Xenoreactive IgM Research Articles

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.

Pre‐treatment of porcine pulmonary xenograft with desmopressin: a novel strategy to attenuate platelet activation and systemic intravascular coagulation in an ex‐vivo model of swine‐to‐human pulmonary xenotransplantation

Von Willebrand factor (vWF) has been proposed as a major contributor to the development of coagulopathy in pulmonary xenotransplantation. Pretreatment of donor swine with 1-deamino-8-d-arginine vasopressin (DDAVP), an analog of vasopressin, can reduce the content of vWF in pulmonary xenografts. Here, we investigate the effects of DDAVP pre-treatment in an ex-vivo perfusion model of pulmonary xenotransplantation. We set up and performed the ex-vivo perfusion using porcine pulmonary accessory lobes and fresh human whole blood (n = 12). Half of the donor swine were given 3 mug/kg DDAVP intravenously for 3 days before ex-vivo perfusion (DDAVP group) and half of them were left untreated (control group). The porcine lung was perfused with fresh blood for 1 h and changes in the following parameters were monitored: pulmonary arterial pressure, pulmonary vascular resistance, blood cell counts, fibrinogen, antithrombin, platelet factor 4, D-dimer, C3a, C4d, and xenoreactive IgM. The release of Galalpha1-3Gal xenoantigen (alphaGal) from porcine lung which had been perfused and retained for 30 min with human blood was assessed by enzyme-linked immunosorbent assay using alphaGal-binding lectin. Both DDAVP and control groups showed typical findings of immediate pulmonary dysfunction: an increase of pulmonary vascular resistance and sequestration of leukocytes and platelets after ex-vivo perfusion. However, in the DDAVP group, the increase of platelet factor 4, C3a, and C4d after perfusion was attenuated compared to that in the control group. The release of alphaGal after blood retention was significantly lower in the DDAVP group than that of the control group. Pre-infusion of DDAVP to the donor swine was beneficial in attenuating platelet activation as well as complement/coagulation activation. These effects of DDAVP are likely to relate to the reduction of porcine vWF content in the xenograft. Therefore, the modulation of vWF secretion in donor lungs could be an additional therapeutic way to reduce systemic coagulopathy in pulmonary xenotransplantation.

FK778 in Experimental Xenotransplantation: A Detailed Analysis of Drug Efficacy

This study examines the efficacy of FK778 regimens for prevention of different phases of xenograft rejection. Antibody and complement tissue depositions were measured by immunofluorescence in a discordant ex vivo rat-to-human heart perfusion model of hyperacute rejection with immunosuppressant-enriched human blood. The concordant hamster-to-rat aortic xenotransplantation model was used to assess host cellular (lymphocyte activation, mixed lymphocyte reaction [MLR]) and humoral responsiveness (xenoantibody production) as well as histologic xenograft rejection. Recipients were treated for 14 days with FK778, tacrolimus, sirolimus or combination regimens at varying doses. Antibody binding during hyperacute rejection was unaffected by the immunosuppressive treatment, but complement deposition was reduced in the following order: tacrolimus > FK778 approximately sirolimus. FK778 most effectively reduced complement factor 5 in vitro. In untreated rats with hamster aortic xenografts, a large infiltrative response was observed within the grafts with extensive myocyte necrosis. Tacrolimus > FK778 approximately sirolimus dose-dependently diminished xenograft infiltration and in the same order reduced vessel-wall myocyte necrosis. Tacrolimus approximately FK778 > sirolimus reduced in vivo lymphocyte CD25 expression and tacrolimus > FK778 approximately sirolimus diminished MLR. Xenoreactive IgM and IgG antibody production levels were vigorously upregulated a few days after transplantation, but were significantly reduced by tacrolimus > FK778 approximately sirolimus. Combination regimens revealed no significant benefit when compared with the corresponding monotherapy groups. FK778 mildly interfered with hyperacute rejection and markedly suppressed acute humoral and cellular aortic xenograft rejection. However, T-cell-dependent host responses were most potently suppressed by tacrolimus, and the overall efficacy of FK778 was similar to that of sirolimus.

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.

