Xenoreactive Antibodies Research Articles

Investigation of potential carbohydrate antigen targets for human and baboon antibodies

The continued presence of a primate antibody-mediated response to cells and organs from alpha1,3-galactosyltransferase gene-knockout (GTKO) pigs indicates that there may be antigens other than Gal alpha 1,3Gal (alpha Gal) 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-alpha Gal antibodies. 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 alpha Gal 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. Both human and baboon sera showed high IgM and IgG binding to alpha Gal saccharides, alpha-lactosamine, and Forssman disaccharide. Human sera also demonstrated modest binding to N-glycolylneuraminic acid (Neu5Gc). When human sera were adsorbed on alpha Gal oligosaccharides, there was a reduction in binding to alpha Gal and alpha-lactosamine, but not to Forssman. WT and GTKO pig sera showed high binding to Forssman, and GTKO pig sera showed high binding to alpha Gal 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. We were unable to identify definitively any saccharides from the selected panel that may be targets for primate anti-non-alpha Gal 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.

Resistance of neonatal porcine Sertoli cells to human xenoantibody and complement-mediated lysis is associated with low expression of α-Gal and high production of clusterin and CD59

Porcine Sertoli cells have an inherent resistance to human xenoreactive antibody and complement-mediated lysis, however, the mechanisms of protection are still unclear. Neonatal porcine Sertoli cells (NPSCs) were isolated from testes of 10 to 15-day-old piglets. Immortalized porcine aortic endothelial cells (iPECs) were used as control cells. An in vitro humoral injury model was used to assess whether NPSCs could resist human xenoantibody and complement-mediated lysis. Expressions of alpha-Gal, clusterin, CD59, CD46, and CD55 were examined to investigate the possible mechanisms of the immunoprotection of NPSCs. Compared with iPECs, NPSCs significantly resisted human natural antibody and complement-mediated lysis when incubated with 20% normal human serum (NHS) in vitro (24.38 +/- 0.50 vs. 53.13 +/- 14.53%, P < 0.01). Mechanistically, NPSCs expressed lower level of alpha-Gal (Gmean: 41.78 +/- 2.39 vs. 95.17 +/- 2.39, P < 0.01), which was correlated with decreased binding of human xenoreactive IgG and IgM. Additionally, NPSCs expressed higher level of clusterin measured by both Western blot and fluorescence-activated cell sorter analysis and expressed more CD59 mRNA detected by reverse-transcription polymerase chain reaction. These data demonstrate that the resistance of NPSCs to human xenoantibody and complement-mediated lysis is associated with low expression of xenoantigen alpha-Gal and high production of the complement inhibitors clusterin and CD59.

Histopathology of xenografts in pig to non‐human primate discordant xenotransplantation

Xenotransplantation could provide a solution to the critical shortage of organs for transplantation in humans. Swine have been proposed as a suitable donor species. Swine organs, however, when transplanted to primates, are rapidly rejected by hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR). Both HAR and AHXR are triggered by xenoreactive natural antibodies directed against a specific epitope (galactose alpha1-3 galactose: Gal) on porcine vascular endothelium. In attempt to prevent HAR and AHXR, alpha1,3-galactosyltransferase gene knockout (GalT-KO) pigs have been produced. GalT-KO pig organs do not express the Gal epitope (antigen), and it therefore can eliminate the anti-Gal antibody--Gal antigen immunoreaction in xenotransplantation. We reported our initial study of kidney transplantation from GalT-KO miniature swine to baboons with either immunosuppression protocol or with a tolerance inducing protocol. Here, we discussed the pathology of xenografts in GalT-KO pig to non-human primate kidney transplantation.

