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

在淋巴细胞发育过程中，V（D）J元件的置换是产生多种T细胞（TCR）和免疫球蛋白（IG）受体的关键过程。扩增激活基因RAG 1和RAG 2通过靶向特定的信号序列切割相邻的DNA来启动这一过程。随后，DNA末端被加工并通过非同源末端连接（NHEJ）途径的因子连接。尽管是短暂的，但RAG诱导的DNA双链断裂启动了涉及各种DNA修复蛋白的DNA损伤反应（DDR），其影响典型DNA修复之外的细胞过程的转录调控。在小鼠模型中有证据表明，RAG蛋白已经在早期淋巴祖细胞中表达，并且还在发育中的NK细胞中靶向序列，对成熟、细胞毒性功能和细胞适应性具有重要的DDR相关作用。具有RAG缺陷的患者的NK细胞更不成熟且反应性更高。我们设计了含有RAG-fate作图报告基因的诱导多能干细胞（iPSC）系，并将其分化为NK细胞，以鉴定人NK细胞发育中的RAG-cleavage，并确定其在NK细胞成熟和细胞毒性功能中的作用。本项目的进一步目标是研究NK细胞在个体发育中有无RAG裂解的差异基因表达和途径调控。更好地了解不同NK细胞群体的发展和潜在的工程可以帮助改善基于NK细胞的免疫疗法。

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.