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

RAG 诱导的 DNA 损伤反应在 iPSC 衍生的 NK 细胞发育中的作用

• 批准号: 328922204
• 负责人: Privatdozentin Dr. Kerstin Felgentreff
• 金额: --
• 依托单位: Universitätsklinik für Kinder- und Jugendmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/328922204?language=en
• 关键词: role; RAG; induced; DNA; damage

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.

V(D)J元件的重组是淋巴细胞发育过程中产生多种T细胞(TCR)和免疫球蛋白(Ig)受体的关键过程。重组激活基因RAG1和RAG2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末端被非同源末端连接(NHEJ)途径的因子加工和连接。尽管RAG诱导的DNA双链断裂是短暂的，但它会引发一种DNA损伤反应(DDR)，涉及多种DNA修复蛋白，从而影响对细胞过程的转录调控，而不是常规的DNA修复。在小鼠模型中，有证据表明RAG蛋白已经在早期的淋巴祖细胞中表达，也在发育中的NK细胞中表达靶序列，对成熟、细胞毒功能和细胞适合性具有重要的DDR相关作用。RAG缺乏患者的NK细胞更不成熟，更具高反应性。我们设计了含有RAG-FATE作图报告的诱导多能干细胞(IPSC)系，并将其分化为NK细胞，以鉴定RAG-裂解在人NK细胞发育中的作用，并确定其在NK细胞成熟和细胞毒功能中的作用。这个项目的另一个目的是研究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节。更好地了解不同NK细胞群体的发展和潜在的工程有助于改进基于NK细胞的免疫疗法。