Medical Biology - Theses
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ItemIntracellular competition regulates B lymphocyte differentiation( 2019)The production of antibodies, with their potential to recognise unique targets and prevent repeat infections, is an important aspect of immune health. In order to generate free antibodies, the cells responsible, B cells, must undergo a differentiation step to transform from lymphoblast to antibody secreting cell (ASC). This differentiation step prevents further antibody modifications and hence the timing for optimal immunity requires a delicate balance between expanding useful clones and providing early protection. How differentiation is controlled to achieve this balance for an effective immune response is of great interest. In this study, the progression from naive B cells to ASC was investigated in the context of an emerging model for competing cell fates. By this model, alternate cell fates, such as division, death and differentiation, are pursued independently in individual cells but are in competition such that events which occur earlier prevent those that require more time from being observed. Evaluation and testing of this model requires careful measurement of distributions of times to fates which is only possible with single cell fate tracking. Here I have developed and applied methods for live cell imaging and analysis for assessing and evaluating cell fate changes over time. Using these methods, several modes of regulating differentiation times were revealed. Low levels of stimulation through CD40 produced a greater proportion of antibody secreting cells per generation as division is slowed and more time is allowed for differentiation, consistent with competing cell fates. A second mechanism was found where increasing division numbers directly reduced the amount of time required for cells to differentiate, without modulating division times, ensuring the natural development of ASC during the ongoing immune response. A direct method of uncensoring was explored where cell cycle inhibitors were used to prevent division, with the hypothesis that more cells would go on to differentiate in the absence of competition. Various inhibitors were assessed for their suitability to this task, and a panel of compounds were found to be suitable for uncensoring underlying differentiation and cell death times. Findings from this study are consistent with the model of independent and competing cell fates, and significantly advance our understanding of how antibody responses are controlled and can be modelled at the cell population level.
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ItemInvestigation of mammary gland development and resident macrophages by 3D and intravital imaging( 2019)The mammary gland is a fascinating organ that develops after birth and is capable of remodelling through multiple rounds of reproduction. The behaviour of mammary epithelial cells and how these interact dynamically with their environment are poorly understood. Cell morphology and arrangement can be addressed by three-dimensional (3D) confocal imaging to provide large-scale, subcellular resolution views of tissue architecture. Further insight can be gained from intravital imaging that allows direct observation of cell behaviour in vivo, but this has rarely been implemented for the normal mammary gland. Mammary ducts are embedded in adipose tissue, making in vivo imaging of mammary ducts extremely challenging. Chapter 3 provides a detailed protocol for an intravital imaging method that was adapted and optimised for the mouse mammary gland. This technique enables high-resolution, 3D intravital imaging of the mammary gland for up to twelve hours. The skin flap surgical technique was modified to expose the entire inguinal mammary gland, allowing rare accessible epithelial structures to be identified. Additional fine microdissection of connective tissue maximised the resolution of imaging. Significant measures were taken to achieve as near to physiological conditions as possible, including creating a sealed environment over the exposed tissue. Strategies used for image analysis are then discussed, including image stabilisation, cell tracking and 3D visualisation. This technique advances our ability to observe mammary cell behaviour in vivo and will enable future investigation of rare events that are spatially and temporally regulated, such as stem cell behaviour, tumour initiation and microenvironment interactions. Mammary gland morphogenesis occurs by migration of terminal end buds through the mammary fat pad. Terminal end buds are large, club-like structures comprising a cap layer and a multi-layered body that give rise to bilayered ducts. Epithelial progenitors within terminal end buds generate mature cells of ducts but how these behave and cooperate to generate the bilayer is not well understood. Chapter 4 describes the lineage-specific behaviours of terminal end bud progenitors as observed by intravital microscopy. Cap cell migration into the body was recorded at high resolution in vivo for the first time. High-dimensional image quantification of cap cell behaviour showed that most cap cells that migrate into the body die rapidly