Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: influence of pre-existing anti-pig antibodies and prevention by the alpha GAL glycoconjugate GAS914

BACKGROUND: Our introductory pig-to-cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay-accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti-pig antibodies. We evaluated the efficacy of GAS914, a Gal alpha 1-3Gal trisaccharide linked to a poly-l-lysine backbone, in inhibition of HAR. METHODS: hDAF transgenic heterotopic heart (n = 15) or life-supporting kidney (n = 8) transplantation included induction with cyclophosphamide or anti-thymocyte globulin, and maintenance with cyclosporine or tacrolimus, steroids and mycophenolate sodium/mofetil. Four doses of GAS914 were given before transplantation. Rejection was confirmed by graft histology, and anti-pig antibody levels were determined in various assays. RESULTS: Four of six heart transplants without GAS914 treatment showed HAR. Nine subsequent transplants with GAS914 pre-treatment, did not show HAR (chi-square, P < 0.05). Two of four kidney transplants without GAS914 treatment ended with HAR. Four subsequent transplants with GAS914 did not show HAR. Animals with HAR showed extremely high antibody levels. Samples just before transplantation showed significantly higher antibody levels in recipients presenting with HAR. In all assays antibody levels were significantly lowered by GAS914 pre-treatment. CONCLUSIONS: HAR of hDAF solid organs could be ascribed to high levels of anti-pig antibodies. It is hypothesized that the hDAF transgene shows a threshold in efficacy, above which an overwhelming attack by antibodies and complement activation cannot be modulated to prevent HAR. HAR does not occur when animals with lower levels are used, or when antibodies are effectively depleted from the circulation by GAS914 treatment.     

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy

BACKGROUND: The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. METHODS: Seven baboons received life-supporting kidney transplants from hDAF transgenic pigs. Anti-Gal antibodies were neutralized by GAS914 or TPC (a Gal PEG glycoconjugate polymer). Group 1 (n=5) underwent a conventional immunosuppressive therapy with FK506, rabbit anti-thymocyte serum/immunoglobulin, mycophenolate mofetil, and steroids. Group 2 (n=2) received an anti-humoral immunity regimen with LF15-0195, Rituxan and cobra venom factor in addition to ATG, FK506 and steroids. Levels of anti-non-Gal antibodies and their mediated complement-dependent cytotoxic activities (CDC) were detected by flow cytometry using Gal knockout (k/o) pig lymphocytes (LC) or endothelial cells (EC) as targets. RESULTS: Continuous infusion of GAS914/TPC significantly reduced anti-Gal antibodies. In Group 1, four of five baboons developed severe acute humoral xenograft rejection (AHXR) and the rejection was associated with either a high level of preformed anti-non-Gal IgG or a marked elevation in induced anti-non-Gal IgG and IgM. Sera collected at the time of AHXR had a high level of CDC to porcine LC/EC from Gal k/o animals. The intensive anti-humoral therapy in Group 2 completely inhibited both anti-Gal and non-Gal antibody production and prevented AHXR. However, this therapy was not well tolerated by the baboons. CONCLUSION: In a pig-to-baboon kidney transplant model, both preformed and induced anti-non-Gal antibodies are strongly associated with the pathogenesis of AHXR when anti-Gal antibodies are neutralized.     

Soluble Galalpha(1,3)Gal conjugate combined with hDAF preserves morphology and improves function of cardiac xenografts

