MICRORNA REGULATION OF NK CELL DEVELOPMENT AND FUNCTION

描述(申请人提供)：本申请的长期目标是定义调节NK细胞发育和功能的分子事件，从而提供对可能被操纵以促进或抑制NK细胞生物学这些方面的途径的洞察。NK细胞是一种天然免疫淋巴细胞，在宿主抵抗感染和介导抗肿瘤免疫反应中起重要作用。具有选择性NK细胞缺陷的罕见患者在生命早期就死于致命的疱疹病毒感染，证明了这些细胞对人类健康的关键重要性。此外，异基因NK细胞可以有效地治疗急性髓系白血病患者，因此NK细胞有望成为一种癌症免疫治疗方法。目前，我们对调节NK细胞功能(细胞因子的产生、细胞毒性、增殖)的分子机制尚不完全了解。MicroRNAs(MiRNAs)是一种小分子调控RNA，主要作用于mRNA的稳定性和/或限制蛋白质的翻译，从而调节关键的细胞过程。最近的研究发现，在静止和激活的小鼠和人NK细胞中都有miRNAs的表达，并表明在体外和体内，全球miRNA缺乏会导致成熟NK细胞功能反应的改变。然而，我们对单个miRNAs如何调控NK细胞生物学的了解有限。在表达并具有调节NK细胞功能的原理的多个候选miRNAs中，miR-155被优先考虑用于深入研究。本申请描述了一项研究miR-155对NK细胞功能调节作用的5年计划。我们假设miR-155调节NK细胞功能的关键方面，包括效应细胞因子(例如，干扰素？)促进或抑制NK细胞激活的重要的生产、细胞毒性和细胞内途径。我们的方法利用了NK细胞特异性的(Ncr1-ICre)条件增益(通过loxP-Stop-loxP过度表达)和miR-155功能模型的丢失(FLOXED)。在AIM 1中，将使用miR-155小鼠模型来确定miR-155对NK细胞功能的调节作用，包括发育/成熟、干扰素？生产和细胞毒性。在目标2中，我们将通过生物信息学、信使核糖核酸图谱和ArgAerte免疫沉淀以及下一代测序来确定它们在NK细胞中的靶mRNAs，从而阐明miR-155的调控机制(S)。这些靶点将在功能实验中进行评估，如AIMS 1和3中所述，并在人类NK细胞中得到证实。在目标3中，我们将利用模型病原体来确定miR-155对NK细胞功能调节的体内意义。目标2与目标1和目标3之间有高度的整合，以深入定义miR-155靶向特定mRNAs的机制，这反过来又调节NK细胞的功能。因此，这些研究将提供关于miR-155如何调节NK细胞激活的新信息，它们通过识别mRNA靶点的作用机制，以及miR-155在体内NK细胞介导的宿主防御中的重要性。因此，这些研究可能为未来加强NK细胞功能作为免疫治疗策略提供参考。

DESCRIPTION (provided by applicant): The long term goals of this application are to define molecular events that regulate NK cell development and function, thereby providing insight into pathways that may be manipulated to promote or inhibit these aspects of NK cell biology. NK cells are innate immune lymphocytes that are important during host defense against infections and mediate anti-neoplastic immune responses. This is exemplified by rare patients with selective NK cell deficiencies that succumb to fatal herpesvirus infections early in life, demonstrating the critical importance of these cells for human health. Moreover, allogeneic NK cells can be an effective treatment for acute myeloid leukemia patients, and thus NK cells hold promise as a cancer immunotherapy approach. Currently, we have an incomplete understanding of the molecular mechanisms responsible for regulating NK cell functionality (cytokine production, cytotoxicity, proliferation). MicroRNAs (miRNAs) are small regulatory RNAs that target mRNA stability and/or limit protein translation, consequently regulating critical cellular processes. Recent studies have identified miRNAs expressed in resting and activated mouse and human NK cells, and have shown that global miRNA deficiency results in altered mature NK cell functional responses in vitro and in vivo. However, our understanding of how individual miRNAs regulate NK cell biology is limited. Of multiple candidate miRNAs that are expressed and have a rationale for regulating NK cell function, miR-155 was prioritized at the highest for in depth study. This application describes a 5 year plan to study the regulatory role o miR-155 on NK cell function. We hypothesize that miR-155 regulates key aspects of NK cell functionality, including effector cytokine (e.g., IFN-?) production, cytotoxicity, and intracellula pathways important for promoting or inhibiting NK cell activation. Our approach utilizes NK cell-specific (Ncr1-iCre) conditional gain (via loxP-STOP-loxP overexpression) and loss (floxed) of function models for miR-155. In Aim 1 miR-155 mouse models will be used to define the regulatory contribution of miR-155 to NK cell function, including development/maturation, IFN-? production, and cytotoxicity. In Aim 2 we will elucidate the regulatory mechanism(s) of miR-155 by defining their target mRNAs in NK cells using bioinformatics, mRNA profiling, and Argonaute-immunoprecipitation followed by next-generation sequencing. These targets will in turn be assessed in functional experiments, described in Aims 1 and 3, and confirmed in human NK cells. In Aim 3 we will define the in vivo significance of miR-155 regulation on NK cell function using model pathogens. There is a high level of integration between Aim 2 and Aims 1 and 3, to deeply define mechanisms whereby miR-155 targets specific mRNAs, which in turn regulate NK cell function. Thus, these studies will provide novel information about how miR-155 regulates NK cell activation, their mechanism of action by identifying mRNA targets, and the importance of miR-155 to NK cell-mediated host defense in vivo. These studies may therefore inform future strategies to augment NK cell functionality as an immunotherapeutic strategy.