Medical Biology - Theses
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ItemSteady-state and emergency dendritic cell development at a clonal level( 2019)Recent clonal fate and single cell RNA-sequencing studies demonstrate that significant lineage imprinting is already in place within individual haematopoietic stem and progenitor cells (HSPCs). Dendritic cells (DCs) represent one such branch of haematopoiesis and are responsible for pathogen-sensing and activation of the adaptive immune response. At the population level, all three major DC subtypes including type 1 conventional DCs (cDC1s), type 2 conventional DCs (cDC2s) and plasmacytoid DCs (pDCs), can be generated from a restricted common DC progenitor (CDP) population downstream of HSPCs. However, recent clonal evidence has suggested earlier subtype-specific imprinting within the CDP and even early HSPC populations. Therefore, the current hierarchical model of haematopoiesis is insufficient to explain the complexity and dynamics of DC development. The aim of this thesis was to investigate the development of DCs at the single cell level. One caveat of most prior single cell lineage tracing studies was that clonal fate was only measured at a single time point. Therefore, questions remain as to whether the fate bias observed at a snapshot in time is consistent with earlier or later times. Here, using cellular barcoding, I develop an experimental and computational framework to allow robust periodical examination of lineage outputs of thousands of transient clones during DC development in vitro. I reveal that single HSPC clones are largely programmed regarding the types of DCs to make (fate), the number of DCs to produce (size), and when DC generation occurs (timing). Together, I define these unique properties as a clone’s cellular trajectory. Importantly, I demonstrate that a large proportion of early HSPCs are already committed towards either cDC or pDC generation, even when clonal output is measured over time. This finding is consistent with and further complements the most recent evidence of DC subtype imprinting during early haematopoiesis. Exogenous administration of Flt3 ligand (FL) is known to preferentially induce ‘emergency’ DC development, and is shown to provide promising therapeutic benefits in various conditions such as infection and cancer. However, how FL signals regulate cell proliferation and differentiation during early DC development is largely unknown. In this thesis, I investigate the clonal aetiology of this process. Using cellular barcoding, I demonstrate that emergency DC generation is predominantly driven by increased expansion of pre-existing HSPC clones that are already primed with DC potential. Consistently, enhanced cell cycle activity is found to be prominent within most early HSPCs after short exposure to FL. In particular, using a single cell multi-omics profiling approach, I identify key cellular and molecular events within a unique group of early HSPCs that are most responsive to FL stimulation, which include increased cell division, maintenance of hyper-proliferative potential and establishment of a DC lineage program. Collectively, the findings presented in this thesis provide new insights into the control and regulation of DC fate within individual HSPCs during steady-state and emergency haematopoiesis, with important implications regarding the maintenance or manipulation of DC generation in health and disease.
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ItemCytokine signalling in haematopoietic cells( 2018)Cytokine receptor signalling is essential for cell survival, proliferation and subsequent differentiation of haematopoietic stem cells (HSCs). Cytokines control development of haematopoietic progenitors into cells of the myeloid, lymphoid and erythroid lineages by stimulating cell cycle progression, proliferation and differentiation as well as by inhibiting apoptosis. My work focusses on Interleukin-3 (IL-3) and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), two essential cytokines in haematopoiesis. Binding of a cytokine to its specific receptor leads to the activation of multiple kinase signalling pathways, including the JAK/STAT, Ras-MAP kinase (MAPK) and PI3-kinase/AKT pathways. In this signalling network, the IkappaB Kinase (IKK) complex plays an important role as a downstream signalling hub. My research investigates how IL-3 mediated IKK activation promotes the survival of myeloid cells and what role this process may play in the development of related diseases, such as myeloproliferative disorders. Using immortalised growth factor (IL-3 or GM-CSF) dependent myeloid progenitor cells (FDMs), as well as employing various in vivo mouse models of haematopoietic development, I was able to show that 1) IKK is a major signalling hub linking IL-3, TNFR1 and p53 signalling to control the survival in haematopoietic cells by describing for the first time a role for the E3 ubiquitin ligase MDM2 downstream of IL-3- or TNFalpha-mediated IKK2 