Uri Galili

Interaction of the natural anti-Gal antibody with α-galactosyl epitopes: a major obstacle for xenotransplantation in humans☆

Cells of nonprimate mammals express an abundance of the carbohydrate structure Gal alpha 1-3 Gal beta 1-4 GlcNac-R (termed the alpha-galactosyl epitope). The natural anti-Gal antibody, which interacts specifically with alpha-galactosyl epitopes, is present in high concentrations in all humans. Here, Uri Galili argues that the interaction between anti-Gal in the serum and alpha-galactosyl epitopes on cells of nonprimate grafts may act as an immunological barrier which prevents xenotransplantation.

Porcine and bovine cartilage transplants in cynomolgus monkey: II. Changes in anti-Gal response during chronic rejection.

The recent advances in avoiding hyperacute rejection by producing transgenic pigs with complement regulatory proteins call for the analysis of posttransplantation changes in anti-Gal activity in the absence of hyperacute rejection. Transplantation of cynomolgus monkeys with porcine or bovine meniscus and articular cartilage enabled the study of anti-Gal IgG response to xenografts that are not subjected to hyperacute rejection. The cartilage implants were kept in suprapatellar pouches of the recipients for 1 or 2 months and anti-Gal activity was measured in the serum at various time intervals after transplantation. Within 2 weeks after transplantation, titer of anti-Gal IgG, in all transplanted monkeys, increased by 20- to 100-fold, as measured in ELISA with synthetic alpha-galactosyl epitopes linked to bovine serum albumin or with mouse laminin. Furthermore, binding of serum anti-Gal to porcine endothelial cells increased by 10-fold or more after transplantation. Complement-mediated cytotoxicity also increased by two- to eightfold after transplantation. The elevated activity of anti-Gal was maintained for the 2-month period during which the grafts were kept in the monkeys, and returned to the pretransplantation level 6 months after graft removal. All these data suggest that the primate immune system responds vigorously to alpha-galactosyl epitopes on xenografts by activating many B lymphocytes that produce increased amounts of anti-Gal IgG, which may also be of high affinity. These antibodies are likely to bind to the xenograft cells, even if these cells express low numbers of alpha-galactosyl epitopes. Such antibody binding may play an important role in chronic rejection of xenografts.

Porcine cartilage transplants in the cynomolgus monkey. III. Transplantation of alpha-galactosidase-treated porcine cartilage.

BACKGROUND Studies on transplantation of porcine meniscus and articular cartilage into monkeys are important for evaluating the possible use of such tissues in humans. In addition, such studies shed light on the chronic xenograft rejection process in primates. Transplantation of porcine cartilage into cynomolgus monkeys for 2 months results in a many-fold increase in anti-Gal activity and in a strong cellular inflammatory response of T lymphocytes and macrophages within the implants. The objective of this study was to determine whether elimination of Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal epitopes) from the xenograft may alter the immune response and the inflammatory reaction. METHODS Porcine meniscus and articular cartilage specimens were treated with recombinant alpha-galactosidase (100 U/ml), and the absence of alpha-gal epitopes was assessed by the binding of the monoclonal anti-Gal antibody M86. The treated cartilage specimens were transplanted into the suprapatellar pouch of cynomolgus monkeys. The immune response to cartilage was monitored in the serum and the inflammatory reaction was assessed in the xenografts, which were explanted after 2 months. RESULTS Incubation with alpha-galactosidase resulted in complete removal of alpha-gal epitopes from the cartilage. The increase in anti-Gal activity in the transplanted monkeys was marginal. However, most monkeys produced antibodies to antigens specific to porcine cartilage. The inflammatory response within the alpha-galactosidase-treated xenografts was much lower than in nontreated cartilage and the proportion of T lymphocytes within the cellular infiltrates was greatly reduced. CONCLUSIONS Treatment of cartilage xenografts with alpha-galactosidase successfully removes alpha-gal epitopes from porcine cartilage. Transplantation of the treated cartilage results in the production of only anti-porcine cartilage-specific antibodies and a reduced inflammatory response consisting primarily of macrophages infiltrating into the cartilage.

A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody.

BACKGROUND The assessment of a-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) expression on various cells and tissues is important for the prediction of anti-Gal-mediated immune rejection of xenografts. This study describes an enzyme-linked immunosorbent assay (ELISA inhibition assay) developed for this purpose, which uses the monoclonal anti-Gal antibody M86. METHODS Cells at various concentrations were incubated overnight with M86 at 1/100 dilution. The cells and bound antibody were removed, and the residual antibody in the supernatant was measured in an ELISA assay with a-gal-bovine serum albumin as a solid phase antigen. The extent of a-gal epitope expression on cells correlates with the subsequent inhibition of M86 binding in ELISA. The inhibition binding curves at various cell concentrations were compared with those of a standard cell line with a known number of epitopes per cell. RESULTS AND CONCLUSIONS The mouse IgM M86 monoclonal antibody was highly specific for a-gal epitopes. Using this antibody in an ELISA inhibition assay with cells at a wide range of concentrations enables the detection of at least 5 x 10(4) and up to more than 5 x 10(7) a-gal epitopes per cell. This assay can be used also for the detection of a-gal epitopes on membranes from tissue homogenates, and thus it enables the determination of the extent of decrease in a-gal epitope expression in animals that are genetically manipulated to alter their carbohydrate make-up.