BACKGROUND: Cytotoxic anti-Galalpha(1,3)Gal antibodies play a key role in the rejection of pig organs transplanted into primates. Regimens reducing anti-Galalpha(1,3)Gal antibodies were associated with severe side effects unable to prevent antibody rebound until soluble synthetic oligosaccharides with terminal Galalpha(1,3)Gal inhibiting antigen binding became available. We displayed kinetics of anti-pig and anti-Galalpha(1,3)Gal IgM and IgG antibody levels using GAS914, a Galalpha(1,3)Gal trisaccharide conjugated to poly-l-lysine, and investigated corresponding changes of parameters of heart function. METHODS: Using a working heart model, hDAF pig hearts were perfused with human blood containing GAS914 (group 1). As controls hDAF pig hearts (group 2) and landrace pig hearts (group 3) were perfused with human blood only. Levels of anti-Galalpha(1,3)Gal (IgM, IgG) and anti-pig antibodies were assessed to prove the effectiveness of GAS914. As parameters of heart function, cardiac output (CO), stroke work index (SWI), coronary blood flow (CBF) and coronary resistance were measured. Creatine phosphokinases, lactate dehydrogenase and aspartate aminotransferase were evaluated as markers of myocardial damage. Histological and immunohistochemical investigations were performed at the end of perfusion. RESULTS: In group 1 an immediate and extensive reduction in both IgM and IgG anti-Galalpha(1,3)Gal was found. Anti-pig antibodies were eliminated accordingly. Antibody binding to GAS914 was complete before the start of organ perfusion. Corresponding to rapid antibody elimination in group 1 GAS914 not only was able to significantly prolong the beating time of the heart in hDAF pigs, but also to clearly improve functional parameters. When switching to the working heart mode hDAF pig hearts perfused with human blood containing GAS914 (group 1) revealed a CO starting at a significantly higher level than hDAF (group 2) and non-transgenic pig hearts (group 3) perfused with human blood only. Similarly, in group 1 SWI was significantly increased at the beginning of perfusion compared to that of group 2 and group 3. The increase in CBF during perfusion and the corresponding fall of coronary resistance occurred without significant differences between the groups revealing the independence of hDAF and GAS914. CONCLUSIONS: Due to an immediate and profound reduction in Galalpha(1,3)Gal-specific antibodies, soluble Galalpha(1,3)Gal conjugates not only prolong survival, but also improve the hemodynamic performance of the heart in DAF pigs.     

Elicited non-anti-alphaGAL antibodies may cause acute humoral rejection of hDAF pig organs transplanted in baboons

The combination of immunosuppression and GAS 914, a polylysine containing alphaGal trisaccharide type 2 (TRI 2), has been associated with the prevention of acute humoral xenograft rejection (AHXR) in human decay accelerating factor (hDAF) pig-to-baboon xenotransplants. The aim of this study was to investigate the role of immunosuppression and GAS 914 to neutralize xenoantibodies before and after xenotransplantation. Eight baboons underwent heteropic heart xenotransplantation with hDAF transgenic pig organs, receiving GAS 914 before and after transplantation. Six baboons (Group A) were treated with an immunosuppression protocol that included cyclophosphamide (CyP), Neoral, ERL, and steroids. The other 2 baboons (Group B) were treated with the same immunosuppression but with a 50% reduction in the doses of CyP. No xenograft from Group A underwent acute humoral xenograft (median survival, 27 days), whereas the 2 in Group B experienced rejection (median survival, 6 days). GAS 914 depleted both immunoglobulin (Ig)M and IgG anti-alphaGAL disaccharide (DI), trisaccharide type 2 (TRI 2), and trisaccharide type 6 (TRI 6), before and after transplantation in Groups A and B. However, cytotoxic antibodies with other anti-pig specificities were elicited by the xenografts in Group B leading to AHXR.     

Administration of GAS914 in an orthotopic pig-to-baboon heart transplantation model

