Doherty Institute - Theses
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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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ItemThe role of inflammatory stimuli in dendritic cell biology( 2017)Dendritic cells (DC) are professional antigen-presenting cells (APC) that capture and present processed protein antigens to T cells when they are immature and mature, respectively. Infection-induced inflammation drives DC activation by two pathways, termed direct and indirect activation. Direct DC activation occurs when DCs “directly” encounter pathogen- or danger-associated molecular patterns (PAMPs or DAMPs), while “indirect” DC activation occurs when DCs encounter secondary inflammatory signals. Direct activation of DC leads to increased expression of major histocompatibility complex class II (MHCII) and co-stimulator molecules such as CD86, as well as the secretion of cytokines. In contrast, while indirect activation of DC elicits a similar expression level of MHCII and co-stimulator molecules, these cells have impaired cytokine production. However, due to the lack of effective markers to discriminate these two DC populations, studies on the characteristics and immunological roles of the two DC populations are limited. In this thesis, we aim to: i) identify markers that discriminate directly and indirectly activated DCs via next-generation RNA sequencing; and ii) assess the role of directly and indirectly activated DCs in vivo under inflammatory conditions. The major findings are as follows: i) CD38 and CD103 are highly expressed in indirectly activated DCs, while CD205 is elevated in directly activated DCs, and these CD molecules could be used as markers to partially distinguish directly activated DCs from indirectly activated DCs; ii) directly and indirectly activated DCs prime CD4 T cell proliferation at a comparable quantity, but with different effector T cell polarisations; and iii) directly activated DCs induce effector memory CD4 T cells upon a secondary challenge, while indirectly activated DCs preferentially induce central memory CD4 T cells. Based on this study, we can describe the specific phenotypes of directly versus indirectly activated DCs, we were able to assess the immune-functional properties of indirectly activated DCs, and assess the formation of directly and indirectly activated DCs in vivo under both steady-state and inflammatory conditions. The discovery of such murine markers to discriminate these DCs may have direct homologs within humans, which would allow for investigation into these differently activated DC in clinical samples. The results will assist rational designing of vaccines to maximise the immune response and strategies to minimise autoimmunity.