Molecular Mechanisms of Natural Killer Cell Cytokine-Activation

描述(由申请者提供)：这项建议的长期目标是培训申请者成为一名独立的学术内科医生-科学家，研究先天性免疫系统及其对血癌的影响。首席研究员(PI)已经完成了以自然杀伤(NK)细胞生物学为重点的博士培训，以及内科和血液肿瘤学的MD培训。本申请描述了一项为期5年的培训计划，该计划将提供有指导的教育经验，旨在发展分子图谱、大规模并行测序、蛋白质组分析、microRNAs(MiRs)操作和小鼠模型生成方面的新科学专业知识。Timothy Ley博士将指导PI的科学和职业发展。他在淋巴细胞细胞毒性、体内遗传小鼠模型和白血病基因组分析领域是公认的领导者。此外，一个由医学科学家专家组成的咨询委员会将提供额外的科学和非科学职业发展指导。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细胞中是转录后调节的。我们假设miRs调节静息NK细胞中的GzmB和PRF1，细胞因子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)我们将定义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miR。2)我们将定义静息和细胞因子激活的NK细胞的mRNA(转录组)和蛋白质(蛋白质组)的表达谱，整合这些数据库来定义NK细胞激活过程中重要分子的调控模式，并确定miRs在转录后调控中的作用。该项目使用的技术包括miR测序、miR微阵列、miR的体外和体内操作、NK细胞特异性Cre小鼠模型的生成、使用Cre-Lox生成MIR缺陷的遗传性小鼠模型以及NK细胞转录组和蛋白质组的全球分析。华盛顿大学为培养内科科学家提供了理想的环境，并将为PI提供宝贵的指导教育经验，以实现他在学术医学方面的职业目标。 这项全面的职业发展计划将培养一名独立的内科科学家，终身研究免疫系统和癌症。由于NK细胞是对多种感染性病原体免疫的关键组成部分，并参与恶性肿瘤的免疫监测，因此这项研究可能会对健康和疾病产生深远的影响。具体地说，更好地了解NK细胞的激活可能会导致基于免疫的新策略来治疗血液系统恶性肿瘤。

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.