Research of xenograft hyperacute rejection by perfusing pig kidneys with human blood in vitro

Objective Ex vivo studies by perfusing pig kidneys with differents blood to evaluate human antiporcine xenograft hyperacute rejection. Methods Pig kidneys were perfused for 30 minutes with pig blood (group 1; n =3),human blood (group 2; n =5),complement heat innactivated human blood (group 3; n =5), and xenoreactive natural antibodies (XNA) -depleted human blood (group 4; n=5). Renal blood flow rate and vascular resistance were caculated very 5 minutes. Histologic and immunohistologic examinations were carried out after perfusion. Results Pig kidneys perfused with human blood, group 2, showed HAR, higher vascular resistance and lower blood flow rate. Kidneys in Group 1,3,4 had no histologic evidence of HAR. Blood flow rate and vasular resistance kept steady. Conclusion The depletion of complement or XNA in human blood protected pig kidneys form HAR. Key words: Xenotransplant; Rejection; Kidney

Identification of preferred carbohydrate binding modes in xenoreactive antibodies by combining conformational filters and binding site maps

Carbohydrates are notoriously flexible molecules. However, they have an important role in many biochemical processes as specific ligands. Understanding how carbohydrates are recognized by other biological macromolecules (usually proteins) is therefore of considerable scientific value. Interfering with carbohydrate-protein interactions is a potentially useful strategy in combating a range of disease states, as well as being of critical importance in facilitating allo- and xenotransplantation. We have devised an in silico protocol for analyzing carbohydrate-protein interactions. In this study, we have applied the protocol to determine the structures of alphaGal-terminating carbohydrate antigens in complex with a panel of xenoreactive antibodies. The most important feature of the binding modes is the fixed conformation of the Galbeta(1,4)Glc/GlcNAc linkage across all of the binding modes. The preferred conformation of the terminal Galalpha(1,3)Gal linkage varies depending on the antibody binding site topography, although it is possible that some of the antibodies studied recognize more than one Galalpha(1,3)Gal conformation. The binding modes obtained indicate that each antibody uses distinct mechanisms in recognizing the target antigens.

Complement regulation in the GalT KO era

A number of institutes have reported on the successful production of alpha-galactosyltransferase knockout (GalT-KO) pigs. After producing such pigs, hyperacute rejection appeared to no longer be a problem. However, acute vascular rejection (AVR)/acute humoral xenograft rejection (AHXR) is defined as a rejection that begins within 24 h after transplantation and gradually destroys the graft. The origin of AVR/AHXR continues to be a controversial topic, but is generally thought to be initiated by xeno-reactive antibodies, including non-Gal antibodies and subsequent activation of the graft endothelium, the complement and the coagulation systems. The complement is activated via the classical pathway by non-Gal antigens and ischemia-reperfusion injury, via the alternative pathway, especially on islets, and via the lectin pathway. Therefore the complement system is still an important recognition and effector mechanism of AVR/AHXR. In addition, quite recently, based on the relationship between complement and coagulation systems, a new pathway has been proposed. All complement regulatory proteins (CRPs) have the ability to regulate complement activation in different ways. Therefore, to effectively protect xenografts against AVR/AHXR, it appears reasonable to employ not only one but several CRPs including anti-complement drugs. Non-Gal antigens, such as the Hanganutziu-Deicher antigen, is still present on GalT-KO grafts. The further assessment of antigens continues to be an important issue in the area of clinical xenotransplantation. The above conclusions suggest that the expression of human CRPs on GalT-KO grafts is necessary. Moreover, multilateral inhibition of complement activation is required in conjunction with the regulation of the coagulation system.

Activation of Complement in the Discordant Heart Xenotransplantation of Pig-to-Monkey Model and the Impact of Intrathymic Inoculation of Xenogeneic Antigen Combined With Whole-Body γ-Radiation

Objective This study investigated changes in complement in discordant heart xenotransplantation using a pig-to-monkey model as well as the impact of intrathymic inoculation (IT) with xenogeneic antigen combined with whole-body γ-radiation (WBI). Methods In this experiment, pigs and monkeys selected as donors and recipients, respectively, were randomly divided into three groups: a blank group (group A), a whole-body irradiation group (group B) and an irradiation plus intrathymic injection group (group C). In every group, monkeys underwent heterotopic heart xenotransplantation. Results The results showed that the survival of donor hearts in group C was significantly longer than that of group A ( P < .01). In mixed lymphocyte reactions, there was a significant reduction of the stimulation index in group C compared with group A. After xenotransplantation, the level of xenoreactive antibody in group C rose slower than that in group A or group B ( P < .01). After rejection, the levels of CD46 and C3 declined greatly. Conclusions These results suggested that pretreatment with IT and WBI induced T-cell immunosuppression, restraining elicited xenoreactive antibody production of both immunoglobulin M and immunoglobulin G classes. However, it did not hinder complement activation via the classical pathway during hyperacute rejection and consequent xenograft rejection.