BACKGROUND: Long-term survival of transgenic cardiac xenografts is currently limited by a form of humoral rejection named acute vascular rejection. Preformed and elicited cytotoxic antibodies against Galalpha(1,3)Gal terminating carbohydrate chains, known as the primary cause of hyperacute rejection, are crucial for this process. We investigated whether GAS914, a soluble, polymeric form of a Galalpha(1,3)Gal trisaccharide would sufficiently minimize xenograft rejection of hDAF-transgenic pig hearts orthotopically transplanted into baboons. METHODS: Orthotopic heart transplantations were performed using hDAF transgenic piglets as donors and four non-splenectomized baboons as recipients. Baseline immunosuppression consisted of tacrolimus, sirolimus, ATG, steroids. In addition two animals received low-dose GAS914, and two animals high-dose GAS914. One of these baboons received high dose GAS914 and cyclophosphamide induction therapy. Serum levels of anti-Galalpha(1,3)Gal IgM and IgG antibodies, and anti-pig antibodies were controlled daily by anti-Galalpha(1,3)Gal enzyme-linked immunosorbant assay and anti-pig hemolytic assays. Histomorphological (hematoxylin and eosin, elastic van Gieson) and immunohistochemical (IgM, IgG) evaluations were performed on tissue specimens. RESULTS: Following low-dose GAS914 therapy survival time was 1 and 9 days, respectively. In baboons treated with high dosages of GAS914 a survival of 30 h and 25 days could be obtained. GAS914 caused an immediate and significant reduction of both anti-Galalpha(1,3)Gal IgM and IgG antibodies. However, sufficient antibody reduction was independent of dosage and form of application of GAS914. A pre-transplant GAS914 treatment was not necessary to effectively reduce antibody levels and prevent hyperacute rejection. In the early postoperative period preformed anti-pig antibodies corresponded predominantly to anti-Galalpha(1,3)Gal antibodies making them susceptible to GAS914. Subsequently, while anti-Galalpha(1,3)Gal antibodies remained low, anti-pig antibodies increased despite of GAS914 application. Corresponding to increased anti-pig antibody titers depositions of IgM and IgG immunoglobulins were detected, which were possibly non-Galalpha(1,3)Gal-specific. CONCLUSIONS: Following orthotopic transplantation of hDAF-transgenic pig hearts into baboons, GAS914 is able to maintain a sufficient reduction of Galalpha(1,3)Gal-specific cytotoxicity to the graft. GAS914 therefore is able to prevent not only hyperacute rejection, but also acute vascular rejection at its beginning, when serum cytotoxicity to the pig heart appears to be predominantly Galalpha(1,3)Gal-specific. A sustained prevention of acute vascular rejection, however, still requires the identification of antibody specificities other than to Galalpha(1,3)Gal.     

Transgenic animals in experimental xenotransplantation models: orthotopic heart transplantation in the pig-to-baboon model

Pig organs are at risk for hyperacute and acute vascular rejection mediated by anti-pig antibodies, mainly binding to the Galalpha(1,3)Gal epitope. Acute cellular rejection is characterized by progressive infiltration of mononuclear cells. There is an ongoing search for immunosuppressive regimens that provide adequate protection against all patterns of xenograft rejection, but have no severe impact on the condition of xenograft recipients. Herein orthotopic heart transplantations were performed from hDAF or hCD46 piglets to nonsplenectomized baboons. Basic immunosuppression consisted of tacrolimus, sirolimus, GAS914, steroids, and ATG. Group 1 received basic immunosuppression. Group 2 was additionally treated with rituximab and group 3 with half-dose cyclophosphamide. Group 4 received cyclophosphamide and an anti-HLA-DR antibody. Three baboons received GAS914 and TPC. Monitoring included the regular assessment of anti-porcine antibodies, blood counts, therapeutic drug monitoring, and graft histology. Two grafts failed due to technical mistakes. In group 1, baboons died after 1 and 9 days. In group 2, maximum survival was 30 hours. In group 3, baboons lived 20 hours, 25 days, and 14 days. Group 4 survival times were 9.5 hours, 5.5 hours, 4 days, 34 hours, and 3 days. An increase of non-Galalpha(1,3)Gal antibodies was observed. Depositions of immunoglobulins and complement revealed a humoral rejection process. No cellular infiltration could be observed. In conclusion, suppressing cellular rejection with half-dose cyclophosphamide together with tacrolimus and sirolimus produced longer graft survival with a good general condition. Prevention of acute xenograft rejection further needs inhibition of non-Galalpha(1,3)Gal cytotoxicity by sufficient depression of B-cell activation.     

Demonstration of the functional importance of the Gal epitope in an ex vivo model of xenotransplantation