Analysis of the immune response induced by a single xenoantigen in vivo

Transgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying murine anti-human MHC immune response. We have previously shown that skin from HLA-DR1 transgenic mice was rejected by control littermates and spleen cells from rejecting mice were able to proliferate to donor cells. The aim of this paper is to analyze the mechanism of recognition of this xenoantigen and the possible involvement of antibody response in anti-HLA-DR1 immune response. Control littermates were immunized with spleen cells from HLA-DR1 transgenic (TG) mice; at indicated times, xenoantigen-specific proliferation and IFNγ production was assessed using APC obtained from HLA-DR1 TG mice. Mixed direct-indirect pathway of xenoantigen recognition was suggested by the following findings: i)T cell response to HLA-DR1 was inhibited adding in culture monoclonal antibodies directed either to donor (HLA-DR) or to recipient MHC (I-A); ii) APC from control mice pulsed with purified DR1 molecules were able to induce proliferation by FVB/N mice immunized with transgenic spleen cells. HLA-DR1 recognition permits DR peptide-specific T cell response by lymphocytes of control littermates immunized with the xenoantigen. In addition, we detected xenoreactive IgM and IgG2 antibodies. Our data suggest that HLA-DR1 xenoantigen may be recognized through direct or indirect pathway and provide additional information on mouse anti-human HLA immune response.

IgM-enriched human introvenous immunoglobulin strongly inhibits complement-dependent porcine cell cytotoxicity mediated by human xenoreactive antibodies.

Treatment with intravenous immunoglobulin preparations consisting of human IgG (IVIgG) prevents hyperacute rejection of pig xenografts transplanted into primates by inhibition of the classical complement pathway. Recent studies indicate that IVIg preparations mainly consisting of human IgM (IVIgM) have a stronger capacity than IVIgG to inhibit the complement system. IVIg preparations also contain xenoreactive antibodies (XAb) binding to pig cells. In the present study, we compared IVIgG and IVIgM for their capacity to inhibit xenogeneic complement activation, with special reference to the roles of IgG and IgM XAb present in these preparations. Xenogeneic complement activation was studied by exposure of pig cells (PK15) to human serum. For some experiments, IVIgG and IVIgM were depleted from XAb by immune absorption. Exposure of PK15 cells to human serum induced surface deposition of C4 and C3 and cytotoxicity, which could be inhibited in a dose-dependent fashion by both IVIgM and IVIgG. The efficacy of IVIgM was more than 10 times higher than that of IVIgG. IgG XAb were detected IVIgG and IVIgM whereas IgM XAb were only present in IVIgM. Depletion of XAb from the IVIg preparations did not modify the protective properties of IVIgG against cytotoxicity induced by human serum, whereas the IVIgM-mediated protection against xenogeneic cytotoxicity was only slightly improved. IgM-enriched IVIg is a potent inhibitor of xenogeneic complement activation and complement-dependent cytotoxicity of human serum to pig cells, irrespective of the presence of cytotoxic xenoreactive IgM antibodies in this preparation. Therefore, IVIgM has a promising therapeutic significance for the treatment of (hyper)acute xenograft rejection.

Continued production of xenoimmune antibodies 6-8 years after clinical transplantation of fetal pig islet-like cell-clusters.

We have monitored the humoral immune responses of 10 type I diabetic patients, xenotransplanted with fetal porcine islet-like cell clusters for up to 8 years after xenotransplantation. We investigated the immunoglobulin subclass distribution as well as specificity differences of xenoreactive antibodies. Hemagglutintion tests, using pig erythrocytes, showed that some patients maintained higher titers of xenoreactive IgM antibodies during the entire follow up period, compared with pretransplant levels. In microcytotoxicity tests all but one patient tested showed higher than pretransplant levels of cytotoxic antibodies against pig peripheral blood mononuclear cells (PBMC) 6-8 years after transplantation. Levels of Gal alpha 1,3Gal specific antibodies, were also high. Antibody dependent cellular cytotoxicity (ADCC) activity against a Gal alpha 1,3Gal expressing human B cell line was detected in four patients while ADCC reactivity against adult pig islet cells was detected in only two patients, 6-8 years after transplantation. Immune sera collected 30 days and 1 year after transplantation showed positive staining of adult pig islet cells in fluoromicroscopy whereas sera from later time points did not. Western blot experiments showed that some patients had IgG1 antibodies reactive against epitopes on pig cells other than Gal alpha 1,3Gal, while xenoreactive IgM and IgG2 antibodies mainly reacted with Gal alpha 1,3Gal-containing epitopes as shown by absorption experiments. These results show that patients continue to produce higher than pretransplant levels of IgM and IgG2 xenospecific antibodies against Gal alpha 1,3Gal for extended time periods following xenotransplantation. Some patients also produce xenoreactive IgG1 antibodies directed against non-Gal alpha 1,3Gal epitopes.