but a small proportion survive long term. Progenitors for the luminal lineages were observed to have contrasting behaviours, with hormone-sensing progenitors being highly migratory. Single cell transcriptomic analysis of terminal end buds is described, providing possible molecular drivers of the distinctive behaviour of hormone-sensing progenitors. This work provides an unprecedented view of mammary stem cell behaviour, making an important contribution to our understanding of how cellular behaviour drives organogenesis. Chapter 5 describes a previously uncharacterised population of resident intra-epithelial macrophages that were revealed by 3D confocal imaging. These cells, termed mammary ductal macrophages, are regularly positioned over the entire mammary gland at all stages of development. They do not migrate but monitor the epithelium by dendrite movement, allowing them to rapidly sense and respond to epithelial damage. Ductal macrophages proliferate in pregnancy to maintain their density on the epithelium in lactation. During involution following weaning, they rapidly phagocytose dying alveolar cells to facilitate remodelling. Breast tumour-associated macrophages are pro-tumorigenic and strongly resemble ductal macrophages, not stromal macrophages. Macrophages are emerging as important targets for breast cancer treatment, therefore, better understanding of parallels between DM function in healthy and perturbed tissue may enable development of improved cancer therapies. Finally, in Chapter 6, the presented results are summarised and their context within the field, wider implications and possible future directions are discussed. Overall, this thesis presents original research that advances our technical ability to address questions of cell dynamics in the mammary gland, provides important insights into mammary stem cell behaviour during morphogenesis, and characterises a novel tissue-resident macrophage population, finding a key role for these in mammary gland remodelling.
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ItemMeasuring B-lymphocyte responses in human health and primary immunodeficiency( 2019)Production of high-quality, specific antibody is essential for lifelong protection against pathogens. Generation of antibodies is the end result of a complex and highly regulated process of B cell development and signalling. Defects anywhere in this process can lead to immune dysregulation, resulting in primary immunodeficiency and/or autoimmunity. This thesis explores a new hypothesis – that the majority of cases of primary immunodeficiency, and likely other complex immune disorders, are caused by the combinatorial effect of multiple small defects in B cell function or ‘health’ that sum to cause disease in patients. Further, that we can measure and model these defects quantitatively in vitro. To test this hypothesis, we focused on Common Variable Immunodeficiency (CVID), a clinically heterogenous primary immunodeficiency, united by antibody deficiency, where most cases are sporadic and, presumably, polygenic. In this thesis, we outline the development of a novel in vitro pipeline and accompanying parametric mathematical modelling tools to identify and measure the functional cause of immunodeficiency in individual patients. These in vitro assays were first developed and calibrated on healthy donors, to measure healthy human B cell responses to T-dependent and T-independent stimulation. These assays measure cell division, death, differentiation, isotype switching and antibody production, to reveal the innate programming of B lymphocytes in response to T-independent and T-dependent stimuli. Here, we observed, for the first time, autonomous programming of the division-burst size (division destiny) in human B cells, a phenomena previously demonstrated in murine lymphocytes. We applied cyton modelling to human lymphocytes for the first time, to explain the parameters underlying the synergy between two signals. Additionally, by testing a number of unrelated healthy donors, we established a healthy donor ‘range’ of B cell responses for comparison to patients. We subsequently applied these assays to a cohort of CVID patients and identified a number of quantitative differences. These included: 1. A striking, severe early survival defect of patient naive B cells; 2. A defect in the ability of patient naive B cells to differentiate to antibody secreting cells and produce switched antibody, and; 3. Reduced proliferation of patient naive B cells in response to T-independent stimulation. We utilised mathematical modelling tools to demonstrate that this reduced proliferation is explained by a combination of multiple small defects in the parameters that control the B cell response (times to divide, times to die etc) that sum together in a non-linear manner to magnify their impact on immune responses. Furthermore, the relative contribution of each parameter to the final immune deficiency was individually determined and found to be unique for each patient. This work significantly improves our understanding of the functional causes of immunodeficiency and offers a clear path toward improved clinical diagnosis and targeted treatment strategies for individual patients.
