Evidence that double transfection to xenoendothelial cells using GPI-anchoring complement regulatory factor (DAF and HRF20) genes is useful for the inhibition of human complement-mediated cytolysis

Abstract: It has been known that xenogeneic cells transfected with regulator of complement activation (RCA) molecules such as DAF ( CD55 ), MCP ( CD46 ) and HRF20 ( CD59 ) resist the lysis by the human complement. However, it has not been demonstrated that the induction of double RCA molecules is more effective than that of single RCA molecule to inhibit the human complement attack. In this study, the authors compared the effect on the protection from complement-mediated lysis between single transfection and double transfection of DAF and HRF20 cDNA to bovine aortic endothelial cells (BAEC) using retro viral vector.
BAEC transfected with DAF or HRF20 cDNA resisted the lysis by human serum in parallel with the intensity of antigen expression, whereas BAEC transfected with both DAF and HRF20 cDNA resisted the lysis by human serum with anti-BAEC antibody, not human serum alone, more effectively than those with DAF cDNA alone. We conclude that the xenogeneic cells doubly transfected with both DAF and HRF20 cDNA are protected from complement mediated lysis more effectively than those singly transfected with DAF cDNA alone.     

Comparative analysis of three genetic modifications designed to inhibit human serum‐mediated cytolysis

Hyperacute rejection (HAR) remains a critical immunologic hurdle in the development of xenogeneic organs for human transplantation. Strategies that simultaneously eliminate both natural antibody reactivity and complement activation on the xenogeneic cell surface may be the best approach to achieve clinical application of xenogeneic vascularized organ transplantation. We have developed multiple lines of genetically manipulated mice to evaluate the combination of different genetic approaches aimed at inhibiting antibody and complement-mediated cell lysis. We utilized transgenic mice expressing the human complement inhibitor, CD59, the human 1,2-fucosyltransferase (H-transferase, HT) and the α1,3-galactosyltransferase (α1,3-GT) knock-out mouse line (Gal KO). Our data show that expression of hCD59 in combination with HT expression or the null phenotype of α1,3-GT are equally effective at preventing human serum-mediated cytolysis. Interestingly, the triple combination affords no additional protective effect. Therefore, coexpression of HT and a complement inhibitor is the most immediate strategy to genetically engineer transgenic pigs to be used as xenogeneic donors.     

CD97-decay-accelerating factor interaction is not involved in leukocyte adhesion to endothelial cells

BACKGROUND: Effective improvement in xenograft survival is achieved using transplants from transgenic pigs expressing human complement (C) regulatory proteins, including decay-accelerating factor (DAF), CD59, and CD46 on endothelial cells (ECs). The aim of this study was to investigate whether human DAF expression in porcine ECs, as well as regulating C activation, can modify intercellular events through its interaction with its receptor, CD97, on human leukocytes. METHODS: Cellular interactions between human leukocytes and porcine ECs were investigated in vitro using ECs from either wild-type or DAF-transgenic pigs. Static leukocyte adhesion and T cell activation assays were performed using porcine ECs as target or effector cells, respectively. The role of the DAF-CD97 interaction was investigated using specific blocking monoclonal antibodies (mAbs) against human DAF and its receptor, CD97, in adhesion assays. RESULTS: Adhesion of U937 or Jurkat T cells, both expressing human DAF and CD97, was quantitatively similar for wild-type and transgenic-DAF-expressing pig ECs. Furthermore, blocking the CD97-DAF interaction did not inhibit xenogeneic leukocyte-endothelium adhesion, whereas blocking the very late antigen 4-vascular cell adhesion molecule-1 pathway reduced this adhesion by 50-80%. Furthermore, DAF and CD97 expression was not up-regulated during tumor necrosis factor-alpha- or lipopolysaccharide-mediated EC activation, unlike the adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule (ICAM)-1. CONCLUSION: We found that high levels of human DAF expressed on ECs abrogates C-mediated cell damage but did not affect the in vitro adhesive properties or antigen-presenting cell function of genetically modified porcine ECs.     

