Microbiology & Immunology - Theses
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ItemInactivation Mechanisms of Therapeutic CD8+ T Cells against a non-Hodgkin B-cell Lymphoma Mouse Model( 2021)Clinical and animal studies have demonstrated the capability of innate or adaptive components of the immune system to eliminate tumour cells. Cytotoxic CD8 T lymphocytes (CTLs) are the main population of the adaptive immune system involved in tumour cells elimination. In clinical studies, autologous tumour associated antigen (TAA)-specific CTLs have been adoptively transferred into patients to eliminate tumour cells expressing the cognate antigen, an approach known as Adoptive Cell Therapy (ACT). In many conditions, these CTLs are functionally impaired. Similar observations have been made in mouse models, including our own. We found that anti-ovalbumin (OVA) OT-I CTL injected into mice were capable of eliminating non-Hodgkin B cell lymphoma cells that express OVA as a model TAA. However, this ACT failed in mice harbouring a large tumour burden because many of the OT-I CTL were eliminated soon after ACT, and even though the surviving ones expanded, they remained functionally impaired. These observations recapitulate successful vs failed outcomes of ACT in the clinic. So far little is known about the extrinsic and intrinsic mechanisms that determine these outcomes. My results show that the mechanisms of CTL inactivation we observe are most likely physiological responses to large target cell burdens and provide experimental system to further understand the mechanisms of inactivation and approaches for the generation of more effective CTL for ACT.
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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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ItemInvestigation of rare actinomycetes for novel antimicrobials( 2020)Nocardia are a genus of ubiquitous environmental bacteria belonging to the phylum Actinobacteria. Genomics has revealed that Nocardia species are endowed with extensive and varied arrays of secondary metabolite biosynthetic gene clusters with the potential to produce natural products that have antibiotic properties. Furthermore, the abundance of such gene clusters within the Nocardia rivals that of Streptomyces, the signature genus among the Actinobacteria, owed to the fact that Streptomyces species have yielded many clinically used antibiotics. This project aimed to address the current antibiotic resistance crisis and the shortfall in new compounds within the drug discovery pipeline. A range of natural product discovery techniques were utilised amongst different Actinobacteria with a particular focus on a collection of species within the generally overlooked genus Nocardia. This study had three primary objectives, the first was to use a traditional, high-throughput, empirical screen of 169 pathogenic actinomycetes predominantly from the genus Nocardia. These isolates were screened for antibiotic activity on 19 distinct growth media against a panel of five highly prevalent, multidrug resistant pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium and Acinetobacter baumannii). Secondly, whole genome sequencing and bioinformatic interrogation of 100 Nocardia species was conducted to assess their genetic potential to biosynthesise natural products. This facilitated the selection of a single Nocardia isolate which possessed a non-ribosomal peptide synthetase locus that appeared to be unique amongst other Nocardia species. The locus was also transcriptionally silent. Bioengineering using promoter refactoring was employed to activate expression of this gene cluster, the product of which might have potential as a novel antimicrobial. Thirdly, by utilisation of liquid chromatography-mass spectrometry (LC-MS), bioinformatics and molecular networking, a metabolomic approach was employed to gain a global secondary metabolic footprint of ten predicted “biosynthetically talented" Nocardia species grown on five distinct media types. This project identified: (i) A Nocardia sp. with activity against multidrug resistant Acinetobacter baumannii. (ii) Two Streptomyces isolates (Streptomyces cacaoi and Streptomyces sp.) which exhibited antimicrobial activity against multidrug resistant Escherichia coli and Acinetobacter baumannii respectively. Secondary metabolite extracts from each of these producing isolates were investigated by LC-MS/MS and the resulting spectra was assessed for uniqueness through a dereplication data platform developed specifically for bacterial natural product identification. No hits for previously discovered metabolites were obtained suggesting that the antimicrobials discovered within this project appear to be unique and have potential as new drug leads for today’s ever-decreasing antibiotic discovery pipeline. (iii) Four distinct families of bioactive secondary metabolites that were produced by multiple Nocardia species following LC-MS/MS and molecular network analysis. The identified secondary metabolites were correlated with genome sequence data to identify their probable biosynthetic origin in Nocardia species.
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ItemThe dynamics of the B cell response during influenza A virus infection( 2020)Although vaccination remains the most effective method of managing influenza epidemics, there is still much that remains to be characterized about humoral immunity against the varying contexts in which influenza infection can occur. With the continuous subversion of humoral immunity by seasonal influenza through antigenic drift and the potential of zoonotic influenza viruses adapting and spreading through human populations through antigenic shift, improving our understanding of B cell immunity against different types of influenza infection could provide important insights into improving management of epidemics and vaccine formulations. In order to understand B cell responses during influenza infection, the well-characterized C57BL/6 mouse model was used to investigate and compare humoral responses in the context of different influenza infection histories. Markers that identified specific B cell subsets such as germinal centre (GC) B cells and plasmablasts were analysed by flow cytometry paired with influenza virus-specific B cell ELISPOT assays to investigate strain-specific antibody secreting responses within the same experiment. As the surface glycoprotein HA is thought to be the immunodominant response for B cell responses against influenza virus, the prediction is that the greater the antigenic differences between the HA of the first and second infecting strains, the more primary-like the response to the second strain would be. Primary and homologous secondary B cell responses in the mediastinal lymph node (MLN) and spleen were first characterized using this model to establish baseline responses against influenza virus before heterosubtypic infection was studied through infection of mice with H1N1A/Puerto Rico/8/34 (PR8) virus followed by H3N2 A/Udorn/305/72 (Udorn) virus 7 weeks later. Unexpectedly, a secondary-like plasmablast, GC B cell and Udorn-specific antibody secreting cells was observed during heterosubtypic infection, with earlier and higher magnitude B cell responses. These findings suggested a possible role for cross-subtype T cell memory in modulating B cell responses. The effect of antigenic drift on the B cell responses during influenza infection was then analysed with the same model. Mice were infected with H3N2 strains isolated between 1972 and 1979, representing different antigenic distance from a virus isolated in 1982 (Ph82). Seven weeks post infection mice were reinfected with Ph82 and the B cell response over the course of infection examined. It was found that infection of strains up to 10 years apart appeared to induce a secondary-like B cell response in the secondary lymphoid organs when compared to baseline primary and secondary responses against Ph82 virus. Prior infection with any H3N2 strain also resulted in minimal viral replication during the secondary challenge when compared to primary infection groups. However, data from both primary antibody inhibition and HA-specific B cell responses appears to suggest a narrower threshold of recognition, around a maximum of 3 years drift before serum and HA-specific responses cease to bind with other strains. Taken together, secondary-like B cell responses in both heterosubtypic and drift models of infection and in the case of drift responses, irrespective of reactivity of HA-specific B cells, appear to refute the hypothesis that virus-specific B cell responses would reflect antigenic relatedness between the HA of the infecting strains. Overall, data from this study identifies the diversity of the overall B cell response against influenza infection in the context of prior exposure to strains of different antigenic properties. Further study into the reactivity of these B cells against different influenza virus components and the role of memory T cells in the observed responses may provide important insights into the nature of host immunity against the ever-shifting target of influenza virus.