Immune Modulation in Xenotransplantation

Authors: Magdalena Boksa Joanna Zeyland Ryszard Słomski Daniel Lipiński

Publish Date: 2014/10/30

Volume: 63, Issue: 3, Pages: 181-192

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Abstract

The use of animals as donors of tissues and organs for xenotransplantations may help in meeting the increasing demand for organs for human transplantations Clinical studies indicate that the domestic pig best satisfies the criteria of organ suitability for xenotransplantation However the considerable phylogenetic distance between humans and the pig causes tremendous immunological problems after transplantation thus genetic modifications need to be introduced to the porcine genome with the aim of reducing xenotransplant immunogenicity Advances in genetic engineering have facilitated the incorporation of human genes regulating the complement into the porcine genome knockout of the gene encoding the formation of the Gal antigen α13galactosyltransferase or modification of surface proteins in donor cells The next step is twofold Firstly to inhibit processes of cellmediated xenograft rejection involving natural killer cells and macrophages Secondly to inhibit rejection caused by the incompatibility of proteins participating in the regulation of the coagulation system which leads to a disruption of the equilibrium in pro and anticoagulant activity Only a simultaneous incorporation of several gene constructs will make it possible to produce multitransgenic animals whose organs when transplanted to human recipients would be resistant to hyperacute and delayed xenograft rejectionAt present transplantation of cells tissues and organs is an effective and frequently the only method to save the lives of patients with endstage organ failure victims of accidents and patients suffering from cancers of the hematopoietic systems or complex immunodeficiencies The main problem in contemporary transplantology is connected with the existing disproportion between the volume of available organs and the number of patients qualified for transplantations Mulligan et al 2008 Neither rationalization of the systems for registering and distributing organs for transplantation nor improving the health in society by promoting healthy lifestyles are sufficiently effective to alleviate the critical deficit of available organs and tissues for transplantations This situation has persisted for many years The problem may be solved thanks to the use of animalorigin cells tissues and organs for transplantation ie xenotransplants The domestic pig Sus scrofa is an optimal donor for transplant organs due to its anatomical and physiological similarities to humans unlimited availability good breeding potential short period to reproductive maturity relatively short pregnancy and high number of offspring Cooper et al 2002 However the considerable phylogenetic distance between this potential donor and the human recipients causes tremendous immunological problems following transplantation It is therefore necessary to introduce genetic modifications and incorporate human genes into the porcine genome which may reduce immunogenicity of xenotransplantationsThe primary cause of transplant rejection in the pighuman system is connected with the Gal antigen Galα13Gal present in glycolipids and glycoproteins found on the surface of porcine cells The Gal antigen is formed as a result of the galactose molecule attaching to Nacetyllactosamine Nlac with the α13glycoside bond and involving the α13galactosyltransferase enzyme Sandrin and McKenzie 1994 Neither the enzyme nor the sugar unit is found in humans or the Old World monkeys Galili et al 1988 Identification and binding of the Gal antigen by xenoreactive antibodies triggers a series of reactions which lead to transplant rejection as a result of “hyperacute rejection” Daniels and Platt 1997The necessary condition to provide organs suitable for interspecific transplantations is to remove the Gal antigen from the surface of xenotransplant cells In 2001 two cooperating research teams one headed by Randall Prather and the other composed of researchers employed at Immerge BioTherapheutics managed to produce pigs with the GGTA1 gene inactivated in the heterozygous system Dai et al 2002 A similar achievement was recorded in the same year by PPL Therapeutics Lai et al 2002 In July 2002 the first piglets with two knockout alleles of the GGTA1 gene were born at PPL Therapeutics Phelps et al 2003 while a similar success was reported by researchers from Immerge BioTherapeutics A small but detectable amount of the Gal antigen is found on the surface of fibroblast cells from porcine embryos with the inactivated gene encoding α13galactosyltransferase in the homozygous system Sharma et al 2003 It is estimated that cells synthesize the Gal antigen at 1–2  in comparison to the amount of the Gal antigen produced in heterozygous cells in relation to the inactivated gene In 2005 the first xenotransplantation was performed with the use of porcine organs with the inactivated α13galactosyltransferase gene Pig hearts were transplanted to baboons subjected to immunosuppression The mean length of xenotransplant survival was 92 days with one of the hearts functioning for 179 days Kuwaki et al 2005 Tseng et al 2005a Yamada et al 2005 None of the organs was rejected as a result of a hyperacute immune reaction Transplant rejections were caused by other less intense reactionsAn alternative to the strategy aimed at rendering the GGTA1 gene inactivate is to incorporate in the porcine genome some human genes encoding enzymes which modify the oligosaccharide structure of glycoproteins and glycolipids found on the cell surface in graft donors The activity of such enzymes should result in the Gal antigen being eliminated or markedly reduced The enzyme α12fucosyltransferase H transferase uses the same acceptor Nlac as α13galactosyltransferase Larsen et al 1990 This enzyme catalyzes the addition of fucose units which leads to the formation of a “H structure” Additional copies of the human H transferase gene incorporated in the donor organisms facilitate modification of surface proteins in donor cells which limits the immunogenicity of organs for transplantation Costa et al 1999 Sharma et al 1996 Both strategies aim to inhibit hyperacute xenograft rejection and were successfully combined The result was