Gal Knockout and Beyond

Recently, Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout (k/o) pigs have been developed using genetic cloning technologies. This remarkable achievement has generated great enthusiasm in xenotransplantation studies. This review summarizes the current status of nonhuman primate experiments using Gal k/o pig organs. Briefly, when Gal k/o pig organs are transplanted into primates, hyperacute rejection does not occur. Although graft survival has been prolonged up to a few months in some cases, the overall results were not better than those using Gal-positive pig organs with human complement regulatory protein transgenes. Gal k/o pig kidneys rapidly developed rejection which was associated with increased anti-non-Gal antibodies. Although the precise mechanisms of Gal k/o pig organ rejection are not clear, it could result from incomplete deletion of Gal, up-regulation of new antigen (non-Gal antigen) and/or production of non-Gal antibodies. Future work in xenotransplantation should place emphasis on further modification of donors, such as combining human complement regulatory genes with Gal k/o, deleting non-Gal antigens and adding protective/surviving genes or a gene that inhibits coagulation. Induction of donor-specific T- and B-cell tolerance and promotion of accommodation are also warranted.     

Warfarin or Low-Molecular-Weight Heparin Therapy does not Prolong Pig-To-Primate Cardiac Xenograft Function

Microvascular thrombosis is a prominent feature in cardiac delayed xenograft rejection (DXR). We investigated the impact of warfarin or low-molecular-weight heparin (LMWH) anti-coagulation on xenograft function using a heterotopic pig-to-primate model. Donor hearts were from CD46 transgenic pigs and baboon immunosuppression included tacrolimus, sirolimus, anti-CD20 and TPC, an alpha-galactosyl-polyethylene glycol conjugate. Three groups of animals were studied. Group 1 (n = 9) was treated with warfarin, Group 2 (n = 13) with LMWH and Group 3, received no anti-coagulant drugs. The median duration of xenograft function was 20 days (range 3-62 days), 18 days (range 5-109 days) and 15 days (range 4-53 days) in Groups 1 to 3 respectively. Anti-coagulation achieved the targeted international normalized prothrombin ratio (INR) and anti-factor Xa levels consistent with effective in vivo therapy yet, no significant impact on median xenograft function was observed. At rejection, a similar histology of thrombosis and ischemia was apparent in each group and the levels of fibrin deposition and platelet thrombi in rejected tissue was the same. Anti-coagulation with warfarin or LMWH did not have a significant impact on the onset of DXR and microvascular thrombosis. However, a role for specific anti-coagulant strategies to achieve long-term xenograft function cannot be excluded.     

Xenograft accommodation is accompanied by intragraft Th2 cytokines and vascular expression of protective genes

Abstract: We have studied the accommodation (survival of an organ graft in the presence of anti-graft antibodies and complement) of heterotopic Golden Syrian hamster heart xenografts transplanted to Lewis rat recipients. The rats were treated with cyclosporine A (15 mg/kg/day i.m.) for the duration of the experiment and for 11 days with cobra venom factor. This regimen resulted in long-term xenograft survival in approximately 75% of cases. Analysis of endothelial cells (and smooth muscle cells) in long-surviving grafts showed expression of "protective" genes: A20, Bcl-x_L, Bcl-2, and hemoxygenase, which we define as genes that prevent endothelial cells from undergoing responses that might lead to graft rejection. Surviving xenografts were also associated with intragraft Th2 cytokine expression. Rejected grafts did not express the protective genes and had a Thl pattern of cytokine expression. These studies indicate a potential mechanism linking molecular and cellular responses to development of xenograft accommodation.     

Preoperative Depletion of C3 Improves the Survival of Guinea Pig-to-Rat Cardiac Xenograft Recipients

Rat strains with congenitally reduced total hemolytic complement activity do not reject cardiac xenografts hyperacutely. Prolongation of graft survival in the guinea pig-to-C6-deficient PVG rat donor/recipient combination has been observed. However, experience with this model has been complicated by a high postoperative mortality from respiratory distress. The authors hypothesized that placement of the xenograft resulted in local activation of complement, which contributed to remote pulmonary injury leading to respiratory dysfunction. To test this hypothesis, an attempt was made to reduce early complement component activation with the use of an antibody to rat C3 in C6-deficient PVG recipients. Six of eight untreated C6-deficient PVG recipients died in the immediate postoperative period with vigorously beating heart grafts, whereas only 2 of 14 C6-deficient recipients pretreated with anti-rat C3 antibody died within 24 h postoperatively. Although pretreatment with antiC3 antibody improved survival of recipients, the duration of cardiac xenograft survival was similar whether the recipients were pretreated or not. The use of anti-C3 antibody in C6-deficient rats is a valid approach to studying xenotransplantation in the absence of hyperacute rejection and has an additional advantage in that it does not require the use of expensive reagents such as cobra venom factor.     

