Phenotypic and functional charakterization of chicken natural killer cells

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In recent years progress has been made in the characterization of NK cells and their receptors. In the chicken we made several mak that recognize NK cells in embryonic spleen, intestine and blood. NK cells can be especially detected in frequent number in the intestinal epithelial layer (IEL). According to the expression of an Fc receptor, they can be further divided into two subpopulations. The most important markers of chicken NK cells are the 28-4 mab directed against CD25, the 8D12 mab recognizing the Fc receptor CHIR-AB1, as well as two mab (20E5 and 1G7) with unknown target antigens. In the proposed project, various aspects of NK cell characterization will be pursued. There is still a paucity of appropriate NK cell markers. Therefore, novel mab against receptors such as CD244, CD226, CRTAM and NCR homologues will be generated and characterized. With the help of these novel mab it may be possible to identify new subpopulations or functional forms of NK cells such as activation markers. In splenocyte preparations, NK like activity is measurable and is enhanced after IL-2 preincubation, however, a NK cell population cannot be detected with the markers available. The phenotype of the responsible cell population will be analyzed by depletion studies of several cell populations and successive cytotoxicity assays. We hypothesize that the cellular population in spleen responsible for the cytotoxicity are gd T cells with NK like functions. The IEL NK cells have not been intensively studied to date, although their frequency is much higher as compared to other tissues. Two subprojects will therefore characterize these cells. In one subproject the distribution and the phenotype of NK cells in various gut regions will be analyzed as well as the response of isolated NK cells towards stimulation with the cytokines IL-2, IL-12, IL-15, IL-18 and IL-12 with regard to their proliferation, cytotoxicity, phenotypic changes and cytokine production. In the second subproject, a microarray analysis will be performed including the two IEL NK cell subpopulations as well as the gd T cell population. These studies should reveal functional features of IEL NK cells as compared to T cells. Finally, an IL-2 transgene in the retroviral RCAS vector will be generated. This system allows a longterm IL-2 overexpression in chickens. The effects of the IL-2 expression on various cell types with particular emphasis to the cytotoxic splenocytes and the intestinal NK cells will be studied. In conclusion, the proposed experiments will generate important novel reagents for the NK cell characterization and they will enhance our knowledge regarding chicken NK cells, with particular emphasis to the intestinal NK cells.