Abstract: The galactose a 1-3 galactose terminal disaccharide (Gal epitope) has been identified as the major porcine xenoantigen recognised by xenoantibody in human plasma. Elimination or suppression of the epitope or antibody will be a major factor in overcoming hyperacute rejection. Inhibition of the antibody by depletion or elimination of the epitope by gene knockout may reveal the importance of other xenoantibodies, and in addition elimination of the epitope may unmask or produce other xenoantibody combinations. This study aims to determine the relative importance of anti-Gal antibody and Gal epitope elimination in a functional model of xenotransplantation, ex vivo perfusion of mouse hearts with human plasma on a Langendorff apparatus. Perfusion of mouse hearts with human plasma depleted of anti-Gal antibody demonstrates a protective effect compared to hearts perfused with undepleted plasma with prolongation of survival time from 24.1 to 44.5 min. Similarly, elimination of the epitope is also protective. Hearts from Gal knockout mice, which were generated by gene targeting of the al,3 galactosyltransferase gene, and hearts from appropriate control mice were perfused with human plasma. Gal knockout mice hearts demonstrated an increase in survival time from 10.2 to 33.8 min compared to control hearts. This was accompanied by a decrease in C3c and IgM, but little change in IgG deposition. The protective effect is incomplete, probably due to the effect of antibodies against non-Gal xenoantigens. There was no functional evidence for generation of neo-antigens in the Gal KO mice that were I recognised by naturally occurring human xenoantibodies.     

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. Results: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.     

Porcine endothelial cell activation in hDAF pig hearts transplanted in baboons with prolonged survival and lack of rejection

Depletion of anti-alphaGal antibodies before and after transplantation with GAS 914, a polylysine containing alphaGal epitopes, together with immunosuppression, has been shown to prevent acute humoral xenograft rejection (AHXR) in hDAF pig-to-baboon xenotransplantation. This therapy was associated with low levels of serum anti-alphaGal antibodies and lack of antipig hemolytic antibodies (APA) during the entire transplant course. In the present study we investigated the condition of xenograft endothelial cells and the presence of other antipig antibodies. No xenograft failed because of AHXR. However, endothelial cell markers of activation, such as CD62, CD106, ET-1, and particularly 5A6/8, were detected at necropsy, along with a lack or scarce deposits of IgM and total absence of complement and fibrin. The endothelial cell markers were negative or slightly positive at biopsy obtained 30 minutes after transplantation. At the time of animal death serum xenoantibodies against pig aortic cells were also detected by immunochemistry whereas anti-alphaGal and APHA were almost absent, suggesting that the presence of non-anti-alphaGal and noncytotoxic xenoantibodies may cause endothelial activation.     

Transgenic expression in pig hearts of both human decay-accelerating factor and human membrane cofactor protein does not provide an additional benefit to that of human decay-accelerating factor alone in pig-to-baboon xenotransplantation

This study investigated whether the coexpression of human decay-accelerating factor (hDAF) and human membrane cofactor protein (hMCP) on porcine organs provides an additional benefit to that of hDAF alone to prevent rejection. Heterotopic heart xenotransplantation was performed in baboons with either hDAF (n=5) or hDAF/hMCP (n=5) transgenic pig organs. The only immunosuppression given was GAS914 (a soluble Gal [alpha1-3] Gal polymer) and cyclosporine A. With the exception of one hDAF organ that failed from a left atrium thrombosis, all xenografts developed acute humoral xenograft rejection. Acute humor xenograft rejection occurred at a median time of 152 hr in hDAF hearts and 162 hr in hDAF/hMCP organs. Recipients of hDAF or hDAF/hMCP hearts did not differ in their patterns of serum antiporcine antibodies or in plasma levels of the soluble terminal complement complex sC5b-9. It is concluded that in this pig-to-baboon heterotopic heart transplant, model expression of hDAF/hMCP does not provide an additional benefit in prevention of rejection to that of hDAF alone.     

Co-stimulation blockade targeting CD154 and CD28/B7 modulates the induced antibody response after a pig-to-baboon cardiac xenograft