异种移植排斥机理的探讨

补体的激活是超急性排斥的中心环节,为了研究经典及旁路途径在这种排斥中的作用,本研究建立了体外超急性排斥模型.选择猪血管内皮细胞为靶,人血清为天然抗体和补体源,用四唑盐法(methyl thagolyl tetragoliam,MTT)行补体依赖的细胞毒反应(complement-dependent cytotoxicity,CDC).人血清能溶解58±5%的猪血管内皮细胞.加入EGTA阻断经典途径后人血清的溶细胞率降为51±3%(P<0.01).同样Clq缺乏的人血清仅溶解37±7%猪血管内皮细胞(P<0.001).人血清50℃加热20min,阻断旁路途径后,其溶细胞率降为42±5%(P<0.001).B因子缺乏的人血清(阻断旁路途径)仅溶42±10%的猪血管内皮细胞(P<0.001)同种猪血清及加热灭活补体的人血清不溶猪血管内皮细胞.将经典途径及旁路途径缺陷的人血清等体积混合,血清的细胞毒作用恢复正常.同样,Clq缺乏人血清和B因子缺乏人血清分别加入Clq和B因子后,血清细胞毒作用亦恢复正常.在这一体外异种超急性排斥模型中,补体经典和旁路两条途径均参与超急性排斥反应.提示抑制猪/人之间的超急性排斥应考虑补体两条途径均激活的问题.     

Profile of Cytokine Production During the Inhibition of Acute Xenograft Rejection

(Received for publication on July 22, 1998; accepted on July 13, 1999)     

Manipulation of the anti-αGal antibody-αGal epitope system in experimental discordant xenotransplantation

Abstract: In 1991, evidence was provided to indicate that hyperacute rejection of pig organs by humans and baboons was initiated by the binding of anti-agalactosyl (αGal) antibodies to the vascular endothelium. A search for suitable donor species that do not express aGal epitopes has demonstrated their absence in birds, including ratites (e.g., ostrich, emu) and a reptile (alligator), and very weak expression in a large rodent (the capybara). Studies on ratites would suggest that they are unlikely to be suitable organ donors for humans. In 1991, it was also proposed that anti-aGal antibody-antigen binding could be prevented either by depletion of antibody using an immunoaffinity column of the specific aGal oligosaccharide, or by the continuous intravenous infusion of the oligosaccharide, leading to binding (and thus inactivation) of the antibody to the aGal hapten. Synthetic aGal oligosaccharides have recently become available in sufficient quantities to allow experience to be gained with immunoaffinity columns, which have demonstrated complete, but temporary, elimination of baboon serum cytotoxicity to pig PK15 cells in vivo. A search for a natural and inexpensive source of aGal has revealed a subfraction of porcine stomach mucin that demonstrates high potency in inhibiting anti-aGal antibodies both in vitro and in vivo. As an alternative therapeutic option, anti-idiotypic antibodies have been produced in mice that are specific for human and baboon anti-aGal antibodies. Their intravenous administration to baboons results in immediate reduction in serum cytotoxicity for 24 hr. Their use in delivering a toxin, such as ricin, to the B lymphocytes that produce the antibody (and express the same idiotype) is suggested. Studies on complement depletion by the administration of purified cobra venom factor (CVF) to baboons have demonstrated that, even in the absence of measurable levels of CH50, both Clq and properdin are deposited on the vascular endothelium, and that IgM, IgG, and IgA bind to the endothelium of a transplanted pig heart. The prior identification of anti-αGal IgM, IgG, and IgA by our group suggests that these antibodies might be playing a role in both classical (by IgM and IgG) and alternative (by IgA) complement pathway activation in this experimental model and in pig-to-human xenotransplantation. CVF contains a terminal aGal structure, and its administration to an otherwise unmodified baboon leads to a massive increase in anti-αGal level, suggesting that anti-CVF antibodies may, at least in part, comprise anti-aGal. The important role of anti-aGal antibodies in xenograft rejection has now been clearly established, and techniques for preventing expression of aGal epitopes in donor animals, either by genetic engineering or by gene therapy, are discussed.     

