DETERMINING THE ROLE OF THE REPLICATIVE HELICASE IN HUMAN NK CELL DEVELOPMENT

项目总结 这一建议建立在令人兴奋的新范式的基础上，该范式由分离的人类NK细胞缺乏症引起 真核DNA解旋酶多种结构成分的亚型突变。发现了 具有惊人相似的NK细胞表型，即终末成熟受损的多个患者队列 导致对严重病毒感染和恶性肿瘤的易感性，强调了这些突变对 人类NK细胞的发育和功能。尽管人类疾病推动了这一令人信服的新见解，但 对于DNA解旋酶复合体在NK细胞功能中的特异性要求还没有确定。在 建议的工作，我们将在NK细胞分化和增殖的背景下定义这一要求，以使 自然杀伤细胞生物学和人类健康的重要进展。 尽管他们在决定移植的结果和成功方面有书面要求，但NK 细胞的发育和获得的功能知之甚少。定义需求的强大工具 人类NK细胞分化是对单基因原因的NK细胞成熟受损患者的研究 导致孤立性NK细胞缺陷(NKD)。使用这种方法，我们和其他人已经确定了新的NKD 由CDC45-MCM-gins DNA解旋酶复合体和相关蛋白质突变引起。在 在拟议的工作中，我们将定义CMG复合体的要求，以及更广泛地说，增殖和细胞 人类NK细胞发育中的分裂。具体地说，我们将1)定义CMG解旋酶突变的影响 用单细胞RNA测序和无偏定量流研究外周血亚群的异质性 细胞学。此外，我们还开发了一种患者突变的模型，这将使我们能够解剖 这些突变对NK细胞从最早的前体细胞向成熟细胞分化的影响。我们会 利用这一点，结合我们验证的体外NK细胞分化系统，来剖析其机制 CMG亚型突变会影响人类NK细胞的发育和溶解功能的获得。最后，我们 3)确定CMG解旋酶突变对人类NK细胞抗病毒反应的影响。vbl.使用 仔细分析基因的表达、表型和功能，我们将确定时间和性质 具有复制体亚型突变的NK细胞发育中间体的发育去调控。 这些目标，我们将提出一种新的人类NK细胞分化的新范式，即特异性 NK细胞终末成熟对增殖和细胞周期调控的要求。这些研究已经 了解NK细胞在特定环境中的分化具有重要临床意义 造血干细胞移植。确定增殖在成熟NK细胞生成中的作用 效应器将使这些细胞能够更好地控制，以防止移植物抗宿主疾病，并促进其自然 抗肿瘤免疫。

PROJECT SUMMARY This proposal builds upon exciting new paradigm defined by isolated human NK cell deficiency resulting from hypomorphic mutations in multiple structural components of the eukaryotic DNA helicase. The discovery of multiple patient cohorts with a strikingly similar NK cell phenotype, namely that of impaired terminal maturation leading to susceptibility to severe viral infection and malignancy, underscores the impact of these mutations on human NK cell development and function. Despite this compelling new insight driven by human disease, the requirement for the DNA helicase complex specifically in NK cell function has not been defined. In the proposed work, we will define this requirement in the context of NK cell differentiation and proliferation to make important advances in both NK cell biology and human health. Despite their documented requirement in determining the outcome and success of transplantation, NK cell development and acquisition of function is poorly understood. A powerful tool in defining requirements for human NK cell differentiation is the study of patients with monogenic causes of impaired NK cell maturation leading to isolated NK cell deficiency (NKD). Using this approach, we and others have identified novel NKD resulting from mutations in the CDC45-MCM-GINS DNA helicase complex and associated proteins. In the proposed work, we will define the requirement for the CMG complex and, more broadly, proliferation and cell division in human NK cell development. Specifically, we will 1) define the effect of CMG helicase mutations on peripheral blood subset heterogeneity using single cell RNA sequencing and unbiased quantitative flow cytometry. Furthermore, we have 2) developed a model for patient mutations that will allow us to dissect the effect of these mutations on NK cell differentiation from earliest precursor to mature cell. We will use this, combined with our validated in vitro NK cell differentiation system, to dissect the mechanism by which hypomorphic CMG mutations affect human NK cell development and acquisition of lytic function. Finally, we will 3) define the effect of CMG helicase mutations on the human NK cell antiviral response. Using careful analysis of gene expression, phenotype and function, we will determine the timing and the nature of developmental deregulation in NK cell developmental intermediates with hypomorphic replisome mutations. These aims we will advance a novel new paradigm in human NK cell differentiation, namely the specific requirement for proliferation and cell cycle control for NK cell terminal maturation. These studies have significant clinical importance for understanding the differentiation of NK cells in the unique milieu following hematopoietic stem cell transplant. Defining the role of proliferation in the generation of mature NK cell effectors will enable the better control of these cells to prevent graft vs. host disease and promote their natural anti-tumor immunity.