68 research outputs found
Expanded human blood-derived γδT cells display potent antigen-presentation functions
No full textCell-based immunotherapy strategies target tumors directly (via cytolytic effector cells) or aim at mobilizing endogenous anti-tumor immunity. The latter approach includes dendritic cells (DC), most frequently in the form of in vitro cultured peripheral blood monocytes-derived DC. Human blood γδT cells are selective for a single class of non-peptide agonists (phosphoantigens) and develop into potent antigen-presenting cells (APC), termed γδT-APC, within 1-3 days of in vitro culture. Availability of large numbers of γδT-APC would be advantageous for use as a novel cellular vaccine. We here report optimal γδT cell expansion (>107 cells/ml blood) when peripheral blood mononuclear cells (PBMC) from healthy individuals and melanoma patients were stimulated with zoledronate and then cultured for 14 days in the presence of IL-2 and IL-15, yielding γδT cell cultures of variable purity (77±21% and 56±26%, respectively). They resembled effector-memory αβT (TEM) cells and retained full functionality as assessed by in vitro tumor cell killing as well as secretion of proinflammatory cytokines (IFNγ, TNFα) and cell proliferation in response to stimulation with phosphoantigens. Importantly, day 14 γδT cells expressed numerous APC-related cell surface markers and, in agreement, displayed potent in vitro APC functions. Day 14 γδT cells from PBMC of patients with cancer were equally effective as their counterparts derived from blood of healthy individuals and triggered potent CD8+ αβT cell responses following processing and cross-presentation of simple (influenza M1) and complex (tuberculin purified protein derivative) protein antigens. Of note, and in clear contrast to peripheral blood γδT cells, the ability of day 14 γδT cells to trigger antigen-specific αβT cell responses did not depend on re-stimulation. We conclude that day 14 γδT cell cultures provide a convenient source of autologous APC for use in immunotherapy of patients with various cancer
Identifying homing interactions in T-cell traffic in human disease
No full textDescription of the molecular mechanisms which regulate the traffic of lymphocyte populations over recent years [for useful reviews see (1, 2)] has significantly enhanced our understanding of the processes underlying acquired immunity and also permitted the development of therapies targeted at specific leukocyte subpopulations. Such therapies are dependent upon a detailed knowledge of the molecular regulation of lymphocyte adhesion to and migration through endothelium in specific tissues. Whereas animal models have been central to understanding the underlying mechanisms, it is crucial to confirm and extend observations in man by using analysis of tissues and in vitro cell-based models. In this chapter, we discuss expertise developed in our laboratory for the isolation of specific lymphocyte and endothelial populations from explanted human liver tissue specimens. We then move on to provide specific examples of assays such as the Stamper-Woodruff assay, the transmigration assay and the tissue-specific endothelial static and flow-based adhesion assays, which can be used to interrogate the tissue-specific adhesion and migration of lymphocyte subsets. Although our own experience is with human liver tissue, the general principles apply to analysing any organ of interest
A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells
No full textThe suppression assay is a commonly performed assay, measuring the ability of regulatory T cells (Treg) to suppress T cell proliferation. Most frequently, Treg are obtained from the peripheral blood or spleen. Lower yields are obtained by isolation from other tissues, rendering downstream suppression assays challenging to perform. Furthermore, the importance of suppressive subpopulations of Treg favours their isolation by fluorescent-activated cell sorting. Here we describe a method to isolate Treg from human tissues, using colorectal cancer tissue as an example. Treg suppressive capacity was further examined by expression of CCR5 to demonstrate the ability of our method to assess the suppressive capacity of regulatory T cell subsets.
To optimise the standard suppression assay to achieve our research aims, the following modifications were made:
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Treg, isolated from tissues, were sorted directly into a well-plate.
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Responder T cells, which had been fluorescently-labelled prior to sorting, were added directly into the well-plate.
