Non-anti-Galα1-3Gal antibody mechanisms are sufficient to cause hyperacute lung dysfunction in pulmonary xenotransplantation

BACKGROUND:Hyperacute lung dysfunction, which is always associated with pulmonary pig-to-primate xenotransplantation is not well understood. The mechanisms associated with its occurrence seem to differ from mechanisms involved in hyperacute xenograft rejection seen in porcine hearts or kidneys transplanted into primates. To determine the contribution of anti-Galα1-3Gal antibodies (αGAb) in such a process, we performed a set of orthotopic pig lung transplants into baboons depleted of αGAb and compared graft function and survival with those receiving only immunosuppression.STUDY DESIGN:Pigs expressing human membrane cofactor protein served as donors. All baboons received triple immunosuppressive therapy. Depletion of αGAb in the experimental group (n = 4) was done by way of immunoadsorption using immunoaffinity membranes. Controls (n = 4) did not undergo immunoadsorption. Orthotopic lung transplants were performed through a left thoracotomy. Main pulmonary artery blood flow and pressure, left pulmonary artery blood flow, and left atrial pressure were recorded.RESULTS:At 1 hour after reperfusion, pulmonary artery graft flows and pulmonary vascular resistances (PVR) were better in animals depleted of αGAb than in controls (605 ± 325.2 mL/min versus 230 ± 21 mL/min; 27.1 ± 41.3 mmHg/L/min versus 63 ± 1 mmHg/L/min). But at 3 hours after reperfusion average graft flows in baboons depleted of αGAb had decreased to 277.6 ± 302.2 mL/min and PVRs had increased 58.3 ± 42.0 mmHg/L/min. On the other hand, controls maintained stable flows and PVRs (223 ± 23 mL/min; 61 ± 3 mmHg/L/min). Survival was ultimately better in control baboons when compared with αGab depleted ones (12.2 ± 3.3 h versus 4.4 ± 3.2 h).CONCLUSION:Unlike heart and kidney xenograft transplants, hyperacute lung xenograft dysfunction seems to be mediated by factors other than αGAb.     

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

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

Failure to deplete anti-Galalpha1-3Gal antibodies after pig-to-baboon organ xenotransplantation by immunoaffinity columns containing multiple Galalpha1-3Gal oligosaccharides

BACKGROUND: The impact of anti-Galalpha1-3Gal (alphaGal) antibodies on the acute humoral xenograft rejection (AHXR) of pig organs transplanted in baboons is unclear. METHODS: Twenty-three baboons underwent heterotopic pig heart transplantation (Tx). Groups A (n = 5) and B (n = 6) received non-transgenic and human decay accelerating factor (hDAF) pig hearts, respectively, without any treatment. Groups C (n = 5) and D (n = 7) were transplanted with non-transgenic and hDAF organs, respectively, and the exclusive treatment was repeated extracorporeal immunoadsorptions (EIA) before and after Tx with an alphaGal column containing disaccharide (DI), trisaccharide (TRI) 2 and pentasaccharide (PENTA) oligosaccharides. RESULTS: In group A, 3 of 5 xenografts underwent hyperacute rejection (HAR). No xenograft from groups B, C and D experienced HAR, most of them failing from AHXR. Immediately after Tx and up to day 2, the level of immunoglobulin (Ig)M and IgG anti-alphaGal DI, TRI2 and TRI6, and anti-pig hemolytic antibody (APHA) antibodies decreased in all the groups by 80 to 96% compared with the concentration present before Tx. From day 3 to AHXR, a sustained increase of anti-alphaGal IgM DI, TRI2 and TRI6, and APHA occurred in all groups. EIA depleted anti-alphaGal IgM and APHA before Tx, but it did not modify the increase of these antibodies after Tx. Baboon serum samples before Tx, pre-incubated in vitro with 1 mg/ml of DI, TRI2 and TRI6, had an average of 93% reduction of anti-alphaGal IgM antibodies specific against each one of these alphaGal oligosaccharides. In contrast, at AHXR, the average reduction after in vitro pre-incubation with either 1 or 5 mg/ml of DI, TRI2 and TRI6 was 40%. CONCLUSIONS: The EIA reduces anti-alphaGal and APHA antibodies, preventing the HAR of non-transgenic pig hearts transplanted in baboons, as does hDAF expression. However, EIA does not modify the level of anti-alphaGal IgM and APHA antibodies after Tx nor the AHXR of either non-transgenic or hDAF pig organs. The increase in anti-alphaGal IgM after Tx was similar for the different antibodies of the anti-alphaGal polymorphism, and was only partially neutralized in vitro with the specific alphaGal oligosaccharide.     

