Microbiology & Immunology - Theses
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ItemThe regulation of MR1 cell surface expression and antigen presentation( 2021)Major histocompatibility complex class I-related protein 1 (MR1) presents vitamin B metabolites that are synthesised by wide range of microbes. Presentation of MR1-metabolite complexes by cells which have encountered bacteria activate the highly abundant innate-like T cells, mucosal associated invariant T (MAIT) cells, which then secrete inflammatory cytokines and perform cytotoxic activity to clear the infection. However, the molecular mechanism that regulates MR1 surface expression remains unclear. In this thesis, I revisited some of the mechanisms regulating MR1 surface expression proposed by previous studies. I found that while a small amount of MR1 associates with invariant chain (Ii), it does not require it for its surface expression nor presentation of antigen (Ag) from intracellular bacteria. Meanwhile, a small degree of modulation of MR1 surface expression by TLR signalling was observed in different primary cell types and cell lines. To characterise the molecular mechanism regulating MR1 surface expression and endocytosis, I employed several experimental approaches. I found that co-immunoprecipitation was not robust enough for the detection of regulatory proteins that interacts with MR1 at the cytoplasmic tail and trialled a genetically incorporated crosslinker that had showed promise. Ultimately, I employed a genome wide CRISPR/Cas9 knockout library screen which revealed that MR1 endocytosis requires alpha subunit (encoded by AP2A1) of the endocytic adaptor protein complex 2 (AP2). I found that AP2A1 interacts with the MR1 cytoplasmic tail and mediates endocytosis of MR1-antigen complexes. I further explored how AP2A1 regulates MR1 cellular behaviour by knocking out AP2A1 from two cell lines using CRISPR/Cas9. These cells showed significantly reduced MR1 internalisation rate, resulting in a higher surface expression level. Impaired MR1 internalisation in AP2A1-deleted cells leads to the accumulation of MR1-ligand complex on the cell surface, in turn causing prolonged activation of MAIT cells even 24 hours after metabolite Ag exposure. Meanwhile, I also found that recycling pathway is not critical for MR1 Ag uptake and presentation. Next, I explored the sorting signal of MR1 at the cytoplasmic tail likely recognised by AP2. I found that MR1 half-life was regulated by a tetrapeptide tyrosine-based motif at the cytoplasmic tail highly conserved in mammals. This motif lacks a feature of canonical tyrosine-based motifs; and when mutated to be canonical motif it accelerated the MR1 internalisation and degradation. In contrast, ablating MR1’s tyrosine residue, MR1 internalisation was impaired. Together, it suggested that MR1 Ag presentation was regulated using a unique non-canonical sorting motif, that allows appropriate MAIT cell activation. While our understanding of the regulatory mechanism of MR1 intracellular trafficking is ongoing, my findings reveal a novel insight into the unique regulation of MR1 surface expression which has important implications in the context of MAIT cell biology.
