Medical Biology

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.

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.

Medical Biology - Theses

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