activation, suggesting crosstalk between the NF-kB signalling and p53 signalling pathways; 2) IKK regulates cellular metabolism through activation of NF-kB- and p53-dependent metabolic target genes by showing that deletion of IKK2 but not IKK1 in hematopoietic cells significantly alters cellular metabolism, impairing oxidative phosphorylation and upregulating glycolysis due to altered expression of p53-dependent metabolic target genes; 3) IKK plays a crucial role during haematopoietic development, regulating myeloid cell proliferation, lineage commitment and survival, showing that deletion of IKK2 but not IKK1 in haematopoietic progenitor cells severely affects haematopoietic development by skewing lineage commitment in vivo, resulting in neutrophilia, elevated circulating interleukin-6 and lethality due to severe gastrointestinal inflammation. The work presented in this thesis provides new important insights into the role of IKK in haematopoietic cells and haematopoietic development and clearly demonstrate that IKK1 and IKK2, the two catalytic subunits of the IKK complex, have distinct functions depending on the context of activation. In the future, this fact could be exploited to develop novel targeted therapies to specifically target a subunit in disease settings such as haematopoietic malignancies where aberrant NF-kappaB activity is frequently observed.
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ItemThe role of HECTD1 and MCL-1 in the regulation of normal and malignant haematopoiesis( 2019)Cellular processes important for haematopoiesis are frequently perturbed in malignant cells. Accordingly, healthy immature progenitor cells and malignant cancer cells often share certain properties, e.g. rapid proliferation and self-renewal. Deciphering these developmental pathways can provide information about the critical drivers involved in neoplastic transformation and sustained cancer cell growth. Results in this thesis addresses the role of two proteins, HECTD1 and MCL-1, in haematopoiesis and haematological malignancies. HECTD1 is an E3 ubiquitin ligase required for mouse embryonic development, as homozygous loss of Hectd1 leads to embryonic lethality. HECTD1 is widely expressed in diverse tissues, including haematopoietic cells. However, its role in adult tissues in vivo has not been described. Therefore, we generated mice in which HECTD1 deletion was restricted to the haematopoietic system of adult mice. Analysis of these mice at steady state revealed small perturbations in certain T cell subsets. However, competitive reconstitution experiments revealed that HECTD1 deletion affects the haematopoietic stem and progenitor cell (HSPC) populations. Serial transplantation assays showed that loss of HECTD1 results in a defect in the self-renewal properties of mouse HSPCs. Interestingly, RNA sequencing of Hectd1-/- HSPCs revealed that HECTD1-deficiency led to increased expression of interferon regulated genes, suggesting that HECTD1 plays a critical role in the maintenance of HSPC populations by negatively regulating the interferon signalling pathway. Additionally, I employed the MLL AF9 mouse model of acute myeloid leukaemia and showed that HECTD1-deficiency significantly delayed the latency of tumour development in vivo compared to control mice. MCL-1 is a pro-survival regulator of the intrinsic apoptosis pathway. MCL-1 expression is integral to the survival of many different blood cell types, and to the development and sustained growth of many haematological malignancies. Recently a highly specific MCL-1 inhibitor, S63845, showing 6-fold higher affinity to human MCL-1 compared to mouse MCL-1 was described. To accurately test the efficacy and tolerability of S63845 in preclinical models of disease, we developed a humanised Mcl 1 (huMcl-1) mouse strain in which the genomic region of the murine Mcl-1 locus was replaced with the coding regions for human MCL-1. These mice are phenotypically indistinguishable from wild-type mice, and the intrinsic apoptotic pathway remains intact in their cells. However, as anticipated, huMcl-1 mice were more sensitive to S63845 than wild-type mice. To test whether malignant cells from the humanised MCL-1 mice also show higher sensitivity to S63845, we generated Eµ-Myc lymphomas on a huMcl-1 background. Lymphoma cell lines derived from huMcl-1;Eµ-Myc mice were ~6 times more sensitive to S63845 in vitro compared to Eµ-Myc lymphoma cells expressing mouse MCL-1. Transplantation of huMcl-1;Eµ-Myc lymphoma cells into huMcl-1 mice and treatment with S63845 resulted in tumour-free survival in >60% of mice. Furthermore, combining low doses of S63845 with sub-optimal doses of cyclophosphamide led to almost complete tumour regression. These results show that our huMcl-1 mouse model represents a valuable preclinical tool to test MCL-1 inhibitors, either alone or in combination with other anti-cancer agents, for a broad range of cancers, allowing accurate prediction of efficacy against tumour cells and on target toxicity to normal tissues.
