB serum, three different affinity‐purified human polyclonal anti‐Gal Ab batches, and two anti‐Gal mAb clones (TH5 and 15.101) as well as Griffonia simplicifolia isolectin B4 and Marasmius oreades agglutinin were examined for reactivity with glycolipid fractions isolated from human and pig (wild‐type and α1,3GalT‐KO) tissues using thin layer chromatogram and microtiter well binding assays. Results: All anti‐Gal‐specific reagents reacted with the pentaglycosylceramide Galα1,3nLc4, and several 6–12 sugar compounds in wild‐type pig kidneys. However, their staining intensity with different αGal antigens varied considerably. Some, but not all, anti‐Gal reagents cross‐reacted with a pure iGb3 glycolipid reference compound. No reactivity with glycolipids isolated from α1,3GalT‐KO pig small intestine or human tissues was found, confirming the specificity of the anti‐Gal reagents in those tissues for α1,3Gal‐epitopes produced by the α1,3GalT (GGTA1). Conclusions: Different anti‐Gal reagents vary in their carbohydrate epitope specificity. Mono‐/polyclonal Abs and lectins have different carbohydrate epitope fine specificity toward pig glycolipids as well as purified Galα1,3nLc4, and iGb3. Despite the difference in αGal specificity, all reagents were completely non‐reactive with glycolipids isolated from α1,3GalT‐KO pig small intestine.