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

确定复制解旋酶在人类 NK 细胞发育中的作用

基本信息

批准号：
9790924
负责人：
Emily Margaret Mace
金额：
$ 40.27万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-24 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9790924
关键词：
Affect Antiviral Agents Antiviral Response Apoptosis Biological CD34 gene CDC45L gene CRISPR/Cas technology Cell Compartmentation Cell Cycle Cell Cycle Arrest Cell Cycle Regulation Cell Differentiation process Cell Maturation Cell Proliferation Cell division Cell physiology Cells Cellular biology Characteristics Clinical Complex DNA DNA Damage DNA biosynthesis Data Defect Development Disease Effector Cell Flow Cytometry Gene Expression Gene Expression Profiling Generations Genes Genetic Transcription Health Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Heterogeneity Homeostasis Human Immune Immune response Immunologic Deficiency Syndromes Immunologics Impairment In Vitro Lead Light Lymphocyte Lymphocyte Subset Lymphoid Lymphoid Cell Lytic MCM10 gene MCM4 gene Malignant Neoplasms Mediating Modeling Molecular Mutation Natural Killer Cells Nature Outcome Pathogenicity Pathway interactions Patients Phase Phenotype Play Precursor Natural Killer Cell Predisposition Process Proteins Regulation Replication Initiation Role Structure System Testing Therapeutic Translating Transplantation Tumor Immunity Viral Virus Diseases Work cell motility cohort graft vs host disease helicase human disease immune reconstitution insight novel peripheral blood prevent response single cell analysis single-cell RNA sequencing success tool

项目摘要

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.

项目摘要该提案以令人兴奋的新范式为基础真核DNA解旋酶多种结构成分中的低形态突变。发现多个患者队列具有非常相似的NK细胞表型，即终末成熟的受损导致对严重病毒感染和恶性肿瘤的敏感性，强调了这些突变对人类NK细胞的发育和功能。尽管这种引人入胜的新见解是由人类疾病驱动的尚未定义对NK细胞功能中特异性DNA解旋酶复合物的需求。在拟议的工作，我们将在NK细胞分化和增殖的背景下定义这一要求，以使 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，但NK还是细胞的发展和功能的获取知之甚少。定义要求的强大工具人NK细胞分化是对NK细胞成熟受损的单基因原因的研究导致孤立的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颖的NKD 由Cdc45-MCM-核DNA解旋酶复合物和相关蛋白的突变产生。在提议的工作，我们将定义对CMG复合物的需求，更广泛地是增殖和细胞人类NK细胞开发部。具体而言，我们将1）定义CMG解旋酶突变的效果使用单细胞RNA测序和无偏定量流量，在外周血子集异质上细胞仪。此外，我们有2）开发了一种用于患者突变的模型，这将使我们能够剖析这些突变对NK细胞从最早的前体到成熟细胞的分化的影响。我们将使用此功能，并与我们经过验证的体外NK细胞分化系统结合使用，以剖析机制型型CMG突变会影响人NK细胞的发展和裂解功能的获取。最后，我们将3）定义CMG解旋酶突变对人NK细胞抗病毒反应的影响。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 NK细胞发育中间体中的发育放松调节与型肌菌群突变。这些目标我们将推进人类NK细胞分化的新型新范式，即特定对NK细胞末端成熟的增殖和细胞周期控制的要求。这些研究有在独特的环境中理解NK细胞的分化的重要临床重要性造血干细胞移植。定义增殖在成熟NK细胞产生中的作用效应子将使这些细胞更好地控制这些细胞，以防止移植与宿主疾病并促进其自然抗肿瘤免疫。