DEFINING THE ROLE OF CELL MIGRATION IN HUMAN NK CELL DIFFERENTIATION

项目摘要 该提案旨在确定人类NK细胞迁移与人类NK细胞迁移之间知之甚少但至关重要的关系。 和差异化。通过整合我们经过验证的人NK细胞分化系统和高度定量的 成像和图像分析，我们将1）确定人NK细胞分化中迁移的要求， （2）分析NK细胞发育中间体与基质细胞之间的分子相互作用。 自然杀伤（NK）细胞是我们身体防御病毒感染和恶性肿瘤所必需的;它们还 影响移植的结果和成功。尽管它们对人类健康的要求有文件记录， NK细胞的发育和功能的获得知之甚少。与辅助细胞的相互作用，如 淋巴基质细胞是人类NK细胞发育所必需的，但这些细胞背后的分子生物学 细胞-细胞相互作用是未知的，并且从未被可视化。利用人NK细胞的体外分化 细胞和定量成像，我们已经描述了发展中的NK细胞和 基质细胞，我们称之为发育突触。此外，我们还定义了不同的迁移 与人NK细胞在基质细胞上进行性成熟相关的表型。解剖 NK细胞发育和基质细胞迁移之间的关系，我们将进行以下具体研究 目的：1）确定人类NK细胞发育中基质上迁移的要求; 机械约束和NK前体细胞的罕见突变，损害NK细胞迁移的患者，我们将 定量测量抑制细胞迁移对人NK细胞分化的影响。进一步 描述粘附和迁移在人类NK细胞发育中的作用，我们将：2）定义 基质细胞对NK细胞发育的物理贡献。这将包括评价备选方案 底物以确定细胞粘附性和底物刚度对NK细胞发育的贡献。 最后，我们将3）确定NK细胞尾足和 发育突触这将描绘细胞中发生的信号转导机制 通过发育突触结合，并定义这是如何与尾足动物形成， 迁移细胞这一最终目标将在原位和体外进行，以关联生理发育 与培养物中形成的突触。 通过这些目标，我们将进一步了解人类NK细胞的接触依赖性需求 在过去的十年里，在这个领域里几乎没有取得什么科学进展。我们的最终目标 是鉴定再现人NK所需的必要和足够的机械和生化信号 细胞发育以有效地产生治疗细胞而不使用基质细胞饲养层。通过了解 在这个过程中细胞迁移的要求，我们将产生临床和科学影响的发现。

PROJECT SUMMARY This proposal aims to define the poorly understood yet critical relationship between human NK cell migration and differentiation. By integrating our validated system of human NK cell differentiation and highly quantitative imaging and image analysis, we will 1) define the requirement for migration in human NK cell differentiation, and 2) dissect the molecular interactions between NK cell developmental intermediates and stromal cells. Natural killer (NK) cells are required for our body's defense against viral infection and malignancy; they also shape the outcome and success of transplantation. Despite their documented requirement in human health, NK cell development and acquisition of function is poorly understood. Interactions with accessory cells such as lymphoid stromal cells are required for human NK cell development, yet the molecular biology behind these cell-cell interactions is not known and has never been visualized. Using in vitro differentiation of human NK cells and quantitative imaging, we have described the contacts formed between developing NK cells and stromal cells, which we termed the developmental synapse. In addition, we have defined distinct migratory phenotypes associated with the progressive maturation of human NK cells on stromal cells. To dissect the relationship between NK cell development and migration on stromal cells, we will pursue the following specific aims: 1) determine the requirement for migration on stroma in human NK cell development; using both mechanical restraint and NK precursors from patients with rare mutations that impair NK cell migration, we will quantitatively measure the effect of inhibiting cell migration on human NK cell differentiation. To further delineate the role that adhesion and migration plays in human NK cell development, we will: 2) define the physical contribution of stromal cells to NK cell development. This will include the evaluation alternative substrates to determine the contribution of adhesiveness and substrate stiffness on NK cell development. Finally, we will 3) define the structural and functional relationship between the NK cell uropod and developmental synapse. This will delineate the mechanism of signal transduction that occurs in cells conjugated through the developmental synapse and define how this is related to the uropod formed in migrating cells. This final aim will be performed in situ and in vitro to correlate the physiological developmental synapse with that formed in culture. Through these aims we will advance our knowledge of the contact-dependent requirements for human NK cell development, a field in which few scientific advances have been made in the past ten years. Our ultimate goal is to identify necessary and sufficient mechanical and biochemical signals required to recapitulate human NK cell development to efficiently generate therapy cells without the use of stromal cell feeders. By understanding the requirement for cell migration in this process, we will generate findings of both clinical and scientific impact.