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ItemInvestigating early haematopoietic specification and platelet-forming lineage development in the mouse embryo( 2016)Embryonic haematopoiesis occurs in three distinct but temporally overlapping waves. Primitive haematopoiesis emerges in the extra-embryonic yolk sac and comprises short-lived primitive erythrocytes. The transient embryonic wave houses progenitors that have been shown in vitro to contain multi-potent potential. Lastly the haematopoietic stem cell-driven definitive wave emerges within the embryo from mid-gestation, giving rise to the full lineage hierarchy seen in the adult. As many studies have utilized in vitro assays to investigate the early waves of haematopoiesis, questions remain outstanding about whether the primitive wave contains greater lineage diversity in vivo than merely erythrocytes, and when and what lineages derive in vivo from second wave progenitors. Focusing on the platelet-forming cell (PFC) it has been observed that polyploid megakaryocyte (Mk) progenitors exist in the E7-8.5 yolk sac and that platelets can be detected within the embryonic circulation at E10.5, however any direct link between the two has not been demonstrated. By analysing PFC potential and in vivo lineage emergence, I discovered a novel primitive PFC lineage that is specified within the primitive haematopoietic wave in a progenitor-independent manner. This lineage was found to release the first embryonic platelets and did so with a diploid genome, and was therefore termed the diploid platelet-forming cell (DPFC). PFCs and platelets were quantified throughout gestation and were found to be dependent on key regulators of conventional Mks at a gene expression and functional level, including Gata1 and Nfe2. A notable exception to this was the THPO-MPL signalling axis, which was dispensable for the specification of both prenatal PFC lineages. Analyses of prenatal PFCs demonstrated a conserved in vitro proplatelet mechanism of platelet formation with adult PFCs. However subsequent studies revealed a novel thrombopoietic mechanism occurring in vivo that differed from conventional adult Mks. Similarly, a difference in the alignment of DPFC cell cycle position and platelet formation was observed in vitro and in vivo. These findings highlighted the distinctions between prenatal and adult PFCs, and contrasts in their behaviours in vitro and in vivo. Looking earlier in development, it has been proposed that the emergence of blood from the mesodermal precursor occurs via a haemangioblast or haemogenic endothelial intermediate. However most studies to date have focused on the in vitro differentiation potential of ES-derived or primary cells, which does not directly address the question. To investigate the pathway of DPFC lineage emergence a direct, high-resolution approach was taken utilizing single cell RNA sequencing on purified yolk sac cells.
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ItemDistinct precursors of the dendritic cell subtypes( 2006-03)Dendritic cells (DC) are antigen-presenting cells that are critical for the initiation and regulation of the immune response. Several DC subtypes within mouse spleen have previously been characterised and these include the plasmacytoid (pDC), and conventional DC (cDC) of the CD8+ and CD8- subtypes. Each subtype appears to have a specialised role in the various arms of immunity and tolerance. Less clear is the process by which these DC develop from haematopoietic precursors, of the precursor stages and branch points from bone marrow (BM) stem cells to each of the peripheral DC subtypes. The research described herein had the aim of identifying and isolating some of the intermediate precursors of DC, downstream of stem cells, and determining whether these differed in the steady-state versus inflammation. Particular was given to DC of the spleen. Experiments that sought the identity of such precursors involved both i) transfer of cell fractions that contained DC precursors into steady-state or inflamed recipient mice to assess their in vivo development at later times, and ii) analysis of an in vitro culture system to question whether it reflected development of the steady-state DC subtypes.
