Gene therapy in transplantation

Gene Therapy volume 6, pages1499–1511(1999)Cite this article

Abstract

Facilitation of solid organ and cell transplantation depends on metabolic and immunologic factors that can be manipulated ex vivo and in vivo using gene transfer technology. Vectors have been developed which can optimally transfer relevant genes to various tissues and organs. Interventions aimed at promoting tissue preservation before transplantation, prevention of oxidative stress and immunological rejection have recently become attractive options using viral and nonviral gene delivery vehicles. Further understanding of the mechanisms involved in tolerance induction as well as the facilitation of xenogeneic engraftment have made possible a variety of avenues that can be exploited using gene transfer technology.

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Introduction

Transplantation medicine is entering a new age and is slowly undergoing an important paradigm shift away from the traditional chemical immunosuppression regimens that dominate the clinic today, to modalities with tissue and cell specificity. This shift promises to be more evident in the years to come when xenotransplantation will be less of an issue for debate and more of a challenge to clinicians, transplantation immunologists and biologists.

Traditionally, graft acceptance has depended on a large number of variables including the overall health of the donor and recipient, the state of the organ or tissue at the time of recovery, and the time between death and recovery which affect the degree of oxidative damage and ischemia,1 the technical success of the transplantation procedure itself and the degree of immunocompatibility between the donor and the recipient.2,3 Much focus has been placed on the means to prevent immune rejection of the graft. Xenobiotic agents have been quite successful, however, the level of associated toxicity of the currently prescribed agents and their potentially life-threatening side-effects that result after years of administration necessitates new, specific therapies. Our aim here is not an exhaustive and comprehensive overview of the different clinical and experimental approaches currently used and being evaluated to prevent graft rejection. Excellent recent reviews have been published and the reader is urged to consult them.4,5 Rather, we will focus on the steps at which one can potentially intervene at the molecular level using gene transfer technology to prevent tissue/organ damage during isolation from the donor and the subsequent preservation. We will also focus on possible novel genetic interventions as well as cell therapies during and after transplantation which could promote engraftment and survival of the tissue/organ as well as confer protection from the immune response. Finally, we will review and discuss existing and potential genetic strategies for facilitating xenotransplantation. The possible targets for intervention to facilitate transplantation by gene transfer to the transplanted organs or to antigen presenting cells are illustrated in Figure 1a and b.

Figure 1
figure1

Molecular pathways for potential intervention using gene transfer strategies. Panel a depicts a transplanted organ or tissue and the possible pathways that can be blocked or modulated to protect it from ischemia/reperfusion injury as well as acute and chronic rejection. Panel B shows interactions between APC and effector T lymphocytes that could be targets for blockade or immunomodulation.

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Vectors for transplantation

Central to the application of gene therapy to facilitate transplantation is the ability to deliver exogenous nucleic acids to cells of various tissues. Although some succsss has been reported in the uptake of ‘naked’ DNA, efficient delivery and persistent expression has been limited to only a few tissues such as muscle. To improve the efficiency and stability of gene delivery, both viral and nonviral vectors are being developed and tested for gene transfer. Nonviral vectors such as liposomes are clearly nonpathogenic, but also are less efficient in the transfer of nucleic acid to cells. In addition, the resulting gene expression is transient, which may or may not be desirable, depending upon the nature of the therapeutic gene to be delivered. Viruses have evolved to deliver efficiently nucleic acid to specific cell types as well as avoid immunosurveillance. Retroviral vectors are able to integrate stably into the host DNA and, in theory, express the transgene for the life of the infected cells. Murine-based retroviral vectors are unable to infect nondividing cells, limiting their use of gene transfer to differentiated cells. However, lentivirus-based vectors, able to infect nondividing cells, are well suited for gene transfer to differentiated cell types. Adenoviral and herpes simplex virus vectors also are able to infect nondividing cells and express transgenes at high levels. The recent development of both adenoviral and HSV-based vectors that do not express any viral genes products may be useful for gene transfer to facilitate transplantation. In addition, adeno-associated virus that is able to infect different nondividing cells with various efficiency, may also be useful for certain applications. A list of both viral and nonviral vectors that potentially could be used for facilitating transplantation is summarized in Table 1.

Table 1 Vectors for gene transfer
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Preservation of organ and tissue integrity and function

As soon as the vital activities of an organ donor cease, there is a race against time to isolate and preserve tissues and organs. The cessation of oxygen supply triggers cellular and tissue degeneration caused by superoxide, free radicals and nitric oxide as well as a cascade of pro-apoptotic events.6,7 At this point, the organ is extremely susceptible to injury, and despite the existence of preservation solutions like University of Wisconsin and Euro-Collins, to date, few organs have been spared even mild ischemia. In fact, this initial lesion may be an important contributing factor to acute and even chronic allograft rejection.8,9 Reperfusion of the organ presents another opportunity for further damage which can recruit host immune cells to the site of injury. Endothelial cells are particularly susceptible to and an early target of preservation/reperfusion injury.10,11