Removal of xenoreactive antibodies by protein-A immunoadsorption: experience in 22 patients.

The presence of naturally occurring anti-Galalpha1-3Gal (antialphaGal) Ab in human serum is believed to be a major factor in the pathogenesis of hyperacute rejection of discordant organ xenografts such as the pig-to-human combination. Galalpha1-3Gal epitopes are expressed on pig tissues and the binding of anti-Galalpha1-3Gal leads to endothelial cell activation and complement-mediated hyperacute graft rejection. Several strategies have been suggested in donor animals or in the xenograft recipient to overcome the anti-alphaGal barrier. Protein-A immunoadsorption (PAIA) was developed for the in vivo removal of circulating Ab and it has been shown to be effective in cases where pathogenic auto or alloAb are present. The aim of our study was to analyze the effect of PAIA on total and xenoreactive serum anti-alphaGal immunoglobulin levels in a group of patients treated with this technique for different diseases. After three consecutive sessions of PAIA, total and xenoreactive IgG and IgM immunoglobulin levels were decreased by more than 50% of pre-treatment levels. So we conclude that PAIA is an effective method to significantly reduce circulating Ab, including xenogeneic IgM and IgG Ab. This mode of therapy might be considered as a tool to overcome hyperacute xenograft rejection. PAIA combined with other therapeutic approaches may well protect the xenograft.

Mechanism of concordant corneal xenograft rejection in mice: synergistic effects of anti-leukocyte function-associated antigen-1 monoclonal antibody and FK506.

The mechanisms of corneal xenogeneic immunoreaction, as well as the potential role of immunosuppressive therapy in the suppression of corneal xenograft rejection, have not been thoroughly explored. BALB/c mice who received orthotopic corneal transplants (Lewis rats donors) were administered intraperitoneally anti-leukocyte function associated antigen-1 (LFA-1) monoclonal antibody (mAb) or FK506 (3 mg/kg/day) or both of these immunosuppressants during a 12-day postoperative period. Histological (hematoxylin-eosin stain) and immunohistochemical evaluations of enucleated eyes were performed. Humoral immune response and delayed-type hypersensitivity (ear-swelling assay) were evaluated. The mean (+/-SD) graft survival time in the untreated control, FK506-treated, anti-LFA-1 mAb-treated, and combined-treatment groups was 5.8+/-0.8, 9.4+/-4.0, 8.7+/-5.0, and 67.7+/-16.4 days, respectively. In the untreated control group, mouse IgG, IgM, and C3 were expressed on the rat corneal grafts during the early postoperative phase. Flow cytometry studies revealed high titers of xenoreactive IgG and IgM antibodies. T helper 1 cytokines were expressed on xenografted corneal beds, and delayed-type hypersensitivity was induced. However, local expression of IgM, C3 and T helper 1 cytokines, serum antibodies of IgG and IgM, and delayed-type hypersensitivity were suppressed in the anti-LFA-1 mAb- plus FK506-treated group. Both humoral and cell-mediated immune reaction play an important role in the initial rejection in rat-to-mouse corneal xenotransplantation. The treatment with anti-LFA-1 mAb in combination with FK506 synergistically suppresses concordant corneal xenogeneic reaction.

Involvement of both the classical and alternate pathways of complement in an ex vivo model of xenograft rejection.

It is now generally accepted that complement activation is critical for the hyperacute rejection of xenografts. Activation of the classical pathway as the result of the interaction of xenoreactive IgM xenoantibodies with the vascular endothelium has been observed in all species combinations examined to date. A number of studies using a variety of species combinations have also implicated alternate pathway involvement; however, these studies do not enable a conclusion to be drawn as to whether the alternate pathway can be activated in the complete absence of classical pathway activation. In this study, human plasma was depleted of both Clq and factor D and then reconstituted with purified Clq or factor D to restore the classical and alternate complement pathways, respectively. The ability of these modified plasmas to prosecute hyperacute rejection was then examined using an ex vivo isolated mouse heart perfusion model based on the Langendorff system. In the mouse to human species combination, both the classical and alternate pathways of complement are independently capable of initiating complement activation and mediating xenograft rejection.