Biophysical characteristics of anti-Gal(alpha)1-3Gal IgM binding to cell surfaces: implications for xenotransplantation

BACKGROUND: Natural antibodies directed against cell surface carbohydrates are thought to be vital to host defense and to initiate the rejection of xenografts and ABO-incompatible allografts. The biophysical properties underlying the association and dissociation of these antibodies from cell surfaces is incompletely understood. We investigated those properties for the binding of Galalpha1-3Gal antibodies to porcine endothelial cell surfaces, because such interactions might be relevant to the clinical application of xenotransplantation. RESULTS AND CONCLUSIONS: The initial rate of binding of anti-Galalpha1-3Gal antibodies to endothelial cells was found to depend on antibody concentration, antibody diffusion, and antigen concentration. The presence of an intact glycocalyx had a greater impact on antibody binding than mobility of antigen in cell membranes. Disruption of glycocalyx increased the amount of antibody bound at equilibrium by more than 50%. Although the binding of anti-Galalpha1-3Gal antibodies to cell surfaces could be inhibited by soluble Galalpha1-3Gal, once bound, some anti-Galalpha1-3Gal could not be dissociated by competitive inhibitors of binding or by denaturation of the bound Ig with chaotropic reagents, but could be dissociated by reduction of disulfide bonds, suggesting that attachment to cell surfaces was, at least in part, by means other than specific reaction with the epitope.     

Anti-Galalpha1-3Gal IgM/IgG antibody levels in infants: do they have a clinical relevance in pediatric xenotransplantation?

BACKGROUND: Anti-Galalpha1-3Gal antibodies (anti-Gal) compose a major obstacle to xenotransplantation. As it is known, there is an immunological window during which infants are thought to have no xenoreactive antibodies. Therefore, we were interested in investigating the occurrence of these antibodies in newborns and infants up to 2 years of age. METHODS: IgM/IgG isotypes of anti-Gal from 74 serum samples of 16 mothers, with the respective cord bloods, and 42 infants of 4 age groups (Group I: day 1-6 months, II: 7-12 months, III: 13-18 months, and IV: 19-24 months) were determined by Enzyme-Linked Immuno-Sorbent Assay (ELISA). A synthetic Galalpha1-3Gal disaccharide-polyacrylamide glycoconjugate was used for coating and monoclonal antibodies were used for the detection of heavy chain isotypes. Antibody concentrations were referred to an internal standard and expressed as arbitrary ELISA units (U). Hemagglutination titers against rabbit erythrocytes (E(R)) were determined in addition. RESULTS: Maternal serum samples showed a wide interindividual variability (IgM: 87 +/- 33 U (mean +/- SD), IgG 59 +/- 39 U) whereas in cord blood no detectable IgM was seen in presence of IgG (52 +/- 34 U). From Group I to IV there was a gradual increase of anti-Gal IgM towards an average of 70% of the adult levels whereas IgG fell to an average of approximately 20% of cord blood levels. Hemagglutination titers followed an increasing tendency with cord blood starting from 1:16 and reaching 1:256 in Group IV. CONCLUSION: The humoral immune response to the Galalpha1-3Gal epitope (alpha-Gal) in infancy follows the generally known development of specific antibodies in humans.     

