During viral infections, CD8+ T cells only respond to a select few epitopes derived from the respective foreign pathogen. These epitopes can be organized into a hierarchy, based on their ability to induce T cell priming. Such phenomenon is known as immunodominance. Cytotoxic T cells can be primed through the direct pathway, or the cross-priming pathway. The latter involves exogenously derived viral epitope presentation by uninfected professional antigen presenting cells. It has been previously reported that Lymphocytic Choriomeningitis nucleoprotein expressed in HEK cells (HEK-NP) could be cross presented to CD8+ T cells. In these studies we have used this same HEK-NP model to study the effects of LCMV-NP cross priming on the LCMV immunodominance hierarchy following viral challenge. Our results provide strong evidence that cross priming is an efficient route with which to induce cell-mediated immunity. We also highlight a regulatory role for cross priming in immunodominance by showing that a single dose of HEK-NP can completely shift the immunodominance hierarchy of a typical LCMV infection. Furthermore, we see that the induction of LCMV-NP cross priming boosts anti-viral immunity to subsequent LCMV infections. This work provides strong support for the physiological role that cross priming plays in normal cell-mediated immune responses. It may also provide relevant information to the realm of immunotherapy. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-10 14:33:18.115

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-08-29 14:53:18.766

γδ T cells belong to the T cell lineage however they possess some innate like properties. γδ T cells recognize non-peptidic microbial and stress induced self antigens in a non-MHC restricted manner and are proposed to bridge the gap between innate and adaptive immunity. Dendritic epidermal T cells are a prototypic population of intraepithelial γδ T cells in murine skin. Found in the basal layer of epidermis in close contact with Langerhans cells and keratinocytes DETC facilitate vital immunological and physiological processes e.g. wound healing, homeostasis, tumour surveillance and regulation of inflammation. The purpose of this thesis was to elucidate whether γδ T cells and in particular DETC play a role in generation of adaptive immune responses to foreign cutaneous antigen (OVA) in the context of skin grafts. Skin grafting has long been established as a means to test cutaneous and epithelial immunity. To answer this question, γδ T cell knock-out mice (TCRδ-/-), transgenic K5mOva mice and a skin grafting model were used. It is shown in this study that in the absence of γδ in the skin and not in the circulation there is a lower rejection rate of OVA expressing skin grafts. This phenomenon is observed in both freshly placed and well healed grafts. To understand which part of the immune response is affected by the absence of γδ T cells the priming and effector phases of the immune response was examined in TCRδ-/- mice. The priming phase was studied using two approaches: the first approach was to test priming to maximal doses of subcutaneous antigen in conjunction with an adjuvant and the second approach involved testing priming to an antigen in the context of skin grafts (graft priming). Using ELISPOTs and CFSE proliferation assays we found that while administration of OVA in conjunction with an adjuvant (QuilA) via the subcutaneous route results in sufficient priming in γδ T cell knockout mice, cross priming to OVA in the context of - freshly placed and well healed skin grafts is impaired in TCRδ-/- mice. By immunizing TCRδ-/- mice prior to skin grafting or by transferring in vitro primed OT-I cells to RAG-/- mice grafted with K5mOVA or TCRδ-/-OVA skin it was shown that 100% of all OVA grafts are rejected regardless of presence or absence of γδ T cells, concluding that effector phase of the immune response is not affected in this model. The inability of DETC to perform the role of cross presentation leads to the hypothesis that DETC indirectly enhance this process by affecting professional antigen presenting cells (APC) of the skin. Based on the contribution of DETC to wound healing it was hypothesized that the migration of dendritic cells (DCs) from the skin grafts to the lymph nodes may be affected. When this hypothesis was tested using hapten sensitization and congenically marked skin grafts it was shown that migration of DCs from skin grafts is not affected by the absence of DETC. In another hypothesis the co-stimulatory markers CD40 and CD86 were examined on migrating DCs found in the skin draining lymph nodes of grafted mice and it was shown that expression levels of those molecules were lower on DCs from TCRδ-/- grafted mice compared to C57BL/6 control mice. In addition using cytometric bead array, we show that the cytokine milieu in TCRδ-/- skin and skin draining lymph nodes is different from that of wildtype C57 skin and this disparate cytokine profile may be contributing to the less efficient cross priming and graft rejection in TCRδ-/- mice.