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Human Treg Suppression Inspector beads (Miltenyi Biotec Ltd., UK) provided a polyclonal stimulus for proliferation and were added to each well at a bead:lymphocyte ratio of 1:2.
This method quantified the suppression of responder T cell proliferation by small numbers of strictly-defined Treg populations isolated from tissues
Editorial: Bringing function to the forefront of cell therapy: how do we demonstrate potency?
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Reactive oxygen species mediate human hepatocyte injury during hypoxia/reoxygenation.
No full textIncreasing evidence shows that reactive oxygen species (ROS) may be critical mediators of liver damage during the relative hypoxia of ischemia/reperfusion injury (IRI) associated with transplant surgery or of the tissue microenvironment created as a result of chronic hepatic inflammation or infection. Much work has been focused on Kupffer cells or liver resident macrophages with respect to the generation of ROS during IRI. However, little is known about the contribution of endogenous hepatocyte ROS production or its potential impact on the parenchymal cell death associated with IRI and chronic hepatic inflammation. For the first time, we show that human hepatocytes isolated from nondiseased liver tissue and human hepatocytes isolated from diseased liver tissue exhibit marked differences in ROS production in response to hypoxia/reoxygenation (H-R). Furthermore, several different antioxidants are able to abrogate hepatocyte ROS-induced cell death during hypoxia and H-R. These data provide clear evidence that endogenous ROS production by mitochondria and nicotinamide adenine dinucleotide phosphate oxidase drives human hepatocyte apoptosis and necrosis during hypoxia and H-R and may therefore play an important role in any hepatic diseases characterized by a relatively hypoxic liver microenvironment. In conclusion, these data strongly suggest that hepatocytes and hepatocyte-derived ROS are active participants driving hepatic inflammation. These novel findings highlight important functional/metabolic differences between hepatocytes isolated from normal donor livers, hepatocytes isolated from normal resected tissue obtained during surgery for malignant neoplasms, and hepatocytes isolated from livers with end-stage disease. Furthermore, the targeting of hepatocyte ROS generation with antioxidants may offer therapeutic potential for the adjunctive treatment of IRI and chronic inflammatory liver diseases
NI-0801, an anti-chemokine (C-X-C motif) ligand 10 antibody, in patients with primary biliary cholangitis and an incomplete response to ursodeoxycholic acid
No full textNI-0801 is a fully human monoclonal antibody against chemokine (C-X-C motif) ligand 10 (CXCL10), which is involved in the recruitment of inflammatory T cells into the liver. The safety and efficacy of NI-0801 was assessed in patients with primary biliary cholangitis. In this open-label phase 2a study, patients with primary biliary cholangitis with an inadequate response to ursodeoxycholic acid received six consecutive intravenous administrations of NI-0801 (10 mg/kg) every 2 weeks. Patients were followed up for 3 months after the last infusion. Liver function tests, safety assessments, as well as pharmacokinetic and pharmacodynamic parameters were evaluated at different time points throughout the dosing period and the safety follow-up period. Twenty-nine patients were enrolled in the study and were treated with NI-0801. The most frequently reported adverse events included headaches (52%), pruritus (34%), fatigue (24%), and diarrhea (21%). No study drug-related serious adverse events were reported. NI-0801 administration did not lead to a significant reduction in any of the liver function tests assessed at the end of the treatment period (i.e., 2 weeks after final NI-0801 administration) compared to baseline. Conclusion: Despite clear pharmacologic responses in the blood, no therapeutic benefit of multiple administrations of NI-0801 could be demonstrated. The high production rate of CXCL10 makes it difficult to achieve drug levels that lead to sustained neutralization of the chemokine, thus limiting its targetability. (Hepatology Communications 2018;2:492-503)
Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation.
No full textBackgroundHypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis.MethodsHuman hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2',7'-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay.ResultsExposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis.ConclusionsCD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury
Autophagy: A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress
No full textThe role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes
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