Microbiology & Immunology - Theses
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ItemThe Development, Homeostasis, and Function of Unconventional T Cells( 2023-09)Unconventional T cells detect non-peptide antigens presented by MHC class-I-like molecules, such as MR1 and CD1. MR1-reactive T cells, CD1-reactive T cells, and gamma-delta T cells represent three broad unconventional T-cell lineages which collectively are abundant and play key roles in the immune response. The frequencies of these cells vary widely between individuals, and the factors that govern their numbers and diverse effector functions are not well-understood. This thesis investigates the mechanisms that controls the development, homeostasis, and function of unconventional T cells and their subsets in the thymus and peripheral tissues. In chapter 3, MR1 and group 1 CD1 (CD1a, CD1b, and CD1c) tetramers were used to isolate and characterise rare unconventional T-cell populations in the human thymus. Using tetramer-mediated enrichment, thymic MR1-reactive T cells were found to comprise Valpha7.2+ and diverse Valpha7.2- subsets that differed in phenotype, binding to antigen-loaded MR1 tetramers, TCR repertoire diversity, frequency, and post-natal expansion. Whilst tetramer-mediated enrichment allowed for isolation of thymic CD1a- and CD1b-reactive T cells, blockade of CD36 expressed on thymocytes was additionally needed in order to detect CD1c-reactive T cells. Group 1 CD1-reactive T cells were highly rare after enrichment and generally resembled other CD4+ thymocytes. These findings highlight diverse MR1 and CD1-reactive T cell subsets in the thymus and forms the basis for understanding their intrathymic generation and developmental pathways. The expansion of MAIT cells in NKT- and gamma-delta T cell-deficient mice was investigated in chapter 4. Although MAIT cells were highly elevated in mice deficient in both NKT and gamma-delta cells, they phenotypically and functionally resembled their counterparts in wildtype mice. Mechanistically, this MAIT cell expansion was due to: 1) increased rearrangement of the MAIT cell TCR alpha chain within developing Tcrd-/- thymocytes, and 2) a higher capacity of peripheral MAIT cells to proliferate in the absence of NKT and gamma-delta cells. Overall, this chapter provides evidence for a shared niche in which MAIT, NKT, and gamma-delta T cells reside and compete for common homeostatic factors, revealing a novel interplay between their steady-state frequencies. In chapter 5, the regulation of peripheral unconventional T cells by the purinergic P2RX7 receptor was examined. Human unconventional T cells expressed P2RX7, whereas their mouse T-bet+, but not RORgt+, counterparts highly co-expressed the ADP-ribosyltransferase, ARTC2, and P2RX7. P2RX7 activation in response to ATP induced death of both mouse and human unconventional T cells ex vivo. Mouse T-bet+ unconventional T cells were highly susceptible to the effects of ARTC2-dependent P2RX7 activation in response to nicotinamide adenine dinucleotide, which resulted in their cell death ex vivo and depletion in vivo. By blocking ARTC2 or P2RX7, this chapter demonstrated the existence of IFN-gamma/IL-4 co-producing unconventional T cells, including MAIT and other non-MAIT/NKT alpha-beta T cell populations, which were selectively regulated by ARTC2-dependent P2RX7 activation. In contrast, this axis did not affect IL-17-producing unconventional T-cell subsets. These findings reveal a unique mechanism that controls the number and functional diversity of unconventional T cells via the selective regulation of their T-bet+ and IFN-gamma/IL-4 co-producing subsets. Overall, this thesis has examined the presence of diverse unconventional T cells in the human thymus, their regulation within a homeostatic niche, and their modulation by P2RX7 activation. Whilst unconventional T cells are collectively abundant, understanding their development and homeostasis will provide insight into why their frequencies vary widely in humans and how their numbers can be finely-controlled. The homeostatic relationships between unconventional T cells and how their functional subsets are regulated will be important considerations in the targeting of one or more unconventional T-cell populations within the immune response. Given their production of functionally-opposing cytokines, findings in this thesis will guide the development of future immunotherapies that leverage the abundance and potent effector functions of unconventional T cells in treating disease.
