published_or_final_version / Medicine / Master / Master of Philosophy

No description available.

No description available.

T helper type--2 (Th2) immunity orchestrates responses against extracellular pathogens under normal conditions and mediates pathogenic responses against innocuous substances when dysregulated, leading to allergic disease. Among the cytokines expressed by Th2 cells, interleukin (IL)--13 has emerged as a critical effector molecule in Th2 responses and common IL13 variants are associated with allergy--related phenotypes in populations of distinct ethnic background. IL13 expression in human T cells is paralleled by extensive IL13 locus remodeling, which results in the appearance of multiple DNase I hypersensitive (HS) sites. Among these, HS4 in the distal IL13 promoter is constitutively present in both naive and polarized Th2 cells, and spans a single nucleotide polymorphism, IL13--1512AC (rs1881457), which is common and strongly associated with total serum IgE levels. This dissertation combines in vitro and ex vivo approaches to characterize the role of HS4 in the regulation of IL13 gene expression and to provide novel insights into the mechanisms that underlie the association between IL13--1512AC and allergic disease.The results showed that HS4 acts as a novel cis--acting element that up--regulates IL13 transcription in activated Th2 cells. The enhancing activity of HS4 mapped within the 3' end of this element and was dependent on binding/recruitment of the transcription factors NF90 and NF45. Moreover, the IL13--1512C risk allele significantly enhanced HS4--dependent IL13 expression by creating a binding site for the transcription factor Oct--1. The increased expression of the --1512C allele was dependent on endogenous levels of Oct--1. Collectively, these results illustrate how a functional variant in an important regulatory element may modulate susceptibility to a common complex disease.

No description available.

[6]-Gingerol, [8]-gingerol, and [10]-gingerol are phytochemical extracts from ginger that are thought to contribute to its health-benefitting properties. The objectives of this investigation were to explore and compare the in vitro anti-proliferative and cytotoxic effects of [6]-, [8]-, and [10]-gingerol on human and mouse mammary carcinoma cells, as well as their ability to inhibit T cell proliferation. [8]-Gingerol and [10]-gingerol induced mammary carcinoma cell death that did not require ROS production or caspase activation. All three gingerols inhibited the proliferation of mammary carcinoma cells and T cells, and the production of IFN-? by T cells. The production of IL-2 and the expression of early T cell activation markers, CD25 and CD69, were significantly decreased by [8]- and [10]-gingerol. The results demonstrated that [10]-gingerol was the most potent, followed by [8]-gingerol, then [6]-gingerol. Consequently, [8]- and [10]-gingerol warrant further investigation for the treatment of breast cancer and the control of inflammation.

No description available.

Access to abstract restricted until May 2016 / Access to thesis restricted until May 2016 / Department of Biology

Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

T cells are a crucial component of the immune system and play an important role in responses to pathogens, tumours, and transplanted tissues. In many human cancers, elevated numbers of tumour-infiltrating CD8+ killer T cells are associated with favourable outcomes, suggesting that enhancing T-cell responses could provide major therapeutic benefit for cancer patients. Thus, identifying factors that can promote protective T-cell responses is of great clinical importance. The cytokine interleukin-2 (IL-2) is a major inducer of T-cell proliferation and differentiation, and is used clinically to treat melanoma and renal cell carcinoma. The first two chapters of this thesis focus on the biochemical mechanisms by which IL-2 induces T-cell proliferation. By using mutant and chimeric cytokine receptors expressed in lymphocyte cell lines, the interplay between Shc and STAT5, two major mitogenic signaling pathways activated by the IL-2 receptor, are investigated, revealing an essential synergy between the two pathways for optimal lymphocyte proliferation. The third chapter of this thesis describes work done to identify cytokines that promote T-cell responses within the ovarian cancer microenvironment. In human diseases such as HIV/AIDS and cancer, high numbers of “polyfunctional” T cells (i.e., T cells capable of multiple effector functions) are associated with favourable outcomes. Using clinical ovarian cancer samples in a novel ex vivo assay, it was found that the ovarian tumour environment inhibits polyfunctional T-cell responses to varying extents among patients. After surveying a large panel of cytokines, the cytokine combination of IL-2, IL-12, and IL-18 was found to overcome the immunosuppressive environment to potently enhance CD8+ T-cell proliferation and polyfunctionality in all patient samples. The polyfunctional profiles induced by these cytokines are associated with protective immunity in various human conditions. Thus, these findings suggest that given the right signals, T cells can become highly polyfunctional effectors in the ovarian cancer microenvironment, which offers promise for the development of effective T-cell based therapies for this clinically challenging disease. / Graduate / 0982