Generation of novel histoculture methods for studying human NK cell development

研究人类 NK 细胞发育的新型组织培养方法的产生

基本信息

批准号：
10310513
负责人：
Emily Margaret Mace
金额：
$ 8.1万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10310513
关键词：
3-Dimensional Adoption Adult Affect Animal Model Architecture Autologous B-Lymphocytes Behavior Biological Biological Models Cell Communication Cell Culture System Cell Differentiation process Cell physiology Cells Complement Complex Cytolysis Cytometry Development Disease Epstein-Barr Virus Infections Flow Cytometry Future Generations Granzyme HIV Health Hereditary Disease Human Human Herpesvirus 4 Image Image Analysis Immune Immunity Immunocompromised Host Immunologic Deficiency Syndromes Impairment In Situ In Vitro Individual Infection Lead Lymphoid Tissue Lytic Measures Mediating Methods Microscopy Modeling Natural Killer Cells Pathogenesis Phenotype Physiological Play Population Process Research Resolution Risk Role Site Source Study models System Technical Expertise Time Tissues Tonsil Tonsillar Tissue Toxic effect Transplantation Validation Viral Virus Diseases base cell killing cell motility cellular imaging chronic infection cytotoxic developmental immunology fighting human disease human model human tissue immune function immune system function improved in vivo innovation insight live cell imaging live cell microscopy microscopic imaging migration multiparametric imaging multiplexed imaging novel novel therapeutics perforin secondary lymphoid organ single cell analysis three dimensional structure two-dimensional

项目摘要

Human natural killer (NK) cells play a critical role in the control of viral infection, particularly herpesviral infections such as Epstein-Barr virus (EBV). Their primary function of killing of virally infected target cells is mediated by the contact-dependent lysis through directed secretion of perforin and granzymes. Despite the demonstrated importance of their development and function, our understanding of how NK cells interact with other cells within tissue is limited by a lack of satisfying models for studying human immunity live and in situ. Significant challenges arise when trying to visualize cells within tissue, however it is highly relevant to human disease to find ways to better define how particularly innate immune cells migrate and exert their cytotoxic function. Our previous research has used 2D cell culture systems to define the importance of cell migration in human NK cell development. In this application, we propose the adoption of previously methods of secondary lymphoid tissue (tonsil) histoculture as a model to study human NK cell migration and function within an in vivolike tissue microenvironment using fast, low-toxicity imaging. We will combine our expertise in human developmental immunology with our technical skills in cutting-edge microscopy and image analysis to visualize human NK cells within autologous tissue and measure migration in 3 dimensions. We will additionally use multi-parametric imaging mass cytometry to localize cells within tissue (Aim 1). Further, we will extend our model to include infection of the tonsils with EBV, which will allow us to measure differences in cell migration and function between infected and uninfected tissue. By integrating infection of tissue histoculture with human EBV we will enable the study of human NK cells killing physiologically relevant targets within tissue (Aim 2). The generation of this novel model will advance future studies of human immunity and enable unprecedented insight into the behavior of immune cells within their tissue microenvironment.

人类天然杀手（NK）细胞在控制病毒感染中起着至关重要的作用，尤其是疱疹病毒感染，例如爱泼斯坦 - 巴尔病毒（EBV）。它们杀死病毒感染靶细胞的主要功能是通过直接分泌穿孔蛋白和颗粒酶的依赖性裂解介导。尽管有我们对NK细胞如何相互作用的理解表现出了其发展和功能的重要性组织中的其他细胞受到缺乏令人满意的模型来研究人类免疫力现场和原位的限制。试图可视化组织中的细胞时会出现重大挑战，但是它与人类高度相关疾病找到更好地定义与先天免疫细胞如何迁移和发挥细胞毒性的方法的方法功能。我们以前的研究使用了2D细胞培养系统来定义细胞迁移的重要性人类NK细胞发育。在此应用程序中，我们建议采用先前的次级方法淋巴组织（扁桃体）组织培养作为研究人NK细胞迁移和功能的模型组织微环境使用快速，低毒性成像。我们将结合人类的专业知识发育免疫学具有我们在尖端显微镜和图像分析方面的技术技能，以可视化自体组织中的人类NK细胞并在3个维度中测量迁移。我们还将使用多参数成像质量细胞术以将细胞定位在组织内（AIM 1）。此外，我们将扩展我们的模型包括用EBV感染扁桃体，这将使我们能够测量细胞迁移的差异感染和未感染组织之间的功能。通过将组织组织培养的感染与人类的感染相结合 EBV我们将使人类NK细胞杀死组织内生理相关的靶标（AIM 2）。这种新颖模型的产生将推动对人类免疫力的未来研究，并使前所未有深入了解其组织微环境中免疫细胞的行为。