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The establishment of immune tolerance through genetic manipulation of haematopoietic stem cells
thesisposted on 2017-02-17, 00:00 authored by Nasa, Zeyad
Autoimmune diseases are incurable and affect about 6-9 % of the population. Treatments of autoimmune diseases include the use of monoclonal antibodies, anti-inflammatory and immunosuppressive drugs, or replacement therapy like insulin for type 1 diabetes. Not all these treatments address the cause, but only aim to reduce symptoms. Autologous bone marrow transplantation (BMT) is currently being trialled to treat autoimmune diseases, however it is associated with high relapse rates. Multiple Sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases of the central nervous system. T regulatory cells (Tregs) have the capacity to supress a wide range of immune responses and play a key role in controlling autoreactive reactions in the periphery, making them an ideal candidate for cellular treatment of autoimmune diseases. They are mainly taken to represent the CD4+ CD25high FoxP3+ T-cells. Their developments happen naturally in the thymus from a separate lineage and their TCR repertoire is mainly self-reactive. The transcription factor Foxp3 is crucial for Tregs development and function. It has been demonstrated that antigen specific T cells transduced with Foxp3 gene have the capability to reduce the symptoms of autoimmune disease. Bone marrow gene modification and subsequent transplantation can be used as a method to express genes, such as T cell receptors linked to Foxp3 gene, in self-renewing BM derived cells. My hypothesis is induce tolerance to autoimmune diseases by the transfer of bone marrow (BM) stem cells, that have been genetically engineered to express self-antigen, into preconditioned mice using a less toxic non-myeloablative chemotherapy regime. In this study I have examined this hypothesis in EAE induced by the self-antigen myelin oligodendrocyte glycoprotein (MOG) and by substituting irradiation which is highly toxic with less toxic non myeloablative regimen drug Treosulfan, as a proof-of-principle that tolerance can be generated with less toxic conditioning. I have shown that when mice are conditioned with a non-myeloablative dose of Treosulfan and received BM cells that have been retrovirally transduced with the autoantigen MOG, they remained EAE free. Furthermore, through this study I have found that using a chemotherapy drug, such as Treosulfan, conditioning promoting a low degree of chimerism at non-myeloablative dose was adequate to promote antigen specific tolerance and protect mice from EAE. In a more clinically relevant scenario, when Treosulfan at non-myeloablative dose was included into a curative protocol for treating mice with established EAE, it resulted in complete remission and proved to be efficient in maintaining disease resistance following subsequent challenge. Taking a different approach but still aimed at promoting tolerance, I have developed a retroviral vector designed to generate antigen specific Tregs. This vector was encoding the Vα3.2 and Vβ11 TCR chains (2D2-TCR) specific for the autoantigen MOG35-55 peptide linked to Foxp3 gene. Therefore, I hypothesized that the introduction of the 2D2-TCR plus Foxp3 into BM stem cells would lead to the generation of T regulatory cells specific for EAE autoantigen, which would impose immune regulation and prevent EAE induction. The generated retroviral constructs were tested in vitro in various cell lines including isolated mouse splenic naïve CD4 cells and found to be able to produce cells expressing 2D2-TCR and Foxp3 as well as other Tregs markers such as CD25, GITR and CTLA-4. Generated retroviruses were also used to transduce BM and create chimeric mice with a quantifiable subpopulation of T cells with MOG35-55 TCR specificity and Foxp3 driven Treg phenotype. However, mice generated were not efficiently tolerant to the induction of EAE. Flow cytometry analysis revealed that a significant population of 2D2-TCR-Foxp3-GFP cells were detected in the thymus but far less in the periphery with lesser Foxp3 expression than that seen in the thymus. This finding suggests that Foxp3 expression alone is not enough to confer Treg cell features and that other epigenetic and transcriptional factors are likely to be involved to ensure their stability and function.