BACKGROUND: The induced antibodies against Galalpha1,3Gal (Gal) and non-Gal epitopes may contribute to delayed xenograft rejection (DXR). We asked whether blockade of the CD40/CD154 and CD28/B7 co-stimulatory pathways modulates the baboon elicited antibody response to pig Gal and non-Gal antigens. METHODS: Eighteen baboons received heterotopic heart transplants from pigs transgenic for human decay-accelerating factor (n = 13) or membrane cofactor protein (n = 5). Ten reference 'conventional therapy' animals received cyclosporin A, cyclophosphamide and mycophenolate mofetil, with (n = 4) or without (n = 6) anti-CD20. Eight 'co-stimulation blockade' animals received anti-CD154 mAb (IDEC-131) and anti-thymocyte globulin, with (n = 4) or without (n = 4) anti-CD20; two of these animals also received CTLA4-Fc. Anti-alphaGal IgG and IgM, anti-non-Gal antibodies and graft histology were assessed serially. RESULTS: Excluding two early graft failures, median graft survival with conventional therapy was 15 days (range 6 to 36 days, n = 8). Anti-Gal IgG antibody remained low through day 6 to 10, only one graft failure was accompanied by significant rise in anti-Gal IgG, and the anti-non-Gal response was weak (n = 2) or absent (n = 7). However many recipients succumbed with infection (n = 4) or coagulopathy (n = 2); DXR and ICOS+ T cells were prevalent in long-surviving grafts. With co-stimulation blockade, excluding three early graft failures, median graft survival was 7 days (range 6 to 11 days, n = 5). This regimen was very well tolerated, but increased anti-Gal antibody titer within 14 days was associated with graft failure in four of six animals. Although an anti-non-Gal response was present in three of six animals during IDEC-131 monotherapy (one strong, two weak), it was absent in both cases with additional CTLA4-Fc treatment. CONCLUSIONS: As used here, CD154 blockade alone does not completely prevent induction of Gal and non-Gal anti-pig antibodies. Our preliminary data suggest that other co-stimulation pathways, including CD28/B7 and ICOS, are sufficient to mediate high-titer anti-non-Gal antibody to porcine antigens in baboons, and contribute significantly to the pathogenesis of DXR.     

The Anti-Non-Gal Xenoantibody Response to Xenoantigens on Gal Knockout Pig Cells Is Encoded by a Restricted Number of Germline Progenitors

Antibodies directed at non-gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into nonhuman primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgV_H genes that encode antibody responses to non-gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3–21 germline progenitor encode xenoantibodies directed at non-gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine α1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of 'anti-non-gal' xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti-non-gal xenoantibodies cross react with carbohydrate xenoantigens. The galactosyltransferase two gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti-non-gal xenoantibodies in primates are encoded by IgV_H genes that are restricted to IGHV3–21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.     

Anti-non-Gal porcine endothelial cell antibodies in acute humoral xenograft rejection of hDAF-transgenic porcine hearts in cynomolgus monkeys

Abstract: Background: Anti‐Galα1–3Gal (Gal) antibodies play a major role in hyperacute rejection and acute humoral xenograft rejection (AHXR) in porcine‐to‐nonhuman primate transplantation. The role of anti‐non‐Gal antibodies in AHXR is less well defined. Methods: Eleven cynomolgus monkeys received a heterotopic heart transplant from a human decay‐accelerating factor transgenic pig, and maintenance immunosuppression with cyclosporin A or tacrolimus, steroids, mycophenolate sodium or mycophenolate mofetil, and in 10 animals the Gal‐containing soluble glycoconjugate GAS914. Six ended with AHXR (6 to 78 day survival) and five did not show AHXR (9 to 36 day survival). Anti‐Gal antibodies were depleted in vivo with GAS914, or in vitro with Gal‐coated Sepharose beads. IgM‐ and IgG‐class anti‐non‐Gal antibodies in serum depleted of anti‐Gal antibodies were measured by flow cytometry using porcine endothelial target cells. Results: Compared with pre‐transplant values, all six recipients with AHXR showed a substantially higher level of anti‐non‐Gal IgM antibodies at rejection; in five animals there was also an increase in IgG‐class antibodies. There was no relevant change in recipients without AHXR. AHXR at time of cessation of heart contraction could be preceeded by a steady increase in antibody level starting 2 to 3 weeks earlier. Conclusions: AHXR is invariably associated with increased circulating anti‐non‐Gal antibodies. These antibodies are not observed in recipients without AHXR, and five of six recipients with AHXR were adequately depleted of anti‐Gal antibodies by maintenance GAS914. This indicates that anti‐non‐Gal antibodies play a significant role in the pathogenesis of AHXR. Also, the assessment of these antibodies could be used as an early monitor of AHXR.     