Anti-Gal alpha 1-3Gal IgM and IgG antibody levels in sera of humans and old world non-human primates

Organs transplanted from pig to primate are rejected within minutes or hours by an antibody-dependent, complement-mediated mechanism [hyperacute rejection (HAR)]. Even after depletion of anti-Galα1-3Gal (Gal) antibody (Ab), for example by extracorporeal immunoadsorption, return of natural Ab is believed to be a major factor in the initiation of acute humoral xenograft rejection. Various non-human primates are used as recipients of pig organs in experimental discordant xenotransplantation (XTx) models. However, anti-Gal IgM and IgG levels in non-human primates may differ from those in humans. Serum levels of anti-Gal IgM and IgG were measured by enzyme-linked immunosorbent assay (ELISA) in humans (n=14), chimpanzees (n=8), baboons (n=214), cynomolgus monkeys (n=29), rhesus monkeys (n=23) and Japanese monkeys (n=6). The mean level of anti-Gal IgM was significantly higher in chimpanzees than in other groups, while in rhesus monkeys it was significantly lower than in other groups, except baboons and Japanese monkeys. The mean human anti-Gal IgG level was higher than in other groups and this difference reached statistical significance except with regard to chimpanzees. The mean anti-Gal IgG level in baboons was significantly lower than that in humans, chimpanzees and cynomolgus monkeys. The measured differences in anti-Gal IgM and IgG levels may affect the kinetics of Ab removal and rate of return in different species, and thus may have relevance for translating work in non-human primate models to the clinical setting.     

Anti-Galalpha1-3Gal antibody levels in organ transplant recipients receiving immunosuppressive therapy

The effect of long-term pharmacologic immunosuppression (PI) on anti-Galalpha1-3Gal (alphaGal) antibody (Ab) levels has not been determined previously in humans. In this study, we measured alpha Gal Ab levels by ELISA in 14 healthy volunteers (controls) and in 70 patients with grafts (kidney, heart, liver) who had received different combinations of PI (including cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil, and steroids) for >3 months. There was great variation in Gal IgM (<80-fold) and IgG (<160-fold). There was no difference in Gal IgM or Gal IgG between any one group and any other. In kidney patients with either high (mean 68%) or low (mean 6%) panel-reactive alloantibodies, there was no difference in alpha Gal Ab level or serum cytotoxicity to pig cells. In vitro immunoadsorption of alphaGal Ab from the serum did not change panel-reactive alloantibody positivity. Therapy with OKT3, a mouse product that might stimulate alphaGal Ab production, led to no significant change in patient Ab levels. We conclude that long-term (>3 months) PI does not reduce Gal Ab levels sufficiently to be of clinical value in xenotransplantation.     

Donor selection for xenotransplantation: detection of Galalpha1-3Gal on different porcine organs.

The expression of the major porcine xenoantigens (Galalpha1-3Gal) in different tissues varies between species. The selection of suitable donors and the interpretation of studies which attempt to prevent hyperacute rejection are dependent on donor expression of Galalpha1-3Gal. Screening of large number of animals to find potential Galalpha1-3Gal negative donors requires a robust, tissue-based and practical method of assessing Galalpha1-3Gal expression. In this study, we have assessed the expression of Galalpha1-3Gal in a variety of pig organs using anti Galalpha1-3Gal antibody. Biopsies of heart, kidney, ear and tail were obtained from 20 outbred pigs. Biopsies were fixed in formalin and stained with a human anti Galalpha1-3Gal antibody obtained from pooled human AB serum passed down a Galalpha1-3Gal immunoadsorbent column. Tissue from all 4 organs from all 20 pigs expressed Galalpha13Gal. This study shows that detection of Galalpha1-3Gal on an ear or tail biopsy is a simple but very reliable method for assessing Galalpha1-3Gal expression on the heart and kidney and facilitates donor selection for xenotransplantation.     

Role of anti-Gal alpha13Gal and anti-platelet antibodies in hyperacute rejection of pig lung by human blood.