Gal alpha(1-3)Gal expression of the cornea in vitro, in vivo and in xenotransplantation

BACKGROUND: To investigate alpha Gal (Gal alpha1-3Gal beta 1-4GlcNAc-R) expression of the porcine cornea in vitro, in vivo and after xenotransplantation. METHODS: Using the GS-IB4 lectin (Griffonia simplifolia I isolectin B4), the expression of alpha Gal was evaluated in the normal porcine cornea and in cultured corneal stromal and endothelial cells of the second passages. The distribution of alpha Gal epitopes was also evaluated in corneal grafts at 4, 7 and 10 days after pig-to-rat orthotopic corneal transplantation. RESULTS: The expression of alpha Gal was mostly confined to the anterior stromal keratocytes of the normal porcine cornea. However, reactivity for GS-IB4 was markedly increased during cell culture passage (P)-by 18.0% (P1) and 39.0% (P2) in cultivated keratocytes, and by 62.1% (P1) and 87.1% (P2) in cultured endothelial cells. The expression of alpha Gal epitopes was gradually enhanced in all corneal layers of the graft after transplantation. CONCLUSION: Alpha Gal expression in the cornea was gradually induced during in vitro culture and after xenotransplantation, suggesting a role of putative Gal-related acute humoral rejection in porcine corneal xenotransplantation.     

Anti-Gal antibody-mediated skin graft rejection requires a threshold level of Gal expression

Abstract: Background: Despite overcoming xenograft hyperacute rejection (HAR), Gal (galactose‐α1,3‐galactose) expression may not be completely eliminated from the α1,3‐galactosyltransferase gene knockout (Gal KO) pig because of alternative galactosyltransferases. Whether low levels of “residual” Gal are still susceptible to either complement fixing or non‐complement fixing antibody beyond the HAR barrier remains unknown. Furthermore, it would be impossible to analyze the immune response specific to low‐level Gal in a xenograft setting given the multitude of xenoantigens that could induce a recipient response. To investigate this question, we therefore used a skin graft model in BALB/c mice where the sole difference between donor and recipient was the expression of Gal, where rejection is caused by passively administered anti‐Gal monoclonal antibody and where HAR does not occur. Methods: Gal expression over time was examined by immunohistochemistry in wildtype‐to‐Gal KO skin grafts. Graft rejection in response to passively administered anti‐Gal monoclonal antibody at early and late time points was studied to determine changes in susceptibility to antibody. To independently test the effect of reduced Gal expression on antibody‐mediated rejection, we used two separate lines of α1,2‐fucosyltransferase transgenic mice as skin donors in the model. These mice have known reduced but different levels of Gal as determined by flow cytometry on peripheral blood leukocytes. Results: Gal expression on skin grafts diminished with time with a corresponding reduction in susceptibility to antibody‐mediated rejection. Skin grafts at day 30 (n = 7) and 150 (n = 11) had a rejection rate of 100% and 45% respectively in response to non‐complement fixing anti‐Gal antibody administered to the recipient. Similar results were demonstrated with a complement fixing anti‐Gal antibody. When α1,2‐fucosyltransferase transgenic mice skin was used in the model, the line with lowest level of Gal expression was resistant to antibody‐induced rejection with a rate 0% (n = 9) vs. 60% (n = 5) in the alternative line with relatively more Gal expressed but still much less than normal mice. Conclusions: Resistance to anti‐Gal antibody‐mediated damage in the model was observed in skin grafts 100 to 150 days post‐grafting but not earlier and was associated with a reduction in Gal expression. It is possible that below a threshold level of Gal expression, the grafts were not susceptible to anti‐Gal antibody.     