Porcine complement regulators protect aortic smooth muscle cells poorly against human complement-induced lysis and proliferation: consequences for xenotransplantation

BACKGROUND: Accelerated atherosclerosis after transplantation has been observed and is characterized by smooth muscle cell proliferation in the graft. Porcine cells are frequently used in models of atherosclerosis and porcine organs are considered for use in transplantation. Complement (C) activation is known to play a major role in rejection of xenografts and is also considered to play a role in the development of atherosclerosis. The aim of this study was to investigate the expression and function of membrane bound regulators of complement (CReg) on porcine aortic smooth muscle cells (PASMC). METHODS: The PASMC were assessed for expression of CReg and susceptibility to lysis by human C by flow-cytometry. The effect of various cytokines on CReg expression and C-susceptibility was investigated. The ability of human C to induce cell proliferation was assessed using the Alamar blue assay. RESULTS: The PASMC only express the CReg membrane cofactor protein (MCP) and CD59 on their cell surface. MCP expression was increased by interleukin (IL)-4. In contrast to porcine aortic endothelial cells (PAEC), PASMC were found to be surprisingly sensitive to C-mediated lysis, mainly due to a low level of expression of CD59. Human C-induced proliferation of PASMC, which was dependent on complete membrane attack complex (MAC) formation. CONCLUSIONS: Endogenously expressed CReg on PASMC poorly protect these cells to human C. Human C can induce proliferation of PASMC. In order to prevent accelerated atherosclerosis in porcine xenografts, increased levels of CReg not only have to be obtained on the endothelial cells but also on the smooth muscle cells.     

Expression of human soluble complement receptor 1 by a pig endothelial cell line inhibits lysis by human serum

The importance of complement activation and naturally occurring anti-pig antibodies in the hyperacute rejection (HAR) observed in models of pig-to-human xenotransplantation is well established. To overcome this, much effort has been dedicated to preparing transgenic pigs by knocking out Galalpha(1-3)Gal expression in these animals, or knocking in the expression of human complement regulatory proteins (CRPs), such as CD59 or decay accelerating factor. A soluble form of another membrane CRP, complement receptor type 1 (CR1), has also been shown to inhibit complement activation. Here, we show that transfection of a pig endothelial cell line with a truncated form of human soluble complement receptor 1 (sCR1) almost completely protected these cells from complement-mediated lysis by human AB serum. Pigs genetically manipulated to express human sCR1 may represent an additional strategy to inhibit HAR of pig-to-human transplanted organs.     

Human CD59 expressed in transgenic mouse hearts inhibits the activation of complement

Porcine-to-human xenotransplantation offers a potential solution to the critical shortage of human organs. The major immunological barrier to xenotransplantation between these species is a rapid rejection process mediated by preformed natural antibodies and complement. Xenogeneic organ grafts are especially susceptible to complement mediated injury because complement regulatory proteins, which ordinarily protect cells from inadvertent injury during the activation of complement, function poorly in regulating activation of heterologous complement. Removal of xenoreactive antibodies or systemic inhibition of complement activity has been shown to prolong graft survival. As an alternative to the systemic inhibition of complement activity, we have established a model system using transgenic animals to test whether the expression of human membrane bound complement regulatory proteins on mouse endothelial cells can inhibit the activation of human complement. CD59, which acts at the terminal stage of complement activation by inhibiting the formation of the membrane attack complex, was used as a paradigm for this model. A CD59 construct containing the putative CD59 gene promoter linked to the CD59 coding region was used to demonstrate expression of the human CD59 protein in various tissues of transgenic mice, including endothelial cells in the heart. In addition, we show that the transgenic CD59 protein is biologically active as determined by the ability to inhibit the formation of membrane attack complex in transgenic mouse hearts perfused ex vivo with human plasma. These results demonstrate that expression of membrane bound complement regulatory proteins can achieve complement inhibition in a xenogeneic organ and suggest that this approach may be useful for successful xenotransplantation between discordant species.     

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.     

Xenogeneic human anti-pig cytotoxicity mediated by activated natural killer cells