Fluorescent microspheres reveal different regional blood flow in hyperacutely rejected nontransgenic and hDAF pig hearts

Classic features of hyperacute rejection show differential severity in the inner compared to the outer myocardium. In the present study, regional blood flow (RBF) measured by fluorescent microspheres served as a marker of the extent of hyperacute rejection. Using a working heart model, hearts of nontransgenic and hDAF transgenic pigs were perfused with human blood. Additionally, hDAF transgenic pig hearts were perfused with human blood containing GAS914 or the GPIIb/IIIa inhibitor tirofiban. Injections of fluorescent microspheres into the donor heart were performed in situ and during perfusion. Reference arterial blood samples were collected from the inferior aorta and the afterload line. Perfusion was terminated before hyperacutely rejected hearts failed to pump against the afterload column. RBF was determined in tissue samples of standardized areas of the left atrium and ventricle. Each specimen was divided into subepicardial and subendocardial tissue samples. Fluorescence intensity was measured using an automated luminescence spectrometer. At the end of perfusion with human blood, hyperacutely rejected nontransgenic pig hearts showed a higher RBF in the subendocardium. In hDAF-transgenic pig hearts perfused with unmodified human blood the subendocardial/subepicardial blood flow ratio changed in favor of the subepicardium. This ratio was not further improved by GAS914. In contrast, tirofiban was able to assimilate subepicardial and subendocardial blood flow. In conclusion, RBF of hyperacutely rejected pig hearts was inhomogeneous. Inhibition of complement activation improved the reduced subepicardial RBF, but depletion of antibodies had no positive effect. The ability of tirofiban to further increase subepicardial RBF affirms thrombosis of subepicardial veins as the defining characteristic of hyperacute rejection.     

Suppression of Natural and Elicited Antibodies in Pig-to-Baboon Heart Transplantation Using a Human Anti-Human CD154 mAb-Based Regimen

Natural and elicited antipig antibodies (Abs) lead to acute humoral xenograft rejection (AHXR). Ten baboons underwent heterotopic heart transplantation (Tx) from human decay-accelerating factor (hDAF) pigs. Depletion of anti-Galalpha1, 3Gal (Gal) Abs was achieved by the infusion of a Gal glycoconjugate from day-1. Immunosuppression included induction of antithymocyte globulin, thymic irradiation, and cobra venom factor, and maintenance with a human antihuman CD154 mAb, mycophenolate mofetil, and methylprednisolone; heparin and prophylactic ganciclovir were also administered. Pig heart survival ranged from 4 to 139 (mean 37, median 27) days, with three functioning for >50 days. Graft failure (n = 8) was from classical AHXR [4], thrombotic microangiopathy [3], or intragraft thrombosis [1], with death (n = 2) from pneumonia [1], or possible drug toxicity (with features of thrombotic microangiopathy) [1]. Anti-Gal Abs (in microg/mL) were depleted by Gal glycoconjugate before graft implantation from means of 41.3 to 6.3 (IgM) and 12.4-4.6 (IgG), respectively, and at graft excision were 6.3 and 1.7 microg/mL, respectively. No elicited Abs developed, and no cellular infiltration was seen. The treatment regimen was effective in maintaining low anti-Gal Ab levels and in delaying or preventing AHXR. The combination of costimulatory blockade and heparin with Tx of a Gal-negative pig organ may prolong graft survival further.     

Coagulation cascade activation triggers early failure of pig hearts expressing human complement regulatory genes