transgenic pigs with the inactivated GGTA1 gene and expressing human H transferase Ramsoondar et al 2003 Apart from preventing hyperacute rejection human transferase expressed in porcine cells might also prevent or weaken delayed xenograft rejection The sensitivity of porcine endothelial cells to lysis caused by the involvement of human natural killer NK cells is reduced as a result of human H transferase expression Artrip et al 1999 Expression of human H transferase in the endothelial cells of pigs also limits adhesion and activation of monocytes Kwiatkowski et al 1999 In this way the strategy involving human H transferase causes reduced binding of antiGal specific antibodies thus preventing the development of hyperacute xenograft rejection This strategy also reduces the response involving NK cells and monocytes It is assumed that in the case of certain types of cells expression of human H transferase may prove sufficient to provide protection against rejection whether a hyperacute or delayed reaction Costa et al 1999Another mechanism facilitating a reduction in the amount of Gal antigen on the surface of transplant donor cells uses αgalactosidase GLA which catalyzes the removal of the αdgalactose unit from the Gal antigen found on the cell surface Incorporating the gene encoding the human GLA enzyme into the porcine genome made it possible to produce organs for transplantations with a reduced amount of the Gal epitope on the cell surface and a limited complementdependent cytotoxicity in comparison with the control cells Zeyland et al 2013 Since human GLA does not eliminate all αdgalactose units from the Gal antigen the suggestion is to simultaneously use GLA and α12fucosyltransferase The additive effect of applying both enzymes was observed in the case of cotransfection of different types of cells with genes encoding human GLA and α12fucosyltransferase In such a situation the expression of the Gal antigen is not observed on the cell surface Jia et al 2004 Osman et al 1997 Yan et al 2003 However in double transgenic animals with confirmed integration of both transgenes in the heterozygous system a complete elimination of the Gal antigen from the cell surface was not observed Zeyland et al 2014The incompatibility of porcine complement regulatory proteins CRPs on donor endothelialium with a human complement resulted in uncontrolled complement activation Therefore the presence of human CRPs on the pig endothelium could stop complement activation in the recipient The complement is mainly regulated by CD46 membrane cofactor protein CD59 membrane inhibitor of reactive lysis and CD55 decay accelerating factor factors These proteins belong to a family of structurally and functionally similar proteins which block complement activation and prevent the formation of a complex attacking the cell membranes To date pigs that express human CD46 hCD46 CD55 CD59 have been produced Chen et al 1999 Cozzi et al 2000 Deppenmeier et al 2006 Diamond et al 1996 Fodor et al 1994 Langford et al 1994 Niemann et al 2001 Waterworth et al 1998 Zhou et al 2005 In 2005 a team of researchers headed by McGregor managed to obtain the longest to date mean survival time of a xenograft originating from monotransgenic animals in the pigtoprimate system McGregor et al 2005 Hearts coming from genetically modified pigs with additional copies of the gene encoding the hCD46 factor were transplanted to baboons and survived on average 96 days The maximum transplant survival time was 137 days Transplant survival time was influenced not only by the type of genetic modification applied but also the type and doses of drugs administered after the procedure It transpired that an increased dose of immunosuppressants tacrolimus sirolimus and a reduced dose of anticoagulants considerably extended the mean survival time of a xenogenic transplant in comparison to that of transplants in groups of animals subjected to other types of therapy Byrne et al 2006 Organs coming from such modified animals are protected against attack from the human complement and hyperacute rejection but are still subjected to processes of delayed xenograft rejection To a considerable degree prevention against the further development of xenograft rejection processes is dependent on the administration of immunosuppressants The newest preparations should be selective in their action so that only processes connected with xenograft rejection could be blocked and which would ensure complete immune response against developing infections Ingelfinger and Schwartz 2005Genetically modified pigs with the inactivated GGTA1 gene facilitate the xenotransplantation of porcine tissues and organs into primates by preventing hyperacute rejection due to preexisting antibodies against the Gal antigen Although pigs that lack Galα13Gal particles ensure prolonged survival of xenografts other epitopes are still expressed and may induce acute humoral xenograft rejection as subsequent rejection However the response to nonGal antigens is not as robust as the response against Gal antigens the nonGal antigens play a significant role and suppression of their production is required Antibodies other than antiGal antinonGal antibodies are found in the pretransplant sera of most primates In 2012 baboons were given hearts coming from pigs produced by crossing animals with the inactivated GGTA1 gene and expression of hCD46 The mean transplant survival time was 94 days but the application of B lymphocyte depletion extended the maximum xenotrasplant survival in the organism of the recipient to 236 days These results indicate a crucial role for B cells in the mechanisms of elicited antinonGal antibodies and xenograft rejection The efficient depletion of B cells was obtained by four doses of the antiCD20 antibody Mohiuddin et al 2012 Another antigen occurring on the surface of porcine cells is cytidine monophosphateNacetylneuraminic acid hydroxylase CMAH which catalyzes the reactions forming the Neu5Gc antigen Nglycolylneuraminic acid Double transgenic pigs have already been produced with inactivated GGTA1 and CMAH genes The lower cytotoxicity of human serum in comparison to peripheral blood mononuclear cells PBMCs coming from double transgenic pigs was shown in that study in comparison to cells from animals with the inactivated GGTA1 gene Lutz et al 2013

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