Molecular Mechanisms of Natural Killer Cell Cytokine-Activation

本提案的长期目标是培养申请人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。主要研究者（PI） 已经完成了博士培训，重点是自然杀伤（NK）细胞的细胞生物学，和医学博士培训， 内科学和血液肿瘤学。此应用程序描述了一个5年的培训计划，将 提供一个指导的教育经验，旨在发展新的科学专业知识的分子 分析、大规模平行测序、蛋白质组学分析、微RNA（miRs）的操作，以及 小鼠模型的产生。Timothy Ley博士将指导Pi的科学和职业发展。他是 在淋巴细胞毒性、体内遗传小鼠模型和基因组学领域的公认领导者 白血病分析此外，一个由医学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。 该研究将评估miR在NK细胞细胞因子活化调节中的作用。 PI在Ley博士实验室的最新工作确定了两种关键的细胞毒性分子，颗粒酶B（Gzm B） 和穿孔素（Prf 1），它们在NK细胞中受到转录后调节。我们假设miR调节 GzmB和Prf 1在静息NK细胞中的表达，并且精氨酸激活释放了它们在翻译中的阻滞。 为了解决这一假设，我们提出以下具体目标： 1)我们将定义静息和丝氨酸激活的NK细胞中的miR表达谱，并评估 可以调节GzmB和Prf 1 mRNA翻译的候选miR。 2)我们将定义静息和静息状态下的mRNA（转录组）和蛋白质（蛋白质组）表达谱， 整合这些数据库，以确定调节NK细胞的模式。 在NK细胞活化过程中重要的分子，并定义了转录后miR的作用， 调控 该项目中使用的技术包括miR测序，miR微阵列，体外和体内 miR的操作，NK细胞特异性Cre小鼠模型的产生，遗传修饰的产生， 使用Cre-Lox的miRs缺陷小鼠模型，以及NK细胞转录组和 蛋白质组华盛顿大学提供了一个理想的环境，以培养医生，科学家，并将培养一个 宝贵的指导教育经验，为PI实现他的职业目标，在学术医学。 相关性（参见说明）： 这个整体的职业发展建议将培养一个独立的医生，科学家的一生， 研究免疫系统和癌症的研究。由于NK细胞是免疫的关键组成部分， 该研究涉及多种传染性病原体，并参与恶性肿瘤的免疫监测 可能对健康和疾病产生深远的影响。具体来说，更好地理解 NK细胞活化的研究可能会导致新的基于免疫的策略来治疗血液恶性肿瘤。

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.