Abstract: Background: Hyperacute rejection (HAR) and early graft failure (EGF) have been described in a minority of pig‐to‐baboon heart transplants using organs transgenic for human complement regulatory proteins (hCRP). Here we investigate the role of coagulation cascade activation in the pathogenesis of HAR and EGF in a consecutive series where a high incidence of these outcomes was observed. Methods: Twenty‐eight naïve wild‐caught Papio anubis baboons received heterotopic heart transplants from pigs transgenic for hDAF (n = 23) or hMCP (n = 5). Immunosuppression consisted of cyclosporine A, cyclophosphamide and MMF (n = 18) or anti‐CD154 mAb (IDEC‐131) and ATG (n = 10). Eleven received anti‐Gal carbohydrates (GAS914, n = 8, or NEX1285, n = 3), of which four also underwent extracorporeal immunoadsorption (EIA), and 12 also received pharmacologic complement inhibitors (C1 INH, n = 9, or APT070, n = 3). Results: Excluding one technical failure, 14 of 27 transplants (11 hDAF, 3 hMCP) exhibited either HAR (n = 10) or EGF (n = 4). Surprisingly, neither complement inhibition (with C1 INH or APT070) nor anti‐Gal antibody depletion with GAS914, NEX1285, or additional EIA consistently prevented HAR or EGF despite low or undetectable complement deposition. Strikingly, most grafts with HAR/EGF exhibited prominent fibrinogen and platelet deposition associated with systemic coagulation cascade activation, consistent with non‐physiologic intravascular coagulation, in many instances despite little evidence for antibody‐mediated complement activation. Conclusion: We conclude that dysregulated coagulation correlates closely with and probably causes primary failure of pig hearts transgenic for hCRP. These data support efforts to define effective strategies to prevent dysregulated coagulation in pig organ xenografts.     

Late CD8+ T cell-dependent xenoantibody production in innate tolerant nude rats after hamster islet grafting but not after hamster heart grafting

BACKGROUND: Xenograft rejection can be provoked by both the innate and adaptive immune compartments and close reciprocal interactions exist between these two systems. We investigated the interdependent roles of T and B lymphocytes in vascularized (heart) and cellular (islet) xenograft rejection in a model with established xeno-nonreactivity of the innate immune system. METHODS: Specific innate xenotolerance was induced in nude rats bearing either a hamster heart or a hamster pancreatic islet graft by a tolerizing regimen consisting of donor antigen infusion, temporary natural killer cell depletion and a 4-week administration of leflunomide. One month after transplantation, syngeneic CD4 and CD8 T cells were adoptively transferred. RESULTS: Both vascular and cellular xenografts were rejected after CD4 T cell reconstitution, corresponding with production of high IgM and IgG xenoantibody titers. Deposition of xenoantibodies and complement was seen in the heart but not in the islet xenografts. After infusion of CD8 T cells, xenohearts underwent a delayed type of rejection without xenoantibody production and xenoislets were not rejected. In xenoislet recipients, CD8 dependent B cells were not tolerized, resulting in the production of IgG xenoantibodies belonging to Th2-dependent isotypes, known not to cause graft rejection, and deposited at the graft implantation site. CONCLUSIONS: We conclude that distinct mechanisms of immune activation underlie xenogeneic reactions against vascular and cellular grafts.     

Characterisation of human natural anti-sheep xenoantibodies

ABSTRACT: Currently, the pig species is regarded as the most likely organ donor for human xenotransplantation in the future. However, it cannot be granted that the pig will be the optimal species of choice. We have studied human anti-sheep antibodies in comparison with anti-pig antibodies. The anti-sheep lymphocytotoxic and hemagglutination titers were in the range 8 to 128 and 2 to 32, respectively, in single individuals, which were considerably lower than the anti-pig titers of these individuals. Perfusion of sheep kidneys with human blood reduced the anti-sheep xenoantibody titers to zero as measured by lymphocytotoxic, hemagglutination, and sheep aortic endothelial cell antibody binding assays. The perfused kidneys showed generalised depositions of human IgM and C3c in the vascular tree and focal depositions of C1q and fibrin. Obliteration of capillaries by human platelets and polymorphonuclear cells were observed. Total neutral glycolipid fractions were isolated from sheep intestinal, pancreatic, and kidney tissues. By using a chromatogram binding assay, a monoclonal anti-Forssman antibody identified a single compound with five sugar residues in all organs. Several glycolipid bands were stained in all organs by the Galα1-specific lectin I-B₄ from Griffonia (Bandeiraea) Simplicifolia. A human AB serum pool showed staining by both IgG and IgM antibodies of the Forssman and Galα1-terminating components as well as some other, not structurally identified, components. The Forssman and Galα1-reactivity in human sera could be eliminated by immunoadsorbtion using Forssman and Galα1–3Gal-immunoadsorbent columns, respectively. Immunostaining of sheep kidney tissue sections showed the presence of Galα1-terminating epitopes by immunoperoxidase and immunogold silver staining techniques. Proximal convoluted tubules showed a strong staining, while thin loops of Henle, collecting ducts, urothelium, and vessels showed a weaker staining. Distal convoluted tubules and thick loops of Henle were completely negative. In summary, human serum contains anti-sheep xenoantibodies reacting mainly with the Forssman and Galα1-determinants in sheep tissues and the anti-sheep antibody titers are lower than the corresponding anti-pig titers.     

