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

• 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
• 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

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.

人类自然杀伤(NK)细胞在控制病毒感染，特别是疱疹病毒感染方面发挥着关键作用。 爱泼斯坦-巴尔病毒(EBV)等感染。它们杀死受病毒感染的靶细胞的主要功能是 通过直接分泌穿孔素和颗粒酶进行接触性裂解。尽管 证明了它们的发育和功能的重要性，我们对NK细胞如何与 组织内的其他细胞由于缺乏令人满意的活体和原位研究人类免疫的模型而受到限制。 当试图可视化组织内的细胞时，出现了重大的挑战，然而这与人类高度相关 寻找更好地定义特别是先天免疫细胞如何迁移和发挥其细胞毒作用的方法 功能。我们之前的研究已经使用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。在本应用中，我们提出了采用以前的二次方法 淋巴组织(扁桃体)组织培养作为研究活体内人NK细胞迁移和功能的模型 组织微环境采用快速、低毒成像。我们将结合我们在人类方面的专业知识 发育免疫学和我们在尖端显微镜和图像分析方面的技术技能来可视化 人NK细胞在自体组织内的迁移，并测量三维迁移。我们还将使用 多参数成像质量细胞术定位组织内的细胞(目标1)。此外，我们将延长我们的 包括扁桃体EB病毒感染的模型，这将使我们能够测量细胞迁移的差异 并在感染和未感染组织之间发挥作用。通过将组织培养的感染与人类相结合 EBV我们将使人类NK细胞杀死组织内生理相关靶点的研究成为可能(目标2)。 这一新模型的产生将推动未来对人类免疫的研究，并使前所未有的 洞察免疫细胞在其组织微环境中的行为。