Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys.

We transplanted kidneys from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy.

BACKGROUND: The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. METHODS: Seven baboons received life-supporting kidney transplants from hDAF transgenic pigs. Anti-Gal antibodies were neutralized by GAS914 or TPC (a Gal PEG glycoconjugate polymer). Group 1 (n=5) underwent a conventional immunosuppressive therapy with FK506, rabbit anti-thymocyte serum/immunoglobulin, mycophenolate mofetil, and steroids. Group 2 (n=2) received an anti-humoral immunity regimen with LF15-0195, Rituxan and cobra venom factor in addition to ATG, FK506 and steroids. Levels of anti-non-Gal antibodies and their mediated complement-dependent cytotoxic activities (CDC) were detected by flow cytometry using Gal knockout (k/o) pig lymphocytes (LC) or endothelial cells (EC) as targets. RESULTS: Continuous infusion of GAS914/TPC significantly reduced anti-Gal antibodies. In Group 1, four of five baboons developed severe acute humoral xenograft rejection (AHXR) and the rejection was associated with either a high level of preformed anti-non-Gal IgG or a marked elevation in induced anti-non-Gal IgG and IgM. Sera collected at the time of AHXR had a high level of CDC to porcine LC/EC from Gal k/o animals. The intensive anti-humoral therapy in Group 2 completely inhibited both anti-Gal and non-Gal antibody production and prevented AHXR. However, this therapy was not well tolerated by the baboons. CONCLUSION: In a pig-to-baboon kidney transplant model, both preformed and induced anti-non-Gal antibodies are strongly associated with the pathogenesis of AHXR when anti-Gal antibodies are neutralized.

Activation of primary porcine endothelial cells induces release of porcine endogenous retroviruses.

BACKGROUND: Endothelial cells form the interface between the porcine graft and the recipient and frequently become activated after xenotransplantation. To evaluate the safety of xenotransplantation further, we assessed the effect of cellular activation on the expression and release of porcine endogenous retroviruses from primary endothelial cells isolated from transgenic and nontransgenic pigs. METHODS: Primary porcine endothelial cells, cultured from pigs transgenic for human decay accelerating factor, were treated with human tumor necrosis factor-alpha, porcine interferon-gamma, or lipopolysaccharide. The release of porcine endogenous retroviruses into the supernatant was monitored at 24-hr intervals (up to 72 hr) by polymerase chain reaction-based reverse transcriptase (PBRT) assay. Activated and unactivated endothelial cells were co-cultured with human cells to investigate the capacity of any virus released from the porcine cells to infect human cells. RESULTS: Virus was not detected in supernatants from quiescent cells by PBRT analysis. The number of viral particles released from endothelial cells was 10 to 5 x 10 viral particles/mL after cellular activation with tumor necrosis factor-alpha, interferon-gamma, or lipopolysaccharide, as shown by PBRT analysis. In contrast, in vitro infection of human cells was observed with unactivated endothelial cells only and was not observed in co-cultures with the activated porcine cells. CONCLUSIONS: Cytokine treatment of primary porcine endothelial cells results in an increase in the release of virus into the supernatant, but the observed increase in viral titer was not mirrored by an increase in infectivity toward human cells.

Serum anti-pig antibodies as potential indicators of acute humoral xenograft rejection in pig-to-cynomolgus monkey kidney transplantation.

BACKGROUND: Hyperacute rejection of solid organ pig xenografts in nonhuman primates has been overcome by using donors transgenic for human complement regulatory proteins, but grafts are still susceptible to humoral (antibody-mediated) rejection. We investigated whether circulating xenoreactive antibodies are a useful indicator of this xenograft rejection. METHODS: Five assays were employed in a retrospective analysis on 20 selected cynomolgus monkey recipients of renal xenografts transgenic for human decay-accelerating factor, with survival between 4 and 60 days. The assays included hemolytic and hemagglutination assays and the measurement of immunoglobulin (Ig)G and IgM binding to porcine endothelial cells and leukocytes, and to the Gal alpha 1-3Gal trisaccharide (Gal) antigen. To assess non-Gal-directed antibodies, sera were absorbed with a Gal-coated resin. A predictive value was defined as an increase in antibody levels before a decline in graft failure (>20% increase in creatinine levels) and humoral rejection in graft pathology. RESULTS: Data on hemolytic anti-pig antibody correlated with those on IgM antibody to endothelial cells, leukocytes, and Gal. In absorbed sera IgM and IgG antibody to endothelial cells and leukocytes correlated with each other, indicative for an elicited antibody response to non-Gal antigens. Sixteen animals showed humoral rejection, and in all but two animals one or more assays was considered of predictive value. On the other hand, increased antibody levels were noted in two animals without signs of rejection in graft pathology and in two cases with cellular xenograft rejection. CONCLUSIONS: It is recommended to use multiple assays (preferably hemolytic, anti-Gal, and anti-endothelial cell) to be able to fully monitor the peripheral antibody responses in pig-to-primate xenograft recipients.

Unexpected immunoresponse to Gal and APA antigens in diabetic type 1 patients receiving neonatal pig islets after 6 years.