Abstract: Even if hyperacute rejection, which is mediated by human natural antibodies (nAb) and complement, could be prevented, xenoreactive human anti-pig cellular responses may lead to delayed and/or chronic xenograft rejection. Among the cell populations participating in such rejection, NK cells have been proposed as an important component. In this study we report the in vitro cytotoxic activity of natural killer (NK) cells obtained from healthy human donors against porcine target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) and purified NK cells (CD16+/CD56+, CD3-, CD20-, CD33-) exhibited little or no cytotoxic activity when tested on porcine phytohemagglutinin (PHA)-stimulated lymphoblasts or bone marrow- or aortic-derived endothelial cell lines in the presence of serum-free medium. Killing was considerably higher in the presence of human decomplemented plasma, containing xenoreactive nAb, or purified Gal(α1,3)Gal-reactive antibodies, suggesting that antibody dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells is an important mechanism involved in xenogeneic cytotoxicity. After incubation of human PBMC for 6 days in the presence of irradiated xenogeneic porcine or allogeneic stimulator cells, or in the presence of exogenous interleukin 2 (IL-2), the cytotoxic activity of the bulk cultures as well as that of isolated NK cells (separated from stimulated bulk cultures) against xenogeneic targets increased considerably, and corresponded to an increased NK-specific lysis of K562 target cells. Cell surface staining and flow cytometry showed that CD16+/CD56+, CD3- NK cells composed ca. 25% of short-term (6 days) xenogeneic, allogeneic, or IL-2 stimulated bulk cultures. In summary, these data suggest that, in contrast to allogeneic cell- mediated killing, xenogeneic human anti-porcine cytotoxicity includes an important contribution from NK cells.     

Characteristics of CD59 up-regulation induced in porcine endothelial cells by alphaGal ligation and its association with protection from complement

Abstract: Background: Activation of endothelial cells may result in proinflammatory and procoagulant changes, or in changes that protect the endothelial cells (EC) from injurious insults. Stimulation of porcine EC with human anti‐porcine antibodies, or lectins from Bandeiraea simplicifolia that bind terminal Galα(1–3)Gal (abbreviated αGal), can induce EC protection from cytotoxicity by human complement. These EC also exhibit up‐regulation of CD59 protein and mRNA expression. Porcine CD59 has been reported to protect porcine cells from human complement. Therefore we investigated the specificity requirements and other characteristics of the induced CD59 up‐regulation, as well as the role of up‐regulated CD59 in lectin‐induced protection of EC from human complement. Methods: Aortic EC were incubated in vitro with αGal‐binding lectins B. simplicifolia lectin I isolectin B4 (IB4) and B. simplicifolia lectin I (BS‐I) and CD59 expression was assessed by flow cytometry and enzyme linked immunosorbent assay (ELISA). Binding requirement was studied using disaccharides containing either αgalactosyl or βgalactosyl moieties to inhibit CD59 up‐regulation. Protection from complement killing was assessed after incubation of EC with human serum as a source of anti‐porcine antibodies and complement. The role of CD59 in lectin‐induced protection was studied in the presence of an anti‐pig CD59 antibody and after removal of CD59 using phosphatidylinositol (PI)‐specific phospholipase C (PI‐PLC). Results: We found that induction of CD59 up‐regulation required specific binding of the lectin to terminal αGal and was not induced either by soluble factors that may be released from EC by stimulation with the lectin or by TNF‐α, IFN‐γ, or IL‐1α. Unstimulated or BS‐I‐treated EC showed little or no expression of decay accelerator factor (DAF). Removal of membrane‐associated CD59 (and other proteins that are associated with the membrane through PI linkage) with PI‐PLC from EC that had been exposed to lectin restored their complement sensitivity to various degrees, depending on the extent of lectin‐induced protection. Cytotoxicity was completely restored in cells that exhibited partial protection induced with lectin at low doses or for a short period of time. However, EC that were fully resistant to complement did not regain sensitivity to complement after removal of CD59. Changes in CD59 expression did not modify the degree of C9 binding. Conclusion: Induction of CD59 expression required specific binding of the lectin to terminal αGal and was not induced by soluble factors that may be released from EC by lectin stimulation. Increased CD59 expression may contribute to this form of protection from complement; however, mechanisms other than CD59 up‐regulation appear to be essential for the development of full protection.     

Human xenoreactive natural antibodies of the IgM isotype activate pig endothelial cells

Abstract: Preformed, xenoreactive natural antibodies (XNA) and complement (C) are involved in the initiation of vascular rejection of organs transplanted between discordant species, presumably by stimulating donor organ endothelial cells (EC). Although C is known to play a role in the activation of EC, it has not been clear whether the antibodies serve only to anchor the initial components of C, and thus permit the C cascade to proceed, or whether the antibodies themselves deliver a signal to the EC. We have tested affinity-purified human IgM containing XNA (IgM-XNA) for its ability to stimulate in vitro the up-regulation of genes in pig EC. Northern blot analysis shows that IgM, which contains XNA, stimulates mRNA accumulation for certain genes (including IL-8, PAI-1, and ECI-7, a new gene that we have found is associated with EC activation), but not others known to be up-regulated in response to TNF, IL-1 or LPS. Our results show that XNA provide a signal to EC, and thus may themselves participate in activation of EC and consequent vascular rejection.     