Among the mechanisms that can contribute to ischemia/reperfusion injury (IRI) one can include neutrophil infiltration, consequent to the effects of proinflammatory cytokines released at the site of injury.12,13,14 Kidneys that were mildly ischemic and then perfused with primed neutrophils underwent acute renal failure.15 In general, neutrophil infiltration begins under the direction of chemotactic factors such as IL-8 and other chemokines.15,16,17,18,19 Antibody-mediated neutralization of the macrophage inflammatory protein-2 (MIP-2) and Kuppfer cell-derived chemokines resulted in a significant decrease in neutrophil accumulation, edema and cellular injury in a hepatic model of IRI.17 Changes in the endothelium at the site of injury result in the up-regulation of endothelial cell-surface adhesion molecules (ie intercellular adhesion molecules; ICAMs: ICAM-1, ICAM-2 and platelet-endothelial cell adhesion molecule; PECAM-1) which will result in neutrophil adherence.11,20,21,22,23 Molecules that are involved in leukocyte-endothelial cell interactions and quite possibly in IRI are indicated in Table 2. Following endothelial attachment, the neutrophils can migrate through the endothelium and come into direct contact with target cells. Stimulated neutrophils will then release mediators such as reactive oxygen species that lead to cell killing. Generally, the accumulation of polymorphonuclear neutrophils within a graft begins within 1 h of transplantation. In heart allografts, there is an up-regulation of E-selectin expression on arterioles between 3 and 6 h followed by the up-regulation of ICAM-1 on virtually all of the endothelial cells by 6–12 h.8 Tumor necrosis factor alpha (TNFα) release is observed immediately following neutrophil infiltration and at about the same time as the infiltration of macrophages and natural killer (NK) cells.8,24 In mouse and rat liver, proinflammatory molecules such as TNFα, IL-1β and activated complement factors are capable of stimulating neutrophil sequestration25,26,27 and antibodies directed to TNFα can prevent hepatic neutrophil sequestration in mice.24

Table 2 Molecules involved in leukocyte–endothelial cell interactions in humans
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The role of cytokines is particularly important in neutrophil-dependent and perhaps -independent IRI as it is in post-transplant rejection. Cytokines, including IL-1 and TNFα can influence neutrophil chemotaxis indirectly by up-regulating the expression and production of chemokines like IL-816 in human hepatocytes, in vitro. In a cardiac allograft model, the primary elaborators of TNFα appear to be resident macrophages as well as macrophages that infiltrate the organ at the same time as do neutrophils.28 Depletion of resident macrophages from allogenic islets to be engrafted into rat liver, as well as Kupffer cells from the recipient liver itself prolonged islet allograft survival, perhaps partly because of the absence of IL-1 and TNFα.29 It was shown that recombinant IL-1 and TNFα were capable of mimicking endotoxemia-stimulated liver damage,25,26 in part by promoting the up-regulation of adhesion molecules involved in neutrophil extravasation.25,26 This was also seen in reperfused human intestine11 as well as in chronic rejection of rat renal allografts.30 Antibodies (Abs) directed to TNFα could prevent neutrophil migration to isolated rat liver sinusoidal endothelial cells previously activated by the supernatant of lipopolysaccharide-stimulated monocytes in vitro.31 The myocardial dysfunction and cardiomyocyte death that occurs in IRI is partly TNFα-dependent as well. In a rat heart allograft IRI model, adenosine-induced prevention of TNFα action protected against IRI and improved post-ischemic myocardial function.28 Blockade of IL-1 binding to its cognate receptor by the interleukin-1 receptor antagonist protein (IRAP) in a rat model of hepatic IRI led to a significant reduction of free radical formation, endothelial cell-adherent leukocytes and overall tissue injury.32,33

The adhesion process is important for neutrophil-mediated events. Monoclonal antibodies (mAbs) against a variety of adhesion molecules including P-selectin, ICAM-1 and leukocyte function antigen-1 (LFA-1) have been shown to reduce the severity and consequences of IRI in rodent models. In a rat lobar liver IRI model, the severity of IRI was reduced following treatment with mAbs against LFA-1 and ICAM-1.34 Furthermore, a reduction of renal dysfunction and tissue damage were observed following the administration of P-selectin mAb in a rat kidney IRI model.34 mAbs against ICAM-1 and LFA-1 were protective for endotoxin-induced liver injury15,25,35 and prevented the adherence of neutrophils to the endothelium as well as their diapedesis following reperfusion in a rat hepatic lobar IRI setting.36 Furthermore, administration of anti-LFA-1 mAbs before reperfusion of heterotopically transplanted allogeneic rat hearts resulted in decreased neutrophil accumulation as well as better contractility compared with control and OKT3 mAb-treated rats.37 Finally, IRI was attenuated, but not completely blocked in a rat lung isograft model using a combination of anti-ICAM-1 and anti-LFA-1 Ab before reperfusion.38

In addition to adhesion molecule up-regulation, cytokines can promote the formation of deleterious reactive oxygen intermediates (ROI) such as superoxide.39 Acute renal failure in humans was prevented40,41 and graft survival was markedly enhanced following the administration of superoxide dismutase, perhaps by inhibiting the effects of ROI following reperfusion.42,43,44 In orthotopic rat liver transplantation where the organ was cold- preserved before engraftment, IRI was significantly diminished following the addition of a modified form of recombinant superoxide dismutase (SOD) in the preservation fluid.45 Furthermore, SOD administration following allogeneic lung transplantation significantly improves the degree of histologic rejection suggesting that prior intervention may have even inhibited or prolonged the onset of rejection.46 More evidence that SOD can confer protection against ischemia reperfusion derives from transgenic mice expressing human copper-zinc SOD in cardiomyocytes and cardiac endothelial cells. Following 30 min of global ischemia, there was an almost complete quenching of the superoxide burst along with a two-fold increase in cardiac contractile function and a significant decrease in infarct size.47 Woo et al48 have demonstrated that adenoviral delivery of SOD to isolated perfused neonatal mouse hearts resulted in significant protection against ischemia reperfusion injury whereas Zwacka et al49 demonstrated the feasibility of using an adenoviral vector to deliver mitochondrial SOD to mouse liver as a means of protecting against acute ischemia reperfusion injury and NF-κB activation. Of all the leukocytes, neutrophils which accumulate in liver following reperfusion are believed to be an important source of ROIs including superoxide.7,50,51

