Tolerization of anti-Galalpha1-3Gal natural antibody-forming B cells by induction of mixed chimerism.

Xenotransplantation could overcome the severe shortage of allogeneic organs, a major factor limiting organ transplantation. Unfortunately, transplantation of organs from pigs, the most suitable potential donor species, results in hyperacute rejection in primate recipients, due to the presence of anti-Galalpha1-3Gal (Gal) natural antibodies (NAbs) in their sera. We evaluated the ability to tolerize anti-Gal NAb-producing B cells in alpha1,3-galactosyltransferase knockout (GalT KO) mice using bone marrow transplantation (BMT) from GalT+/+ wild-type (WT) mice. Lasting mixed chimerism was achieved in KO mice by cotransplantation of GalT KO and WT marrow after lethal irradiation. The levels of anti-Gal NAb in sera of mixed chimeras were reduced markedly 2 wk after BMT, and became undetectable at later time points. Immunization with Gal+/+ xenogeneic cells failed to stimulate anti-Gal antibody production in mixed chimeras, whereas the production of non-Gal-specific antixenoantigen antibodies was stimulated. An absence of anti-Gal-producing B cells was demonstrated by enzyme-linked immunospot assays in mixed KO + WT --> KO chimeras. Thus, mixed chimerism efficiently induces anti-Gal-specific B cell tolerance in addition to T cell tolerance, providing a single approach to overcoming both the humoral and the cellular immune barriers to discordant xenotransplantation.

In vivo T-cell depletion enhances production of anti-GALalpha1,3GAL natural antibodies in alpha1,3-galactosyltransferase-deficient mice.

BACKGROUND: It has been reported that T-cell depletion by in vivo treatment with monoclonal antibodies results in polyclonal B-cell activation. However, its effects on B cells responding to Galalpha1,3Gal (Gal) epitopes remain unknown. METHODS: alpha1,3-Galactosyltransferase-deficient (GalT-/-) mice were treated with depleting anti-CD4 and CD8 monoclonal antibodies. The kinetics of anti-Gal natural antibodies (NAb) and total immunoglobulin levels in their sera were evaluated. The frequencies of anti-Gal NAb-producing cells were determined in the various tissues of GalT-/- mice by enzyme-linked immunospot assay. RESULTS: In vivo T-cell depletion led to significant increases in both anti-Gal IgM and total IgM levels in sera of GalT-/- mice, but did not influence either anti-Gal IgG or total IgG levels. An increased frequency of anti-Gal and total IgM-producing cells was observed in the spleens and bone marrow of T-cell-depleted GalT-/-mice but not in peritoneal cavity cells. CONCLUSION: In vivo T-cell depletion facilitates anti-Gal IgM production, suggesting that T cells deliver inhibitory signals to B cells responding to Gal.

A murine model of antibody-mediated hyperacute rejection by galactose-alpha(1,3)galactose antibodies in Gal o/o mice.

BACKGROUND: In pig-to-primate/human xenografts, hyperacute rejection of primarily vascularized organs usually occurs in 10-60 min and is due to the reaction of the recipients' natural antibodies with antigens expressed on the donor endothelium, the fixation of complement, and ultimately vascular stasis and hemorrhage. Surprisingly, the major target of the natural antibodies is the disaccharide galactose-alpha(1,3)galactose (Gal alpha(1,3)Gal), which is found on many different molecules in pig tissues and reacts with naturally occurring human anti-pig IgM and IgG antibodies. There are a number of strategies to remove/block/alter Gal alpha(1,3)Gal expression in pig tissues, all of which involve the expression of transgenes in pigs. To overcome the difficulty of preclinical studies using primates, we describe a model of hyperacute rejection of heart transplants to Gal o/o mice, which are similar to humans in that they have anti-Gal alpha(1,3)Gal antibodies. METHODS: Gal o/o mice received skin or heart grafts from Gal+ mice or rats, and additional antibody and complement were provided; hyperacute rejection was monitored by observation and histology. RESULTS: Gal alpha(1,3)Gal+ mouse tissues (skin or heart) are not rejected by Gal o/o mice. This was not unexpected, as mice do not utilize alloantibody/complement systems satisfactorily in experimental transplantation studies. However, with the addition of anti-Gal alpha(1,3)Gal antibody and complement, hyperacute rejection of hearts can occur in 10-20 min; it is mediated by IgM, not IgG, antibodies and leads predominantly to tissue hemorrhage. CONCLUSION: Gal alpha(1,3)Gal antigen modification by expression of the H transferase cDNA leads to "indefinite" survival (>120 min) and no hyperacute rejection, which shows that this model is suitable for the study of antibody-mediated rejection of relevance to pig-to-human xenografts.

