Microbiology & Immunology - Theses
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ItemAntibody-mediated passive immunity against Helicobacter pylori(University of Melbourne, 2008)
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ItemThe role of T cells in the immunopathology of Helicobacter pylori infections in mice(University of Melbourne, 2007)
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ItemThe role of dendritic cells in CD8+T cell priming after epidermal HSV-1 infection(University of Melbourne, 2006)
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ItemInduction of immune responses by lipopeptide vaccines(University of Melbourne, 2006)
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ItemFunctional and cellular heterogeneity of the myeloid cell system( 2019)Cells of the myeloid lineage form the innate part of the immune system and are characterized by a high level of functional plasticity, which is required to address the diverse set of functions of these mononuclear cells. Monocytes, Macrophages and dendritic cells (DC) are collectively categorized as the mononuclear phagocyte system (MPS), to highlight their functional equipment that specializes them to the phagocytosis of pathogens as a starting point to elicit an immune response. Besides this role, cells of the MPS are also involved in a wide variety of homeostatic functions including early development and regulation of physiological processes. However, the multitude of mechanisms required to acquire this functional plasticity remains poorly understood. The work that has been performed in the scope of this dissertation aimed to advance current knowledge of the causes and consequences of functional and cellular plasticity of the myeloid immune system. High-dimensional characterization of the effects of a Western diet on myeloid immune cell progenitor cells revealed a long-term transcriptional and epigenetic reprogramming of the myeloid cell compartment. The formation of an innate immune memory in myeloid progenitor cells leads to lasting inflammatory priming of monocytes, which may directly contribute to the progression of myeloid cell-associated diseases. In addition, single-cell RNA-seq elucidated unreported cellular heterogeneity of the monocyte and dendritic cell compartment in human peripheral blood. A combination of phenotypic and transcriptional analyzes resulted in a precise categorization of the human DC compartment consisting of pDCs, cDC1, two cDC2 subsets, and a deeply characterized preDC subset. Furthermore, a universal strategy for the integration of cellular atlases was conceptualized and applied to establish a consensus map of the human DC and monocyte cell space. This thesis provides mechanistic insights into the cellular composition of myeloid cells and their functional plasticity, which will form the foundation for further investigations into the dynamic changes of the immune cell compartment during diseases and will be critically relevant for designing effective treatments for a wide variety of pathologies linked to myeloid cells.
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ItemThe molecular and cellular basis of antigen recognition by unconventional T cells( 2019)Unconventional T cells are evolutionarily conserved populations of T cells, many of which recognise non-peptide antigens in the context of monomorphic antigen presentation molecules related to the major histocompatibility complex (MHC). These MHC related molecules, known as CD1 and MR1 present lipid and small metabolite antigens respectively. Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that are highly abundant in humans and recognise the potent riboflavin biosynthesis derived metabolite 5-OP-RU presented by MR1. The majority of MAIT cells in the peripheral blood of humans express the CD8 co-receptor, a molecule also expressed by other T cells that contributes to antigen recognition and T cell activation. In contrast to other T cells, a role for CD8 on MAIT cells has not been formally tested. The first chapter of results in this thesis examines whether CD8 is able to bind MR1, and if CD8 is important for the activation of MAIT cells and other MR1-reactive T cells. The data revealed that CD8 binds directly to MR1, in a manner concordant with MHC class I, and that this interaction is important for the functional response of MAIT cells. Specifically, both isoforms of CD8 (CD8aa and CD8ab) bound to MR1 tetramers and the CD8-MR1 interaction could be abrogated by mutating MR1 in the putative CD8 binding site. The effects of the CD8-MR1 interaction were examined on primary