Approaches to analyses of cytotoxic cells. And studies of their role in H. pylori infection

Abstract: Helicobacter pylori infection causes chronic gastritis that may progress to peptic ulcers or gastric adenocarcinoma and thereby cause major world-wide health problems. Previous studies have shown that CD4+ T cells and the production of the cytokine IFN- × are important components of the immune response to H. pylori in humans. However, the roles of NK cells and CD8+ T cells ¡V which are major IFN- × producers and have cytotoxic function - are less clear.The aim of this thesis was to develop methods to study NK cells and CD8+ T cells in the context of human H. pylori infection, and to study their role in the immune response toH. pylori in infected subjects.To this end, we first evaluated ways to activate NK cells by H. pylori antigens. This was done by purifying NK cells from peripheral blood, and stimulating the cells by combinations of stimulatory cytokines such as IL-12 and lysate from H. pylori. Furthermore, parallel experiments were performed when the NK cells were separated from the bacterial antigens by an epithelial cell layer. To develop methods to activate and study H. pylori-reactive CD8+ T cells, we initially evaluated whether dendritic cells (DC), B cells or monocytes pulsed with H. pylori antigens could efficiently activate CD8+ T cells from H. pylori infected or non-infected individuals. In order to study proliferation of CD8+ T cells in cell cultures, the cells were stained using the fluorescent dye CFSE, which allows analysis of proliferation of single cells in a mixed cell population. Furthermore, the presence of cytotoxic activity among the H. pylori-reactive CD8+ T cells was analysed mainly by the cytotoxicity-related molecules granzyme A and B, and IFN- ×. This was done using intracellular analysis of granzyme B and IFN- × expression, and by analysing the secretion of granzyme A and B into the supernatants by H. pylori-activated CD8+ T cells.Our results show that highly purified NK cells can be activated by H. pylori antigens, and that there is a synergistic effect of H. pylori and IL-12 in the activation of NK cells. Furthermore, we show that in H. pylori-infected individuals, there are H. pylori-reactive memory CD8+ T cells that proliferate, produce IFN- × and secrete granzyme A after activation. We show that these cells can be activated both by B cells and DC pulsed with H. pylori antigens, but for practical purposes, B cells are preferable to use as APC.In conclusion, in this thesis we show that cytotoxic cells may contribute to the immunity against H. pylori in infected individuals by production of IFN- ×; but there are also indications that cytotoxic activity is involved. These findings may be of importance for the further study of NK-cell and cytotoxic T lymphocytes (CTLs) activity in subgroups of H. pylori-infected individuals, especially in relation to protection against H. pylori-induced gastric cancer development.

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