In silico analysis of antibody–carbohydrate interactions and its application to xenoreactive antibodies

Antibody–carbohydrate interactions play central roles in stimulating adverse immune reactions. The most familiar example of such a process is the reaction observed in ABO-incompatible blood transfusion and organ transplantation. The ABO blood groups are defined by the presence of specific carbohydrates expressed on the surface of red blood cells. Preformed antibodies in the incompatible recipient (i.e., different blood groups) recognize cells exhibiting host-incompatible ABO system antigens and proceed to initiate lysis of the incompatible cells. Pig-to-human xenotransplantation presents a similar immunological barrier. Antibodies present in humans recognize carbohydrate antigens on the surface of pig organs as foreign and proceed to initiate hyperacute xenograft rejection. The major carbohydrate xenoantigens all bear terminal Galα(1,3)Gal epitopes (or αGal). In this study, we have developed and validated a site mapping technique to investigate protein–ligand recognition and applied it to antibody–carbohydrate systems. This site mapping technique involves the use of molecular docking to generate a series of antibody–carbohydrate complexes, followed by analysis of the hydrogen bonding and van der Waals interactions occurring in each complex. The technique was validated by application to a series of antibody–carbohydrate crystal structures. In each case, the majority of interactions made in the crystal structure complex were able to be reproduced. The technique was then applied to investigate xenoantigen recognition by a panel of monoclonal anti-αGal antibodies. The results indicate that there is a significant overlap of the antibody regions engaging the xenoantigens across the panel. Likewise, similar regions of the xenoantigens interact with the antibodies.

Intrasplenic Preconditioning: A Model for the Study of Xenostimuli Accommodation

Discordant xenotransplantation, the grafting of organs from one phylogenic species to another, results in hyper-acute rejection (HAR). HAR is associated with the deposition of recipient preformed xenoreactive natural antibodies and complement on the endothelium of the donor organ, leading to activation and apoptosis of the endothelium, an event associated with xenograft rejection. Endothelial resistance to HAR, termed "accommodation," an active protection of graft endothelium, may be achieved by previous stimulation of endothelial cells by discordant xenoantibodies. Forty-eight male Wistar rats were used to evaluate HAR induction in an isolated, dually perfused in-situ rat liver transfused with human blood. This ex-vivo model served to mimic rat-to-human liver xenotransplantation. Preconditioning of the liver endothelium was induced by rat intrasplenic injection of human blood (n=8) or effluent of previously xenotransfused rat liver (n=8), i.e., high versus low xenoantibody solution, each undertaken 1d before liver xenotransfusion. Two other groups were not preconditioned. Preconditioned and non-preconditioned rats were perfused directly with human blood, and eight rats were used as controls (non-preconditioned Krebs-perfused). Eight rats were perfused directly with human blood, and eight rats were used as controls. The effluent that exited these first-line livers was used to perfuse the second-line livers. Portal and hepatic artery perfusion pressures, resistances, rates of oxygen extraction, lactic acid and pH, and wet-to-dry weight ratio values were significantly increased in livers xenotransfused with blood indicating HAR, compared with unchanged values in livers perfused with Krebs solution. Portal pressure and resistance were best protected from HAR by the blood preconditioning in the blood perfused group, while the hepatic artery perfusion system was better protected by the perfusate precondition-blood perfused group. The physiologic effects of HAR were attenuated in most second-line livers. Attenuation of HAR in rats' livers is achieved by preconditioning with xenoantibodies and/or by "filtering out" xenoantibodies present in the circulation, and is suggestive of accommodation. This novel method may be useful in future studies aimed at refining methods for accommodating xenotransplantation.

