Formation of human NK cell repertoires: role of HLA class I and KIR gene polymorphism

自然杀伤（NK）细胞在肿瘤监测过程中，在病毒感染的早期控制和恶性细胞的根除中发挥重要作用。在人类中，KIR和NKG2基因家族的HLA I类特异性NK受体在这一过程中起着至关重要的作用。这些受体以不同的组合在NK细胞上表达，并决定针对HLA I类缺陷靶细胞的特异性。目前尚不清楚NK细胞库的形成主要是受干细胞生态位和HLA配体的局部表达的外源性影响，还是受遗传因素如多态性KIR基因的内源性影响。在本项目中，这些问题将在几个体外模型的NK细胞分化。为此，将早期造血祖细胞与HLA I类转染的鼠基质细胞共培养并分化为成熟NK细胞。或者，间充质干细胞（MSC）将用作辅助细胞，使得能够在人干细胞龛中遵循NK细胞分化过程。为了更仔细地剖析受体获得的过程，NKG2A和选定的KIR基因将被RNAi敲低，并在造血祖细胞阶段稳定过表达。 作为替代干细胞来源，我们将采用诱导多能干细胞（iPS）用于NK细胞分化。将使用一组NKG2A和KIR特异性抗体以及功能标志物，通过流式细胞术分析，在克隆基础上分析NK库的形成和效应子功能的获得。这些实验应该有助于了解在多大程度上的程序NK库的形成是硬连线，或者如果相反KIR配体和干细胞生态位作为主要的外源性决定因素影响这一过程。重要的是，这些结果可以作为一个模型，以更好地了解造血干细胞移植后的NK细胞重建，并可能为未来采用特异性体外生成和扩增的NK细胞的免疫学应用铺平道路。

Natural killer (NK) cells serve an important function in the early control of virus infection and the eradication of malignant cells in the course of tumor surveillance. In humans, HLA class I-specific NK receptors of the KIR and NKG2 gene families play a crucial role in this process. These receptors are expressed on NK cells in different combinations and determine the specificity against HLA class I-deficient target cells. It is currently unclear if the formation of NK cell repertoires is primarily exogenously influenced by the stem cell niche and the local expression of HLA ligands or endogenously by genetic factors such as the polymorphic KIR genes. In the present project these questions will be addressed in several in vitro models of NK cell differentiation. To this end, early hematopoietic progenitors will be co-cultured with HLA class I-transfected murine stroma cells and differentiated to mature NK cells. Alternatively mesenchymal stem cells (MSC) will be employed as accessory cells enabling to follow the NK cell differentiation process in a human stem cell niche. To dissect the process of receptor acquisition more closely, NKG2A and selected KIR genes will be knocked down by RNAi as well as stably overexpressed at the hematopoietic progenitor stage. As an alternative stem cell source, we will employ induced pluripotent stem (iPS) cells for NK cell differentiation. The formation of NK repertoires and the acquisition of effector function will be analyzed on a clonal basis by multicolor flow cytometry analysis, using a panel of NKG2A and KIR-specific antibodies as well as functional markers. These experiments should help to understand in how far the program of NK repertoire formation is hard-wired or if rather KIR ligands and the stem cell niche as major exogenous determinants influence this process. Importantly, the results could serve as a model to better understand NK cell reconstitution following hematopoietic stem cell transplantation and might pave the way for future immunotherapeutic applications employing specific in vitro generated and expanded NK cells.