Cotransplantation of porcine islets and Sertoli cells into preimplanted subcutaneous devices improve metabolic control in type 1 diabetic patients, and survive grafted for more than 4 years. We report here, further assessment of the endocrine and porcine nature of the surviving cells and the immune responses elicited toward Gal alpha(1,3)-Gal beta(1,4)-GlcNAc (Gal) antigen in patients who received a second and third transplants. No immunosuppressive drugs were administered. We were able to immunostain insulin- and glucagon-positive cells in all biopsies of patients and Sertoli cell markers in 60.9% of biopsies. Additionally, all biopsies tested, amplified the porcine COII gene. Patients demonstrated an increase in antipig antibodies in response to the first transplant with a decreasing response toward the second and third transplants. In all transplants, the IgG levels promptly returned to basal values after 3-4 months. The long-term survival of porcine cells and the reduced humoral immune response to multiple transplants indicate a form of tolerance. We have not been able to find CD25-positive cells, indicating that it is probably an immune accommodation of the graft.

Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor.

BACKGROUND: The expression of human complement regulators has been proved as an effective strategy to overcome hyperacute rejection in discordant xenogeneic organ transplantation. In this study, we tested the hypotheses that expression of triple transgenes for human complement regulators and provide more effective protection to the transplanted pig tissues. METHODS: Pigs transgenic for human complement regulatory proteins, human CD59 (hCD59) and human membrane cofactor protein (hMCP), have been generated using large genomic constructs. Heterozygous human decay-accelerating factor (hDAF) transgenic pigs, from a previously established line, were bred with hCD59 or hCD59 plus hMCP pigs to produce animals that expressed both hCD59 and hDAF, or expressed triple transgenes hCD59, hDAF and hMCP. RESULTS: All three transgenes were widely expressed in most of the tissues analyzed, but the expression of hMCP was at low levels. In cytotoxicity assays on porcine peripheral blood mononuclear cells, the expression of a single transgenic protein, hCD59, or hCD59 in combination with hMCP provided similar protection against human complement-mediated damage as the single expression of hDAF. However, the expression of triple transgenic proteins or double hCD59 and hDAF transgenic proteins provided greater protection than either hCD59 or hDAF alone. CONCLUSIONS: Thus, pigs transgenic for multiple transgenes provide a greater degree of human complement regulation and hence might be more suitable for xenotransplantation.

Anti-pig antibody levels in naïve baboons and cynomolgus monkeys.

Anti-pig antibodies (APA) were analysed in serum from 28 naïve wild-caught baboons (originating from Kenya) and 31 naïve captive-bred cynomolgus monkeys (13 from the Philippines and 18 from Mauritius), using a haemolytic assay with pig erythrocytes (APA), flow cytometry on the porcine lymphoma T-cell cell line L35, and enzyme linked immunosorbent assay (ELISA) using alpha-Gal type II and type VI antigen. This was extended in baboon samples by the evaluation in two laboratories (Imutran, Cambridge, UK and Immerge, Boston, USA), and by antibody absorption using either immobilized alpha-Gal type II or alpha-Gal type VI. Anti-porcine antibodies were demonstrated in all assays with substantial variability within and between the three non-human primate groups. Immunoglobulin (Ig)M antibody levels tended to be similar to or higher than those in a pooled normal human standard serum while IgG levels tended to be lower. Highest antibody levels were recorded in Mauritius cynomolgus monkeys. There were statistically significant correlations between assays for IgM or IgG class anti-Gal antibodies using either alpha-Gal type II or alpha-Gal type VI as antigen, both for different assays and two laboratories involved. Also, significant correlations were observed between the anti-Gal and L35 binding assays. Baboon sera before and after absorption to immobilized alpha-Gal type II or type VI were analysed for anti-Gal type VI or type II antibody: levels were almost undetectable indicating that most anti-Gal antibodies react to epitopes shared between alpha-Gal type II and type VI oligosaccharides. Finally, the relation between APA and outcome of porcine heart xenotransplantation in cynomolgus monkeys and baboons showed no apparent relation between pre-transplant APA levels and the occurrence of hyperacute rejection (HAR) when compared with non-immunological cause of organ/recipient dysfunction or acute humoral xenograft rejection during the first 4 days post-transplantation or survival exceeding 4 days post-transplantation.

Production and characterization of a pig line transgenic for human membrane cofactor protein.

A pig line transgenic for human membrane cofactor protein (hMCP) has been established. Offspring from the founder were produced by crossing the founder with pigs heterozygous for the human decay accelerating factor (hDAF) transgene. As a result, pigs transgenic for both hMCP and hDAF have been produced. Ribonuclease protection assay (RPA) indicated that hMCP was expressed in all the tissues analysed. In addition, immunohistochemical results indicated a high level of expression of hMCP on neural tissues and islets where hDAF was absent or weakly expressed. C3 fragment deposition and cytotoxicity assays indicated that hMCP expression alone on pig endothelial cells and peripheral blood lymphocytes (PBLs) provided protection against human complement mediated damage. However, we did not find that porcine endothelial cells expressing both hDAF and hMCP were better protected than those expressing hDAF alone. The expression of hMCP on tissues where hDAF is not expressed could provide these tissues with protection against human complement mediated lysis.