Molecular Mechanisms of Natural Killer Cell Cytokine-Activation

描述（由申请人提供）：本提案的长期目标是培养申请人成为一名独立的学术医师-科学家，研究先天免疫系统及其对血癌的影响。首席研究员（PI）已经完成了自然杀伤细胞（NK）细胞生物学的博士培训，以及内科和血液肿瘤学的医学博士培训。此应用程序描述了一个为期5年的培训计划，该计划将提供指导教育经验，旨在开发分子分析，大规模平行测序，蛋白质组学分析，microrna （miRs）操作和小鼠模型生成方面的新科学专业知识。蒂莫西·莱伊博士将指导派的科学和职业发展。他是淋巴细胞毒性、体内遗传小鼠模型和白血病基因组分析领域公认的领导者。此外，医学家专家咨询委员会将提供额外的科学和非科学职业发展指导。该研究将评估miRs在调节NK细胞细胞因子激活中的作用。Ley博士实验室的PI最近的工作发现了两个关键的细胞毒性分子，颗粒酶B （GzmB）和穿孔素（Prf1），它们在NK细胞中受到转录后调节。我们假设miRs调节静止NK细胞中的GzmB和prf1，并且细胞因子激活释放了它们在翻译中的阻滞。为了解决这一假设，我们提出了以下具体目标：1)我们将确定miR在静息和细胞因子激活的NK细胞中的表达谱，并评估可能调节GzmB和Prfl mRNA翻译的候选miR。2)我们将定义静止NK细胞和细胞因子激活NK细胞的mRNA（转录组）和蛋白质（蛋白质组）表达谱，整合这些数据库来定义NK细胞激活过程中重要分子的调节模式，并定义miRs在转录后调节中的作用。该项目使用的技术包括miR测序、miR微阵列、体外和体内miRs操作、NK细胞特异性Cre小鼠模型的生成、使用Cre- lox生成miRs缺失的遗传小鼠模型，以及NK细胞转录组和蛋白质组的全局分析。华盛顿大学为培养医学科学家提供了理想的环境，并将为PI实现他在学术医学方面的职业目标提供宝贵的指导教育经验。

DESCRIPTION (provided by applicant): 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 Prf 1 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 educational experience for the PI to realize his career goals in academic medicine. 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 immuno-surveillance 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 strategies to treat hematologic malignancies.