Medical Biology - Theses
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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 studies of dendritic cells in type 1 diabetes: non-obese diabetic (NOD) mouse model( 2012)Dendritic cells (DC) are highly efficient antigen presenting cells and are important in regulating immune defense and tolerance. Both conventional DC (cDC) and plasmacytoid DC (pDC) are found in lymphoid tissues, such as the thymus and spleen. cDC can be further segregated into two populations: the CD8α+Sirpα- (CD8+) and CD8α-Sirpα+ (CD8-) cDC. Thymic cDC play an important role in the negative selection of auto-reactive T cells and the induction of naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTregs). The splenic cDC, particularly the CD8+ cDC, can cross-present auto-antigen in steady state and delete auto-reactive T cells in peripheral tissues. In addition, pDC have also been shown to induce or regulate immune tolerance in various autoimmune disease models via cross talk with regulatory T cells, producing anti-inflammatory cytokines, including TGF-β, IL-10 or typtophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO) which can inhibit the activity of T cells. Human type 1 diabetes (T1D) mellitus is an autoimmune disease that results from the autoimmune destruction of β-cells in the pancreas. The non-obese diabetes (NOD) mouse is an important animal model for T1D and shares many pathological features with human T1D. Both genetic and environmental factors are believed to play an important role in T1D development. However, the mechanisms for disease initiatory and the loss of immune tolerance remain unclear. In addition, infectious pathogens such as Lymphocytic Choriomeningitis virus (LCMV), mycobacterial reagents (e.g. BCG, CFA) or CpG oligonucleotide can delay or prevent the onset of diabetes in NOD mice. Again, the mechanisms for these effects remain elusive. Until recently, no thorough studies have been performed on the phenotype and function of steady state DC subsets in autoimmune T1D mouse models. This has prompted us to investigate whether altered functions of DC subsets contribute to the break of immune tolerance in autoimmune mouse models. In this study, characteristics of steady state DC subsets in the lymphoid organs of NOD mice were determined and compared with that of the diabetes resistant control mice NOR, Idd11 congenic mice (C57BL/6 x NOD intercross mice which have similar genetic background as NOD mice, but have a difference diabetes incidence level), as well as C57BL/6 mice. In addition, the frequency and the number of nTregs in NOD mice were also analyzed. A reduced percentage of splenic CD8+ cDC and an increased number of pDC in the thymus and spleen were found in NOD mice. Data showed that a CD8+ cDC deficiency, which is associated with diabetes onset in NOD mice, maps to the Idd11 locus. Further analysis of alterations in DC function and their potential contribution to the impairment of immune tolerance in NOD mice was carried out. A novel auto-antigen cross-presentation assay using islet-specific glucose-6-phosphate catalytic subunit-related protein (IGRP) was established to test the cross-presentation capacity of NOD DC. The results demonstrated a comparable cross-presentation ability between NOD and NOR cDC on a per cell basis. However, a lower capacity to cross-present cell associated auto-antigen in the spleen was found in NOD mice. Moreover, an elegant protocol, in which cells with the ability to cross-present can be deleted by horse cytochrome c (cyt c) treatment, was employed for the detection of cross-presenting cells. Results showed that the cells in NOD mice were insensitive to cyt c treatment, implying that the reduced percentage of the CD8+ cDC in NOD spleen was most likely due to the absence of the cDC subset with cross-presentation potential. These observations suggested that the reduced number of cross-presenting CD8+ cDC in NOD mice might result in a deficiency in maintaining peripheral immune tolerance with an accumulation of auto-reactive T cells in the peripheral, which could lead to the development of diabetes in NOD mice. pDC is a subset of DC that can protect the host from viral infection through the production of large amounts of type-1 interferon, including IFN-α, IFN-β etc., to inhibit the replication of viruses. The results from this study showed that pDC in NOD mice produced higher levels of IFN-α and IL-3 upon TLR ligand stimulation. Subsequent analysis showed that high dose of IFN-α could inhibit the proliferation of pathogenic CD8+ T cells. In addition, IL-3 could promote the development of CD103+ cDC with the ability to cross-present auto-antigens for the deletion of auto-reactive T cells in the peripheral tissues. These observations may help to explain the findings, which showed that infection by microbial agents could delay or prevent the onset of diabetes in NOD mice. Moreover, this study also revealed a reduction in the number of T regulatory cells in pancreatic lymph nodes, but not in the thymus and spleen of NOD mice. However, the ratio of Foxp3+ Tregs to CD4+ T cells was lower in thymus, spleen as well as in pancreatic lymph nodes of NOD mice. These findings suggests that the reduced ratio of Tregs to CD4+ T cells, rather than reducing absolute number of Tregs may result in less efficient control of auto-reactive T cells, and these may contribute to the development of diabetes in NOD mice. Overall, this study thoroughly examined and compared the phenotype, the number and the function of individual resident DC subsets in the thymus and spleen of NOD, NOR and Idd11 congenic mice. This study provides novel evidence for the importance of cross-presenting CD8+ cDC in the maintenance of peripheral immune tolerance. In addition, the distinct capacities of NOD pDC to produce IL-3 and much higher level of IFN-α in response to TLR activation were first demonstrated in this study. The effects of these two cytokines on T cells and DC function further suggested a potential mechanism for pDC in the prevention of T1D upon microbial stimulation. Further investigation of DC biology in T1D patients will be crucial for developing an effective approach for T1D immunotherapy.
