Browsing by Author "Cross-Najafi, Arthur A."

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    Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation
    (Frontiers Media, 2023-07-17) Cross-Najafi, Arthur A.; Farag, Kristine; Isidan, Abdulkadir; Li, Wei; Zhang, Wenjun; Lin, Zhansong; Walsh, Julia R.; Lopez, Kevin; Park, Yujin; Higgins, Nancy G.; Cooper, David K. C.; Ekser, Burcin; Li, Ping; Surgery, School of Medicine
    Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.
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    Current Barriers to Clinical Liver Xenotransplantation
    (Frontiers Media, 2022-02-23) Cross-Najafi, Arthur A.; Lopez, Kevin; Isidan, Abdulkadir; Park, Yujin; Zhang, Wenjun; Li, Ping; Yilmaz, Sezai; Akbulut, Sami; Ekser, Burcin; Surgery, School of Medicine
    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.
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    Porcine UL-16 Binding Protein 1 Is Not a Functional Ligand for the Human Natural Killer Cell Activating Receptor NKG2D
    (MDPI, 2023-11-07) Lopez, Kevin J.; Spence, John Paul; Li, Wei; Zhang, Wenjun; Wei, Barry; Cross-Najafi, Arthur A.; Butler, James R.; Cooper, David K. C.; Ekser, Burcin; Li, Ping; Surgery, School of Medicine
    Natural killer (NK) cells play a vital role in xenotransplantation rejection. One approach to induce NK cell immune tolerance is to prevent the NK cell-mediated direct killing of porcine cells by targeting the interaction of the activating receptor NKG2D and its ligands. However, the identity of porcine ligands for the human NKG2D receptor has remained elusive. Previous studies on porcine UL-16 binding protein 1 (pULBP-1) as a ligand for human NKG2D have yielded contradictory results. The goal of the present study was to clarify the role of pULBP-1 in the immune response and its interaction with human NKG2D receptor. To accomplish this, the CRISPR/Cas9 gene editing tool was employed to disrupt the porcine ULBP-1 gene in a 5-gene knockout porcine endothelial cell line (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin, 5GKO). A colony with two allele mutations in pULBP-1 was established as a 6-gene knockout pig cell line (6GKO). We found that pULBP-1-deficient pig cells exhibited a reduced binding capacity to human NKG2D-Fc, a recombinant chimera protein. However, the removal of ULBP-1 from porcine endothelial cells did not significantly impact human NK cell degranulation or cytotoxicity upon stimulation with the pig cells. These findings conclusively demonstrate that pULBP-1 is not a crucial ligand for initiating xenogeneic human NK cell activation.
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    Strategies to induce natural killer cell tolerance in xenotransplantation
    (Frontiers Media, 2022-08-22) Lopez, Kevin J.; Cross-Najafi, Arthur A.; Farag, Kristine; Obando, Benjamin; Thadasina, Deepthi; Isidan, Abdulkadir; Park, Yujin; Zhang, Wenjun; Ekser, Burcin; Li, Ping; Surgery, School of Medicine
    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.