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ItemTranscriptomic diversification along the monocyte-macrophage continuum.( 2019)Current models of innate immune responses describe hard wired, gene-centric signalling networks, with limited capacity to define the molecular mechanisms underpinning transcriptomic diversity. It is well established that a transcriptional spectrum of responses accompanies acute macrophage activation, however it is unclear whether this spectrum originates in monocytes and to what extent it continues throughout reinfection. The contribution of molecular mechanisms such as enhancers and alternative transcription start sites is also undetermined. Given the critical roles that myeloid cells play in directing acute infection and priming adaptive immunity, it is important the regulation of their responses be understood. This thesis employed bioinformatic analysis of Cap Analysis Gene Expression (CAGE) and microarray data to describe transcriptional diversity along the monocyte-macrophage continuum. Using CAGE to map transcription start sites for capped RNAs, this thesis has shown that pathogen-specific transcriptional diversification commences early in monocyte infection (Chapters Three and Four) and continues throughout acute macrophage infection (Chapter Five). Transcriptomic diversity during acute infection was the product of kinetic and pathogen-specific engagement of distinct transcription start sites. Engagement of multiple transcription start sites drove responsiveness by regulating expression amplitude in functionally focused inflammatory gene sets and diversifying secondary response networks via expression of distinct protein isoforms. Chapter Six extended these studies of acute infection, demonstrating that transcriptional phenotypes continue to diversify during reinfection. These findings highlight the importance of studying innate immune responses at the isoform level and prompt the need to revise current models of innate immune signalling, such that monocyte-macrophage biology should no longer be modelled as a series of static states, but rather, as a continually evolving continuum.
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ItemThe Transcription Factor T-bet in the Control of Germinal Centre Dynamics in Malaria( 2019)With reductions in the global malaria burden stalled, this preventable and curable infectious disease caused by the Plasmodium parasites, remains a public health challenge that affects the world’s most vulnerable populations. Naturally acquired immunity plays an important role in protection from disease; however, there is long-standing evidence that it requires years of repeated infections to develop. The reasons for this are largely elusive, but immuno-epidemiological studies support that protective antibodies and memory B cells are short-lived and inefficiently generated to infection. Moreover, recurrent infections are associated with an expansion of atypical memory B cells that may have impaired function. Histological analyses revealed significant disorganisation of the spleen in severe malaria patients, which led to the concept that acute infection may undermine the acquisition of B cell memory. T helper 1 pro-inflammatory responses induced by blood-stage infection were subsequently shown to compromise the induction of humoral immunity by inhibiting effective T follicular helper (Tfh) cell differentiation and germinal centre (GC) reactions. The relative contribution of the T helper 1 lineage-defining transcription factor, T-bet, in CD4+ T cells and B cells to GC development in malaria, was investigated using the P. berghei ANKA blood-stage infection model. T-bet expression in CD4+ T cells limited the differentiation of Tfh cells that supported GC development in the spleen. This led to an impaired generation of antibody-secreting cells and memory B cells following infection. In addition to its impact on CD4+ T cells, T-bet was highly up-regulated in GC B cells elicited by infection, and limited the magnitude of the GC response in a B cell-intrinsic manner. Strikingly, T-bet expression in the B cell compartment modulated the transcriptional landscape of GC B cells to promote the GC dark zone program but constrained light zone development. In particular, T-bet suppressed expression of the regulator of G-protein signaling 13, which down-regulates the responsiveness of B cells to migrate towards the chemokine CXCL12, for effective dark and light zone transition within the GC. T-bet-driven dark zone skewing of the GC reaction following malaria infection associated with enhanced somatic hypermutation of GC B cells, and improved the avidity of antibodies against the parasite. Therefore, this thesis supports a model in which malaria-elicited inflammation mediated by T-bet, exquisitely modulates the dynamics of the GC reaction, promoting GC B cell dark zone polarization that promotes the generation of B cells with increased affinity for antigen, consequently enhancing affinity maturation. This provides novel insight into the cellular mechanisms that underlie the development of humoral immune responses in malaria, and has implications for other chronic infections and autoimmune disease that are characterised by a similarly potent inflammatory milieu.