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ItemFunctional characterisation of Caspase-9 in haematopoiesis( 2012)Caspases are a family of cysteine-aspartic proteases that play essential roles in programmed cell death (apoptosis), programmed necrosis (necroptosis), and inflammation. This work aims to clarify additional reported functions of caspases, and to enhance our understanding of the functional roles of caspases in the blood (haematopoietic system). By genetically dissecting the apoptotic pathway, I show that caspase activation is not required for megakaryocytes to form platelets from their cytoplasm. Rather the opposite is true, apoptotic caspase activation must be restrained for megakaryocytes to survive and produce platelets. In addition, platelets are fully functional without the initiator Caspase-9. Caspase-9-deficient platelets maintain blood clotting (hemostasis), and are capable of facilitating thrombin generation via the exposure of membrane phospholipid phosphatidylserine – supporting the notion that platelet apoptosis and platelet activation are biochemically distinct processes. Herein, I also show that the Bcl-2 regulated caspase cascade is critical for haematopoietic stem cell maintenance. A novel relationship between apoptotic caspase activation and type-1 interferon production – a cytokine known to regulate ‘stem-ness’ – is established. Together, this research refines previously described biological functions for caspases, and provides new insight into the role of caspases in cell death and the physiological consequence of their genetic or pharmacological inhibition.
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ItemCritical roles for the transcription factor PU.1 in early lymphopoiesis in adult mice( 2013)PU.1 is a central regulator of foetal haematopoiesis. Conditional knockout of PU.1 in adult mice demonstrated disrupted myeloid and lymphoid developments. While this suggests a role of PU.1 in early haematopoietic progenitors, the progenitor stage at which PU.1 is important remain unknown. Moreover, the source of immature myeloid cells that contribute to the development of acute myeloid leukaemia (AML) in PU.1fl/flMxCre+ mice is still poorly defined. With the use of PU.1fl/-MxCre+RAG1+/gfp and PU.1fl/flRosa26CreERT2 mice that allows the inducible inactivation in stem cells and early lymphoid progenitors, I identify that PU.1 plays a critical role in lymphoidprimed multipotent progenitors (LMPPs), and deficiency in PU.1 results in a developmental block at the transition from LMPPs to common lymphoid progenitors. In addition, I demonstrate that the aberrant myeloid cells that eventually develop into AML originate from the PU.1 deficient Lin-Sca-1+c-Kit+ (LSK) cells upon transplantation. Results from PU.1fl/-RAG1cre mice suggest that PU.1 deficient LMPPs within the LSK cells are the most likely candidate that forms these abnormal cells. Gene expression studies using PU.1 deficient LSK cells revealed distinct difference in total transcriptomes when compared to the wild-type LSK controls. Earlier studies reported that specific deletion of PU.1 in B cells revealed its dispensable role in B cell development in the bone marrow. I demonstrate similar findings by using PU.1fl/-RAG1cre mice that delete PU.1 in an earlier stage of B cell progenitors. I further investigate the importance of PU.1 in interacting with IRF4 and IRF8. Deficiencies in both PU.1 and IRF4/8 revealed distinct consequences for B cell development. Strikingly, the pre-B cells from PU.1/IRF4 deficient mice are hyperproliferative to IL-7, and express elevated level of pre-BCR. Both PU.1 and IRF4/8 deficient mice develop pre-B acute lymphoblastic leukaemia, from which isolated cells can be maintained in culture supplemented with IL-7. I also demonstrate that there is a dose dependent pattern observed from a range of PU.1/IRF4 deficient mice in which lack of one copy of both would result in leukaemia. These leukaemic cells expressed low levels of Spib, Izkf1 (Ikaros) and Blnk, and by using ChIPseq, my collaborators and I show that Spib and Ikaros are transcriptionally activated by PU.1 and IRF4/8. Restoration of Spi-B and Ikaros expression in these leukaemic cells reduce their IL-7 dependent growth in culture. In conclusion, these findings demonstrate that PU.1 is functionally important in LMPPs, and together with IRF4/8, they are essential transcriptional regulators as well as tumour suppressors in early B cell development.
