Xenotransplantation: Current Status in Preclinical Research

Abstract

The increasing life expectancy of humans has led to a growing numbers of patients with chronic diseases and end-stage organ failure. Transplantation is an effective approach for the treatment of end-stage organ failure; however, the imbalance between organ supply and the demand for human organs is a bottleneck for clinical transplantation. Therefore, xenotransplantation might be a promising alternative approach to bridge the gap between the supply and demand of organs, tissues, and cells; however, immunological barriers are limiting factors in clinical xenotransplantation. Thanks to advances in gene-editing tools and immunosuppressive therapy as well as the prolonged xenograft survival time in pig-to-non-human primate models, clinical xenotransplantation has become more viable. In this review, we focus on the evolution and current status of xenotransplantation research, including our current understanding of the immunological mechanisms involved in xenograft rejection, genetically modified pigs used for xenotransplantation, and progress that has been made in developing pig-to-pig-to-non-human primate models. Three main types of rejection can occur after xenotransplantation, which we discuss in detail: (1) hyperacute xenograft rejection, (2) acute humoral xenograft rejection, and (3) acute cellular rejection. Furthermore, in studies on immunological rejection, genetically modified pigs have been generated to bridge cross-species molecular incompatibilities; in the last decade, most advances made in the field of xenotransplantation have resulted from the production of genetically engineered pigs; accordingly, we summarize the genetically modified pigs that are currently available for xenotransplantation. Next, we summarize the longest survival time of solid organs in preclinical models in recent years, including heart, liver, kidney, and lung xenotransplantation. Overall, we conclude that recent achievements and the accumulation of experience in xenotransplantation mean that the first-in-human clinical trial could be possible in the near future. Furthermore, we hope that xenotransplantation and various approaches will be able to collectively solve the problem of human organ shortage.

Keywords: coagulation dysfunction; genetically modified pigs; immunological rejection; non-human primate; xenotransplantation.

Figure 1

Antibody-mediated xenograft rejection. (A) Hyperacute rejection. Hyperacute rejection of vascularized porcine xenografts in untreated primates is triggered by the binding of preformed antibodies to the xenoantigenic epitopes (predominantly α1,3Gal) on the surface of donor endothelial cells. The binding antibody deposition induces the activation of complement proteins and formation of the membrane attack complex, leading to lysis of endothelial cells destruction of the graft vasculature and subsequent graft failure. Loss of endothelial barrier function contributes to bleeding, leading to tissue ischemia and necrosis. (B) Acute humoral xenograft rejection. Acute humoral xenograft rejection can be induced by low levels of natural and elicited xenoreactive antibodies. The binding of xenoreactive antibodies to endothelial cells results in complement activation, vascular endothelial activation, and injury caused by antibody-dependent cell-mediated cytotoxicity. Innate immune cells are recruited by activated endothelia and proinflammatory signals. Simultaneously, human antipig antibodies are triggered by natural killer cells and macrophages. MAC, membrane attack complex; C, Complement; NK, natural killers.

Figure 2

Cellular-mediated rejection. (A) Natural killer (NK) cells-mediated rejection. Xenoantibodies bind to donor endothelial cells with their Fab portion. The Fc fraction of the antibody is recognized by FcRs located on the surface of NK cells, triggering the signaling cascade that leads to NK cell destruction. The release of lytic granules (marked as dark spots) leads to pig endothelial cells lysis. The activating NK cell receptors recognize their ligands on the donor cells and trigger lytic granule release. The inability of swine leukocyte antigen (SLA) class I to interact with human inhibitory NK-cell receptors makes porcine cells highly susceptible to human NK-cell-mediated lysis. (B) Macrophages-mediated rejection. Macrophages can be activated by cytokines [e.g., interferon gamma (IFN-γ)] that are produced by xenoreactive T cells contributing to the amplification of the T-cell response (not shown). Macrophage also can also be activated by signals mediated by the Fc receptor for IgG (FcγR) upon interaction with xenoreactive-antibody-coated porcine cells. Macrophages secrete proinflammatory cytokines [e.g., tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, and IL-6) that augmented cytotoxicity of macrophages. (C) T-cell response in xenograft rejection. The direct pathway refers to the recognition of antigens presented by pig antigen-presenting cells (APCs) by recipient T cells. The T cell is activated by interaction between T-cell receptors (TCRs) and the SLA I and II peptide complexes. This interaction results in T-cell-mediated cytotoxicity that is directed against the xenograft vascular endothelium. The indirect pathway refers to the recognition of donor-derived peptides on recipient APCs by recipient T cells. The interaction between primate TCRs and major histocompatibility complex (MHC) and porcine peptide complexes leads to primate T-cell response, including cytokines production and induction of B-cell activation.

Figure 3

The coagulation cascade related to xenotransplantation. (A) Coagulation cascade in primates. Black arrows designate cascade amplification steps. The coagulation cascade is initiated by tissue factor (TF) (extrinsic pathway) or negatively charged surface contact (intrinsic pathway). TF is expressed by vascular subendothelial cells. When endothelium is damaged, TF is exposed to the circulation and forms complexes with factor VIIa, activating factors V and X. Factor Xa converts prothrombin to thrombin. Thrombin then cleaves fibrinogen into fibrin monomers and activates factor XIII, which cross-links fibrin monomers into an insoluble clot. In response to shear stress, von Willebrand Factor (vWF) binds to glycoprotein 1b (GPIb) on platelets leading to platelet activation and adhesion (83). Activated platelet bind to fibrinogen to mediate platelet aggregation and endothelial adherence. Red lines show the natural inhibitors of coagulation. (1) Tissue factor pathway inhibitor (TFPI) inhibits the activation of factor Xa and formats TFPI/Xa, which subsequently inhibits the TF/VIIa complex. These processes consequently prevent the formation of thrombin (81). (2) In the thrombomodulin (TBM)–protein C (PC) pathway, TBM serves as a cofactor in the thrombin-induced activation of PC. Endothelial protein C receptor (EPCR) is a receptor for PC that enhances its activation. The activated PC (aPC), together with cofactor protein S (PS), suppressing factors Va and VIIIa, thereby downregulating thrombin formation and suppressing coagulation cascade (84). (3) Soluble antithrombins (AT) inhibits factors XIa, IXa, Xa, VIIa, and thrombin by targeting serine proteases (82). (B) Dysregulated coagulation in pig-to-primate xenotransplantation. Red and black arrows designate incompatibility between pig and primates. When pig endothelium is activated, pig TF is expressed and released into the circulation. After interaction with the pig endothelium, recipient platelets and peripheral blood mononuclear cells (PBMCs) express primate tissue factor (hTF). The porcine TF (pTF) pathway inhibitor is an ineffective inhibitor of the human Xa factor and may ineffectively shut down the activation of the major TF. Pig TBM (pTBM) binds only weakly to primate thrombin, leading to levels of activated PC that are insufficient to inhibit coagulation, resulting in thrombotic microangiopathy in pig grafts within a matter of weeks (85). Porcine vWF spontaneously could aggregate primate platelets through GPIb receptors even in the absence of shear stress (86). After aberrant porcine GPIb–primate vWF interaction, platelets are activated. Small vessels in the graft become occluded by fibrin and platelet aggregation.