MAIT cells, where MR1 tetramers bound more strongly to CD8+ compared CD8- MAIT cell subsets. Importantly, mutating MR1 tetramers reduced CD8+ MAIT cell engagement to CD8- MAIT cell levels. To determine the effect of the CD8-MR1 interaction on function, primary MAIT cells were activated in the presence of wild type or mutant MR1. In line with the importance of CD8 on MR1 recognition, the cytokine secretion by CD8+ MAIT cells was decreased in the absence of CD8 binding to MR1. Furthermore, the data here establishes that CD8 is vital for the recognition of MR1 presenting less potent, lower affinity antigens such as folate derivatives. Similarly, low affinity MR1-antigen recognition and cytokine secretion by other MR1-reactive T cells was completely abrogated in the absence of CD8 binding. Thus, CD8 is an important co-receptor for the function of MAIT cells and may expand the diversity of ligands recognised by MAIT and other MR1-reactive T cells. CD1b-restricted T cells are a subset of unconventional T cells in humans that recognise lipid antigens derived from endogenous and microbial sources that are presented by CD1b. Comparatively little is known about the biology of CD1b-restricted T cells, particularly autoreactive CD1b-restricted T cells that recognise endogenous lipid antigens such as phospholipids. The second chapter of results examines autoreactive TCR recognition of CD1b, including the breadth of permissive endogenous lipid antigens that are bound by mammalian CD1b. Autoreactive CD1b-restricted T cells were identified from healthy blood donors using CD1b tetramers presenting heterogeneous mammalian lipids and the autoreactive T cells expressed distinct T cell receptors (TCRs) which were cloned to generate autoreactive T cell lines. Different CD1b restricted T cell lines exhibited altered staining reactivity with CD1b tetramers loaded with different mammalian phospholipid antigens and strikingly, some autoreactive T cell lines recognised CD1b in an antigen independent manner. An activation assay using the autoreactive T cell lines cocultured with a series of mutant CD1b expressing cell lines revealed several CD1b binding ‘hotspots’ along the a1 and a2 helices that were critical for TCR-mediated activation. Using soluble proteins, autoreactive TCR permissive ligands were isolated from mammalian CD1b by separating ternary TCR-CD1b-lipid complexes from binary CD1b-lipid complexes that did not bind to the TCR using size-exclusion chromatography. Mass spectra analysis of the fractionated proteins revealed an abundance of phospholipids, particularly phosphatidylcholine as permissive CD1b lipid antigens. These data suggest autoreactive TCR antigen-specificity is more diverse than previously described and that these TCRs may adopt novel docking modes onto CD1b to recognise distinct endogenous antigens. In the final chapter of results, the phenotype and functional characteristics of M. tuberculosis-reactive CD1b-restricted T cells was investigated. Glucose monomycolate (GMM) is a cell wall lipid expressed by pathogenic Mycobacteria, including M. tuberculosis, that is presented by CD1b to T cells. GMM-reactive T cells were first isolated directly ex vivo from the peripheral blood of latent M. tuberculosis infected donors using CD1b tetramers. Herein, an optimised CD1b tetramer enrichment was developed to isolate very small frequencies of GMM-reactive T cells from healthy donor blood directly ex vivo. In contrast to M. tuberculosis infected patients, GMM-reactive T cells from healthy donors expressed a diverse TCR repertoire. GMM-reactive T cells were exclusively CD4+CD8- and most cells expressed the memory marker CD45RO. Similar to other unconventional T cells, GMM-reactive T cells from healthy donors expressed the transcription factor promyelocytic leukaemia zinc finger (PLZF). In vitro stimulation revealed GMM-reactive T cells secrete both TNF and IFNg, similar to MAIT cells. To characterise these cells further, single-cell transcriptomic analysis was performed on GMM-reactive T cells isolated using CD1b-GMM tetramers. GMM-reactive T cell expressed unique transcriptomic signatures that were accurately distinguishable from the transcriptomes of natural killer T (NKT) cells and CD4+ T cells, indicating that these cells may be functionally distinct from other unconventional and conventional T cells populations. In summary, these data describe a role for the CD8 co-receptor on MAIT and other MR1-reactive T cells and expand on the limited knowledge of CD1b-restricted T cells in healthy blood in regard to TCR repertoire, antigen-specificity and phenotype.