BACKGROUND: Previous work has shown that antibodies against porcine antigens are an important trigger of hyperacute lung rejection (HALR). The relative importance of Gal alpha1,3Gal epitopes and other antigens, such as those expressed on pig platelet membranes or lung itself, has not been defined. This study compares the efficiency of three anti-pig antibody depletion strategies, and their efficacy with regard to attenuation of HALR. METHODS: Plasma pooled from three human donors was adsorbed against Gal alpha1,3Gal disaccharide or porcine platelet extract (PPE), or passed through pig lung vasculature. Whole blood reconstituted using adsorbed plasma was then used to perfuse piglet lung, and results were compared with unmodified human blood. RESULTS: Depletion of lung-reactive anti-Gal alpha1-3Gal antibodies was most efficient with the alphaGal column (99% +/- 0.5% vs 87% to 93% +/- 11% for PPE and 92% to 95% +/- 8% for lung, p < 0.01 vs alphaGal column). PPE column tended to be more efficient (77% to 84% +/- 12%) in removing anti-PPE antibodies than pig lung (66% to 70% +/- 14%) or the alphaGal column (56% to 63% +/- 16%, p < 0.05). Lung survival and function with each antibody depletion strategy was improved relative to unmodified controls (mean survival > or = 146 minutes vs 8 minutes for controls). Although alphaGal and lung adsorption yielded more consistent lung protection (survival beyond 2 hours) than did PPE, no approach proved significantly superior. Complement C3a elaboration at 10 minutes was attenuated > 80% by each adsorption strategy, an effect that was most pronounced in the lung adsorption group (95%, p < 0.01). Histamine elaboration was blunted significantly by PPE adsorption but not in other groups (p < 0.05). Platelet but not leukocyte sequestration was decreased with antibody depletion compared with the nondepleted group (44% to 50% vs 82%, p < 0.01). CONCLUSIONS: Each antibody depletion strategy tested significantly prolongs lung xenograft survival and function compared with unmodified human blood, but none was sufficient to reliably prevent HALR. Depletion of antibodies against both alphaGal and additional cell membrane antigens, or control of antibody-independent pathogenic pathways, may be necessary to consistently prevent HALR.     

Importance of the Gal alpha1-3 Gal antigen in discordant islet xenotransplantation: immunosuppression, which inhibits porcine islet xenograft rejection in ordinary mice, is equally effective in Gal-knockout mice

BACKGROUND: Islet xenotransplantation will most likely be performed in diabetic patients treated with immunosuppressive drugs. The importance of the galactosyl alpha(1-3) galactose (Galalpha1-3Gal) antigen in immunosuppressed islet xenograft recipients has not been studied. METHODS: Fetal porcine islet-like cell clusters (ICCs) were transplanted into the renal subcapsular space of both Gal-knockout mice and ordinary mice. Transplantations were performed in untreated mice and mice immunosuppressed with cyclosporine A (CsA) plus 15-deoxyspergualin (DSG). Studies were also performed in immunosuppressed Gal-knockout mice that had been actively immunized against Galalpha1-3Gal. Evaluation was performed 12 days after transplantation using morphologic techniques. The levels of serum immunoglobulin (Ig)G and IgM to the Galalpha1-3Gal antigen or to the ICCs were determined. RESULTS: No difference in the morphologic appearance could be seen between ordinary mice and Gal-knockout mice. No deposits of IgG, IgM, or C3 could be detected. Almost no difference could be seen between immunosuppressed Gal-knockout mice and immunosuppressed ordinary mice. In immunosuppressed, immunized Gal-knockout mice, the results were similar. In ordinary mice treated with CsA+DSG, the levels of anti-Gal IgM were lower than they were in untreated mice, whereas the levels of anti-Gal IgG were similar. In Gal-knockout mice (including immunized animals) treated with CsA+DSG, the levels of anti-Gal IgG and IgM were lower than they were in untreated Gal-knockout mice. CONCLUSIONS: After renal subcapsular transplantation, antibodies against Galalpha1-3Gal have no major influence on islet xenograft rejection in the pig-to-mouse model. Immunosuppression, which inhibits rejection in the pig-to-mouse model, is equally effective when transplantation is performed across the Galalpha1-3Gal barrier.     

Spectrotype analysis and clonal characteristics of human anti-Gal alpha1-3Gal antibodies