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ItemThe developmental pathways of splenic dendritic cells( 2011)Dendritic cells (DC) are professional antigen presenting cells, specialised in the activation of naïve T-cells. DC can be further subdivided into circulating plasmacytoid DC (pDC), and conventional DC (cDC), which are functionally distinct subsets. cDC can be further subdivided into peripheral cDC, and lymphoid tissue resident cDC. In this thesis, we focus on the developmental pathways of murine splenic DC – that is, resident cDC and pDC. DC potential has been found in multiple early precursors, and an immediate resident cDC precursor has been described in the spleen. Two precursors have been described in the bone marrow that may form the developmental bridge between the upstream precursors, and the committed pre-cDC. We and others have described common DC progenitors (CDP), which are c-kitintflt3+M-CSFR+, and negative for lineage antigens (lin-). CDP are restricted to differentiation into cDC and pDC. Another precursor, the macrophage-DC progenitor (MDP) has been described as an intermediate stage on the pathways to macrophage and DC development. The definition of a macrophage-DC progenitor includes potential for both macrophages and steady state cDC within single progenitors. In these studies, the ability of a single progenitor to give rise to both macrophages and steady state DC, and thus the existence of a macrophage DC progenitor, has not been established. Here, we have here investigated the developmental potential of the putative MDP populations. Contrary to previous data, we find that the populations defined in the literature as ‘MDP’ are not restricted to the macrophage and DC lineages, but rather retain potential for other haematopoietic lineages. To detect single progenitors with both macrophage and steady state DC potential, we have developed a clonal assay using M-CSF and flt3-ligand, the cytokines that drive macrophage and steady state DC development respectively. We find no evidence of a progenitor with both macrophage and DC potential within the progenitors previously described as ‘MDP’. To determine whether an MDP exists outside of the populations described as such in the literature, we have examined alternative fractions of the bone marrow for candidate ‘MDP’. We find a population within the lin-M-CSFR+CD16/32lowc-kitint/highsca-1-flt3+ BM fraction gives rise to macrophages and steady state cDC on a clonal level. However, this population is not restricted to these lineages. Thus, we find no evidence for a macrophage DC progenitor as a common intermediate on the pathways to macrophage and DC development. In addition, we have investigated the pathways to plasmacytoid DC development. We find that flt3 ligand (FL)-driven differentiation of pDC occurs via multiple developmental pathways. We have described an intermediate precursor on the pathway from development of common lymphoid progenitors to pDC. Furthermore, we have established the validity of using a history of expression of recombinase-activating gene 1 and the presence of D – J rearrangements at the immunoglobulin heavy chain locus as a marker of developmental history. We here demonstrate that the presence of these markers in pDC indicates a developmental history distinct from those pDC that lack these markers. The description of pDC in vivo both with and without a history of RAG1 expression, or with and without D – J rearrangements thus indicates the operation of multiple developmental routes to pDC in vivo. We have also investigated the role of external factors in the pathways of DC development. We find that FL plays an important role in directing multipotent cells into the DC lineage. We further show that the cytokines interleukin-10 and granulocyte-macrophage colony stimulating factor promote DC development. In this thesis, we have clarified the pathways to the development of the resident dendritic cell subsets, and described some of the factors involved in regulating these pathways. The elucidation of the pathways to steady state resident DC development will form the basis for understanding the regulation of these pathways, and the adaptation of these pathways under conditions of infection.