Induction of Human T-Cell Tolerance to Pig Xenoantigens via Thymus Transplantation in Mice with an Established Human Immune System

Thymus xenotransplantation has been shown to induce tolerance to porcine xenografts in mice and to permit survival of alpha1,3Gal-transferase knockout porcine kidney xenografts for months in nonhuman primates. We evaluated the ability of porcine thymus xenotransplantation to induce human T-cell tolerance using a humanized mouse (hu-mouse) model, where a human immune system is preestablished by implantation of fetal human thymus tissue under the kidney capsule and intravenous injection of CD34(+) hematopoietic stem/progenitor cells. Human T-cell depletion with an anti-CD2 mAb following surgical removal of human thymic grafts prevented the initial rejection of porcine thymic xenografts in hu-mice. In these hu-mice, porcine thymic grafts were capable of supporting human thymopoiesis and T-cell development, and inducing human T-cell tolerance to porcine xenoantigens. Human T cells from these mice responded strongly to third-party pig, but not to the thymic donor swine leukocyte antigen (SLA)-matched pig stimulators in a mixed lymphocyte reaction (MLR) assay. Anti-pig xenoreactive antibodies declined in these hu-mice, whereas antibody levels increased in nontolerant animals that rejected porcine thymus grafts. These data show that porcine thymic xenotransplantation can induce donor-specific tolerance in immunocompetent hu-mice, supporting this approach for tolerance induction in clinical xenotransplantation.

Resistance of porcine Sertoli cells to xenoreactive antibodies mediated complement lysis

Objective To investigate whether porcine Sertoli cells eould resist xenoreactive antibodies mediated complement lysis. Methods Sertoli cells were isolated from testes of 10 to 15 day-old landrace pigs. Α-Gal expression on Sertoli cells was measured by FACS and cytoimmunofluorescence. The binding of human se-rum IgG and IgM with Sertoli cells was assayed by FACS. After the incubation of the cultured Sertoli cells with 20% human B serum in vitro, the cellular lysis and cytotoxicity assay were detected with CytoTox-ONETM homogeneous membrane integrity assay and MTT method, and then activation of the complement cascade was examined by immunohistochemistry and immunofluorescence. The SV40-porcine endothelium cells line (SV40-PED) was served as control cells. Results α-Gal expression was found on Sertoli cells by FACS and cytoimmunofluorescence. After incubation with 20% human serum, cellular lysis ratio of the SV40-PED was 53. 13% ± 14.53%, while lysis ratio of the Sertoli cells was significantly lower (24.38% ±0.50%, P<0.01), the viability of Sertoli cells and SV40-PED were 98.73% ± 18.84% and 52.43% ± 8.08%, respectively. With immunohistochemistry and immunofluorescence, C3c and C4d were found binding on both the Sertoli cells and SV40-PED cells, however, C5b-9 was only detected on SV40-PED cells. Conclusion In vitro, compared with the SV40-PED, Sertoli cells could resist xenoreactive antibodies of human serum mediated complement lysis by preventing the C5b-9 formation. Key words: Sertoli cells; α-Gal; Complement

Expression of Tissue Factor and Initiation of Clotting by Human Platelets and Monocytes After Incubation With Porcine Endothelial Cells

Intravascular thrombosis remains a major barrier to successful pig-to-primate xenotransplantation. However, the precise factors initiating thrombosis are unknown. In this study, we investigated the contribution of recipient platelets and monocytes. Primary pig aortic endothelial cells (PAECs) were incubated with combinations of fresh or heat-inactivated human plasma, platelets, or monocytes, after which they were separated and analyzed individually by flow cytometry for tissue factor (TF) expression and for their ability to clot recalcified normal or factor-VII-deficient plasma. Procoagulant porcine TF was induced in PAECs only by fresh human plasma, and not by heat-inactivated plasma, platelets, or monocytes. In contrast, procoagulant human TF was induced on platelets and monocytes after incubation with PAEC, irrespective of whether the plasma was present or not. In addition, human platelets caused the shedding of procoagulant TF-expressing aggregates from PAEC. This work defines a cell-based in vitro assay system to address complex interactions among PAECs, human platelets, and monocytes. The induction of procoagulant TF on PAECs by fresh human plasma was most likely dependent on xenoreactive natural antibody and complement present in fresh human plasma. In contrast, the shedding of procoagulant platelet-PAEC aggregates, induced by human platelets, and the induction of procoagulant TF on human platelets and monocytes by PAEC, occurred independently of these factors. These results suggest that different mechanisms may contribute to the initiation of thrombosis after xenotransplantation, some of which may not be influenced by the further manipulation of the immune response against pig xenografts.