Taken together, these observations suggest that it may be feasible to block (1) cytokine–receptor interactions, (2) neutrophil activation and chemotaxis, and (3) ROI formation or combinations of the above in preventing or minimising IRI. Table 3 illustrates potential therapeutic proteins which could be delivered to the tissue or organ before transplantation. Recent observations show that such approaches may be feasible as adenovirally mediated catalase gene transfer to human and porcine islets in vitro reduced the effects of oxidant stress.52 A potentially important area of study that requires further definition is the compatibility between gene delivery vehicles and preservation solutions, as it is anticipated that any gene engineering of the donor organ or tissue will be performed in solution immediately following organ procurement before reperfusion.

Table 3 Potential interactions and biochemical pathways that can be inhibited minimizing ischemia/reperfusion injury as well as prolonging allograft survival
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Inhibition of leukocyte-mediated rejection

As soon as an organ, a tissue or a homogeneous cell population derived from a genetically mismatched donor is transplanted it is immediately scrutinised by the host immune system. The integrity of the graft is important since any damage it may have incurred during procurement and preservation will almost certainly act as a trigger for cellular infiltration. These cells include macrophages, NK cells, neutrophils, a small population of dendritic cells (DC) as well as activated endothelial cells in the graft or in the immediate surrounding area that is to act as its site or environment of engraftment. In fact, the surgical trauma that the host environment is subject to during the engraftment procedure can be considered a contributing factor to the activation of a local immune response mediated by antigen-nonspecific processes that may include complement, neutrophils, NK cells and macrophages. These cells may then initiate an inflammatory cascade resulting in an infiltrate whose constituent cells such as T and B lymphocytes, as well as macrophages and DC can then mediate more specific antigen-dependent processes. As organs are revascularised, host leukocytes begin migrating, tethered to the endothelium through cell-surface adhesion molecules of the selectin family.53 In response to proinflammatory cytokines like TNFα and IL-1, endothelial cells express E- and P-selectins, the latter which can also interact with neutrophils.15 Further adhesion is mediated by integrins including LFA-1/ICAM-1 and very late antigen-4/vascular cell adhesion molecule-1 (VLA-4/VCAM-1) which promote interactions between leukocytes and endothelial cells.15,25,54,55 Table 4 illustrates potential interactions that can be blocked by proteins whose genes could be delivered by one or more of the vectors indicated in Table 1.

Table 4 Antagonism of cytokine-receptor and costimulatory interactions, and its effects on allograft survival
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Of all the adhesion molecule interactions, Springer et al56 have suggested that the ICAM-1-LFA-1 and the LFA-3-CD2 interactions may be the most important in graft rejection. Although this may be an oversimplification, a series of experiments as well as experimental therapies have yielded promising results. A short-course treatment of mice and rats with a daily injection of anti-ICAM-1 and anti-LFA-1 Ab prolonged cardiac allograft survival.57 A similar significant prolongation of renal allograft survival was noted in non-human primates treated with anti-ICAM-1 Ab for 12 days following transplantation.58 ICAM-1 antisense therapy led to a prolongation of allogeneic islet survival and when combined with an anti-ICAM-1 Ab, the survival of the islets was indefinite.4 Macacque monkeys receiving an allogeneic heart were able to sustain its survival for up to 95 days following peri-operative administration of an anti-LFA-1 Ab.4 A combined regimen of anti-ICAM-1 and anti-LFA-1 Ab in mice and rats undergoing transplantation of allogeneic small bowel prolonged graft survival and in some instances, indefinitely.59 Kaplon and colleagues60 have developed a protein reagent that is a fusion composed of the extracellular domain of LFA-3 and an immunoglobulin carboxy-domain (LFA-3TIP). With this reagent, they demonstrated prolongation of baboon cardiac allograft survival for a short time60 and more recently, this reagent was used to show protection against immune rejection of human skin allografts in an immunodeficient mouse/human chimera.61 Finally, clinical trials with an anti-ICAM-1 Ab in renal transplantation demonstrates its potential as it significantly improved renal function and prolonged graft survival in treated patients relative to the untreated control group.4

Leukocytes migrate to the site of tissue injury where they are further activated by locally produced chemokines. Chemokines are potent immunoattractants that are fairly resistant to degradation and are sequestered by proteoglycans on the endothelium15 ensuring their concentration at the site of inflammation. They promote adhesion to the endothelium in addition to acting as chemoattractants and leukocyte activators.15 The interactions between chemokine ligands and their receptors on leukocytes can also be a target of blockade by antibodies or soluble receptors, either alone or in combination with other inhibitors of leukocyte–endothelium interactions. A potential strategy could involve the ex vivo perfusion of an organ with viral or nonviral vectors encoding inhibitors of endothelium–leukocyte interactions as well as soluble chemokine receptors that act as decoys for the chemokines. In fact, such an ex vivo approach has been successfully demonstrated in murine liver, heart and islet perfusion settings where the perfusion solution was supplemented with adenoviral vectors encoding β galactosidase. Even when the perfusion was carried out at 4°C, reporter gene activity was evident as early as 1 h following perfusion.62,63,64,65