Xenoantigens and xenoantibodies: their modification.

The hyperacute rejection of pig organs by primates, including humans, results from antibody-mediated complement activation. Protection from complement injury still leads to delayed rejection, which results from other mechanisms that may also be dependent on the presence of antibody. Anti-pig antibodies are directed largely, if not entirely, against alpha-galactose (alpha Gal) epitopes on pig vascular endothelium. Prevention of antibody-antigen binding is being attempted by methods that (1) alter antigen expression on the donor organ or (2) deplete the potential recipient of anti-alpha Gal antibody. To date, most progress has been made in depleting antibody by extracorporeal immunoadsorption using immunoaffinity columns of synthetic alpha Gal oligosaccharides. A pig organ transplanted into a recipient baboon depleted of antibody functions for several days--in contrast to minutes to hours in unmodified baboons. The return of antibody, however, results in rejection of the graft. Pharmacologic immunosuppressive agents are relatively ineffective for prolonged suppression of anti-alpha Gal production. Total-body irradiation and splenectomy are proving more successful. Studies are continuing with the aim of achieving a state of B cell tolerance toward alpha Gal epitopes.

Oocyte Gal alpha 1,3Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse.

The Gal alpha 1-->3Gal structure is displayed on the zona pellucida glycoprotein ZP3 on murine oocytes. This trisaccharide has been implicated in sperm-zona pellucida adhesive events thought to be essential to fertilization in the mouse. To determine directly if this molecule is required for fertilization, we have generated mice that are deficient in a gene (alpha 1,3GT) encoding the UDP-Gal:beta-D-Gal-alpha 1-->3Gal-galactosyltransferase enzyme responsible for Gal alpha 1-->3Gal synthesis and expression. These mice develop normally and exhibit no gross phenotypic abnormalities. The Gal alpha 1-->3Gal epitope is absent from the vascular endothelium and other tissues in alpha 1,3GT (-/-) adult mice. By contrast, alpha 1,3GT (-/-) mice, like humans, develop naturally occurring anti-alpha-galactoside antibodies normally absent in wild type mice. Female alpha 1,3GT (-/-) mice yield oocytes that are devoid of the Gal alpha 1-->3Gal epitope; however, these mice are fully fertile. These observations indicate that the Gal alpha 1-->3Gal moiety is not essential to sperm-oocyte interactions leading to fertilization or to essentially normal development. They further suggest that alpha 1,3GT (-/-) mice will find utility for exploring approaches to diminish anti-Gal-dependent hyperacute xenograft rejection, which presents a major barrier to the use of porcine and other non-primate organs for xenotransplantation in humans.

P-selectin glycoprotein ligand-1 and E-selectin ligand-1 are differentially modified by fucosyltransferases Fuc-TIV and Fuc-TVII in mouse neutrophils.

P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are the two major selectin ligands on mouse neutrophils. Transfection experiments demonstrate that each ligand requires alpha1,3-fucosylation for selectin-binding. However, the relative contributions made by the two known myeloid alpha1, 3-fucosyltransferases Fuc-TVII or Fuc-TIV to this alpha1, 3-fucosylation are not yet clear. To address this issue, we have used mice deficient in Fuc-TIV and/or Fuc-TVII to examine how these enzymes generate selectin-binding glycoforms of PSGL-1 and ESL-1 in mouse neutrophils. Selectin binding was analyzed by affinity isolation experiments using recombinant, antibody-like forms of the respective endothelial selectins. We observe essentially normal binding of E- or P-selectin to PSGL-1 expressed by Fuc-TIV-deficient neutrophils but find that PSGL-1 expressed by Fuc-TVII-deficient neutrophils is not bound by E- or P-selectin. By contrast, E-selectin binds with normal efficiency to ESL-1 on Fuc-TVII-deficient neutrophils but exhibits an 80% reduction in its ability to bind ESL-1 isolated from Fuc-TIV-deficient neutrophils. The same specificity with which Fuc-TVII and Fuc-TIV generate selectin-binding forms of PSGL-1 and ESL-1 was found in transfection experiments with CHO-Pro(-)5 cells. In contrast, each fucosyltransferase alone could generate selectin-binding glycoforms of each of the two ligands in CHO-DUKX-B1 cells. Our data imply that in mouse neutrophils and their precursors, Fuc-TVII exclusively directs expression of PSGL-1 glycoforms bound with high affinity by P-selectin. By contrast, Fuc-TIV preferentially directs expression of ESL-1 glycoforms that exhibit high affinity for E-selectin. This substrate specificity can be mimicked in CHO-Pro(-)5 cells.