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ItemThe role of CD8+ tissue-resident memory T cells in melanoma immune surveillance.( 2018)In addition to its role in protecting the body from infection, the immune system can prevent the development of cancer in a process termed tumour immune surveillance. During this process, immune cells can either recognise and completely eliminate cancerous cells, or can suppress the outgrowth of malignant cells without completely eradicating them. This latter mode of control, designated ‘cancer-immune equilibrium’, can be sustained for extended periods of time in a manner dependent upon adaptive immune cells such as T cells. The vast majority of human cancers are spawned from epithelial tissues. However, long-lived CD8+ circulating memory T (TCIRC) cells such as effector memory T (TEM) cells and central memory T (TCM) cells are typically excluded from epithelial tissue compartments in the absence of robust inflammation. In contrast, CD8+ tissue-resident memory T (TRM) cells are a population of non-migratory immune cells that permanently occupy epithelial tissue sites without recirculating. CD8+ TRM cells provide efficacious protection against peripheral viral and bacterial infections and have recently been identified in a variety of human solid tumours, where they associate with improved disease outcome. However, a direct role for TRM cells in promoting natural immunity to cancer has yet to be demonstrated. In this thesis, we examined the contribution of CD8+ TRM cells to peripheral cancer immune surveillance and the mechanisms through which these cells protect against tumour progression. In order to study the peripherally localised anti-tumour immune response, we developed and characterised an orthotopic epicutaneous (e.c.) model of melanoma in mice that targets tumour growth to the outermost layers of skin. We found that a portion of mice receiving tumour cells e.c. remained free of macroscopic cancer long after inoculation, in a manner that depended upon immune cell mediated control. Spontaneous protection from progressive tumour development was associated with the formation of melanoma-specific CD69+CD103+ CD8+ skin TRM cells, whereas mice genetically deficient in TRM cell formation were highly susceptible to tumour growth. Importantly, tumour-specific skin TRM cells could protect against tumour development independently of TCIRC cells. Closer inspection of macroscopically tumour-free mice revealed that many harboured occult melanoma cells in their skin long after e.c. inoculation. These dormant melanoma cells were retained in the epidermis, where they were dynamically surveyed by tumour-primed CD8+ skin TRM cells. Ablation of skin TRM cells from macroscopically tumour-free mice that were initially protected from tumour development triggered late-stage tumour outgrowth, demonstrating that CD8+ TRM cells can suppress cancer progression by promoting a state of subclinical cancer-immune equilibrium. Further, our findings suggest that the cytokine tumour necrosis factor (TNF) may play a role in the induction and maintenance of this equilibrium state. Overall, we show that CD8+ TRM cells contribute to immune surveillance of peripherally localised cancers by upholding tumour-immune equilibrium. As such, our findings elucidate how cancers arising in epithelial compartments are subject to long-term and ongoing immune suppression. Collectively, our work provides critical insight and the impetus necessary to exploit CD8+ TRM cells as targets of cancer immunotherapies in order to improve solid cancer treatments in patients.
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ItemAnalysis of C-peptide-specific CD4+ T cells in the peripheral blood of people with type 1 diabetes( 2018)Uncovering the primary antigen targets in type 1 diabetes (T1D) is essential to our understanding of disease pathophysiology. Despite the clear role of CD4+ T cells in orchestrating the immune destruction of the pancreatic cells, what they are targeting in human T1D has remained poorly defined. Most knowledge of in vivo T-cell responses in T1D derives from studies in mouse models, and translating results to humans has been limited to analysis of peripheral blood. However, only 3% of the total T cells in the body reside in the peripheral blood. Prior work at this institute by Mannering and colleagues on islet-infiltrating CD4+ T cells in humans, complemented by other similar studies, provided insight into the resident T-cell population of the target organ in subjects with T1D. These studies have concurred that a cleavage product of proinsulin, C-peptide, is a target antigen of islet-infiltrating CD4+ T cells. Because these studies were done in just a handful of deceased organ donors with T1D, they led to the question how relevant is C-peptide as an autoantigen in T1D more generally? Given the pancreas is not routinely accessible, to address this question, evidence of C-peptide as a target of CD4+ T cells was sought from the peripheral blood in subjects with T1D. The main obstacle to assessing T-cell targets in the peripheral blood is the lack of a sufficiently sensitive and reproducible T-cell assay. In Chapter 3, the CFSE-based proliferation assay was optimised for detection of C-peptide-specific CD4+ T-cell responses. The CFSE-based proliferation assay was demonstrated to have comparable reproducibility as compared to other currently available T-cell assays, and greater sensitivity than the commonly used ELISpot assay. In Chapter 4, using the CFSE-based proliferation assay, >60% of people with recent-onset T1D were shown to have a detectable peripheral C-peptide-specific CD4+ T-cell response. The response was disease specific because few control subjects were positive. Analysis of cloned C-peptide-specific CD4+ T cells revealed they were restricted by HLA alleles strongly associated with T1D risk, namely HLA-DQ8, -DQ2, -DQ8trans, -DQ2trans and HLA-DR4. This added further support to the notion that they were pathogenic. In Chapter 5, the hypothesis that autoantibodies to C-peptide may be detected in the serum of people with T1D, was tested. Using solid-phase ELISA, it was found, unlike C-peptide-specific CD4+ T cells, C-peptide autoantibodies are not detectable in the serum of subjects with T1D in a disease-specific manner. Together, these findings indicate that proinsulin C-peptide is commonly a target of autoreactive CD4+ T cells in newly-diagnosed T1D. Hence, C-peptide is a promising candidate for biomarker development and antigen-specific immunotherapy.
