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

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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.