Xenotransplantation: Where are we in 2008?

Xenotransplantation holds promise to solve the ever increasing shortage of donor organs for allotransplantation. In the last 2 decades, major progress has been made in understanding the immunobiology of pig-into-(non)human primate transplantation and today we are on the threshold of the first clinical trials. Hyperacute rejection, which is mediated by pre-existing anti-alpha Gal xenoreactive antibodies, can in non-human primates be overcome by complement- and/or antibody-modifying interventions. A major step forward was the development of genetically engineered pigs, either transgenic for human complement regulatory proteins or deficient in the alpha1,3-galactosyltranferase enzyme. However, several other immunologic and nonimmunologic hurdles remain. Acute vascular xenograft rejection is mediated by humoral and cellular mechanisms. Elicited xenoreactive antibodies play a key role. In addition to providing B cell help, xenoreactive T cells may directly contribute to xenograft rejection. Long-term survival of porcine kidney- and heart xenografts in non-human primates has been obtained but required severe T and B cell immunosuppression. Induction of xenotolerance, e.g. through mixed hematopoietic chimerism, may represent the preferred approach, but although proof of principle has been delivered in rodents, induction of pig-to-non-human primate chimerism remains problematic. Finally, it is now clear that innate immune cells, in particular macrophages and natural killer cells, can mediate xenograft destruction, the determinants of which are being elucidated. Chronic xenograft rejection is not well understood, but recent studies indicate that non-immunological problems, such as incompatibilities between human procoagulant and pig anticoagulant components may play an important role. Here, we give a comprehensive overview of the currently known obstacles to xenografting: immune and non-immune problems are discussed, as well as the possible strategies that are under development to overcome these hurdles.

Resistance to anti-xenogeneic response by combining α-Gal silencing with HO-1 upregulation

Background A major barrier to clinical xenotransplantation is preformed xenoreactive natural antibodies (XNA) found in higher primates which react to Galα(1,3)Gal (α-Gal) epitopes found on lower species. Accommodation of organs to xenogeneic recipients involves upregulation of cytoprotective genes and resistance to complement dependent cytotoxicity (CDC). Methods To develop methods of increasing these organ-protective effects, we established an in vitro CDC model utilizing human serum as the source of XNA and porcine endothelial cells (pEC) as targets. Results Using this system we demonstrated that downregulation of α-Gal epitopes by siRNA silencing of α1,3-galactosyltransferase (α-GT) led to marginal protection from CDC while α-Gal silencing combined with Griffonia simplicifolia isolectin B4 (GS-IB4), a lectin that specifically binds to α-Gal epitopes, led to complete protection. Interestingly, α-Gal silencing and GS-IB4 mediated effects were not associated with inhibition of XNA binding to cells, but with significant decreased E-selectin expression and cytoprotective gene HO-1 upregulation. PI3K inhibitor LY294002 could block the elevation of HO-1 protein expression and reverse the protective effect of α-Gal silencing and GS-IB4 against CDC. Conclusion These data support the use of combination approaches targeting independent accommodation mechanisms to synergistically enhance donor organ survival in a xenogeneic setting.

Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: Potential implications for disease

Human heterophile antibodies that agglutinate animal erythrocytes are known to detect the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc). This monosaccharide cannot by itself fill the binding site (paratope) of an antibody and can also be modified and presented in various linkages, on diverse underlying glycans. Thus, we hypothesized that the human anti-Neu5Gc antibody response is diverse and polyclonal. Here, we use a novel set of natural and chemoenzymatically synthesized glycans to show that normal humans have an abundant and diverse spectrum of such anti-Neu5Gc antibodies, directed against a variety of Neu5Gc-containing epitopes. High sensitivity and specificity assays were achieved by using N-acetylneuraminic acid (Neu5Ac)-containing probes (differing from Neu5Gc by one less oxygen atom) as optimal background controls. The commonest anti-Neu5Gc antibodies are of the IgG class. Moreover, the range of reactivity and Ig classes of antibodies vary greatly amongst normal humans, with some individuals having remarkably large amounts, even surpassing levels of some well-known natural blood group and xenoreactive antibodies. We purified these anti-Neu5Gc antibodies from individual human sera using a newly developed affinity method and showed that they bind to wild-type but not Neu5Gc-deficient mouse tissues. Moreover, they bind back to human carcinomas that have accumulated Neu5Gc in vivo. As dietary Neu5Gc is primarily found in red meat and milk products, we suggest that this ongoing antigen-antibody reaction may generate chronic inflammation, possibly contributing to the high frequency of diet-related carcinomas and other diseases in humans.

Antibody–ligand docking: insights into peptide–carbohydrate mimicry

Despite the enormous clinical importance for xenotransplantation, very little is known about the 3D structural basis for natural antibody recognition of the major carbohydrate xenoantigen, its derivatives and their peptide counterparts. Fundamentally, understanding the nature of peptide–carbohydrate cross-reactivity is necessary to allow a rational design of useful inhibitors. To satisfy this need, we have initiated a project to investigate the structural aspects of antibody recognition of carbohydrate xenoantigens and their peptide mimics by molecular docking. We aim to analyse critical ligand–protein interactions with a focus on identifying the degree of structural carbohydrate mimicry exhibited by peptide ligands. In this paper, we present docking simulations of complexes between a prototypical xenoreactive monoclonal antibody and two ligands: the major carbohydrate xenoantigen, terminal galactose-α(1,3)-galactose epitope [Galα(1,3)Gal] and a peptide inhibitor Galpep1 (DAHWESWL).

Normal human serum contains high levels of anti‐Galα1‐4GlcNAc antibodies

Natural xenoreactive antibodies (Abs) directed against the Bdi-epitope (Gal alpha 1-3Gal beta) on the cells of non-primate mammals take part in hyperacute rejection of xenotransplanted organs. We found that some Abs, which were one-step affinity purified on Bdi-Sepharose, cross-reacted with the disaccharide Gal alpha 1-4GlcNAc beta. The epitope Gal alpha 1-4GlcNAc has not been identified on mammals or bacterial polysaccharides yet. To isolate the antibodies of the corresponding specificity the disaccharide was immobilized on Sepharose and antibodies were affinity purified from pooled serum of blood group O individuals. These one-step purified Abs cross-reacted with Bdi, but after a prior absorption step on Bdi-Sepharose no cross-reactivity with Bdi was observed any longer. Surprisingly, the quantity of anti-Gal alpha 1-4GlcNAc isolated from the same serum pool, 4-7 microg/ml, was equal to that of anti-Bdi or more. Independently of ABO blood groups all the tested healthy donors had anti-Gal alpha 1-4GlcNAc Abs at a similar level. Monospecific anti-Gal alpha 1-4GlcNAc Abs were not cytotoxic towards porcine cells. 1. The actual concentration of monospecific, xenoreactive Gal alpha 1-3Gal beta Abs in blood may be considerably lower than the value referred to in the literature for 'anti-alpha Gal' or 'anti-Galili' antibodies. 2. Anti-Gal alpha 1-4GlcNAc Abs seem not to be important for xenotransplantation.