Alpha-galactosyl epitope-mediated activation of porcine aortic endothelial cells: type II activation.

BACKGROUND Xenoreactive natural antibodies (XNAs) and complement mediate hyperacute rejection of discordant xenografts. Inhibition of complement alone results in some prolongation of graft survival, but delayed xenograft rejection still precludes long-term graft survival. In vitro data provide evidence for the direct proinflammatory activation of endothelial cells (ECs) by XNAs. These antibodies are primarily directed against galactose alpha(1-3)-galactose (alpha-gal), the major xenoantigen in the pig to primate xenotransplant model. Previous studies have shown EC activation by XNAs but failed to address the question of whether alpha-gal-specific ligands can induce EC activation. The aim of this study was to investigate whether agonist binding to the alpha-gal epitope by alpha-gal-specific lectins as compared with XNAs or elicited xenoreactive antibodies can directly elicit type II porcine aortic EC (PAEC) activation (i.e., activation that requires protein synthesis). METHODS AND RESULTS The tetravalent, alpha-gal-binding Bandeiraea simplicifolia lectin I (BS-I), the wholly alpha-gal-specific BS-I isolectin B4, and elicited primate anti-pig xenoreactive antibodies (decomplemented cynomolgus monkey anti-porcine serum) induced E-selectin protein expression in PAECs. This induction was alpha-gal-specific, as preincubation with synthetic alpha-gal carbohydrate or adsorption of lectin or serum to rabbit, but not human, red blood cells removed the activating component. E-selectin expression, induced by BS-I, was inhibited in the presence of genistein, a tyrosine kinase inhibitor, and by mepacrine, an inhibitor of phospholipase A2. Human and primate XNAs lacked this activity when tested at relevant concentrations; however, stimulation of PAECs with affinity-purified human XNA (IgM and IgG) resulted in slightly increased interleukin-8 and P-selectin mRNA levels but had no apparent effects on E-selectin transcription. BS-I strongly induced E-selectin, P-selectin, intercellular adhesion molecule-1, and interleukin-8 mRNA in an NF-kappaB-dependent manner. CONCLUSIONS Several agonists that specifically bind to alpha-gal can evoke type II EC activation. Hence, anti-Gal antibodies may contribute directly to xenograft rejection in the absence of complement activation.

Changes In Cell Surface Glycosylation In α1,3-galactosyltransferase Knockout And α1,2-fucosyltransferase Transgenic Mice

BACKGROUND Inactivation of the alpha1,3-galactosyltransferase (GalT) gene by homologous recombination (knockout [KO] mice) and competition for the enzymes N-acetyllactosamine substrate by transgenically expressed alpha1,2-fucosyltransferase (H-transferase) are two genetic approaches to elimination of the Gal alpha1,3Gal (alphaGal) epitope, which is the major xenoantigen in pigs against which humans have preformed antibodies. Such genetic manipulations often have unpredictable results. METHODS A panel of 19 selected lectins was used to characterize the changes in cell surface glycosylation in GalT KO and H-transferase transgenic mice, compared with nontransgenic littermate controls. RESULTS GalT KO mice showed complete elimination of the alphaGal epitope, as reported previously. Surprisingly, however, this was associated with only a modest increase in N-acetyllactosamine residues and had little other effect on the pattern of lectin binding. In contrast, the pattern of lectin binding to H-transferase transgenic mouse cells was more profoundly disturbed and indicated, in addition to the expected expression of H substance and suppression of the alphaGal epitope, that there was a marked reduction in alpha2,3-sialylation and exposure of the normally cryptic antigens, sialylated Tn and Forssman antigens. Similar changes in lectin reactivity with porcine aortic endothelial cells were induced by neuraminidase treatment. CONCLUSIONS Lectins were able to bind underlying carbohydrate structures (sialylated Tn and Forssman antigens) that are normally cryptic antigens on H-transferase transgenic mouse spleen and cardiac endothelial cells, probably as a consequence of the reduction in the electronegativity of the cell surface due to reduced sialylation. As humans have preformed anti-Tn and anti-Forssman antibodies, it is possible that these structures may become targets of the xenograft rejection process, including hyperacute rejection.

Differential expression of alpha-GAL epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) on pig and mouse organs.

BACKGROUND Expression of the alpha-gal epitope in mice can be completely eliminated by disruption of the alpha1,3 galactosyltransferase gene. As an initial step for assessing the feasibility of this approach in the pig, it was of interest to compare the expression of alpha-gal epitopes in pig and mouse organs. METHODS Membranes from pig and mouse organ homogenates were analyzed for alpha-gal epitope expression by Western blots, enzyme-linked immunosorbent assay (ELISA), immunostaining of tissues, and ELISA inhibition assay. RESULTS Immunostaining of Western blots with human anti-Gal detected alpha-gal epitopes on glycoproteins from pig organs but not on glycoproteins from the corresponding mouse organs. ELISA with membrane homogenates and immunostaining of tissue sections demonstrated a much higher binding of human anti-Gal to alpha-gal epitopes on pig membranes than on mouse membranes. ELISA inhibition assay with monoclonal anti-Gal indicated that alpha-gal epitope expression in pig organs is up to 500-fold higher than in mouse organs. CONCLUSION Expression of alpha-gal epitopes in pig organs is many fold higher than in mouse organs. The abundance of these epitopes in pigs raises the question of whether pigs can properly develop without expression of alpha-gal epitopes.