Any immune response requires some degree of orchestration so that appropriate cells are activated to perform a specific function at a defined time, either alone, as a homogeneous population or in concert with other cells. Cytokines are particularly important in this regard, especially in the context of transplantation. These and other immunoregulatory signaling molecules can be released by resident antigen-presenting cells within the organ to be transplanted or by host antigen-presenting cells, endothelial cells and fibroblasts at the site of transplantation and revascularisation. In most cases, a proinflammatory cascade is established with IL-1 and TNFα at the summit. These cytokines will then activate antigen presentation, either by endothelial cells or by other antigen-presenting cells, in particular dendritic cells, as well as a plethora of other signaling cascades culminating in the influx of T and B lymphocytes. Blockade of cytokine binding to their receptors, either by mAb, decoy receptors or antagonists has beneficial effects in prolonging allograft survival in a number of settings. Injection of a soluble form of the type I IL-1 receptor, which transmits the biologic actions of IL-1, to mice receiving allogeneic hearts prolonged their survival from a mean of 12 days to 17 days.66 Combination therapy with cyclosporin A and the interleukin-1 receptor antagonist protein (IRAP), a naturally occurring competitive antagonist of IL-1 binding to its receptors, significantly prolonged the survival of rat heart allografts with IRAP treatment alone having a statistically significant effect. Rats treated with IRAP alone showed a decrease in graft-infiltrating leukocytes.67 More recently, Dana et al68 demonstrated a statistically significant prolongation of allogeneic corneal transplants in mice following topical application of IRAP to the grafts. Inflammation of the IRAP-treated corneas was also significantly-reduced as compared with the control grafts.68

The administration of cytokines that can down-regulate or suppress an amplification of the initial immune response has also markedly improved allograft survival, as will be discussed below. Possible mechanisms include prevention of class II MHC expression and adhesion molecule up-regulation on endothelial cells.69,70 Of all the cytokines, transforming growth factor β1 (TGF-β1) and the viral homologue of IL-10 (vIL-10; encoded by the Epstein–Barr virus) have already been evaluated for allotransplant prolongation following gene transfer to the organ to be engrafted. Murine cardiac allograft survival was prolonged following the gene transfer of TGF-β1 using an expression plasmid or with vIL-10 using a retrovirus as vehicle.71,72 Additionally, vIL-10 expression from an adenoviral vector in the same cardiac allograft model also prolonged graft survival.73 In rat liver allografts, adenovirally delivered vIL-10 led to a significant decrease in the in vitro alloreactivity of peripheral blood leukocytes at 7 days after transplantation,74 and TGF-β1 gene transfer using the same vehicle down-regulated TNFα and IFNγ production early after orthotopic transplantation of rat allogeneic liver grafts.75 IL-4 is another cytokine that has been associated with immunosuppressive properties, but to date its effects on allograft survival remain unclear. IL-4 treatment of rats prolonged the survival of allogeneic neonatal hearts,76 and similar prolongation of graft survival was seen in mice transplanted heterotopically with an allogeneic heart from a mouse transgenic for IL-4.77 Combined treatment of IL-10 and IL-4 of non-obese diabetic (NOD) mice (a strain that develops autoimmune diabetes) prevents the recurrence of diabetes following syngeneic islet transplantation78 whereas adenoviral gene transfer of IL-4 and IL-10 to NOD syngeneic islets fails to protect them from autoimmune destruction.79

These results, obtained from experiments using cytokines with immunosuppressive characteristics, although promising, indicate that neither cytokine is able to prolong allogeneic organ survival as effectively or as long as treatments aimed at blockade of co-stimulation or adhesion. A potential strategy could be gene delivery of immunoregulatory cytokines such as vIL-10 and/or TGF-β1 along with co-stimulation and adhesion blockade using either multicistronic gene delivery vehicles or co-infection with multiple vectors.80,81

Traditionally, the main reason why allogeneic grafts were rejected was because of MHC mismatch. Although today, we know that there are many factors other than MHC discordance which contribute to graft rejection, a number of recent studies have been initiated whereby donor MHC-encoding cDNAs have been transferred to host-derived cells and the cells then transferred back to the recipient before organ transplantation. Madsen et al82 initially demonstrated the potential of this approach in the prolongation of murine cardiac allograft survival following the injection of genetically engineered autologous fibroblasts expressing donor class I or class II MHC genes. More recently, it was shown, in different experimental models, that specific tolerance can be induced following the reconstitution of the host bone marrow with genetically modified host hematopoietic cells engineered to express donor MHC genes.83,84 Another interesting approach to induce donor-specific tolerance has been attempted by intrathymic injection of autologous cells engineered to express donor MHC. Knechtle et al85 reported donor-specific unresponsiveness to MHC-disparate liver and cardiac transplants when autologous myoblasts and myotubes were engineered to express donor class I MHC. In a similar approach, these investigators injected cDNA encoding donor class I MHC antigen to rat thymus along with anti-lymphocyte serum. In these rats, allogeneic liver survival was significantly prolonged and interestingly, passive transfer of splenocytes from tolerant rats to naive recipients prolonged cardiac allograft survival.86 Although thymic injection is not a routine procedure in humans, a potential area worth investigating could be the identification of an easily isolatable cell type that could migrate to, or naturally reside in the thymus. Candidates include thymic epithelial cells and perhaps thymic DC. Once identified, this cell type could be expanded ex vivo and genetically engineered to express not only donor-type MHC, but other immunoregulatory molecules that can impact on donor-specific tolerance through central mechanisms.

