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.
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.
Although liver transplantation is the gold-standard therapy for end-stage liver disease, the shortage of suitable organs results in only 25% of waitlisted patients undergoing transplants. Three-dimensional (3D) bioprinting is an emerging technology and a potential solution for personalized medicine applications. This review highlights existing 3D bioprinting technologies of liver tissues, current anatomical and physiological limitations to 3D bioprinting of a whole liver, and recent progress bringing this innovation closer to clinical use. We reviewed updated literature across multiple facets in 3D bioprinting, comparing laser, inkjet, and extrusion-based printing modalities, scaffolded versus scaffold-free systems, development of an oxygenated bioreactor, and challenges in establishing long-term viability of hepatic parenchyma and incorporating structurally and functionally robust vasculature and biliary systems. Advancements in liver organoid models have also increased their complexity and utility for liver disease modeling, pharmacologic testing, and regenerative medicine. Recent developments in 3D bioprinting techniques have improved the speed, anatomical, and physiological accuracy, and viability of 3D-bioprinted liver tissues. Optimization focusing on 3D bioprinting of the vascular system and bile duct has improved both the structural and functional accuracy of these models, which will be critical in the successful expansion of 3D-bioprinted liver tissues toward transplantable organs. With further dedicated research, patients with end-stage liver disease may soon be recipients of customized 3D-bioprinted livers, reducing or eliminating the need for immunosuppressive regimens.
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and its advanced form, nonalcoholic steatohepatitis (NASH), are prevalent liver diseases with no effective treatment available. NAFLD is marked by increased lipid accumulation in the hepatocytes, lobular inflammation and fibrosis. However, the effects of lipid accumulation in other hepatic lineage cells, such as liver endothelial cells (LEC), intrahepatic cholangiocytes (IHCHOLs), and hepatic stellate cells (HSCs), on NAFLD/NASH progression are less understood. METHODS: Human HSCs, IHCHOLs, and LECs were isolated from liver explants of healthy donors and further characterized with immunofluorescence staining. The cells were treated with different concentrations of free fatty acids (FFAs, oleic acid and palmitic acid 2:1) for 48 hours, and then examined for lipid accumulation with staining of BODIPY493/503. A cell viability assay and LDH cytotoxicity assay were performed. Real-time PCR was performed to monitor gene expression in LECs after treatment. RESULTS: Lipid accumulation confirmed by BODIPY493/503 staining was observed following treatment, especially with higher FFA concentrations. No statistically significant difference in cell viability or cytotoxicity of the cells was observed under different treatment concentrations of FFA. However, a negative trend in cell viability was observed with an increment of FFA dosage. FFA treatment upregulated the expression of the genes in LEC that are related to angiogenesis (CDH5), epithelial-mesenchymal transformation (TGF-β2 and TGF-β3), cellular stress (P53), inflammation (IL-32) and immunoglobular cell adhesion molecules (ICAM-1 and VCAM-1) that are key to monocyte adhesion to endothelial cells in response to a local inflammatory signal. CONCLUSION: Using an in vitro cellular model system, we examined lipid accumulation and cellular stress in different human liver cell types, especially LECs, under dose-dependent FFA treatment. This could be further developed to delineate adaptive responses within different hepatic lineage cells in the progression of NAFLD/NASH. IMPACT: A better understanding of the involvement of hepatocytes and other hepatic lineages in the progression of NAFLD/NASH will augment the development of targeted and effective treatments.
Background: Xenotransplantation offers a prospective solution to organ shortage. The elimination of major xenoantigens in pig cells prevents hyperacute xenograft rejection (HXR), driven by preformed antibodies. However, acute xenograft rejection (AXR), driven by immune cell activation,continues to be a barrier in pig-to-human xenotransplantation. Natural killer (NK) cells, with inhibitory and activating receptors, play unique roles in AXR by promoting graft rejection or tolerance. NK cell tolerance occurs naturally in utero where human leukocyte antigen (HLA)-E and HLA-G are present. Expressing HLA-E/G in xenografts may provide immune protection from human NK cell cytotoxicity. Methods: This study aims to demonstrate the use of HLA class I molecules in inducing human NK cell tolerance. 5-gene-knock-out (5GKO) porcine endothelial cells (pECs) were transfected with HLA-E and HLA-G genes. Transfected cells were stained with HLA-E and/or HLA-G antibody. HLA class I expressing 5GKO cells were isolated by flow sorter. Genetically modified pECs were co-cultured with human peripheral blood mononuclear cells (PBMCs) for E:T ratio of 10:1 for 2 hours. PBMCs were collected and stained with fluorochrome-conjugated antibodies. NK cell degranulation was accessed by the percentage of CD107a expression in the CD3-CD56+population. Co-localization of HLA-E and HLA-G on pECs was imaged by immunofluorescence microscopy and quantified by Image-Pro. Results: 5GKO pEC lines expressing HLA-E, HLA-G, and co-expressing HLA-E and HLA-G were successfully established. Co-expression of HLA-E and HLA-G in 5GKO pECs reduced human NK cell degranulation by 50% compared to the 21% reduction achieved with 5GKO alone (p<0.0001). Co-localization intersection of 5GKO.HLA-E.HLA-G with stimulated PBMCs was not significantly different than when cultured with media (0.067 vs. 0.059, p=0.57). Conclusion and Impacts: Co-expression of HLA-E and HLA-G in 5GKO pECs significantly reduced human NK cell degranulation, compared to 5GKO.HLA-E, 5GKO.HLA-G, and 5GKO pECs. However, co-localization of HLA-E and HLA-G didn’t change when cultured with stimulated PBMCs. This study provides insight into the interactions between HLA molecules that promote an immunotolerant phenotype.
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