Human peripheral blood leukocyte-reconstituted severe combined immunodeficient mouse: analysis of the human immune response against porcine islet transplantation.

Human peripheral blood leukocyte (PBL)-reconstituted severe combined immunodeficient (SCID) mice (Hu-PBL-SCID) were used as a model to study xenograft rejection in humans. SCID mice were reconstituted with human PBL using a protocol that included a booster injection with anti-human CD3 antibody-primed cells. This protocol enhanced chimera establishment in SCID mice and resulted in the detection of higher levels of human Ig when compared with SCID mice receiving unprimed PBL alone. Human xenoreactive natural antibodies (XNA), both IgM and IgG subtypes, which recognized porcine islets (PI), were detected in sera of Hu-PBL-SCID by cytofluorometric analysis. Pretreatment of porcine cells with GS-IB4 lectin inhibited the XNA binding, demonstrating the specificity of the XNA from Hu-PBL-SCID. Western blot analysis showed that XNA from normal human serum and Hu-PBL-SCID serum recognized similar xenoantigens on PI, indicating that Hu-PBL-SCID contained a XNA repertoire representative of normal human serum. Immunofluorescent staining of the tissue sections revealed that both human IgG and IgM bound in vivo to the PI engrafted beneath the kidney capsule of Hu-PBL-SCID. In addition, mouse complement (C3) was detected on xenografted PI. The function of xenografted islets were monitored by measuring porcine insulin concentration using a radioimmunoassay. Porcine insulin concentration in the sera of both Hu-PBL-SCID and plain SCID xenografted with PI was similar for up to 14 days after transplantation, after which the insulin levels in Hu-PBL-SCID decreased, thereby indicating rejection. Therefore, PI transplanted into the Hu-PBL-SCID should be a useful model for the study of cellular as well as acquired humoral immune response against xenoislets.

Selective IgM depletion prolongs organ survival in an ex vivo model of pig-to-human xenotransplantation.

In the pig-to-primate model, xenograft hyperacute rejection (HAR) is mediated by antibody and complement. Previous studies have implicated xenoreactive IgM natural antibody (nAb) as the predominant immunoglobulin involved in HAR. To further evaluate the role of IgM, we selectively reduced IgM levels in human blood, without changing IgG and IgA levels, and then used this blood to perfuse porcine hearts ex vivo. Specific IgM depletion was accomplished with an immunoabsorption column containing sheep anti-human IgM (mu-chain specific) conjugated to Sepharose beads. Human blood was separated into plasma and cellular components. For control experiments, those components were unmodified and recombined in the perfusion system. For experiments with IgM reduced blood, the plasma was passed through the IgM column. Immunoabsorption resulted in approximately 90% reduction in xenoreactive IgM levels, as measured by ELISA. Porcine hearts perfused with unmodified human blood survived 25 +/- 5.6 min (n=5). Porcine hearts perfused with human blood containing reduced levels of IgM survived 229 +/- 45.2 min (n=4; P<0.01). Organ survival was negatively associated with xenoreactive IgM nAb levels measured immediately before perfusion (r=-0.83; P=0.01), and not with IgG nAb levels (r=-0.21; P=0.62). The ability of plasma from IgM-depleted blood to elicit complement activation, measured by iC3b binding to porcine aortic endothelial cells in vitro, was also strongly associated with IgM xenoreactive nAb levels (r=0.92; P<0.0001). Control hearts perfused with unmodified human blood showed typical widespread histologic features of HAR, while porcine hearts perfused with IgM-reduced blood demonstrated milder and less uniform changes. Immunopathological analysis of heart tissues obtained at the completion of each study showed similar deposition of IgG between groups but markedly less IgM, C3, C4, and C9 in the IgM reduction group. These results suggest that selective IgM reduction delays HAR with prolongation of survival and that xenoreactive IgM may be the predominant immunoglobulin involved in HAR in the pig-to-human combination.