Inhibition of costimulation

In addition to interacting with the MHC–peptide complex on antigen-presenting cells, antigen-specific T lymphocytes require co-stimulatory signals to become fully activated. In the absence of co-stimulation, the T lymphocytes will either enter into a state of energy, or they may be primed for apoptosis. Upon initial activation, following antigen presentation, T lymphocytes up-regulate the ligand for the CD40 antigen (CD40L/CD154). The CD40 molecule is expressed primarily on cells with a capability to present antigen such as dendritic cells, B lymphocytes, macrophages, fibroblasts and endothelial cells.87 Once ligated by CD40L through CD40, antigen-presenting cells (APC) respond by producing pro-inflammatory cytokines such as IL-1β, TNFα, IL-6 and IL-8,88,89,90,91 by modulating the expression of leukocyte adhesion molecules92,93,94 and by upregulating the expression of anti-apoptotic genes.87,95,96 Concurrently, the (APC) will produce IL-1297 and recent data suggest that IL-12 further enhances the cell surface expression of CD40L on T lymphocytes98 acting like a feed-forward mechanism enhancing the function of the (APC) as well as the state of activation of the responding T lymphocyte. Finally, ligation of CD40 stimulates the cell-surface expression of another set of important co-stimulatory molecules on (APC), CD80 and CD86 (B7-1 and B7-2).99,100,101

For full activation, a T lymphocyte must interact with CD80 or CD86 via the CD28 molecule. In addition, T cells also possess another cell-surface antigen called cytotoxic T lymphocyte antigen-4 (CTLA-4) that can bind with even higher affinity to CD80 and CD86. Unlike CD28-mediated T cell co-stimulation, signaling through CTLA-4 appears to attenuate and perhaps abrogate activation.101 None the less, blockade of both the CD40–CD40L and the B7–CD28 pathways has been attempted with encouraging results in numerous transplantation models, as reviewed immediately below.

Although initial attempts to induce permanent acceptance of allografts with blockade of CD40–CD40L alone failed, significantly prolonged allograft survival was achieved. Administration of an antibody against CD40L in conjunction with either small lymphocyte infusion or with T cell-depleted splenocytes prolonged murine islet allograft survival.102,103 In addition, the same antibody regimen prolonged murine cardiac allograft survival.104 In a recent experiment, Kirk et al105 showed that administration of rhesus monkeys with an anti-CD40L antibody for up to 28 days resulted in prolonged kidney allograft survival for more than 90 days, however, a second course of antibody administration was required to further prolong the allograft. What was more striking was the prolongation of these allografts to more than 180 days when a combination of CD40–CD40L and B7–CD28 blockade was employed.105 Moreover, in a murine skin or a cardiac allograft model, long-term acceptance was achieved using a combined blockade of the CD40–CD40L and B7–CD28 pathways.106 Finally, long-term acceptance of xenogeneic cardiac or skin grafts was achieved in rat-to-mouse and pig-to-mouse models along with a marked inhibition of a cellular response to xenoantigens using a combined regimen of CD40–CD40L and B7–CD28 blockade.107

In 1992, Lenschow et al108 showed for the first time, that it was possible to induce long-term xenogeneic engraftment as well as donor-specific tolerance to alloantigens by co-stimulation blockade. Using a fusion protein consisting of the extracellular domain of CTLA-4, which binds CD80 and CD86, and the Fc portion of IgG1 to stabilize the protein and prolong its half-life in vivo, they were able to prolong the survival of human pancreatic islets in mice. The protein fusion, called CTLA-4Ig, has been tested since then in numerous allogeneic and xenogeneic models, either alone or in combination with xenobiotics or antibodies against adhesion molecules or CD40L to prolong graft survival, in most cases with some success and in a few cases with indefinite survival of the graft.106,109,110,111,112 In a set of experiments where murine islet allografts were coated with CTLA-4Ig, transplantation led to prolongation of graft survival to more than 150 days in 50% of the hosts which also exhibited donor- specific tolerance despite the presence of a noninvasive CD4+ and CD8+ T lymphocyte peri-islet accumulation.113 The importance of the B7–CD28 pathway in allograft acceptance was further demonstrated by Lenschow et al114 who used Abs against CD80 and CD86, alone or in combination to prolong murine allogeneic islet survival. Interestingly, the effects of the anti-CD86 antibody on allograft survival were more pronounced than those of the anti-CD80 antibody.114 Recently, CTLA-4Ig has been used to prolong pancreatic islet allograft survival in a monkey model, where in addition to prolongation of allograft survival, donor-specific hyporesponsiveness was observed in vitro, as well as suppression of humoral responses.115

More recently, transfer of CTLA-4Ig cDNA to allogeneic as well as xenogeneic tissues destined for transplantation, has yielded encouraging results. Co-transplantation of syngeneic myoblasts stably transfected with CTLA-4Ig cDNA together with allogeneic islets prolonged their survival relative to unmodified myoblasts.116 Transplantation of allogeneic mouse islets expressing the human CTLA-4Ig cDNA following gene gun delivery also prolonged allograft survival significantly (67 versus 13 days, unmodified islets).117 Using a replication-defective adenoviral vector, Olthoff et al118 introduced murine CTLA-4Ig cDNA into cold-preserved rat liver which was then transplanted into an allogeneic host. Following revascularization, there was indefinite prolongation of allograft survival with no apparent reperfusion injury, and with donor-specific tolerance achieved. An adenoviral vector was also used to deliver CTLA-4Ig cDNA to rat hearts whose survival was prolonged significantly when transplanted into an allogeneic host.119 Additionally, prolonged survival of allogeneic mouse islets expressing CTLA-4Ig was recently observed.120 Preliminary studies using gene transfer of CTLA-4Ig to solid organs for allogeneic or even xenogeneic transplantation are encouraging and should be extended. Clearly, however, co-stimulation will most likely involve mediators in addition to B7–CD28 or CD40–CD40L. Once these are identified, gene therapy vectors that can carry multiple transgenes may become extremely useful in modifying cells and tissues for the purpose of prolongation of allo- or xenogeneic graft survival and perhaps even acceptance. The xenogeneic barrier, however, poses another level of difficulty, above and beyond cell-mediated rejection and will be discussed below.