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ItemDefining programs of cell death that can be harnessed to impact on outcomes of chronic viral infection( 2019)Pathogens causing chronic infections have successfully evolved mechanisms to subvert host immunity. Excessive and inappropriate inflammation together with attrition of repeatedly overstimulated high affinity T cells leads to abrogated immunity and persistence of pathogens such as HIV and HBV. T cell exhaustion has been touted as a prelude to T cell deletion during these infections, however, studies indicate that high affinity T cell clones are deleted at the onset of infection. The T cells that remain have lower affinity for pathogen epitopes and hence their response is weaker and more easily antagonised by inhibitory networks, including T-regulatory (Treg) cells. The killing of immune effector cells during chronic overwhelming infections is juxtaposed to the pathogen’s attempts to promote survival of infected target cells. Keeping infected cells alive is imperative for the maintenance of a microbial replicative niche. In this body of work, I dissected the role of host cell molecules and how they contribute to the death and survival of immune and infected cells. Necroptosis did not contribute to the loss of highly functional virus-specific CD8+ T cells during the course of infection. In contrast, when I interfered with death receptor signalling there was a modest rescue of functional CD8+ T cells. This gain in immune function, however, did not translate to improved viral control. The same mechanism I used to promote the survival of T cells made infected target cells refractory to death receptor mediated killing and therefore, offset any gain in immune function. Whilst examining the role of necroptosis in chronic infection, I made the discovery that the necroptotic inducer molecule, RIPK3, has additional non-necroptotic roles. Ripk3-/- mice cleared LCMV with enhanced kinetics compared to wild-type mice and mice that lacked the necroptotic executioner MLKL. I found that in the absence of RIPK3, chronically infected mice had impaired IFNβ responses. Excessive and prolonged IFNβ production is known to impair immunity. This may partially explain why mice lacking RIPK3 had enhanced numbers of granzyme B expressing T cells and controlled infection better than WT animals. The host-viral dynamics that favour displacement of highly functional cells with poorly activated cells makes the immune system highly vulnerable to inhibition through the activity of Treg cells. I next investigated the role of Treg cells in immune dysfunction during chronic infections and I was particularly interested in the cell death and cell survival pathways that contributed to the turnover and accumulation of these cells. I utilised mice with a Treg-specific deletion of Casp8. These mice had twice as many Treg cells as wild-type mice at steady state. Surprisingly, when these mice were infected with chronic LCMV, only 25% of the animals survived to 145 days post infection. Moribund animals succumbed to overt T cell activation and autoimmunity due to a precipitous drop in Treg cell numbers. Survivors, intriguingly, eliminated LCMV in most organs consistent with a massive gain in immune function. The death of the Treg cells was due to necroptosis. When I ablated the necroptotic pathway, through the deletion of Mlkl, I completely prevented the loss of Treg cells and the fatal immune pathology in Treg conditional caspase-8 deficient mice. I found that differential expression of RIPK3 and MLKL in Treg cells made them highly susceptible to necroptosis during chronic infection compared to Tconv cells. This was also the case for human Tregs and I was able to preferentially kill these cells, over Tconv cells, in vitro by driving necroptosis with a clinical stage caspase-8 antagonist called emricasan. Necroptosis is a lytic form of cell death that promotes inflammation and it has been implicated in chronic liver disease. I initially investigated if necroptosis in the liver contributed to the control of chronic LCMV, HBV or the malaria parasite Plasmodium berghei. Ablation of necroptosis had no impact on liver-pathogen dynamics and no impact on general liver function and architecture. In many cell types caspase-8 inhibits death receptor induced necroptosis. So, I reasoned that this molecule must be inhibiting induction of necroptosis in the liver of infected animals. I examined this by infecting mice that had a conditional loss of caspase-8 within hepatocytes. Despite abundant, infection driven, death ligands I observed no necroptosis in the liver. Even drug induced ablation of NF-ĸb survival signalling, downstream of TNF, failed to promote liver necroptosis in the aforementioned scenarios. The liver’s inability to undergo necroptosis was confirmed in mice with a human chimeric liver. I showed this refractoriness was due to liver repression of RIPK3 in humans and mice. The work conducted in this thesis provides important insights into the cell death pathways that are engaged in diverse cell types during chronic viral infections and I provide evidence that antagonising them therapeutically may lead to better clinical outcomes.