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研究先天免疫系统及其对血癌影响的独立学术医生-科学家。主要研究者（PI）已完成博士培训，重点是自然杀伤（NK）细胞的细胞生物学，以及内科和血液肿瘤学的MD培训。该申请描述了一个为期5年的培训计划，该计划将提供指导性的教育经验，旨在开发分子分析，大规模平行测序，蛋白质组学分析，microRNA（miR）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，一个由医学专家组成的咨询委员会将提供更多的科学和非科学职业发展指导。该研究将评估miR在NK细胞细胞因子活化调节中的作用。Ley博士实验室的PI最近的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调节静息NK细胞中的GzmB和Prf 1，并且精氨酸激活释放了它们的翻译阻断。为了解决这一假设，我们提出了以下具体目标：1）我们将定义静息和精氨酸激活的NK细胞中的miR表达谱，并评估可能调节GzmB和Prfl mRNA翻译的候选miR。2)我们将定义静息和尼古丁激活的NK细胞的mRNA（转录组）和蛋白质（蛋白质组）表达谱，整合这些数据库以定义NK细胞激活过程中重要分子的调控模式，并定义miR在转录后调控中的作用。该项目中使用的技术包括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。华盛顿大学提供了一个理想的环境，以培养医生科学家，并将促进PI实现他在学术医学的职业目标的宝贵的指导教育经验。 这个整体的职业发展建议将培养一个独立的医生，科学家的研究免疫系统和癌症的一生。由于NK细胞是对许多传染性病原体免疫的关键组成部分，并且参与恶性肿瘤的免疫监视，因此所提出的研究可能对健康和疾病产生深远的影响。具体而言，更好地理解NK细胞活化可能导致新的基于免疫的策略来治疗血液恶性肿瘤。

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.