Gene transfer of the adaptor Lnk (SH2B3) prevents porcine endothelial cell activation and apoptosis: implication for xenograft's cytoprotection

Chatelais M, Devallière J, Galli C, Charreau B. Gene transfer of the adaptor Lnk (SH2B3) prevents porcine endothelial cell activation and apoptosis: implication for xenograft’s cytoprotection.
Xenotransplantation 2011; 18: 108–120. © 2011 John Wiley & Sons A/S. Abstract: Background: Targeting protective gene expression to porcine endothelium by genetic modification of the donor could improve xenograft survival by controlling cell activation and death. We previously found that, in endothelial cells (EC), the molecular adaptor Lnk (SH2B3) is a negative regulator of cytokine signaling. We also have shown that Lnk is upregulated in pig EC (PAEC) in response to tumor necrosis factor‐α (TNF) and xenoreactive natural antibodies (XNA) binding. The present study investigated whether ectopic expression of human Lnk using gene transfer may be efficient to control signaling pathways associated with inflammation and apoptosis in porcine aortic endothelial cells (PAEC). Methods: Endothelial cells cultures were established from WT and Gal^?/? pigs and transduced with a recombinant adenovirus encoding human Lnk. Phenotype and functions of transduced PAEC expressing Lnk were analyzed by flow cytometry, western blot and XNA and complement‐dependent assays. The regulatory functions of Lnk toward inflammation were assessed in TNF‐activated EC, and the protective functions were tested toward TNF‐induced apoptosis and anoïkis. Apoptosis assays included DNA content analysis and caspase‐3/7 activity. Results: First, we found that as a result of adenoviral transduction, human Lnk was efficiently and similarly expressed in EC from WT or Gal^?/? pigs. Lnk expression or EC transduction caused no significant change in the binding of XNA (IgG and IgM) to PAEC and has no effect on complement activation and C5b‐9 formation. We demonstrated that expression of human Lnk efficiently inhibits TNF signaling in PAEC and decreases VCAM‐1 induction by 46.3 ± 1.2% compared to controls (n = 6, **P < 0.01). Furthermore, expression of Lnk was associated with a significant decrease in the percentage of caspase‐3/7‐dependent apoptosis caused by TNF in the presence of actinomycin D and also reduces cell death by anoïkis by 25.0 ± 1.9% compared to controls (n = 5, **P < 0.01). Conclusions: Together, these findings indicate that the signaling adaptor Lnk is effective to reduce PAEC activation and apoptosis. Thus, Lnk is a potential candidate for the modulation of signaling pathways to protect vascular EC from inflammation in xenotransplantation.     

Human complement regulatory proteins protect swine lungs from xenogeneic injury

BACKGROUND: Pulmonary xenotransplantation is not possible because of hyperacute lung injury, the pathogenesis of which is unknown. This study evaluates complement-dependent pathways of pulmonary injury during heterologous perfusion of swine lungs. METHODS: Lungs from unmodified swine and swine expressing human decay-accelerating factor and human CD59 (hDAF/hCD59 swine) were perfused with either human plasma or baboon blood. Pulmonary vascular resistance and static pulmonary compliance were measured serially, and swine lung tissue were examined by light microscopy. Complement activation was assessed by serial measurements of baboon plasma C3a-desArg concentrations. RESULTS: Perfusion of unmodified swine lungs with human plasma and baboon blood resulted in hyperacute lung injury within minutes of perfusion. However, function was preserved in swine lungs expressing human decay-accelerating factor and human CD59. In both study groups, xenogeneic perfusion with baboon blood resulted in at least a sevenfold increase in plasma C3a-desArg levels suggesting transient activation of complement. CONCLUSIONS: Lungs from swine expressing human decay-accelerating factor and human CD59 were resistant to injury during perfusion with human plasma and baboon blood, indicating that complement mediated some of the features of xenogeneic acute lung injury.     