Galalpha1-3Gal (anti-Gal), a polyclonal so-called natural antibody (Ab), is present in large amounts in human serum not only as IgG-, but also as IgM- and IgA-isotypes. It has gained a particular interest in the context of xenotransplantation, because the endothelial pig cells express the terminal Galalpha1-3Gal determinant on several adhesion molecules. Little is known of it's function and direct examination of the structure of the Ig genes responsible for coding anti-Gal is lacking. We used the technique of isoelectric focussing (IEF)/affinity immunoblotting for direct analysis of the clonal distribution and spectrotype analysis of IgM- and IgG anti-Gal. By single cell analysis of magnetic bead and fluorescent-activated cell sorter (FACS) isolated mature anti-Gal bearing human B cells from whole blood we analyzed the VH gene families involved in anti-Gal production. Oligoclonal and individually distinct IgG banding patterns were found with isoelectric points between 4 and 9. IgM spectrotypes revealed to be more uniform with a polyclonal banding pattern of more than 12 bands at a pH between 4.7 and 7. IgG- and IgM-banding patterns over a period of 6 months remained unchanged. Single cell polymerase chain reaction (PCR), with all family specific primers, revealed the use of the VH2f gene family for the IgG2 isoptype. The differences found in the spectrotype banding patterns of IgG and IgM could be explained by the suggestion that anti-Gal IgM were produced by the use of unmutated germline genes and the possibility of the absence of somatic mutations. The greater clonal heterogeneity in the IgG population could be explained by somatic hypermutations during the switch from IgM to IgG. The use of this VH2f gene family, which is also involved in the generation of Abs against bacterial pathogens, could mean that this is a predominant region used for the generation of such natural occurring antibodies.     

Localization and identification of T-(Gal beta 1-3GalNAc alpha 1-O-R) and T-like antigens in experimental rat bladder cancer.

A mouse monoclonal antibody and a rabbit polyclonal antibody against the T-antigen (Gal beta 1-3GalNAc alpha 1-O-R) were used to study the distribution of T-antigens in an experimental rat bladder cancer model. Neoplasia was induced in 28 rats by intravesical installation of N-nitroso-N-methylurea (NMU) dissolved in acetate buffer. Fifteen rats were installed with acetate buffer, and served as controls. Urothelial samples were taken from all animals, the atypia was graded and detailed data on the location of the antibody binding structures were obtained by immunohistochemical methods. In addition, Western Blots of glycoproteins and thin-layer-chromatography (TLC) immunostainings of glycolipids extracted from normal and malignant tissue were performed to characterize the molecules presenting T-antigens. Examination of the histologic distribution of T-antigens showed that both the monoclonal and the polyclonal reagents reacted with atypical cells in proportion to the grade of atypia, but showed no reaction in invasive cells. These results confirm previously obtained data on the T-antigen using peanut (arachis hypogaea) agglutinin (PNA), and support the structure identity as being the classical O-linked mucin type T-antigen. Western blots of tumor glycoproteins showed that the monoclonal and the polyclonal antibody reacted with epitopes different from that of PNA, but all the probes correlated with atypia. In addition PNA, as the only anti-T reagent, bound to glycolipid. By using well characterized and highly specific immunological reagents the present study shows that the T-antigen is a highly selective marker of urothelial atypia.     

Pig kidney transplantation in baboons treated intravenously with a bovine serum albumin-Galalpha1-3Gal conjugate

The maintenance of depletion of antibody (Ab) reactive with Galalpha1-3Gal (Gal) on pig vascular endothelial cells by the intravenous (i.v.) infusion of a synthetic Gal conjugate has been proposed as a means of delaying Ab-mediated rejection of transplanted pig organs in primates. We have therefore studied the effect of the continuous i.v. infusion of bovine serum albumin conjugated to multiple synthetic Gal type 6 oligosaccharides (BSA-Gal) on anti-Gal Ab levels and on graft survival in baboons undergoing pig kidney transplantation. Group 1 baboons (n=3) underwent extracorporeal immunoadsorption of anti-Gal Ab, a cyclophosphamide (CPP)-based immunosuppressive regimen, and a non-transgenic pig kidney transplant. Group 2 (n=2) were treated identically to Group 1 but, in addition, received a continuous i.v. infusion of BSA-Gal. Group 3 (n=2) were treated identically to Group 2, but without CPP. A single baboon (Group 4) underwent extracorporeal immunoadsorption, a CPP-based regimen, and continuous i.v. BSA-Gal therapy for 28 days, but did not receive a pig kidney transplant. Two of the transplanted pig kidneys in Group 1 were excised on post transplant days 7 and 13 for a rejected ureter, and disseminated intravascular coagulation (DIC), respectively. The third baboon died of sepsis on day 6. All transplanted ureters and kidneys showed some histopathologic features of acute humoral xenograft rejection. Group 2 baboons were euthanized on days 8 and 11, respectively, for liver failure. At autopsy, there were histopathological features of widespread liver necrosis, but the pig kidneys and ureters showed no features of rejection. The pig kidneys in Group 3 baboons were excised for renal vein thrombosis (day 9) and DIC (day 12); there was no histological signs of rejection in the pig kidneys or ureter, although there were focal areas of modest liver injury in one baboon on biopsy. The single Group 4 baboon showed no biochemical or histological features of liver injury. Anti-Gal Ab levels returned in Group 1, but were maintained at negligible levels in the baboons in Groups 2 to 4 that received BSA-Gal therapy. Continuous i.v. therapy with BSA-Gal is largely successful in maintaining depletion of circulating anti-Gal antibodies and in preventing or delaying Ab deposition and acute humoral xenograft rejection in porcine grafts, but may be associated with liver injury when administered in the presence of a pig kidney transplant and CPP therapy. The mechanism of the hepatic injury remains uncertain.     