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ItemTargeting regulators of natural killer cell homeostasis in cancer immunotherapy( 2018)The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory receptors and from cytokines such as IL-15. The ability to recognise tumour cells in the absence of antigen presentation has garnered significant interest in NK cells as novel targets for immunotherapy development. However, successful developments in this area have led to limited success. This is, in part, due to the lack of understanding of the underlying mechanisms governing inhibitory and stimulatory pathways in NK cells. In this work, we aimed to identify the key regulator of NK cell proliferation in order to further our understanding of NK cell activity both in the steady-state and in the setting of inflammation. Here, we have identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signalling in NK cells. IL-15 is the main driver of NK cell proliferation, survival, differentiation and function, and thus a highly relevant checkpoint in NK cell homeostasis. We found Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFNγ production and cytotoxicity toward tumours, in vitro. This was associated with increased JAK-STAT signalling in Cish-deficient NK cells. Cish-deficient mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity, uncovering CIS as a potent intracellular checkpoint in NK cell-mediated tumour immunity. Under homeostatic conditions, phenotypic changes in NK cells lacking Cish were observed in vivo. This included an increase in terminally differentiated NK cells as well as increased expression of cell cycle markers, suggesting that under steady-state conditions, CIS also plays a role in maintaining IL-15 driven regulation of NK cells in vivo. Additionally, the changes observed in steady state Cish-deficient NK cells manifested in a lower activation threshold, evidenced by the redundancy of exogenous IL-15 to induce augmented production of inflammatory cytokines and cytotoxicity when stimulated ex vivo. These data suggest that Cish not only regulates NK cell responsiveness to IL-15, but may also play a role in maintaining an activation threshold, consequently regulating effector functions in vivo. Furthermore, inhibition of CIS was found to be conserved between human and mouse NK cells, emphasising its potential role as a novel immunotherapy target for the treatment of human cancer.
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ItemA journey of synthetic chemistry towards immunogenic glycolipids and non-lipidic antigens( 2018)Microbes, both pathogenic and commensal, produce a wide range of glycolipids that act as unique molecular signatures. The ability of the human immune system to fight infection as well as to modulate commensal organisms are active areas of research. Microbial glycolipids are known to interact with the immune system though discrete protein families including CD1 and Mincle. The main challenge in the study of such systems is the difficulty in, and often impossibility of, obtaining pure, homogeneous material from natural sources. We synthesised four classes of molecules of both natural and unnatural origin to investigate their potential to modulate the human immune system through the CD1 and Mincle axes. Chapter 2 describes the synthesis of a range of cholesteryl α-glucosides that are found in members of the Helicobacter family, including the prominent gut bacterium Helicobacter pylori. As part of this work we investigated the effect of remote protecting groups on the sugar on the stereochemical outcome of glucosylation reactions. In chapter 3 we designed and synthesised a set of purely synthetic glycolipids drawing upon the structures of known Mincle agonists. We investigated these compounds for their ability to signal through Mincle as a prelude to the development of improved vaccine adjuvants that promote cellular and humoral immunity. Chapter 4 discloses the total synthesis of α-glucosyl and α-glucuronosyl diglycerides, found in both pathogenic and commensal organisms relevant to human health. Finally, we prepared a set of analogues of the unique, non-lipidic synthetic CD1d-restricted effector, PPBF, to explore structure activity relationships for T cell activation. In collaboration with immunologists, the synthetic glycolipids and non-lipidic antigens have been studied for their ability to activate CD1d-restricted natural killer T cells or for their ability to stimulate signalling through Mincle.