Fucosyl transferase (H) transgenic heart transplants to Gal-/- mice

BACKGROUND: We have previously described the rejection of Gal+ mouse hearts by mice lacking Gala(1,3)Gal (Gal-/-) and demonstrated this to be a model of xenogeneic hyperacute rejection (HAR) which would occur in pig-to-human/primate xenotransplantation, where Gal+ antibody (Ab) and complement (C') mediate HAR. To reduce the amount of Gal present we used fucosyl transferase (H) as a transgene, H transferase competes for the same substrate as Gal transferase and reduces Gal expression by >90%. METHODS: Gal-/- mice received a heart graft from C57BL/6 Gal+ or H transgenic mice and additional Gal Ab and C' provided; HAR was monitored by direct observation for up to 90 min, or by palpation thereafter. When grafts were rejected they were examined macro- and microscopically. RESULTS: H transgenic mice were used as donors to Gal-/- mice; it was found that: 1) C57BL/6 or H transgenic hearts were not rejected by Gal-/- recipients within 90 min in the absence of additional Gal Ab. 2) If additional Gal Ab and C' were provided as fresh normal human serum (NHS), Gal+ (C57BL/6) grafts were rejected by Gal-/- mice in approximately 34 min, whereas H transgenic hearts mostly lasted up to 17 hr, but were then rejected. The histological appearances showed features of both Arthus and Shwartzmann phenomena. 3) Mice hyperimmunized with Gal with anti-Gal titers of >1:20,000, rejected Gal+ grafts in 31 min; the survival was prolonged to 75 min with the H transgenic hearts. CONCLUSION: The presence of the H transgene in donor hearts transplanted to naive Gal-/- mice delays the onset of HAR, but rejection ultimately occurs; if the mice are hyperimmune earlier rejection occurs. The expression of the H transgene alone is insufficient to avoid HAR in the Gal-/- mouse model; the presence of other transgenes and techniques will be required to give an appropriate increase in survival of pig-to-human/primate grafts.     

An ELISA technique for quantitation of human xenoantibodies binding to pig cells: Application in patients with pig kidneys extracorporeally connected to the circulation

ABSTRACT: A quantitative ELISA technique for determination of human anti-pig xenoantibody number in serum samples has been established using pig lymphocytes and pig/rabbit erythrocytes as target cells and a pool of serum from human blood group AB donors. The number of low affinity antibodies binding to the cells was determined by quantitation following the use of aqueous washing of the cells and separation of bound and unbound antibodies with the phthalate oil method. The efficiency of different soluble Galal-3Gal-terminating di- and tri-saccharides to inhibit antibody binding was tested and found to vary between 70–90% at a saccharide concentration of 10 mg/ml. The assay was used to evaluate the antibody changes in two patients who, after plasmapheresis treatments, had pig kidneys extracorporeally connected to their blood circulation. The number of anti-pig IgM/IgG antibodies bound to each pig lymphocyte were reduced from 5,600/13,200 to 1,300/3,100 in patient 1 and from 1,200/6,500 to 500/2,100 in patient 2 by three consecutive daily plasmapheresis treatments. Although the lymphocytotoxic titers were reduced to very low levels, the antibody numbers still present in the blood of patient 1 caused a hyperacute rejection of the pig kidney. However, the antibody levels in patient 2 did not cause rejection of this kidney during 15 min perfusion time. A strong anti-pig antibody response 3 weeks after the perfusion experiment was found in patient 1 as shown by 27,600/245,300 IgM/IgG molecules bound to pig lymphocytes corresponding to an increase of lymphocytotoxic titer from 8 to 512. The second patient showed a much weaker immune response with 1,400/19,800 IgM/IgG antibodies corresponding to a lymphocytotoxic titer increase from 8 to 32. The use of this quantitation technique enables more accurate investigation of antibody bindine to xenoeenic tareet cells than conventional titration techniaues.