The role of RAG-induced DNA damage response in iPSC-derived NK cell development

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Recombination of V(D)J elements is a crucial process for generation of diversified T cell (TCR) and immunoglobulin (Ig) receptors in lymphocyte development. Recombination activating genes RAG1 and RAG2 initiate this process by targeting specific signal sequences to cleave adjacent DNA. Subsequently, DNA ends are processed and joined by factors of the non-homologous end-joining (NHEJ) pathway. Although transient, RAG-induced DNA double strand breaks launch a DNA damage response (DDR) involving various DNA repair proteins, which impact on transcriptional regulation for cellular-processes beyond canonical DNA repair. There is evidence in mouse models that RAG proteins are already expressed in early lymphoid progenitors and also target sequences in developing NK cells with important DDR-related effects on maturation, cytotoxic function and cellular fitness. NK cells of patients with RAG-deficiency are more immature and hyperresponsive. We engineered induced pluripotent stem cell (iPSC) lines containing a RAG-fate mapping reporter and differentiate them into NK cells to identify RAG-cleavage in human NK cell development and to define its role in NK cell maturation and cytotoxic function. Further aims of this project are to investigate differential gene expression and pathway regulations in NK cells with and without RAG-cleavage in their ontogeny. A better insight into the development of different NK cell populations and potential engineering can help improving NK cell-based immunotherapies.