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

项目摘要 该建议建立在令人兴奋的新范式基础上，该范式由以下原因导致的孤立的人类NK细胞缺乏症定义： 真核DNA解旋酶的多个结构组分中的亚晶突变。的发现 多个患者队列具有惊人相似的NK细胞表型，即终末成熟受损 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细胞发育和功能。尽管这一引人注目的新见解是由人类疾病驱动的， 在NK细胞功能中对DNA解旋酶复合物的特异性要求尚未确定。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， NK细胞生物学和人类健康的重要进展。 尽管他们在确定移植的结果和成功方面有记录的要求， 细胞发育和功能的获得知之甚少。一个定义需求的强大工具， 人类NK细胞分化是对具有NK细胞成熟受损的单基因原因的患者的研究 导致孤立的NK细胞缺乏症（NKD）。使用这种方法，我们和其他人已经确定了新的NKD 由CDC 45-MCM-GINS DNA解旋酶复合物和相关蛋白的突变引起。在 建议的工作，我们将定义CMG复合物的要求，更广泛地说，增殖和细胞 在人类NK细胞发育中的分裂。具体来说，我们将1）定义CMG解旋酶突变的影响 使用单细胞RNA测序和无偏定量流对外周血亚群异质性的研究 细胞仪此外，我们还建立了一个病人突变模型， 这些突变对NK细胞从最早的前体细胞分化为成熟细胞的影响。我们将 利用这一点，结合我们经过验证的体外NK细胞分化系统，来剖析其机制， 亚型CMG突变影响人NK细胞发育和溶解功能获得。最后我们 将3）确定CMG解旋酶突变对人NK细胞抗病毒应答的影响。使用 仔细分析基因表达，表型和功能，我们将确定时间和性质， 亚型复制体突变的NK细胞发育中间体的发育失调。 这些目标，我们将提出一种新的人类NK细胞分化的新模式，即特异性的NK细胞分化。 NK细胞终末成熟需要增殖和细胞周期控制。这些研究 对于理解NK细胞在以下独特环境中的分化具有重要的临床意义： 造血干细胞移植确定增殖在成熟NK细胞生成中的作用 效应子将能够更好地控制这些细胞，以防止移植物抗宿主病，并促进它们的自然生长。 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.