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ItemCharacterisation of mice deficient for the pro-survival BCL-2 family member A1/BFL-1( 2018)Apoptosis is important for the development and maintenance of a healthy immune system. The intrinsic apoptotic pathway is regulated by the BCL-2 family of proteins, which consists of both pro-survival and pro-apoptotic members. The balance between these two sub-groups determines whether a cell lives or dies. The levels of different BCL-2 family proteins is influenced by the signals that an immune cell receives – for example, cytokine signalling drives pro-survival protein expression, whilst cellular stress from cytokine deprivation upregulates pro-apoptotic proteins. Studies using knockout mouse models have highlighted the importance of some of the pro-survival proteins in different haematopoietic cell types, such as BCL-2 in mature lymphocytes and MCL-1 in haematopoietic stem cells. The pro-survival protein A1/BFL-1 is highly expressed in many different immune cell subsets and is upregulated after immune cell activation. However, little is known about the physiological importance of A1/BFL-1 in the immune system. This is because the presence of three murine A1 isoforms complicates the generation of a knockout mouse model. We have generated a completely A1-deficient mouse strain through sequential gene targeting in embryonic stem cells. This thesis presents an analysis of A1’s role in the haematopoietic system through the study of these A1-/- mice. A1/BFL-1 is reportedly expressed in B and T lymphocytes, neutrophils, mast cells, and conventional dendritic cells (cDCs). We characterised the cell numbers for these populations in the A1-/- mice in the steady state. There were no major differences found when compared to wild-type mice, although there was a small but significant decrease in cDC numbers in the spleen, and also small reductions in memory T cell populations. Given that A1/BFL-1 is upregulated by immune cell activation, we proceeded with in vitro activation assays with the various different immune cell subsets. Despite the reduction in memory T cells, in vitro survival of T cells after activation was unperturbed. A1-/- cDCs, however, had a marked survival disadvantage in tissue culture. We further characterised the A1-/- mice in response to immune challenge in vivo, with influenza infection, LCMV infection, and T cell-dependent immunization. The A1-/- mice responded normally to all of these models. We reasoned that A1/BFL-1 may have overlapping roles with other pro-survival BCL-2 family proteins. To this end, we generated compound mutant mice that are deficient for A1 and heterozygous for BCL-2, BCL-X or MCL-1. We focused on the lymphocytic compartment of these mice, based on the co-expression of these pro-survival proteins in B and T lymphocytes. Again, we found no overt differences in the compound mutant mice when compared to control mice. It is likely that complete deletion of BCL-2/BCL-X/MCL-1 in combination with A1 loss is required to decipher these overlapping roles. Overall, despite high expression of A1/BFL-1 in different immune cell types, loss of A1 is tolerable in mice and, therefore, A1 is a redundant pro-survival BCL-2 family member in the haematopoietic system.
