Phenotypic and functional charakterization of chicken natural killer cells

近年来，对NK细胞及其受体的研究取得了一定的进展。在鸡的体内，我们制备了几种识别胚胎脾、肠和血液中的NK细胞的MAK。特别是在肠上皮层(IEL)中，NK细胞数量最多。根据Fc受体的表达，它们可以进一步分为两个亚群。鸡NK细胞最重要的标志是针对CD25的28-4单抗、识别Fc受体CHIR-AB1的8D12单抗以及靶抗原未知的两种单抗(20E5和1G7)。在拟议的项目中，将研究NK细胞特征的各个方面。目前尚缺乏合适的NK细胞标记物。因此，针对CD244、CD226、CRTAM和NCR同系物等受体的新型单抗将被开发出来并进行鉴定。在这些新的单抗的帮助下，有可能确定新的NK细胞亚群或功能形式，如激活标记。在脾细胞制备中，NK样活性是可测量的，并在IL-2预孵育后增强，然而，利用现有的标志物无法检测到NK细胞群。负责细胞群体的表型将通过对几个细胞群体的耗竭研究和连续的细胞毒性分析来分析。我们推测，脾中负责细胞毒作用的细胞群是具有NK样功能的gd T细胞。尽管与其他组织相比，IEL NK细胞的频率要高得多，但到目前为止还没有得到深入的研究。因此，两个分项目将描述这些单元的特点。在一个子项目中，将分析NK细胞在不同肠道区域的分布和表型，以及分离的NK细胞对细胞因子IL-2、IL-12、IL-15、IL-18和IL-12的增殖、细胞毒性、表型变化和细胞因子产生的反应。在第二个子项目中，将进行微阵列分析，包括两个IEL NK细胞亚群以及gd T细胞群体。这些研究应该揭示IEL NK细胞与T细胞相比的功能特征。最后，在逆转录病毒RCAS载体中产生IL-2转基因。这个系统允许IL-2在鸡中长期过度表达。IL-2的表达对各种细胞类型的影响，特别是对细胞毒脾细胞和肠道NK细胞的影响将被研究。总之，拟议的实验将产生重要的NK细胞表征的新试剂，它们将增进我们对鸡NK细胞的了解，特别是对肠道NK细胞的了解。

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.