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ItemIdentifying novel compounds to eliminate latent HIV infection( 2019)There are currently an estimated 36.9 million people living with human immunodeficiency virus (HIV) (PLWH) worldwide. In the past few decades, the advent of antiretroviral therapy (ART) has significantly reduced the number of deaths associated with this virus. However, ART is not curative. The persistence of HIV latently infected CD4+ T-cells presents the major barrier towards a cure for HIV. Latently infected T-cells are formed when the virus integrates into the host genome of infected cells without ensuing productive infection. Due to these latently infected cells, viral gene expression and production infection rebounds from the integrated viral DNA if ART is ceased. Thus, ART must currently be taken life-long, posing a tremendous economic burden. The “shock and kill” approach is an extensively studied cure strategy that involves the use of pharmacological agents termed latency reversing agents (LRAs) to reactivate or “shock” the latent virus to express viral RNA and proteins. Following the reactivation of latently infected cells, the production of HIV proteins and viral particles was proposed to result in the elimination of these cells through immune-mediated clearance or cytopathic events. Results from clinical trials that involve a single LRA to reactivate latently infected cells in PLWH have not yielded any significant impact on the HIV DNA reservoir. This can be attributed to a number of different reasons that include the potency of the LRAs to reactivate latency, the failure to elicit an effective immune response and the inhibition of T-cell clearance by cytopathic viral proteins. There is clearly a need for more potent LRAs as well as novel strategies that will result in the clearance of these latently infected cells once reactivated. In this thesis, we investigate several novel pro-apoptotic compounds in isolation as well as in combination with LRAs to clear latently infected cells. We have also developed two new methods in which to study the effects of LRAs and pro-apoptotic drugs on latently infected cells. Dual-fluorescent reporter viruses have proven to be useful tools in studying latent HIV infection in vitro. Here we have modified a dual-fluorescent reporter HIV aiming to improve its functional characteristics in a pre-activation model of HIV latency. The new virus termed, DuoAdvance, contains two fluorescent viral reporters: a latent GFP reporter driven by elongation factor 1-alpha (EF1-alpha) and a productive E2 Crimson reporter driven by the HIV long terminal repeat (LTR) (Chapter 2). Using DuoAdvance, we demonstrate that DuoAdvance can successfully infected Jurkat T-cell lines. In a pre-activation model of HIV latency in primary resting CD4+ T-cells, DuoAdvance infection resulted in little to no latent GFP expression. Subsequent analysis of the GFP negative population of cells revealed DuoAdvance infection can result in the production of latently infected cells carrying latent provirus but the expression of the GFP latency reporter was perturbed. Due to the partial expression of this GFP latent reporter in primary resting T-cells, DuoAdvance is limited to use in dividing T-cell lines and potentially a post-activation model of HIV latency using activated CD4+ T-cells, where better expression of the GFP latency and E2 Crimson reporters were seen. Latency reversing agents can reactivate latent HIV but the effects on decreasing HIV DNA in PLWH has been less encouraging. In this thesis, we examine the effects of different pro-apoptotic drugs combined with different LRAs on decreasing HIV DNA in cultures of CD4+ T-cells from PLWH on ART ex vivo. Here we tested a number of LRAs together with several phosphoinositide-3 kinase (PI3K) inhibitors: IPI-443, IPI-3063 and wortmannin, as well as a B-cell lymphoma-2 (Bcl-2) inhibitor venetoclax as our pro-apoptotic drugs. The LRA romidepsin combined with all pro-apoptotic drugs resulted in synergistic decreases in the levels of integrated HIV DNA in the PLWH CD4+ T-cells ex vivo (Chapter 3). Additionally, several other LRA and pro-apoptotic combinations also decreased integrated HIV DNA in CD4+ T-cells ex vivo. All drugs were able to induce HIV viral transcription. Interestingly, we show that the pro-apoptotic drugs alone also led to an increase in HIV transcription and a decrease in HIV DNA. These data demonstrated the select combinations of pro-apoptotic drugs and LRAs together or pro-apoptotic drugs alone can result in a decrease in HIV integrated DNA in CD4+ T-cells from PLWH on ART ex vivo. However, we were unable to distinguish if there was selective death of the reactivated latently infected cells with minimal impacts on uninfected T-cells also in the cell cultures. In order to explore this, we developed a new approach to detect selective cell death (Chapter 4). This method involves the use of PrimeFlow, a HIV RNA in situ hybridisation method combined with branched-DNA technology, together with a cell death stain and analysis of stained cells using flow cytometry. Using this approach, we were able to demonstrate selective cell death in ACH2 T-cell lines treated with a combination of the PMA LRA, and