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ItemHarnessing unconventional T-cells for vaccines and immunotherapies in pre-clinical animal models( 2023-03)Unconventional T-cells represent a heterogenous population of CD3+ T-cells that recognise protein and non-protein antigens through MHC-unrestricted mechanisms. Unconventional T-cells also undergo cytokine- or surface receptor-mediated activation independent of T-cell receptor ligation, a characteristic that is commonly associated with innate immune cells and permits rapid responses to stimuli. These cells have diverse effector responses following activation, including direct cytolytic activity against target cells, proinflammatory cytokine production to mediate other immune responses, and antigen presentation to conventional CD4+ and CD8+ T-cells. Several unconventional T-cell subsets have also been explored as immunotherapeutics, in part due to our ability to readily expand them pharmacologically, with some expressing highly conserved public T-cell receptors. Current knowledge gaps with unconventional T-cell immunotherapies includes our understanding of the frequency and phenotype of therapeutic cells in different tissue compartments, and how this is impacted by changes in pharmacological expansion protocols. Additionally, several unconventional T-cell subsets can augment conventional T- and B-cell responses associated with humoral immunity. However, the contribution of these unconventional T-cell populations to conventional adaptive immune responses against protein vaccines or viral infection is not well understood. The overarching aim of this thesis is to characterize unconventional T-cell in the context of immunotherapeutics and during vaccine-elicited immune responses, in pre-clinical animal models. Vgamma9Vdelta2 T-cells are a subset of unconventional T-cells that recognises endogenous and exogenous phosphoantigens and have garnered significant interest for immunotherapies to treat cancer and infectious diseases. While studies in pre-clinical animal models have shown promise, the clinical efficacy with Vgamma9Vdelta2 T-cell therapy has been limited. In Chapter 2, we characterised the Vgamma9Vdelta2 T-cell population at steady-state and following in vivo pharmacological expansion in pigtail macaques. We found the tissue distribution of pharmacologically expanded Vgamma9Vdelta2 T-cells changed based on the antigen administration route. Additionally, our pharmacological expansion protocol drove marked CCR6 downregulation and granzyme B upregulation in expanded Vgamma9Vdelta2 T-cells. Our results highlight how changes to pharmacological expansion protocols can alter the phenotype and tissue distribution of the expanded cell population, which is important to consider as this will likely impact therapeutic efficacy. Lymph nodes are a critical site of adaptive immune responses and the generation of antigen specific Tfh and BGC cells following vaccination. However, vaccine draining lymph node identification to study these responses is hindered by anatomical variations in lymphatic drainage between individuals, and lymph nodes being arranged in clusters with only a subset draining the vaccine site. To improve the identification of vaccine draining lymph nodes in preclinical animal models, we developed a vaccine strategy to label draining lymph nodes with tracking dyes (Chapter 3). We show that protein vaccines co-formulated with tattoo ink accurately labels vaccine draining lymph nodes in both mice and nonhuman primates. Ink-containing lymph nodes had higher frequencies of antigen specific BGC and Tfh cells compared to lymph nodes without ink. Furthermore, the ink coformulation was compatible with flow cytometry-based assays and did not alter the vaccine immune response serologically or at the B- and T-cell level. Unconventional T-cells are capable of humoral immune responses through multiple mechanisms including conventional antigen presentation, co-stimulatory signalling to Tfh cells, and providing both cognate and non-cognate B-cell support. Whether different unconventional T-cell subpopulations significantly contribute to the humoral immune response following vaccination or viral infection has not been well established. In Chapter, 4, we evaluated the contribution of unconventional T-cells to conventional adaptive immune responses elicited by vaccines or influenza infection, using transgenic mice that individually lack gamma delta T-cells, MAIT cells, and NKT cells. We found transgenic animals had comparable serological, Tfh, and BGC responses following immunisation with clinically-relevant vaccine formulation or subclinical influenza infection. Our findings indicate these unconventional T-cell subpopulations are not individually essential for mounting a robust humoral immune response to protein vaccines or viral infection. These results also raise questions about compensation between these unconventional T-cell populations, or a potential lack of unconventional T-cell recruitment to vaccine- or viral-mediated immune responses. Collectively, we evaluated unconventional T-cells in the context of immunotherapies and conventional humoral immune responses, in preclinical animal models. Better animal model characterisation will likely improve clinical translatability of candidate vaccines and therapeutics. Future utilisation of and improvements to the labelling techniques described here will help interrogate vaccine responses in regional draining lymph nodes in preclinical animal models. Our findings also raise important questions about modifying in vivo Vgamma9Vdelta2 T-cells treatment protocols to improve therapeutic cell delivery to target tissues, and modifying vaccine formulations to better recruit different unconventional T-cell populations as part of the humoral immune response.
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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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