Immunomodulation using dendritic cells

Dendritic cells (DC) are now regarded not only as the initiators and regulators of immune responses, but also as potentially powerful tools for the therapeutic manipulation of immune reactivity in cancer, infectious disease, allograft rejection and autoimmunity.121

DC are high specialized mobile sentinels derived from CD34+ hematopoietic progenitors, that convey antigens from peripheral sites, such as the skin or other nonlymphoid tissues, to T cells in secondary lymphoid organs. The shape and mobility of DC are well-suited to their roles in antigen (Ag) capture, and presentation to rare, Ag-specific T cells expressing receptors that recognize Ag bound to major histocompatibility complex (MHC) molecules. Mature DC synthesize high levels of IL-12, and are rich in MHC products, and many accessory molecules that enhance intercellular adhesion (eg CD54, CD58) and co-stimulation (eg CD40, CD86).

The natural immunologic adjuvant properties of DC have been the focus of research on these cells for so long that their importance in tolerance is often overlooked. A role for DC in tolerance induction was recognized initially in the context of intra-thymic self-tolerance. It is now evident that DC may also be important in regulation of peripheral self-tolerance. In experimental models, DC tolerogenicity has been manifested by the suppression of T cell responses in T cell ontogeny, allo-, tumor- and autoimmunity.122 Several agents, eg UV-B irradiation, IL-10, transforming growth factor β (TGFβ), or the chimeric fusion protein CTLA-4-Ig that blocks the B7–CD28 costimulatory pathway, can render DC tolerogenic. Mechanisms underlying the tolerogenic properties of DC include induction of anergy, a shift to a Th2 predominant response, apoptosis, or induction of regulatory (‘veto’) cells.122,123

Since DC constitutively express MHC antigens, home to T-dependent areas of recipient lymphoid tissue, and interact selectively with host T cells, they are attractive targets for genetic manipulation to improve the outcome of allograft survival. Candidate genes include viral (v) IL-10, TGFβ, Fas L (CD95L) and CTLA4Ig.

In principle, use of viral vectors to modify DC in vitro reduces the administration of viral proteins, in contrast to direct in vivo administration of viral vectors, thus minimizing the generation of antiviral Abs that can prevent repetition of treatment. Of the transfection methods available, gene delivery to DC by replication-defective Ad vectors is the most efficient. Primary cultures of murine myeloid DC or DC progenitors can be adenovirally transduced to express CTLA-4Ig123 or TGFβ.124,125 CTLA-4Ig-transduced DC exhibit selective and marked reductions in cell surface staining for CD86, the capacity to induce alloantigen-specific T cell hyporesponsiveness, and enhanced survival in non-immunosuppressed, allogeneic hosts.123 The in vivo presentation of alloantigens by donor DC in the absence of co-stimulation plus local production of immunosuppressive molecules, such as CTLA-4Ig, is likely to promote the inhibition of anti-donor reactivity, and potentiate tolerance induction, with minimization of systemic immunosuppressive effects. Retroviral transduction of replicating immature DC to express vIL-10 has also been achieved. These cells exhibit marked reductions both in cell surface MHC and costimulatory molecule expression and in T cell allostimulatory activity. Moreover, retroviral delivery of vIL-10 to the DC promotes the induction of T cell hyporesponsiveness.126 Using genetically engineered DC to manipulate anti-donor immune reactivity may be a feasible approach to improved therapy of allograft rejection.

In addition to dendritic cells, other APC such as macrophages can be harnessed to induce tolerance to alloantigens. In fact, recently Zhang et al127,128 demonstrated the utility of Fas-deficient APC in inducing tolerance against adenovirus and furthermore, they achieved alloantigen-specific T cell tolerance following adenoviral gene transfer of Fas ligand. Using this approach, it may be feasible to use genetically engineered DC or other APC expressing Fas ligand along with specific alloantigens or even autoantigens as a means of achieving tolerance.

Inhibition of the immune response using death ligands

One of the major mechanisms by which activated lymphocytes are eliminated and the extent of immune reactivity regulated is by apoptosis, triggered by the ligation of cell surface Fas antigen by its ligand (FasL/CD95L).129,130 The role of FasL in conferring immune privilege to sites such as the testis by genetic and functional criteria compelled a number of groups to evaluate the feasibility of using it to provide immunoprotection to cells and organs against immune cell infiltration and destruction.130,131,132 Initial attempts to protect allogeneic islets either by overexpressing FasL in myoblasts for co-transplantation, or by engineering the islets themselves to express FasL, however, have led to conflicting results. The survival of islet allografts transplanted with myoblasts overexpressing FasL was significantly prolonged,133 whereas transgenic mice overexpressing FasL in pancreatic β cells experienced accelerated islet destruction.134 Similarly, mice transplanted with FasL-expressing islet allografts mounted a vigorous and predominantly neutrophil-mediated destruction of the islets.135 In contrast, it appears that FasL expression determines the survival of allogeneic corneal transplants in a mouse model where 45% of FasL+ orthografts were accepted but none of the FasL-negative corneas.136 Moreover, rat liver allograft survival was prolonged in animals receiving organs overexpressing FasL.137,138 Recent results suggest that the form of the FasL may be important in conferring protection or susceptibility to apoptosis. It appears that soluble FasL variants confer protection from apoptosis whereas cell-membrane bound molecules confer susceptibility.139 Indeed, recent data support this hypothesis based upon a statistically significant prolongation of allogeneic islets overexpressing soluble human FasL.120 Further evidence supporting the role of FasL as an immunoprotective molecule derives from observations that rat renal allogeneic graft survival is prolonged when FasL is delivered to the donor kidney by an adenoviral vehicle.140