venetoclax or IPI-443 PI3K inhibitor pro-apoptotic drug in a latently infected T-cell line. However, due to the elaborate staining procedure and large cell loss from the multi-step staining procedure, further investigation is required to move this staining approach into testing these drugs upon inducing the selective death of latently infected CD4+ T-cells from PLWH ex vivo. In summary, we have developed two new methods to investigate the effects of LRAs and/or pro-apoptotic drugs on HIV latency. Although further work is required to optimise these methods for use of the novel DuoAdvance fluorescent reporter virus with primary resting CD4+ T-cells for drug testing, or for use of the novel PrimeFlow assay to study the selective impact of these drugs upon latently infected CD4+ T-cell samples from PLWH ex vivo. Most importantly, our work demonstrates novel combinations of pro-apoptotic drug and LRA combinations that can decrease HIV integrated DNA in cultures of CD4+ T-cells from PLWH on ART ex vivo. This has important therapeutic implications for using these drug combinations to deplete latently infected cells in PLWH on ART and additional studies that investigate these combinations in a clinical setting is warranted. In conclusion, our work demonstrates that latency reversal combined with a drug-based strategy to promote apoptosis can eliminate HIV latently infected CD4+ T-cells from PLWH on ART ex vivo and thus this approach holds important potential to lead to HIV remission off ART in PLWH.
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ItemEvolutionary Dynamics of Successful Clones of Methicillin-Resistant Staphylococcus aureus in Australia and New Zealand( 2019)Staphylococcus aureus is a common cause of bacterial infections in humans and a leading nosocomial pathogen, associated with significant morbidity, mortality and economic impact. A cornerstone in the evolution of staphylococcal lineages that infect humans has been their remarkable ability to rapidly and efficiently develop or acquire mechanisms of antimicrobial resistance, impacting effective disease management, prevention, and eradication. Improving these measures or developing novel approaches requires a comprehensive understanding of the infecting agent and while S. aureus has been extensively studied there remains considerable gaps in our knowledge surrounding this pathogen, especially concerning population dynamics. What drives the emergence and/or persistence of certain staphylococcal lineages? What evolutionary pathways and molecular mechanisms are being utilised and under what circumstances? What environmental and host factors have the greatness influence on bacterial population adaptation? And ultimately, what are the consequence of these population level changes and what impact do these have on staphylococcal disease? This thesis represents three projects undertaken to strengthen our understanding of the evolutionary dynamics surrounding staphylococcal population adaptation, using a combination of comparative genomics and detailed phenotypic profiling to provide insight into antibiotic-resistant lineages of S. aureus that circulate in Australia and New Zealand. The first project investigated the long-term persistence of the globally disseminated, multidrug resistant hospital associated methicillin-resistant S. aureus (MRSA) lineage ST239 in Australia. From this work, it has been identified that the ST239 MRSA population circulating in Australia represents not one, but two genetically distinct clades; the previously unrecognised introduction of the Asian-Australian clade followed by successful local expansion in the state of Victoria has contributed to the persistence of ST239 by supplementing the diminishing population size of the local clone, the Australian clade. The ability of the Asian-Australian clade to spread following its introduction is owed to the reduced susceptibility this population developed against anti MRSA antibiotics, namely the glycopeptides and daptomycin, prior to its importation. This same phenotype has convergently emerged in the local Australian clade. However, this adaption has come at a cost as both clades were found to have reduced replicative fitness and impaired pathogenic potential, the latter occurring through loss of functionality or reduced expression of a staphylococcal global virulence regulatory system, the accessory gene regulator. The second project considered a different evolutionary circumstance; the rapid emergence of a novel MRSA lineage in the New Zealand community. This region has a high incidence of staphylococcal skin and soft tissue infection and over the last two decades has seen a significant shift in the local molecular epidemiology of S. aureus, with the emergence of multiple fusidic acid resistant clones. This work has focused on the predominant MRSA lineage, an ST5 clone locally referred to as AK3. Genomic investigation of this lineage has found that AK3 represents a single phylogenetic clade that has arisen through local population expansion, having emerged from a resident fusidic acid susceptible methicillin-susceptible S. aureus upon the acquisition of a novel chimeric SCCmecIVa-fusC II mobile element harbouring the fusidic acid resistance determinant