Characterization of Natural Human Anti-Non-Gal Antibodies and Their Effect on Activation of Porcine Gal-Deficient Endothelial Cells

The generation of Galalpha1-3Gal (Gal) transferase deficient pigs has increased the interest in non-Gal antigens potentially representing important targets for xenoreactive antibody binding leading to xenograft rejection. The present study addressed the levels and immunoglobulin isotypes of preformed human anti-non-Gal antibodies and their potential to activate porcine endothelial cells. Porcine endothelial cells lacking the Gal epitope (Gal-/-) were used to measure immunoglobulin (Ig) M and IgG subclass anti-non-Gal antibodies, using sera from 80 blood donors and pooled human AB serum. Antibodies specific for the non-Gal Hanganutziu-Deicher (HD) xenoantigen were measured by enzyme-linked immunosorbent assay. Activation of Gal-/- and Gal+/+ endothelial cells by human serum was measured, in the presence or absence of complement inhibitors, by E-selectin cell-surface expression using flow cytometry. Anti-non-Gal antibody levels varied considerably among individual sera and comprised approximately 10% of total anti-porcine antibodies without sex or age differences. Among the IgG subclasses only IgG1 and IgG2 were detected. Human serum-induced E-selectin expression on Gal-/- cells was less than 20% compared with Gal+/+ cells, correlated with anti-HD IgM and IgG antibody levels (P=0.027 and 0.032, respectively), and was largely complement-independent in accordance with the lack of IgG3 anti-non-Gal antibodies. In contrast, E-selectin upregulation on Gal+/+ cells was reduced in complement blocking experiments. Preformed anti-non-Gal antibodies, in particular anti-HD antibodies, were present in all human sera samples, activated porcine endothelial cells, and may therefore play a role in xenograft rejection using organs from GalT-/- pigs.

Induction of regulatory cells and control of cellular but not vascular rejection by costimulation blockade in hamster‐to‐rat heart xenotransplantation

In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T-cell activation and promotes a long-term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4(+)T cell-mediated xenograft rejection. Hamster hearts were transplanted into LEW.1A rats receiving an accommodation-inducing treatment consisting of a short course administration of LF15-0195 and a daily administration of cyclosporine A (CSA). To achieve long-term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad-CTLA4Ig) was added to the accommodation induction protocol. On day 40 post-transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti-CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined. Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4(+) xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad-CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non-donor-specific suppressive activity dependent on IL-2, NO, and IDO. These regulatory cells were different from those previously identified after Ad-CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti-donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival. CD28/B7 blockade delays CD4(+) T cell-mediated rejection after CSA withdrawal in accommodated recipients of hamster heart xenografts. In addition, a sustained expression of CTLA4Ig has the potential of inducing cellular regulatory mechanisms. However, such treatment does not prevent the development of xenoreactive IgM antibodies that participate in vascular rejection processes in a complement-dependent manner.

Reactivity of Human Natural Antibodies to Endothelial Cells From Gal??(1,3)Gal-Deficient Pigs

Xenoreactive human natural antibodies (NAb) are predominantly directed against galactose-alpha(1,3)galactose (Gal). Binding of immunoglobulin (Ig) G and IgM NAb activates porcine endothelial cells (pEC) and triggers complement lysis responsible for hyperacute xenograft rejection. In vitro, IgG NAb induce human natural killer (NK) cell-mediated lysis of pEC by antibody-dependent cell-mediated cytotoxicity (ADCC). The present study examined the levels of anti-porcine NAb in a large number of individuals and addressed the functional role of non-Gal anti-porcine NAb. Sera from 120 healthy human blood donors were analyzed for the presence of anti-porcine NAb by flow cytometry using porcine red blood cells (pRBC), lymphoblastoid cells (pLCL), and pEC derived from control or Gal-deficient pigs. Xenogeneic complement lysis was measured by flow cytometry using human serum and rabbit complement. ADCC was analyzed by chromium-release assays using human serum and freshly isolated NK cells. Human IgM binding to pRBC was found in 93% and IgG binding in 86% of all samples. Non-Gal NAb comprised 13% of total IgM and 36% of total IgG binding to pEC. NAb/complement-induced lysis and ADCC of Gal-deficient compared to Gal-positive pEC were 21% and 29%, respectively. The majority of anti-Gal and non-Gal IgG NAb were of the IgG2 subclass. The generation of Gal-deficient pigs has overcome hyperacute anti-Gal-mediated xenograft rejection in nonhuman primates. Non-Gal anti-porcine NAb represent a potentially relevant immunological hurdle in a subgroup of individuals by inducing endothelial damage in xenografts.