Molecular Mechanisms of Natural Killer Cell Cytokine-Activation

这项建议的长期目标是培训申请者成为一名独立的学术医生- 研究先天免疫系统及其对血癌影响的科学家。首席调查员(PI) 已完成以自然杀伤(NK)细胞生物学为重点的博士培训，并在 内科和血液肿瘤学。本申请描述了一项5年期培训计划，该计划将 提供有指导的教育经验，旨在发展新的分子科学专业知识 简介、大规模并行测序、蛋白质组分析、microRNAs的操作(MiRs)以及 一代老鼠模型。Timothy Ley博士将指导PI的科学和职业发展。他是 在淋巴细胞细胞毒性、体内遗传小鼠模型和基因组学领域公认的领导者 白血病的分析。此外，由医学科学家专家组成的咨询委员会将提供额外的 科学的和非科学的职业发展指导。 这项拟议的研究将评估MIR在调节NK细胞细胞因子激活中的作用。 莱伊博士实验室的PI最近的工作确定了两个关键的细胞毒性分子，颗粒酶B(GzmB) 和穿孔素(Prf1)，在NK细胞中是转录后调节的。我们假设MIR调节 GzmB和Prf1在静息的NK细胞中被激活，而细胞因子的激活释放了它们在翻译过程中的阻断。 为了解决这一假设，我们提出了以下具体目标： 1)我们将确定静息和细胞因子激活的NK细胞中miR的表达谱，并评估 可能调节GzmB和Prf1 mRNA翻译的候选miRs。 2)我们将确定静息和蛋白质组的mRNA(转录组)和蛋白质(蛋白质组)的表达谱 细胞因子激活的NK细胞，整合这些数据库以定义调控模式 在NK细胞激活过程中重要的分子，并定义miRs在转录后的作用 监管。 项目中使用的技术包括miR测序、miR微阵列、体外和体内 操纵miRs，产生NK细胞特异性的Cre小鼠模型，产生遗传 利用Cre-Lox建立MIR缺陷的小鼠模型，并对NK细胞转录组和 蛋白质组。华盛顿大学为培养内科科学家提供了理想的环境，并将培养 为国际医学会实现其学术医学的职业目标提供了宝贵的教育经验。 相关性(请参阅说明)： 这一总体职业发展建议将培养一名独立的内科科学家终身 研究免疫系统与癌症的关系。因为NK细胞是免疫的关键成分 众多感染性病原体，并参与恶性肿瘤的免疫监测，研究 这一提议可能会对健康和疾病产生深远的影响。具体地说，更好地理解 NK细胞的激活可能导致新的基于免疫的治疗血液系统恶性肿瘤的策略。

The long term goal of this proposal is to train the applicant to become an independent academic physician- scientist studying the innate immune system and its impact on blood cancers. The principal investigator (PI) has completed PhD training focused on the cellular biology of natural killer (NK) cells, and MD training in internal medicine and hematology-oncology. This application describes a 5 year training program that will provide a mentored educational experience aimed at developing new scientific expertise in molecular profiling, massively parallel sequencing, proteomic analysis, manipulation of microRNAs (miRs), and the generation of mouse models. Dr. Timothy Ley will mentor the Pi's scientific and career development. He is a recognized leader in the field of lymphocyte cytotoxicity, in vivo genetic mouse models, and genomic analysis of leukemia. Furthermore, an advisory committee of medical scientist experts will provide additional scientific and and non-scientific career development guidance. The proposed research will evaluate the role of miRs in the regulation of NK cell cytokine activation. Recent work by the PI in Dr. Ley's laboratory identified two critical cytotoxic molecules, granzyme B (GzmB) and perforin (Prf1), that are post-transcriptionally regulated in NK cells. We hypothesize that miRs regulate GzmB and Prf1 in resting NK cells, and that cytokine-activation releases their block in translation. To address this hypothesis, we propose the following specific aims: 1) We will define the miR expression profiles in resting and cytokine-activated NK cells, and evaluate candidate miRs that may regulate GzmB and Prfl mRNA translation. 2) We will define the mRNA (transcriptome) and protein (proteome) expression profiles of resting and cytokine-activated NK cells, integrate these databases to define the mode of regulation of molecules important during NK cell activation, and define the role of miRs for post-transcriptional regulation. Techniques utilized in the project include miR sequencing, miR microarrays, in vitro and in vivo manipulation of miRs, the generation of a NK cell-specific Cre mouse model, the generation of genetic mouse models deficient in miRs using Cre-Lox, and the global analysis of the NK cell transcriptome and proteome. Washington University provides an ideal setting to train physician-scientists, and will foster an invaluable mentored eduational experience for the PI to realize his career goals in academic medicine. RELEVANCE (See instructions): This overall career development proposal will train an independent physician-scientist for a lifetime of research studying the immune system and cancer. As NK cells are key components of immunity to numerous infectious pathogens, and are involved in the immunosurveillance of malignancy, the research proposed may have far reaching consequences for health and disease. Specifically, a better understanding of NK cell activation may lead to novel immune based stategies to treat hematologic malignancies.