THE NATURE OF THE SURFACE CARBOHYDRATES ON PORCINE CELLS WHICH ARE NOT RECOGNIZED BY HUMAN NATURAL ANTIBODIES. • 125

There is a severe shortage of donor organs in the United States. This shortage is even more pronounced in Pediatric patients. A possible solution to this problem may be xenotransplantation using porcine organs. However, a barrier to cross-species transplantation exists in which naturally occurring antibodies bind predominantly to Galα1-3Gal, initiating a rapid and severe rejection of the porcine graft. Even though this initial rejection may be avoided by current therapies, another complication is that the presence of Galα1-3Gal stimulates the production of a very strong elicited humoral response. The initial barrier may be smaller in young patients because natural antibodies in these patients are present in only very low levels. For this reason, xenotransplantation is thought to be more feasible for young patients. However, the presence of the predominant xenogeneic antigen, Galα1-3Gal, poses a threat even in pediatric patients, as it may stimulate the production of elicited antibodies. One possible way to alleviate the humoral barrier posed by xenotransplantation is to use pigs which possess very little Galα1-3Gal recognized by natural human antibodies. Such pigs have been found to exist naturally. Although very little antibody binds to the cells of“low-antigen” pigs, the cells were found to express normal levels of Galα1-3Gal. The mechanism underlying this surprising observation was explored using an enzyme linked dot blot assay and photodensiometric scanning to determine binding of human natural antibodies and lectins to porcine cells. Using this assay, xenoreactive IgM showed greater binding to“high-antigen” cells than low-antigen cells, while the lectin GS-I-B4, which is specific for Galα1-3Gal, bound to high-antigen cells the same as to low-antigen cells. When low-antigen platelets were digested with neuraminidase followed by β-galactosidase, greater binding to low-antigen platelets was seen, suggesting that other non-antigenic carbohydrates may hinder antibody binding to low-antigen porcine cells. The reduced immunologic response to low-antigen organs may be attributed in some degree to the arrangement of the determinant on the organ rather than a reduced amount of antigen. This information may be used to specifically modify porcine organs by transgenic technology which is already available.

Identification of porcine membrane antigens involved in the cytotoxic response mediated by human xenoreactive antibodies

Abstract: The identification of xenoantigens on the surface of endothelial cells is important for understanding the mechanism of hyperacute rejection and development of abrogating methods. The objective of our study was to identify the porcine antigens that, when bound by xenoreactive antibodies in human serum, result in cytotoxicity of porcine cells. Human AB and O sera were adsorbed with porcine aortae, erythrocytes, platelets, and a broad spectrum of immobilized carbohydrate moieties (Synsorbs). Aortae and erythrocytes were able to adsorb the xenoreactive antibodies that were cytotoxic to porcine cells (LLC‐PK1), determined using an MTT cytotoxicity assay. Only carbohydrates having the αGal(1–3)βGal(1–4) moieties (Synsorbs 90, 115) were able to significantly reduce cytotoxicity with both types of sera. Western blots of porcine aortic endothelial cells (PAEC) and LLC‐PK1 cell membrane extracts probed with unadsorbed sera indicate the binding of xenoreactive IgM to approximately 17 and 11 antigen bands, respectively, having molecular weights ranging from 20–133 kDa. Anti‐IgG development showed 8 and 11 antigen bands on PAEC and LLC‐PK1 membrane preparations, respectively. When blots were performed using adsorbed AB sera, the binding to all antigens was still observed. When Synsorb 90 bound antibodies were used to probe the blots, the majority of antigens were still detected. This suggests that the binding of xenoantibodies to the most prominent antigens, as detected by Western blot procedures may not be the ones to which cytotoxic xenoreactive antibodies bind. Alternative approaches are required to identify such antigens.

