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.

项目概要该提案建立在令人兴奋的新范式之上，该范式是由孤立的人类 NK 细胞缺陷引起的真核 DNA 解旋酶多个结构成分的低等位突变。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 细胞的发育和功能。尽管这一令人信服的新见解是由人类疾病驱动的， NK 细胞功能中对 DNA 解旋酶复合物的具体要求尚未确定。在提出的工作中，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方面的重要进展。尽管 NK 在确定移植结果和成功方面有记录要求，人们对细胞发育和功能获得知之甚少。定义需求的强大工具人类 NK 细胞分化是对 NK 细胞成熟受损的单基因原因患者的研究导致孤立性 NK 细胞缺陷 (NKD)。使用这种方法，我们和其他人已经鉴定出新型 NKD 由 CDC45-MCM-GINS DNA 解旋酶复合物和相关蛋白的突变引起。在在拟议的工作中，我们将定义 CMG 复合体的要求，更广泛地说，是增殖和细胞人类 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 测序和无偏定量流分析外周血亚群异质性细胞计数术。此外，我们2）开发了一个患者突变模型，使我们能够剖析这些突变对 NK 细胞从最早的前体细胞分化为成熟细胞的影响。我们将使用它，结合我们经过验证的体外 NK 细胞分化系统，来剖析其机制亚形性 CMG 突变影响人类 NK 细胞的发育和溶解功能的获得。最后，我们 3) 定义 CMG 解旋酶突变对人类 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，我们将确定发生的时间和性质具有低等态复制体突变的 NK 细胞发育中间体的发育失调。这些目标我们将推进人类 NK 细胞分化的新范式，即特异性 NK 细胞终末成熟的增殖和细胞周期控制的要求。这些研究有对于了解 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。定义增殖在成熟 NK 细胞生成中的作用效应器将能够更好地控制这些细胞，以预防移植物抗宿主病并促进其自然生长抗肿瘤免疫力。