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  • Publication
    The invitro risk stratification of endothelial specific immune reactions post allogenic transplant
    (University College Dublin. School of Medicine, 2022) ;
    0000-0002-3358-7544
    Allogeneic haematopoetic stem cell transplantation (HSCT) is the only curative option for certain leukaemias. However, severe HSCT-related-complications such as graft versus host disease (GvHD) contribute to mortality and morbidity of HSCT patients. Endothelial cell (EC) damage is the primary event in many of these and ensues from toxicity of conditioning chemo-radiotherapy and the alloreactivity of donor T lymphocytes, a subset of which are specific to the vascular endothelium. Some of these endothelial-specific effectors cannot be controlled by conventional regulatory mechanisms, which suggests the previously unmet clinical need for an intervention with these T cells. Our project aims to develop an individualised in vitro monitoring of adverse EC-specific T cell reactions to predict and minimise endothelial-related complications in the course of HSCT. A prerequisite for this in vitro monitoring are patient-specific EC. Since bona fide EC are not available in sufficient amounts, mesenchymal stem or stromal cells (MSC) from bone marrow serve as an alternative for the generation of EC-like cells. MSC from various sources are transduced by lentiviral vectors containing TERT and CDK4 genes and subsequently differentiated into EC-like cells (eMSC) to generate stable individualised endothelial cell lines. The eMSC can be induced by growth factors (GF), hypoxia and shear stress (SS) and they are phenotypically characterised through dry lab and wet lab experiments for EC markers. 3D spheroids culture can enhance the endothelial differentiation of MSC and induce CD31 expression on MSC. As for bona fide EC, eMSC give rise to alloreactive T lymphocyte reactions.. In vitro immortalisation of MSC by overexpression of TERT and CDK4 genes does not change the phenotype of eMSC from experimental and bioinformatics data, allowing the generation of robust, stably growing cell lines for long-term use. The lentiviral transduction still allows eMSC to become antigen-presenting cells for CD8+ T cells. In a second part of this project we investigated whether unmodified 3rd MSC and their extracellular vesicles (EV) can protect EC against the cytotoxicity of the aforementioned CD8+ cytotoxic T lymphocytes (CTL). Both, MSC and MSC-EV could efficiently protect EC against allogeneic CTL. Priming of MSC with hypoxia and Interferon gamma (IFN-¿) , as it had been observed to be of help in other immune reactions, could not enhance MSC- and MSC-EV-mediated immunomodulation of CTL activity. The underlying mechanism of immunomodulation of MSC-derived EV was investigated by mass spectrometry. In the future, bone marrow-derived eMSC from HSCT recipients and donors’ CD8+ T cells can be used for in vitro monitoring of adverse EC-specific alloreactions in the course of HSCT. This monitoring system also allows to test EC protectants and their mechanism of action. Overall, MSC qualify as endothelial-like cell sources for the detection of and the adjuvant protection against EC-specific immune reactions thus enabling a personalised medicine approach to transplant-related complications.
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