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ItemDiscovery of the cells that express the antigen-presenting molecule MR1 in vivo( 2021)Major histocompatibility complex class I-related protein 1(MR1) is a monomorphic antigen-presenting molecule that is highly conserved across animal species. It presents vitamin B-related metabolite antigens, produced by a broad range of bacteria and yeast, to mucosal-associated invariant T cells (MAIT cells). This induces the activation of inflammatory and cytolytic MAIT cells to resolve microbial infections. The MR1-MAIT cell axis has been implicated in immunity against a range of major bacterial pathogens primarily in mucosal tissues. MR1 is also essential for the development and expansion of MAIT cells and can trigger anti-cancer responses. MR1 is thought to be expressed at very low levels ubiquitously in many cell types, but due to the difficult nature of detecting MR1, this has not been systematically investigated. Importantly, it is not known if the expression of MR1 varies among cell types in vivo or if it changes during pathological conditions. This project aims to address these unknowns by using a novel genetically altered mouse model that reveals the expression of MR1 by a fluorescent reporter. The fidelity of this model to report MR1-expressing cells has been validated by several means including quantitative real-time polymerase chain reaction (qPCR) and surface detection of MR1 after exposure to MR1 metabolite ligands. By employing the model, a range of expression levels of MR1 in diverse cell types with different tissue distributions in mice have been revealed. Overall, tissue-resident macrophages in the lungs and peritoneal cavity (PerC) had the highest MR1 expression. Factors that could influence MR1 expression in the healthy steady state were investigated. It was found that MR1 expression increased in mice with age up to 7 months, while there was no difference seen between the sexes. Bone marrow (BM) chimeras were used to reveal that MR1 expression in tissue-resident macrophages was not restricted to those originating from the embryonic precursors, but also in BM-derived macrophages. Then intriguingly, MR1 expression was not elevated in any cell type during pulmonary infection with Legionella longbeachae, but on the contrary, it was significantly downregulated in alveolar macrophages (AMs). Overall, this work reveals that MR1 has a cell type- and tissue-restricted expression profile in vivo, with tissue-resident macrophages expressing the highest levels, indicating that these cells may be the most potent MR1 antigen-presenting cells in vivo. Lung and peritoneal macrophages are instructed to express MR1 from the local tissue environment during their differentiation rather than from their precursor origins, and infection rapidly switches off its expression. This suggests that these innate MR1-presenting cells are already equipped with MR1 prior to infections, in order to rapidly activate MAIT cells upon microbial metabolite detection.
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ItemMucosal-associated invariant T (MAIT) cells and their function in bacterial infection( 2021)Mucosal-associated invariant T (MAIT) cells are a subset of innate-like alpha/beta T cells that recognize riboflavin metabolites presented by the monomorphic major histocompatibility complex (MHC) class I related protein-1 (MR1). The most potent antigen known to date is 5-(2-oxopropylidineamino)-6-D- ribitylaminouracil (5-OP-RU). MAIT cells are abundant in mucosal tissues and blood in humans. Upon bacterial infection, MAIT cells expand rapidly, with production of cytokines, including interferon-gamma (IFN gamma), tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-17 (IL-17), and cytotoxic granzymes. Their phenotype indicates that they play an important role in immunity. Previous studies showed a protective role in local infections, involving a single organ or tissue, but the mechanisms of MAIT cell-mediated protection in systemic infections are not fully understood. This study aimed to elucidate MAIT cell activation, protective role in primary infection and potential for a MAIT cell-based systemic vaccination to protect against Francisella tularensis live vaccine strain (LVS) and Legionella longbeachae. F. tularensis is a gram-negative intracellular bacterium which can cause systemic infection, in mice and humans. In this study, F. tularensis LVS was used to induce systemic infection in C57BL/6 (wild type) mice and in Mr1 -/- mice, which lack MAIT cells. A combination of CpGcombo (fused oligos for CpG-B and CpG-P) and synthetic 5-OP-RU antigen was used to vaccinate mice. Bacterial load, survival rate, and MAIT cell response kinetics and post-infection cytokine profiles were examined. MAIT cells expanded systemically and showed Th1-like cellular profile after infection with F. tularensis LVS. In several organs, C57BL/6 mice showed better control of bacterial burden compared with Mr1 -/- mice. Vaccination of MAIT cells with CpGcombo plus 5-OP-RU, but not CpGcombo alone, protected mice from infections by an otherwise lethal dose of F. tularensis LVS and L. longbeachae. Thus, MAIT cells displayed a protective role against systemic infections and potential to be boosted to protect against local and systemic infections. This study also showed that post infection, MAIT and non-MAIT alpha/beta-T cells manifest different contraction kinetics, indicating that these two groups could be regulated by different viability mechanisms. Specifically, the in vivo data illustrated that loss of receptor-interacting serine/threonine-protein kinase 3 (RIPK3, a key regulator of apoptosis and necroptosis), but not mixed-lineage kinase domain like pseudokinase (MLKL, a signaling molecule involved in necroptosis), preferentially increased MAIT cell abundance in a cell-intrinsic manner. In summary, the results in this thesis demonstrated that MAIT cells are critical in immune protection against systemic F. tularensis LVS infection, and are long lived with a differently regulated cell death and survival. These findings may inform the future development of vaccination strategies targeting MAIT cells.
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