Enhancement of antigen presentation of influenza virus hemagglutinin by the natural human anti-Gal antibody

Immunogenicity of inactivated virus or subviral vaccines may be enhanced by complexing with an IgG antibody. Such antibody would increase the uptake, processing and presentation of the vaccines antigens by antigen presenting cells (APC), via the adhesion of the antibody-vaccine complex to Fc-receptors on macrophages and other APC. A natural antibody in humans, which may be generally exploited for this purpose, is the natural anti-Gal antibody. This antibody is ubiquitously produced as 1% of circulating IgG in humans and Old World primates, and it interacts specifically with the carbohydrate epitope Gal alpha 1-3 Gal beta 1-4 GlcNAc-R (termed the alpha-galactosyl epitope). This epitope is synthesized in large amounts in cells of nonprimate mammals and New World monkeys by the glycosylation enzyme alpha 1,3 galactosyltransferase. Here we describe in vitro studies on the ability of anti-Gal to bind to alpha-galactosyl epitopes on influenza virus propagated in mammalian cells, and to enhance presentation by APC of viral hemagglutinin antigenic determinants to specific helper T cell clones. The various approaches for achieving alpha-galactosyl epitope expression on virion and subviral vaccines are discussed.

Inhibition of anti-Gal IgG binding to porcine endothelial cells by synthetic oligosaccharides

Rejection of pig-to-human or pig-to-primate xenografts is mediated by the natural anti-Gal antibody, which interacts with alpha-galactosyl epitopes (i.e., Gal alpha1-3Gal beta1-4GlcNAc-R) abundantly expressed on porcine cells. The objective of this study was to determine the ability of various synthetic oligosaccharides to inhibit the binding of anti-Gal IgG molecules to porcine endothelial cells in vitro. Such inhibition ultimately may help to reduce or to prevent the in vivo antibody-dependent cell cytotoxicity (ADCC) reaction. In the absence of complement-mediated hyperacute rejection, the ADCC induced by anti-Gal IgG molecules is likely to cause the chronic rejection of xenografts. The synthetic free alpha-galactosyl epitope (Gal alpha1-3Gal beta1-4GlcNAc) was found to be 300-fold more effective than melibiose or alpha-methyl galactoside in inhibiting anti-Gal binding to porcine endothelial cells, and to prevent >90% of the antibody binding at a concentration of 1 mM. The disaccharide Gal alpha1-3Gal was ten-fold less effective than the free alpha-galactosyl epitope. Accordingly, the affinity of the disaccharide to anti-Gal, as measured by equilibrium dialysis, was seven-fold lower than that of the trisaccharide. The effective concentration of oligosaccharides inhibiting anti-Gal is independent of the antibody affinity, but is dependent on the concentration of the antibody. Based on the small difference in affinity between Gal alpha1-3Gal beta1-4GlcNAc and Gal alpha1-3Gal beta1-4GIc, and the large difference in the price of N-acetyllactosamine vs. lactose, it is suggested that lactose may be considered as an appropriate starting material for synthesizing large amounts of a trisaccharide that effectively neutralizes anti-Gal.

Differential host-dependent expression of α-galactosyl epitopes on viral glycoproteins : a study of eastern equine encephalitis virus as a model

The carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-galactosyl) is abundantly expressed on cells of non-primate mammals, prosimians and New World monkeys, where it is synthesized by the enzyme alpha 1,3-galactosyltransferase (alpha 1,3GT). Old World monkeys, apes and humans lack alpha 1,3GT and hence do not synthesize alpha-galactosyl epitopes. Instead, these species produce a natural antibody, anti-Gal, which interacts specifically with alpha-galactosyl epitopes and which constitutes up to 1% of circulating immunoglobulins in humans. We have used eastern equine encephalitis (EEE) virus as a model to examine the differential expression of alpha-galactosyl epitopes on the glycoproteins of virus propagated in cells that either produce or lack alpha 1,3GT. As predicted, virus propagated in Vero cells (derived from the African green monkey, an Old World monkey) did not express alpha-galactosyl epitopes. In contrast, virus propagated in mouse 3T3 cells (EEE3T3) expressed approximately 80 alpha-galactosyl epitopes per virion on both the E1 and the E2 envelope glycoproteins. Thus, expression of the alpha-galactosyl epitope on virions paralleled that on host cells. The binding of anti-Gal antibody to these epitopes on EEE3T3 virions partially neutralized virus infectivity, raising the possibility that anti-Gal production in hosts may influence the initial infectious stage of viruses expressing alpha-galactosyl epitopes.