Gal alpha 1,3Gal expression on porcine pancreatic islets, testis, spleen, and thymus

Abstract:  Gal α 1,3Gal (Gal) is the first target in antibody-mediated rejection of pig-to-non-human primate xenograft. Its expression may vary between organs and constituents of organs. Gal expression was studied in pancreas, testis, spleen and thymus of 22 pigs, with ages ranging from 1 to 22 months. The immunoperoxidase technique using the biotinylated lectin, Griffonia simplicifolia (IB4), was used. In the pancreas, neither endocrine (islet cells) nor exocrine cells expressed Gal. The Sertoli cells in the testis were negative. The spleen capsule and trabeculae did not stain for Gal, although both splenic T and B lymphocytes expressed Gal (B > T). Thymocytes were weakly positive, whereas thymic epithelial cells were negative for Gal. No age-related differences were seen in any tissues. Porcine islets of Langerhans, Sertoli cells, and the splenic and thymic structural frameworks did not express Gal, and therefore, should be relatively resistant to anti-Gal antibody-mediated rejection. The availability of pigs deficient in Gal as a source of islets may therefore not be beneficial in extending islet graft survival in non-human primate models.     

Isolation and partial characterization of Gal alpha-containing polyglycosylceramides from porcine tissues

Abstract:  Mammalian cell surface carbohydrate antigens are present both as glycoproteins and glycolipids. Of the glycolipids, polyglycosylceramides (PGC) have very long carbohydrate chains extending out from the cell surface. Hereto, Gal α -terminating xenoantigens in pig tissues have been identified in glycoproteins and short chain glycolipids but no studies of the complex PGC have been performed. In this communication, we describe the isolation and partial characterization of PGC from pig erythrocytes, small intestinal mucosa, kidney and liver. The mucosa, kidney and liver PGC fractions contained a complex pattern of Gal α antigens as shown by immunostaining using the Griffonia Simplicifolia isolectin B₄ while no reactivity was found with the erythrocyte PGC fractions. The mucosa PGC fractions stained strongly for blood group A antigens while the erythrocyte PGC fractions were negative. The presence of Gal α -terminating PGC compounds in porcine tissue adds further complexity to the distribution of this xenoantigen. Due to the long carbohydrate chains, PGC will be important targets for the Gal α xenoantibodies in pig to human xenotransplantation.     

Production of human monoclonal antibodies against Galalpha(1-3)Gal

The hyperacute rejection observed in models of pig-to-human xenotransplantation is mainly because of the presence of natural antibodies in human blood with specificity for the Galα(1–3)Gal (Gal) carbohydrate moiety present on the surface of porcine endothelial cells. Human monoclonal anti-Gal antibodies could be of use both in the study of the basic mechanisms of hyperacute rejection as well as in its clinical prevention. In the present study we prepared 42 heterohybridomas (human–mouse) secreting antibodies with specificity for the Gal epitope. All of the antibodies produced were of the IgM isotype, according to a dot-blot assay. Twenty-seven antibodies were further characterized, and shown to be specific for Gal by different methods, including an enzyme-linked immunosorbent assay, in which the plates were sensitized with mouse laminin as a source of Gal. Specificity was also confirmed using purified Gal carbohydrate in a hemagglutination inhibition assay. The antibodies were shown to mediate lysis of Gal-expressing rabbit erythrocytes in the presence of complement. However, the heterohybridomas themselves were shown to express Gal, a result of the mouse P3x63Ag8.653 hybridoma cells used during hybridoma generation. The presence of this epitope on the surface of anti-Gal-producing cells, and on the antibody itself, represents a limitation to the production of high affinity anti-Gal antibodies.     

Complete Inhibition of Acute Humoral Rejection Using Regulated Expression of Membrane-tethered Anticoagulants on Xenograft Endothelium

Xenotransplantation promises an unlimited supply of organs for clinical transplantation. However, an aggressive humoral immune response continues to limit the survival of pig organs after transplantation into primates. Because intravascular thrombosis and systemic coagulopathy are prominent features of acute humoral xenograft rejection, we hypothesized that expression of anticoagulants on xenogeneic vascular endothelium might inhibit the process. Hearts from novel transgenic mice, expressing membrane-tethered fusion proteins based on human tissue factor pathway inhibitor and hirudin, respectively, were transplanted into rats. In contrast to control non-transgenic mouse hearts, which were all rejected within 3 days, 100% of the organs from both strains of transgenic mice were completely resistant to humoral rejection and survived for more than 100 days when T-cell-mediated rejection was inhibited by administration of ciclosporin A. These results demonstrate the critical role of coagulation in the pathophysiology of acute humoral rejection and the potential for inhibiting rejection by targeting the expression of anticoagulants to graft endothelial cells. This genetic strategy could be applied in a clinically relevant species such as the pig.