The Role of Unconventional T Cells in the Immune Response to the Gastric Pathogen Helicobacter pylori
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Helicobacter pylori (HP) is a gastric pathogen that is a known causative agent of gastritis, and peptic ulcers. It is a co-factor for the onset of gastric cancer and for these reasons is classified as a type I carcinogen. HP infection elicits an inflammatory Th1 driven immune response, during which, classical T cells have been considered the primary orchestrators, although this action is rarely effective in clearing infection. Ultimately, the aim of studies in this thesis was to assess the importance of previously overlooked unconventional T cell populations through examination of their immunological responses in the gastric mucosa to HP and to HP derived antigens (Ags). Firstly, using flow cytometry we phenotypically characterised lymphocyte populations paying particular attention to unconventional T cell subsets [natural killer T cells (NKT cells) and gamma delta+ T cells] in the human gastric epithelium (EP) and lamina propria (LP) layers and compared their frequency in HP infection. Results showed that CD4+ and CD8+ classical T cell numbers were significantly higher and lower respectively in the LP of H. pylori infected individuals. Moreover, a significant reduction in the percentage of CD56+ NK cells was found in the LP of H. pylori positive subjects compared to control samples. The numbers of NKT cells were low in both the EP and LP of control subjects and the frequencies were unchanged in HP infected subjects. Furthermore, when gamma delta+ T cells were investigated, numbers were markedly higher in the H. pylori infected EP and not in the LP. In the next experiments, we investigated immunological responses to H. pylori antigenic stimulation through proliferation, cytokine and cytotoxicity assays. Here, a variety of HP derived Ags were used to stimulate PBMCs, single cell suspensions derived from the EP, LP or NKT cell clones. The cytokine response of gamma delta+ T cells to HP derived Ags was also investigated using gamma delta+ T cell clones. The HP derived antigenic stimuli included whole cell extract, cytoplasmic Ags, crude membrane Ags, outer membrane proteins, inner membrane proteins from strains 26695 and J99 and LPS derived from strains NCTC11637 and CCUG17874. A lack of proliferation as measured by the BrdU ELISA was observed in response to many of the HP derived Ags used following antigenic stimulation of NKT cells, PBMCs or cell suspensions from the gastric EP and LP layers. In cytokine studies, using the Th1/Th2 11plex FlowCytomix kit, small increases in cytokines (IL-8, IFN-gamma and others) were observed following stimulation of NKT cells with some of the HP derived Ags (whole cell extract from HP 26695 and others) while results using PBMCs or gastric lymphocyte populations were variable. In these experiments, when the gamma delta+ T cell clones were stimulated with the HP derived Ags, higher levels of IFN-gamma, IL-2, IL-4, IL-5, IL-8, IL-12p70, IL-1beta, TNF-alpha and TNF-beta were observed consistent with a Th1 and Th2 cytokine pattern. Furthermore, using the LDH assay, a lack of cytotoxic responses was seen following NKT cell stimulation with the HP derived Ags. In the final chapter, gene expression profiling of RNA isolated from gastric EP and LP derived CD2+ cells from HP infected and uninfected gastric mucosa was undertaken using the Affymetrix system in conjunction with the Karolinska Institute in Sweden. The findings from these studies generated a large database of novel genes differentially expressed in HP infection in key immunological areas such as cytotoxicity, cancer progression, inflammation, cytokine production and others. Moreover, we categorised the top 20 most significantly up-regulated and down-regulated genes in the EP and the LP in HP infection relative to controls. Together, this work contributed to gaining further knowledge about HP pathogenesis and immunity and gave important insights into the role of unconventional T cells in HP infection.
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