Inhibition of apoptosis

Although the role of FasL as a molecule that can act as a signal transducer remains unclear, signaling through Fas is necessary for apoptosis induction and blockade of the Fas–FasL interaction may confer protection to allogeneic grafts. This protection could be conferred either by overexpressing antagonists in donor tissue, or by introducing transgenes encoding antagonists to the site of transplantation in the host. One possibility is a soluble chimeric Fas-Ig fusion.141 Indeed, data from a study where Fas-mediated cytotoxic activity by a T cell clone against sensitized recipient target cells could be prevented by addition of the Fas-Ig fusion protein, suggest that a local, high level transfer of the Fas-Ig cDNA to allografts may be feasible in preventing FasL-dependent destruction. Alternatively, signaling-defective, dominant-negative variants of Fas, mutant downstream effectors such as FADD (Fas-associated death domain protein)-a docking protein involved in linking the intracytoplasmic domain of Fas to the death effector molecules,142,143 inhibitors of the proapoptotic cascade like crmA protein encoded by cowpox,144 and bcl-2 family members145,146,147,148,149,150 may all be useful in suppressing Fas-triggered apoptosis induction (Table 3). In fact, bcl-2 and another member of the bcl family (Bcl-xL) have demonstrated protective effects in models such as bone marrow transplantation following chemotherapy-induced myelosuppression,151 and in hypoxic cell necrosis in rat liver following liposome-mediated transfection.152

Xenotransplantation

The current need for increases in the number of human organ transplants is making it extremely challenging to keep pace with the demand if surgeons continue to rely on human cadaveric or liver donor tissues and organs. Although still in its infancy and subject to great debate, the use of animal organs promises to revolutionize transplantation medicine in general. Clearly, many issues need to be addressed before this treatment becomes clinically feasible. This section will briefly summarize the current experimental approaches to prevent rejection of xenografts and will suggest other possibilities for future study. It is worth noting that excellent reviews have already been published on the topic that this section will summarize.153,154,155

The major immediate immunological hurdle that a discordant vascularised xenograft will most probably face is a complement-mediated hyperacute rejection following the binding of preformed, naturally occurring antibodies against xenoantigens. These antibodies exist in non-human primates as well as in humans. This antibody-mediated hyperacute rejection of porcine donor organs has been shown to occur in non-human primate recipients.156,157,158 In fact, almost all of the human Abs bind to an oligosaccharide pig xenoepitope defined as Gal-α(1,3)-Gal-β(1,4)-GlcNAc-protein, also known as the α-galactosyl epitope.159,160 Complement-mediated lysis of the xenograft occurs by the classical pathway culminating in the formation of the membrane attack complex. This hyperacute rejection occurs within minutes. Recent studies indicate that the removal of circulating natural xenoantibodies can prolong xenograft survival significantly. In a pig-to-baboon kidney xenotransplant, removal of the Abs along with immunosuppression resulted in prolongation of graft survival up to the time killed which was 13 days after the transplant.158

Although Ab-dependent complement activation appears to be the most important determinant of hyperacute rejection, it is possible that the alternative pathway can also be involved. Therefore, alternative strategies have been devised to inhibit or prevent hyperacute rejection in the presence of xenoreactive Abs. At the cell surface exist regulatory proteins that modulate the binding of complement cascade proteins and that can regulate their activation. Decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 are all involved in the regulation of complement activation. Presently, a number of groups are generating transgenic pigs that will express human DAF, MCP and CD59, alone or in combinations, with the objective of interfering with the activation of host-derived complement against vascularised pig xenografts.161 In a pig-to-primate model and in the absence of immunosuppression, a cardiac xenograft derived from a human DAF-transgenic pig survived up to 5 days compared with about 2 from non-transgenic donors.162 Furthermore, in another pig-to-primate model, hearts from double-transgenic pigs expressing human DAF and CD59 survived hyperacute complement-mediated damage even when expressed at low levels.163 Finally, competition of complement binding by a soluble complement receptor (CR1) suppressed the destruction of pig cells and prolonged pig xenograft survival.164 The genetic manipulation of pig organs and tissues is certainly feasible and gene transfer of one or all of the genes encoding DAF, MCP or CD59 at the time of organ procurement where concurrent strategies aimed at preventing or suppressing ischemia/reperfusion injury is a potential modality.