fusC. This phenomenon was not restricted to ST5, with the other newly emerged lineages having acquired fusC via structurally distinct SCC and SCCmec elements. Indicating that, the unregulated use of fusidic acid in the region has supported the emergence and expansion of these novel lineages and is potentially contributing to the high local incidence of staphylococcal infection. Mobile elements can greatly influence staphylococcal populations, therefore the third project focused on exploring the evolution of the multidrug resistant plasmid family pSK1 and the role it has played in augmenting antimicrobial resistance and biocide tolerance in the ST239 MRSA population in Australia. Modelling the evolutionary history of this plasmid identified that it emerged during the late 1970s and over the following four decades has undergone significant structural change, involving a combination of chromosomal integration, transposon loss/gain, structural inversion and deletion events. When aligned to a phylogenetic model of the ST239 population, it became apparent that these changes in plasmid configuration represented a clear pathway of step wise adaptation; the shortened, chromosomally integrated plasmid structural variants having emerged on multiple occasions in a convergent manner. Further, these changes correlated with the development of enhanced tolerance against chlorhexidine, an important finding as it implicates biocide use as a factor potentially influencing MRSA evolution. Collectively, this work enhances our understanding of how antibiotic-resistant lineages of S. aureus evolve and adapt; exemplifying the evolutionary pathways facilitating adaptation in staphylococcal populations and the circumstances under which they are used. Further, it provides detailed insight into two highly successful lineages of MRSA. This knowledge can be exploited to improve measures that reduce the burden of antibiotic-resistant staphylococcal infection. Importantly, this work enhances and reinforces our awareness about the consequences of antimicrobial overuse and misuse.
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ItemMAIT cell diversity, function and impact on dendritic cells( 2019)T cells represent an important component of the immune system. Whilst early studies were largely focused on the role of conventional CD8+ and CD4+ T cells that recognize peptide-antigens in association with MCH molecules, more recently, T cells that recognize other types of antigens have been described. Mucosal associated invariant T (MAIT) cells are such a cell population and belong to the broad family known as ‘unconventional’ T cells, due to their non-peptidic antigen recognition characteristics. MAIT cells are defined by their recognition of microbial vitamin B2 metabolites presented by MHC related protein 1 (MR1). Upon antigen recognition they immediately display effector functions, like secreting cytokines and expression of cytotoxic proteins. Whilst the majority of MAIT cell studies have focused on the role of MAIT cells to bacterial infections, however their function within the immune system and interaction with other immune cells is still unknown. This thesis focuses on the role that MAIT cell activation has on other immune cells like dendritic cells (DCs) and other T cells. Furthermore, the full potential of MR1-recognition by other T cell subsets was also examined, revealing that MR1-reactive T cells may extend beyond what is currently describe as MAIT cells. The first chapter of this thesis investigates the role of MAIT cell activation on DCs in an in vivo mouse model. MAIT cells were activated by intratracheal injection of the activating MAIT cell antigen 5-amino-6-D-ribitulaminouracil/ methylglyoxal (5-A-RU/MeG). This activation of MAIT cells led to migration of DCs from the lung to the mediastinal lymph node (medLN) as well as DC maturation in an MR1-dependent manner. Furthermore, production of the chemokines CCL17 and CCL22 was induced by MAIT cell activation, which suggests that MAIT cells are able to modulate the immune system far more than previously thought. The possible role of MAIT cell induced DC maturation on initiation of a CD8+ T cell response is analyzed within the second result chapter. No enhanced antigen-specific CD8+ T cell response to the model antigen ovalbumin (OVA) was observed by additional MAIT cell activation. Besides MAIT cells, recently more MR1-reactive T cells were identified. By using antigen-loaded MR1 tetramers, a population of FOXP3+ T-bet+ T cells was identified in human thymus that can bind to MR1 tetramers. In the third chapter this FOXP3+ T-bet+ T cell population was further characterized by analysis of their phenotype as well as their TCR usage. The results in this chapter will serve as a basis for further investigation of the diversity of MR1-recognition within the T cell pool. In conclusion, this thesis reveals a new role of MAIT cells that may be used to manipulate their functions to treat different diseases like autoimmune diseases or cancer. Moreover, the knowledge of MR1-reactive T cell diversity is extended including a potential regulatory role of MR1-reactive T cells and MAIT cells. In summary, this thesis extends the current knowledge of MAIT cell biology.