THE HUMORAL IMMUNE RESPONSE IN HUMANS FOLLOWING CROSS-PERFUSION OF PORCINE ORGANS

A major question in xenotransplantation is the nature of the humoral response that would occur following the transplantation of a xenogeneic organ into an immunosuppressed recipient as such a response could mediate delayed types of injury to the graft. To begin to address this issue we characterized the changes in the properties of xenoreactive antibodies occurring in patients exposed to porcine organs under conditions simulating transplantation. In two patients whose blood had been cross-perfused through porcine livers as a treatment for hepatic failure, the titer of xenoreactive IgM increased by four-fold and the titer of xenoreactive IgG increased by sixty-fold within ten days after perfusion procedures. The xenoreactive IgM and IgG antibodies were specific for Gal alpha 1-3Gal based on binding to porcine endothelial cells and bovine thyroglobulin, which express this determinant, and on the decrease in binding following treatment of porcine endothelial cells or bovine thyroglobulin with alpha-galactosidase. The sequential addition to endothelial cells of amounts of serum known to saturate antibody-binding sites obtained before and ten days after perfusion of porcine organs revealed no increase in binding of IgM above the level observed with serum obtained before perfusion, suggesting that new determinants were not identified. Moreover, the functional avidity of binding to porcine endothelial cells of IgM in serum obtained before and ten days after perfusion of porcine organs was unchanged. Even at later times, the presence of newly elicited antibodies against porcine aortic endothelial cell targets was not detected. Thus, exposure to porcine antigens in a vascularized organ results in increases in the levels of xenoreactive IgM and IgG antibodies--however, these antibodies exhibit properties similar to natural antibodies.

THE HUMORAL IMMUNE RESPONSE IN HUMANS FOLLOWING CROSS-PERFUSION OF PORCINE ORGANS

A major question in xenotransplantation is the nature of the humoral response that would occur following the transplantation of a xenogeneic organ into an immunosuppressed recipient as such a response could mediate delayed types of injury to the graft. To begin to address this issue we characterized the changes in the properties of xenoreactive antibodies occurring in patients exposed to porcine organs under conditions simulating transplantation. In two patients whose blood had been cross-perfused through porcine livers as a treatment for hepatic failure, the titer of xenoreactive IgM increased by four-fold and the titer of xenoreactive IgG increased by sixty-fold within ten days after perfusion procedures. The xenoreactive IgM and IgG antibodies were specific for Gal alpha 1-3Gal based on binding to porcine endothelial cells and bovine thyroglobulin, which express this determinant, and on the decrease in binding following treatment of porcine endothelial cells or bovine thyroglobulin with alpha-galactosidase. The sequential addition to endothelial cells of amounts of serum known to saturate antibody-binding sites obtained before and ten days after perfusion of porcine organs revealed no increase in binding of IgM above the level observed with serum obtained before perfusion, suggesting that new determinants were not identified. Moreover, the functional avidity of binding to porcine endothelial cells of IgM in serum obtained before and ten days after perfusion of porcine organs was unchanged. Even at later times, the presence of newly elicited antibodies against porcine aortic endothelial cell targets was not detected. Thus, exposure to porcine antigens in a vascularized organ results in increases in the levels of xenoreactive IgM and IgG antibodies--however, these antibodies exhibit properties similar to natural antibodies.

Xenoreactive natural antibodies in the world of natural antibodies: typical or unique?

Xenoreactive natural antibodies are thought to be responsible for initiating the hyperacute rejection of porcine organs transplanted into primates. Progress has been made in recently years in the characterization of the specificity and functions of these antibodies. Xenoreactive natural antibodies recognize Galα1-3Gal, a carbohydrate related to the blood group A and blood group B antigens. The presence of Galα1-3Gal may not be sufficient to allow the binding of xenoreactive natural antibodies under physiological conditions; rather, the clustering of Galα3-3Gal determinants may dictate the extent to which xenoreactive natural antibodies attach to surfaces on which Galα1-3Gal is expressed. The predominant role of xenoreactive natural antibodies in the pathogenesis of hyperacute rejection in porcine-to-primate xenotransplantation involves the activation of complement. Complement activation is mediated by IgM, not IgG xenoreactive natural antibodies. Based on functional avidity, thermal binding optima, thermal liability and homogeneity of binding interactions, xenoreactive natural IgM appear to be members of a family of natural antibodies which includes isohaemagglutinins. The classification of xenoreactive antibodies and isochaemagglutinins into a family of antibodies may provide further insight into the nature and physiological function of xenoreactive natural antibodies.

Characterization and affinity isolation of xenoreactive human natural antibodies.