Another approach to prevent hyperacute graft rejection involves diversion of xenoepitope oligosaccharide synthesis away from the α-galactosyl type to the oligosaccharide whose terminal residues define the O antigen in human blood to which Abs do not exist. This method involves the genetic engineering of xenogeneic cells to express α-(1,2)-fucosyltransferase.159 This enzyme adds fucose residues to a nascent oligosaccharide. In both pig and human, the substrate for the fucosyltransferase is identical, therefore, by overexpressing the enzyme, the amount of xenoantigen would be significantly reduced. Immunologically, the remodelled oligosaccharide (α-1,2-fucosyl lactosamine) becomes the non-antigenic H-epitope of the antigen defining the human universal donor O blood group, blocking natural antibody-mediated rejection. Indeed, experimental data in vitro demonstrate that as much as 80% of the oligosaccharide is in the fucosylated form in genetically modified cells.159,160,165,166 Moreover, a transgenic mouse overexpressing the enzyme has been generated. It too shows a diversion to the fucosylated variant to a level as high as 80%.159,165 A potential gene therapy strategy would be to introduce the fucosyltransferase to pig organs, preferably at the time of organ procurement, although recent advances in animal cloning and transgenics may obviate this in the future.167,168 A recent novel approach to inhibit the production of anti-xenoepitope antibodies involves the establishment of molecular chimerism. In this approach, bone marrow-derived cells from mice deficient in the galactosyltransferase (the enzyme involved in the synthesis of the α-galactosyl xenoepitope) were infected with a retrovirus encoding the porcine homologue. The mice were then reconstituted with the genetically engineered cells. There was no detectable xenoreactive antibody in their sera up to 51 weeks after transplantation. Moreover, the sera from these mice were unable to kill porcine cells in vitro.169

Although hyperacute rejection may be ultimately prevented, there still remain the obstacles of acute, delayed and chronic graft rejection processes that appear to depend on non-humoral factors and involve cells and processes common to allograft rejection as well as other humoral reactions like antibody-dependent cell-mediated cytotoxicity (ADCC). Of particular importance is the role of the NK cell, especially in acute graft rejection. NK cells normally respond in an MHC-non-restricted manner and recent data suggest that allogeneic tissue may be recognized and targeted by NK cells for destruction.170 Proinflammatory cytokines culminating in the infiltration of the graft by macrophages, T cells and B cells are most likely involved at the early phase of acute as well as delayed rejection. Neutrophils are also potentially important.15 This is clearly an area of study that requires intensive research to further define the cellular determinants of xenograft rejection following the suppression or prevention of hyperacute rejection. Recently, Tran et al171 have engineered primary bovine endothelial cells, which do not express Fas, to express FasL under the control of a tetracycline-inducible promoter. Their objective was to prevent the survival of Fas-positive, xenoreactive lymphocytes in a vascularized xenograft model in vitro. Indeed the engineered endothelial cells were able to kill Fas-expressing cell lines as well as human peripheral blood monocytes.171 One could envision the engineering of xenogeneic tissues so that FasL could be expressed in an endothelial cell-specific manner as an approach for eliminating activated xenoreactive T lymphocytes. Ultimately, the number of variables involved in allo- and xenograft survival will most likely necessitate a patient-specific therapeutic approach. One can envision a ‘mix-and-match’-type gene therapy which could involve genes that interfere with the pathways discussed in this review. Whether intervention in these pathways can prolong xenograft survival in humans is speculative at present, but is certain to lead to insightful information that will improve understanding at the fundamental level of not only xenograft, but allograft rejection as well.

Future prospects

Transplant immunology has made great advances in the past few years, but many issues need to be resolved for a complete understanding of the cellular processes underlying allograft rejection. These processes will also be encountered in xenograft rejection once the hurdle of hyperacute rejection is surmounted. The development of non-immunogenic gene delivery vehicles with the potential for long-term, stable integration in the host genome is presently an area of intense investigation. The need for targeted, regulatable and tissue-specific expression of therapeutic or preventive genes will be even greater in the not too distant future. Immunoregulatory cytokines are at the forefront of research into local immunosuppression and tolerance induction, but other important molecules and interactions need to be evaluated including modifiers of adhesion as well as modulators of neutrophil and NK cell function and activation. Cell therapy, other than bone marrow and islet transplantation, is an area that is in its infancy in the context of transplant immunology. Recently, however, there has been some progress in the genetic modification of the very cells that initiate and propagate an immune response to achieve tolerance to allogeneic or xenogeneic tissues. Finally, the need to define the factors involved in ischemia/reperfusion injury and to modify them in favor of minimizing tissue damage is critical as pre-activation of an immune response leading to graft rejection is highly probable at this time. Early genetic intervention at the time of organ procurement, as well as post-transplant infusion of genetically modified cells may be the transplantation immunologist’s answer to their quest for the Holy Grail.

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Acknowledgements

The authors would like to acknowledge the insightful comments of Dr Adrian Morelli on this review. This work was supported in part by Public Health Services grant DK 44935 to PDR and AI 41011 to AWT. NG is a recipient of a postdoctoral fellowship award from the Juvenile Diabetes Foundation International as well as a prize from the Fonds pour la formation de chercheurs et a l’aide a la recherche (Fonds FCAR) from the provincial government of Quebec, Canada.

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Affiliations

  1. Department of Molecular Genetics and Biochemistry, University of Pittsburgh, School of Medicine, Pittsburgh, 15261, PA, USA

    N Giannoukakis, A W Thomson & P D Robbins

  2. Department of Surgery, University of Pittsburgh, School of Medicine, Pittsburgh, 15261, PA, USA

    A W Thomson

  3. Department of Thomas E Starzl Transplantation Institute, University of Pittsburgh, School of Medicine, Pittsburgh, 15261, PA, USA

    A W Thomson

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  1. N Giannoukakis
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  2. A W Thomson
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  3. P D Robbins
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Correspondence to P D Robbins.

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Giannoukakis, N., Thomson, A. & Robbins, P. Gene therapy in transplantation. Gene Ther 6, 1499–1511 (1999). https://doi.org/10.1038/sj.gt.3300981

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Keywords

  • transplantation
  • gene therapy
  • tolerance
  • allograft
  • rejection

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