Search

Eliminating major xenoantigens in pig cells has drastically reduced human antibody-mediated hyperacute xenograft rejection (HXR). Despite these advancements, acute xenograft rejection (AXR) remains one of the major obstacles to clinical xenotransplantation, mediated by innate immune cells, including macrophages, neutrophils, and natural killer (NK) cells. NK cells play an ‘effector’ role by releasing cytotoxicity granules against xenogeneic cells and an ‘affecter’ role on other immune cells through cytokine secretion. We highlight the key receptor-ligand interactions that determine the NK cell response to target cells, focusing on the regulation of NK cell activating receptor (NKG2D, DNAM1) and inhibitory receptor (KIR2DL1-4, NKG2A, and LIR-1) signaling pathways. Inhibition of NK cell activity may protect xenografts from cytotoxicity. Recent successful approaches to reducing NK cell-mediated HXR and AXR are reviewed, including genetic modifications of porcine xenografts aimed at improving pig-to-human compatibility. Future directions to promote xenograft acceptance are discussed, including NK cell tolerance in pregnancy and NK cell evasion in viral infection.

Preclinical trials of pig-to-nonhuman primate liver xenotransplantation have recently achieved longer survival times. However, life-threatening thrombocytopenia and coagulation dysregulation continue to limit preclinical liver xenograft survival times to less than one month despite various genetic modifications in pigs and intensive pharmacological support. Transfusion of human coagulation factors and complex immunosuppressive regimens have resulted in substantial improvements in recipient survival. The fundamental biological mechanisms of thrombocytopenia and coagulation dysregulation remain incompletely understood. Current studies demonstrate that porcine von Willebrand Factor binds more tightly to human platelet GPIb receptors due to increased O-linked glycosylation, resulting in increased human platelet activation. Porcine liver sinusoidal endothelial cells and Kupffer cells phagocytose human platelets in an asialoglycoprotein receptor 1-dependent and CD40/CD154-dependent manner, respectively. Porcine Kupffer cells phagocytose human platelets via a species-incompatible SIRPα/CD47 axis. Key drivers of coagulation dysregulation include constitutive activation of the extrinsic clotting cascade due to failure of porcine tissue factor pathway inhibitor to repress recipient tissue factor. Additionally, porcine thrombomodulin fails to activate human protein C when bound by human thrombin, leading to a hypercoagulable state. Combined genetic modification of these key genes may mitigate liver xenotransplantation-induced thrombocytopenia and coagulation dysregulation, leading to greater recipient survival in pig-to-nonhuman primate liver xenotransplantation and, potentially, the first pig-to-human clinical trial.

Xenotransplantation (i.e. cross-species transplantation) using genetically-engineered pig organs could be a limitless source to solve the shortage of organs and tissues worldwide. However, despite prolonged survival in preclinical pig-to-nonhuman primate xenotransplantation trials, interspecies coagulation dysregulation remains to be overcome in order to achieve continuous long-term success. Different platelet aggregometry methods have been previously used to study the coagulation dysregulation with wild-type and genetically-engineered pig cells, including the impact of possible treatment options. Amongst these methods, while thromboelastography and rotational thromboelastometry measures the change in viscoelasticity, optical aggregometry measures the change in opacity. Recently, impedance aggregometry has been used to measure changes in platelet aggregation in electrical conductance, providing more information to our understaning of coagulation dysregulation in xenotransplantation compared to previous methods. The present study reviews the merits and differences of the above-mentioned platelet aggregometers in xenotransplantation research.

Corneal transplantation is the only option to cure corneal opacities. However, there is an imbalance between supply and demand of corneal tissues in the world. To solve the problem of corneal shortage, corneal xenotransplantation studies have been implemented in the past years using porcine corneas. The corneal xenografts could come from 1) wild type pigs, 2) genetically-engineered pigs, 3) decellularized porcine corneas, and 4) decellularized porcine corneas that are recellularized with human corneal cells, eventually with patients’ own cells, as in all type of xenografts. All approaches except, the former would reduce or mitigate recipient immune responses. Although several techniques in decellularization have been reported, there is still no standardized protocol for the complete decellularization of corneal tissue. Herein, we reviewed different decellularization methods for porcine corneas based on the mechanism of action, decellularization efficacy, biocompatibility, and the undesirable effects on corneal ultrastructure. We compared 9 decellularization methods including: (i) sodium dodecyl sulfate, (ii) triton x-100, (iii) hypertonic saline, (iv) human serum with electrophoresis, (v) high hydrostatic pressure, (vi) freeze-thaw, (vii) nitrogen gas, (viii) phospholipase A(2), and (ix) glycerol with chemical crosslinking methods. It appears that combined methods could be more useful to perform efficient corneal decellularization.