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ItemRole of the chemokines CCL17 and CCL22 in the immune defence against Salmonella infection( 2019)The chemokines CCL17 and CCL22 are both ligands of the chemokine receptor CCR4, which is expressed on dendritic cells (DC) and a variety of different effector T cells including regulatory T cells (Treg). Both chemokines are mainly produced by DC, but also by macrophages. CCL17 promotes numerous inflammatory and allergic diseases, whereas CCL22 is rather associated with an immunosuppressive milieu. These differential roles are reflected by preferential recruitment of distinct subsets of T cells to site of inflammation. While CCL17 facilitates chemotaxis of effector T cells and supports DC-T cell interactions as well as DC migration towards CCR7-ligands, CCL22 induces chemotaxis of Treg cells. In addition, CCL22 signalling induces a more rapid desensitisation and internalisation of CCR4 than CCL17, suggesting biased agonism of CCL17 and CCL22. The functionality of CCL17 and CCL22 should, therefore, be considered in combination as well as individually in the context of immune-related diseases. The role of CCL17 and CCL22 in infectious diseases has not been well understood. The central hypothesis was that CCL17 and CCL22 play important but potentially different roles during bacterial infection. This was modelled using a well-studied bacterial pathogen, Salmonella enterica serovar Typhimurium (STM). It was hypothesised that CCL17 expression may direct the migration of STM-infected DC from the gut to draining lymph nodes a key bottleneck in early infection that controls bacterial dissemination to systemic sites. It was further hypothesised that CCL22 may play a role in immune regulation through the induction of Treg cells. These regulatory cells may have downstream effects on Th1 responses, which are critical for the control of Salmonella infection. In the first part of the thesis, the role of CCL17+ DC in the transmission of STM was investigated. Histological analysis of CCL17 reporter mice revealed that CCL17-expressing cells co-localised with Salmonella in the dome area of Peyer’s patches (PP). Further, CCL17-expressing DC contributed to dissemination of STM from PP to the mesenteric lymph nodes (mLN). Within the mLN, STM were found within CCL17+ DC as well as in other DC, monocytes and macrophages. Analysis of the STM+ DC subpopulations revealed that all DC subsets carried STM, but the CD103+ CD11b- DC could be identified as the main STM-containing population. STM infection triggered upregulation of CCL17 expression in specific intestinal DC subsets in a tissue-specific manner. Interestingly, the CD103+ DC subsets upregulated CCL17 in the PP, whereas CD103- DC subsets upregulated CCL17 in the mLN. In the second part of this thesis, the role of CCL17 and CCL22 in the induction of antigen-specific CD4+ T cell responses was investigated. CCL17/CCLL22 double-deficient, CCL17- and CCL22 single-deficient, and wild type mice were analysed after live-attenuated STM TAS2010 vaccination, vaccination/challenge and in steady-state. Mice deficient in both chemokines, CCL22 and CCL17, demonstrated a reduction of effector Treg cells. This promoted an enhanced STM-specific Th1 immune response characterised by an expansion of Th1 T cells, resulting in a more favourable effector Treg/activated Tconv ratio and a significantly improved vaccine efficacy to challenge with virulent Salmonella. In conclusion, the work presented within this thesis showed the contribution of CCL17+ DC in the dissemination of STM and identified CCL22 as a potential target to improve vaccine approaches.