Promotion of xenogeneic hematopoietic chimerism in rodents by mononuclear phagocyte depletion.

The successful establishment of tolerance toward pig tissues in primates through hematopoietic progenitor cell engraftment is restricted by the rapid disappearance of these cells in the recipient following infusion. We developed and tested the hypothesis that phagocytes of the reticuloendothelial system are responsible for the rapid clearance of infused pig hematopoietic cells using a mouse model. Mice received non-myeloablative conditioning and, on various days, were injected with medronate-encapsulated liposomes (M-L) or control blank liposomes, followed by the intravenous infusion of miniature swine hematopoietic cells. M-L were well-tolerated in mice (n=100) at levels that deplete mononuclear phagocytes. Depletion of mononuclear phagocytes in normal Balb/c mice as well as in severe combined immune deficient mice increased the accumulation of pig hematopoietic cells in the bone marrow (BM) by 10-fold when measured 24 h after the infusion of the cells. Colony-forming unit analysis showed an increased accumulation of pig hematopoietic progenitors in the BM of mice that were infused with medronate-liposomes. We conclude that depletion of mononuclear phagocytes by M-L has the potential to lower the barrier to the establishment of mixed chimerism and tolerance induction in xenotransplantation.

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice.

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.

Blockade of CD28-B7, but not CD40-CD154, prevents costimulation of allogeneic porcine and xenogeneic human anti-porcine T cell responses.

Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 microg/ml) was considerably more inhibitory than anti-CD154 mAb (100 microg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-gamma by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-gamma and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.

Pharmacologic immunosuppressive therapy and extracorporeal immunoadsorption in the suppression of anti-alphaGal antibody in the baboon.

The aim of this study was to deplete baboons of anti-(alpha)galactosyl (alphaGal] antibody and attempt to maintain depletion by pharmacologic immunosuppressive therapy (PI). In 12 experiments, involving nine baboons, repeated extracorporeal immunoadsorption (EIA) was carried out by plasma perfusion through immunoaffinity columns of synthetic alphaGal trisaccharide type 6. Five of the baboons were immunologically naive and four had undergone various procedures at least 6 months previously. All, however, had recovered lymphohematopoietic function and (with one exception) had levels of anti-alphaGal antibody within the normal range. Eleven protocols included continuous i.v. cyclosporine (to maintain whole blood levels of approximately 1,600 ng/ml). In addition, in ten protocols, the baboon received one or more of the following drugs: cyclophosphamide (1-20 mg/kg/day), mycophenolate mofetil (70-700 mg/ kg/day), brequinar sodium (1-12 mg/kg/day), prednisolone (1 mg/kg/day), melphalan (0.15-0.6 mg/kg/day), methylprednisolone (125 mg/day x3), and antilymphocyte globulin (ATG) (50 mg/kg/day x3). EIA was carried out on 1-9 occasions in each study and was temporarily successful in removing all antibody. When no PI was administered, antibody returned close to pre-EIA levels within 48 hr. Cyclosporine alone delayed the rate of antibody return only slightly. While EIA was continuing on a daily or alternate day schedule, antibody levels (both IgM and IgG) were maintained at 20-45% of pre-EIA levels. Once EIA was discontinued but PI maintained, IgM rose to 40-90% and IgG to 30-60% of pre-EIA levels. In vitro testing demonstrated significant cytotoxicity to pig cells at these antibody levels. We conclude that i) EIA utilizing columns of alphaGal trisaccharide is successful in temporarily depleting baboons of anti-alphaGal antibody, but ii) none of the PI regimens tested suppressed antibody production to levels which would be expected to prevent antibody-mediated rejection of pig xenografts. Additional strategies will therefore be required if xenotransplantation is to become a clinical reality.

The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing.

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.