Natural Abs, which are thought to provide an initial defense against invasive microorganisms, include isohemagglutinins, anti-phosphatidylcholine Abs, and anti-alpha-galactose Abs. We have evaluated the physiologic properties of the fraction of human natural Abs that bind to porcine endothelial cells and that would, as a result, initiate the rejection of a porcine organ transplanted into a human. The concentration of xenoreactive IgM in the serum varied widely in the population (5 to 105 micrograms/ml), but was highly dependent on the concentration of IgM in the serum (r = 0.85). Despite this variation and the potential diversity of epitopes recognized, human xenoreactive natural Abs exhibited surprisingly homogeneous binding characteristics, both in one individual and in the population. The apparent avidity determined by using a direct ELISA yielded a functional dissociation constant of 10(-8) M to 10(-10) M, depending on the temperature used. This high functional Kd apparently results from polyvalent interactions between the IgM and the porcine cell surface. Although the xenoreactive IgMs were absorbed by structurally diverse molecules such as ssDNA and thyroglobulin, about 80% of the xenoreactive Abs were specific for the terminal alpha-galactose determinant. A method was developed for affinity isolation of xenoreactive natural Abs by using a thermal extraction procedure. The method quantitatively accounts for all xenoreactive IgM, yielding functional IgM as evidenced by Ag binding and complement activation. Given the overlapping specificity of xenoreactive Abs in the population and the homogeneity of the functional Kd, the natural humoral immunologic barrier to xenotransplantation may be far less formidable than previously thought.

Evidence that polyreactive antibodies are deposited in rejected discordant xenografts.

The rejection of organs transplanted between phylogenetically disparate species is thought to be initiated by the binding of naturally occurring antibodies of the recipient to the endothelium lining the blood vessels of the donor organ. We recently showed that among the xenoreactive natural antibodies in human serum that react with porcine endothelial cells and endothelial cell--derived glycoproteins are polyreactive antibodies. To test whether polyreactive natural antibodies are present in rejected xenografts we developed a series of hybridoma-derived antibodies specific for the polyreactive human monoclonal antibody 103, which we have shown to bind efficiently to porcine endothelial cells. Using these antiidiotypic reagents we detected antibodies bearing the 103 idiotype in a panel of human sera and on the surface of lymphocytes in the spleen of humans, rhesus monkeys, and baboons. The antiidiotypic reagents reacted in a pattern similar to the distribution of IgM, with immunoglobulin deposits in tissues obtained from pig-to-rhesus monkey and pig-to-baboon xenografts. Analysis of immunoglobulin eluted from these sites showed that they antibodies display the antigen-binding features of natural antibodies. Our findings are consistent with the hypothesis that idiotypes of polyreactive natural antibodies are broadly crossreactive. They also suggest that the polyreactive and xenoreactive IgM, which have been detected based on in vitro assays, may be deposited in a xenogeneic organ graft.

THE ROLE OF NATURAL ANTIBODIES IN THE ACTIVATION OF XENOGENIC ENDOTHELIAL CELLS

Hyperacute rejection of organ xenografts is thought to be mediated by the reaction of naturally occurring antibodies and complement of the recipient with blood vessels in the donor organ. We have suggested previously that the pathogenesis of hyperacute rejection might involve the activation of endothelial cells in the graft. To evaluate the potential role of natural antibodies and complement in hyperacute xenograft rejection, sixteen human sera were tested for variation in the ability to activate porcine endothelial cells as manifested by the release of biosynthetically labeled heparan sulfate from the cells. It was then asked to which extent such variation might reflect differences in natural antibody titer and/or complement activity. The sera mediated release of 3.6-57% of endothelial cell-associated heparan sulfate. Heparan sulfate release correlated significantly with the titer, in the sera, of IgM antibodies that bound to cultured endothelial cells (P = 0.0008) or to a triad of glycoproteins believed to represent the major targets of natural antibodies in porcine to primate xenografts (P = 0.001); correlation was also observed with the total concentration of IgM (P = 0.0046). The release of heparan sulfate did not correlate with corresponding properties of serum IgG, with anti-swine hemagglutination or with isohemagglutination titers. Heparan sulfate release correlated with deposition on endothelial cells of iC3b (P = 0.0095), but not with serum complement activity. Our findings indicate that in the reaction between human serum and xenogeneic endothelial cells, it is the concentration of xenoreactive IgM and not differences in complement activity that limits the ensuing pathophysiologic events.