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ItemFunctional characterisation of Bcl-G( 2011)Apoptosis is a controlled and precise form of cell death necessary for maintaining tissue homeostasis and normal development. Perturbations of this cell death process contribute to a wide range of diseases such as cancer and autoimmunity. The Bcl-2 family regulates the intrinsic pathway of apoptosis and members share homology at one or more of the four different Bcl-2 Homology (BH) domains. They can be subdivided into the pro-survival proteins, the multi-domain pro-apoptotic proteins and the BH3-only proteins. The physiological functions and mechanism of action of the main players of the Bcl-2-regulated apoptotic pathway have been studied extensively using mouse models. However, the roles played by less prominent Bcl-2 family members are not as well characterised. In this thesis, we described the characterisation of Bcl-G, an evolutionarily conserved novel Bcl-2 family member implicated in cancer. Human BCL-G produces two major isoforms, BCL-GL and BCL-GS. BCL-GS only contains a BH3 domain while BCL-GL also contains a BH2 domain. While BCL-GL exhibited little killing ability, in vitro over-expression studies suggested that BCL-GS kills cells by binding BCL-XL. The mouse Bcl-G gene only produces one isoform, which contains both the BH2 and BH3 domains. Little is known about its functions and roles in apoptosis. To characterise the tissue distribution and subcellular localisation of mBcl-G, we produced monoclonal antibodies specific for this protein. Bcl-G was found to be predominantly cytoplasmic when over-expressed in HeLa cells and is present in a wide range of mouse tissues including spleen, thymus, lung, intestine and testis. Anti-Bcl-G immunohistochemistry revealed that it is expressed highly by some dendritic cell (DC) subtypes and certain epithelial cell types including those lining the gastrointestinal tract. To study the functions of mBcl-G and gain clues on its human orthologue, I generated and characterised Bcl-G-deficient mice. Bcl-G-1- mice were born viable, developed normally and had similar numbers of DCs and other immune cell subtypes when compared to their wild-type counterparts. Notably, Bcl-G-deficient cells were normally sensitive to the range of apoptotic stimuli tested. I also analysed the gastrointestinal tract of the Bcl-G-1- mice but did not observe any abnormalities in the gross morphology of the stomach, small intestine and colon. However, Bcl-G loss attenuated the colitis response to acute dextran sodium sulphate (DSS) administration, resulting in reduced immune infiltration and destruction of crypt architecture. The role mBcl-G plays in DSS-induced colitis is unclear and currently under investigation. To gain clues on the processes in which Bcl-G is involved in, I conducted a search for binding partners using two different approaches: co-immunoprecipitation coupled with mass spectrometric analysis (IP-MS) and yeast two-hybrid screening. Importantly, no Bcl-2 family member, whether it is pro-survival or pro-apoptotic, was pulled-down in either screen. In fact, in vitro over-expression studies revealed that Bcl-G’s BH3 domain was incapable of binding Bcl-XL and activating apoptosis when placed in the context of BH3-only protein Bim. These results raise doubts that mBcl-G interacts with the pro-survival proteins and is involved in the Bcl-2-regulated apoptotic pathway. Instead, Trappc6b, a subunit of the Trafficking protein particle (Trapp) complex, was identified as a potential Bcl-G binding partner. Mammalian Trapp is a multi-subunit protein complex involved in intracellular membrane traffic. This finding proposes a novel role of this Bcl-2 family member in protein trafficking and vesicle transport. In a second project, I have studied how BH3-only members activate the essential effectors Bax and Bak. Two models have been proposed, but the issue remains controversial. The indirect activation model suggests that BH3-only proteins simply neutralize all of the pro-survival proteins, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak by direct binding. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of pro-survival relatives but lost interaction with Bax. Analysis of these mice showed that Bim's pro-apoptotic activity is not solely determined by its ability to engage its pro-survival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.
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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.