IgG2 anti-Galalpha1-3Gal does not induce porcine aortic endothelial cell accommodation in vitro

BACKGROUND: Xenografts that have been protected from hyperacute rejection (HAR) are termed accommodated if they are not then rejected despite the presence of xenoantibody. It has been proposed that IgG may confer resistance to complement dependent cytotoxicity (CDC), a conventional in vitro marker of accommodation. We hypothesized that noncytotoxic IgG2 anti-Galalpha1-3Gal was responsible for this effect. METHODS AND RESULTS: We purified IgG anti-Galalpha1-3Gal from pooled human normal immunoglobulin and three sera, by elution from protein G and Galalpha1-3Gal-R immunoadsorbents. The eluates were IgM free and > or =95% IgG2. They bound to Galalpha1-3Gal, porcine aortic endothelial cells (PAEC) and lymphocytes. It was not possible to block IgM binding to PAEC or lymphocytes using IgG anti-Galalpha1-3Gal (200 microg/ml). The eluates were noncytotoxic in micro-CDC assays. To investigate accommodation, PAEC were cultured with subsaturating doses of the four IgG eluates for up to 144 hr. Resistance of nontrypsinized PAEC to CDC by human serum was measured in a cell viability assay. PAEC were not rendered resistant to CDC in any of the experiments. To investigate the possibility that accommodation might be induced by non-Galalpha1-3Gal IgG, the experiments were repeated using HNIg, again with no protection demonstrated. CONCLUSIONS: Using primary PAEC monolayers, we were unable to induce resistance to CDC with human normal immunoglobulin and its IgG2 anti-Gabeta1-3Gal subset. This contradicts previous experiments using trypsinized, immortalized cells. Although resistance to CDC is not an ideal marker of accommodation, the detrimental effects of IgG make it unlikely that it will become a useful clinical means of inducing accommodation.     

The role of anti-Galalpha1-3Gal antibodies in acute vascular rejection and accommodation of xenografts

BACKGROUND: A major impediment to the transplanting of porcine organs into humans is the susceptibility of porcine organs to acute vascular rejection, which can destroy a vascularized xenograft over a period of hours to days. Acute vascular rejection of porcine-to-primate xenografts is thought to be triggered by binding of xenoreactive antibodies to the graft. We tested whether antibodies, binding to Galalpha1-3Gal epitopes in porcine tissue, initiate this phenomenon. METHODS AND RESULTS: Specific depletion of anti-Galalpha1-3Gal antibodies from the blood of baboons, using extracorporeal perfusion of separated plasma through columns of Sepharose beads covalently linked to the antigenic trisaccharide, Galalpha1-3Galbeta1-4GlcAc, averted the development of acute vascular rejection in porcine organs transgenic for human decay-accelerating factor and CD59. More importantly, after immunodepletion was stopped and Gala1-3Gal antibodies were allowed to return, these same organs continued to function and remained pathologically normal and thus seemed to achieve a state of accommodation. CONCLUSION: These results demonstrate that anti-Galalpha1-3Gal antibodies cause acute vascular rejection and suggest that depletion of these antibodies leads to accommodation of the donor cardiac xenograft and could supply an important model for additional study.