An engineered bifunctional recombinant molecule that regulates humoral and cellular effector functions of the immune system

BACKGROUND: Humoral and cellular defense mechanisms mediate the rejection of transplanted cells, tissues, and organs after allogeneic or xenogeneic transplantation. Inhibition of complement and T-cell costimulation are strategies aimed at increasing transplant survival. METHODS: Engineered novel fusion proteins that contain the functional domains of human CD152 (hCTLA4) or porcine CD152 (pCD152) and human CD59 (hCD152-hCD59, pCD152-hCD59) were developed to form bifunctional chimeric proteins that retain the effector functions of both moieties. Porcine aortic endothelial cells and murine Balb/3T3 cells were transduced or transfected to express the novel fusion proteins. RESULTS: Fluorescence-activated cell sorter analysis of hCD152-hCD59 transduced primary porcine aortic endothelial cells or hCD152-hCD59 and pCD152-hCD59 transfected Balb/3T3 cells determined that the molecules were expressed on the cell surface, and that they retained conformational epitopes. We demonstrate that hCD152-hCD59 and pCD152-hCD59 chimeric proteins inhibit complement-mediated cell lysis. In addition, hCD152-hCD59 or pCD152-hCD59 expression resulted in a significant reduction in T-cell activation as the result of CD152 engagement of porcine CD86 or murine CD80 in when Jurkat cells were cocultured with the hCD152-hCD59 or pCD152-hCD59 expressing cells. Antibody-blocking experiments or phosphatidylinositol phospholipase C removal of the glycosyl-phosphatidylinositol-linked molecules resulted in increased serum-mediated cytolysis and eliminated the costimulatory blockade. CONCLUSIONS: These data illustrate that a single molecule can confer resistance to humoral and cellular immune attack.     

Additive effects of CD59 expression in Gal knockout mice in vitro but not in an ex vivo model

Abstract: Transgenic expression of the human complement regulatory molecule CD59 in mice and genetic deletion of the major xenoantigen galactose α 1,3 galactose (Gal KO) each resulted in partial protection of spleen cells from lysis by human serum. These protective effects were additive when the two genetic modifications were combined. However, when the effects of these genetic modifications were examined in an ex vivo model in which mouse hearts were perfused with human plasma, it was Gal KO which was the modification which determined protection. CD59 expression alone was not protective and CD59 expression in combination with Gal knockout did not result in a significant additional increase in protection over and above that provided by Gal knockout alone. The likely explanation for this discrepancy between the in vitro and ex vivo data is that the H2-K^b promoter used to drive CD59 expression results I in substantially less expression on endothelium than on spleen cells.     

Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection

Abstract: Research in pig-to-primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell- and tissue-based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human α1,2-fucosyltransferase (H transferase, HT) in pigs modifies the cell-surface carbohydrate phenotype resulting in reduced Galα1,3-Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum-mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro-inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.     

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.     

Xenogeneic ex vivo hemoperfusion of rhesus monkey livers with human blood

Abstract In order to copy the clinical situation of concordant xenotransplantation, Rhesus Monkey livers were hemoperfused with human blood. Changes of immunological (TNFα, IL-1β, IL-2, IL-2R, IL-6, IFNy, TXB2, 6kPGF1α, sICAM-1, SELAM-1, sHLA-I-Ag) and pathophysiological (GOT, GPT, LDH, CK) parameters were followed. Our experiment proves that all phenomena start in the first hour of xenogeneic blood circulation. Xenogeneic rejection in our concordant system is surprisingly severe. Preformed natural antibodies only cannot be the reason of such a damage [5, 6]. We think that beside other important immunological mechanisms, humoral mediators play a considerable role at the beginning of a xenogeneic rejection.     

Expression of Complement Regulatory Proteins in Accommodated Xenografts Induced by Anti-α-Gal IgG1 in a Rat-to-Mouse Model

Anti-graft antibodies are often associated with graft rejection. Under special conditions, grafts continue to function normally even in the presence of anti-graft antibodies and complement. This condition is termed accommodation. We developed a xenograft accommodation model in which baby Lewis rat hearts are transplanted into Rag/GT-deficient mice, and accommodation is induced by repeated i.v. injections of low-dose anti-α-Gal IgG₁. The accommodated grafts survived a bolus dose of anti-α-Gal IgG₁, while freshly transplanted second grafts were rejected. To study the mechanism of anti-α-Gal IgG₁-mediated accommodation, both real-time PCR and immunohistochemical staining revealed elevated expression of DAF, Crry and CD59 in the accommodated grafts. In vitro exposure of rat endothelial cells to anti-α-Gal IgG₁ also induced the up-regulation of DAF, Crry and CD59, as revealed by Western blot analyses, and was associated with an acquired resistance to antibody and complement-mediated lysis in vitro . Collectively, these studies suggest that the up-regulation of complement regulatory proteins may abrogate complement-mediated rejection and permit the development of xenograft accommodation.