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ItemThe interface of nanotechnology and the human immune system( 2017)Harnessing nanomaterials for the benefit of human health has the potential to improve drug delivery, vaccination and diagnostic imaging. However, a greater understanding of the interaction between nanomaterials and the human immune system is required to improve the clinical translation of nanomedicines. Knowledge of the bio-nano interface has arisen largely from studies in cell lines and rodent models, and our poor understanding of bio-nano interactions in primary human systems remains a key knowledge gap in the development of clinical applications of nanomedicine. This thesis uses novel nano-engineered materials to characterise how material properties influence biological outcomes in primary human samples. By investigating the human bio-nano interface, this research has the potential to improve the rational design of novel nanomedicines. Blood is the first tissue encountered by nanomedicines following intravenous administration – the most common delivery method in the clinic. Therefore, human blood was used as both a source of primary blood cells to examine cell association, targeting and activation, and of plasma for the formation of complex biomolecular coronas. Flow cytometry and confocal microscopy were employed to characterise the role of key physicochemical properties of nanoparticles: charge, reactive surface chemistry, and targeting with antibodies and antibody fragments. A range of nano- engineered particles were developed including caveospheres, hyperbranched polymers (HBP), star polymers and pure PEG particles. Attempts were also made to determine how the biomolecular corona formed in human blood influences the observed bio-nano interactions. Using antibody-capture caveosphere nanoparticles, CD4+ and CD20+ human immune cells could be targeted within mixed cell populations following antibody- functionalisation. Moreover, functionalisation with anti-CCR5 antibodies enabled nanoparticle internalisation into HIV-tropic, non-phagocytic CD4+ T cells, a key hurdle in the delivery of nanoparticle-based anti-HIV therapeutics. Nanoparticle charge defined clear patterns of HBP association with blood cells. These patterns varied for nanoparticles of different material and size, and were not defined by the plasma biomolecular corona that forms in blood. Follow up studies demonstrated cationic, but not anionic or neutral, HBPs activated the myeloid subset of dendritic cells – an important cell target for vaccine applications. The effect of surface chemistry was examined using star polymers. Engineering thiol-reactive pyridyl disulfides onto star polymers directed their association with cancer cell lines, platelets (without activating them) and distinct immune cells subsets. Further studies using preclinical polymer vaccine nanoparticles demonstrated clear differences in blood phagocyte clearance based on brush vs. linear architectures of PEG. Lastly, immunologically stealth particles were functionalised with bispecific antibodies to evaluate cell targeting in the presence of complex biomolecular coronas and the impact of targeting moieties on particle stealth properties. Targeted stealth particles demonstrate potential for the targeted delivery of therapeutics or imaging agents in the presence of plasma coronas, with high specificity and only minimal disruption to particle stealth properties. Phagocytic uptake of PEG particles was dependent on the plasma biomolecular corona. Taken together, these findings further our understanding of the interactions between nano-engineered materials and the human immune system. Ultimately, the development of comprehensive human bio-nano principles will contribute to the rational design of novel nanomedicines.
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ItemInvestigating the interactions between dendritic cells, T cells and B cells mediated by targeting Clec9A( 2016)Dendritic cells (DC) are endowed with an array of receptors that can be exploited for immunotherapy. Targeted delivery of antigen to CD8α+ DCs via Clec9A in vivo induces versatile immune responses, most notably potent thymus-dependent humoral responses even in the absence of adjuvant. However, the basis of the immunogenicity of Clec9A-targeted antigen remains incompletely understood. This thesis describes the complex interactions between CD8α+ DCs and T and B cells mediated by Clec9A to promote and/or regulate immunity. Characterization of CD4+ T cells responding to Clec9A-targeted antigens revealed that they had the phenotype, localization pattern and effector functions consistent with T follicular helper cells (TFH) that provide B cell help. Furthermore, targeting Clec9A primed long-lived memory CD4+ T cells capable of robust secondary TFH responses, even in the absence of adjuvant. Thus, in the steady-state Clec9A-targeted CD8α+ dendritic cells are capable of stimulating CD4+ T cells to promote the development of fully polarized TFH cells. Strikingly, Clec9A was also found to mediate direct interactions between CD8α+ DCs and B cells. B cells were rapidly activated through recognition of native antigen presented on the surface of CD8α+ DCs upon Clec9A-targeted immunization. Direct activation of B cells by CD8α+ DCs was critical for optimal Clec9A-mediated antibody responses as it enabled B cells to effectively acquire help from cognate CD4+ T cells at the T/B borders within the spleen and lymph nodes. Thus, the effective triad of interactions mediated by Clec9A drives potent antibody responses in the steady-state. Unlike TFH and B cells that were potently activated in the steady-state, cross-priming of cytotoxic lymphocytes (CTLs) by Clec9A-targeted antigen required co-administration of adjuvant. In contrast to B cells, Clec9A-mediated primary CTL responses were impaired by the presence of CD4+ T cells. Clec9A-mediated MHC II-restricted presentation favoured the expansion of pre-existing Foxp3+ regulatory T cells (Tregs) in the steady-state, which presumably impaired non-Tregs capacity to activate CD8α+ DCs. Collectively, the data presented in this thesis reveal the versatile capacity of CD8α+ DCs to interact with various cell types to promote immunity/tolerance and reinforces the notion that targeting Clec9A in vivo is a promising strategy to exploit for immunotherapy.
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