Modeling the influence of sex on natural killer cell networks

说明(申请人提供)：接种疫苗是预防与传染病相关的发病率和死亡率的最有效方法之一，但仍有许多病毒感染，如人类免疫缺陷病毒(HIV)和巨细胞病毒(CMV)，因此仍然迫切需要持久的、广泛交叉保护的疫苗。尽管疫苗开发的大部分焦点都集中在适应性免疫反应上，但自然杀伤(NK)细胞是一种先天的淋巴细胞反应者，可以迅速而有力地杀死感染病毒的细胞。最近的数据表明，NK细胞具有获得性免疫反应的特征--包括对同一抗原的第二次挑战做出更快、更有力的反应的能力。因此，它们在疫苗或治疗环境中建立早期感染控制方面显示出巨大的希望，在这些环境中，适应性免疫反应被证明太少和太晚。在成功地激发这种反应之前，病毒感染细胞的NK细胞识别必须 在详细的水平上被理解。然而，NK细胞是一个高度异质性的细胞群体，具有多种功能，并具有复杂的表面受体镶嵌。荧光细胞术的局限性阻碍了对所有这些特征的同时检测。为了解决这个问题，我们建议使用一种新的技术-飞行时间细胞测量(CyTOF)。CyTOF可以同时检测多达40个细胞标志物，与传统的12-18个荧光细胞仪的限制相比，这是一个相当大的进步。因此，我们将使用CyTOF来全面检测NK细胞在杀伤病毒感染细胞过程中的表型和功能。我们将通过开发体外病毒抑制试验来确定对HIV、流感和CMV至关重要的效应NK细胞的特定群体，在这些试验中，来自健康捐赠者的NK细胞识别并杀死感染这些病毒的自体细胞。CyTOF的高度参数、深度剖析能力，应用于这一特定的、受控的抗病毒NK细胞功能的询问，将使我们能够深刻地了解哪些NK细胞正在积极识别和杀死病毒感染的细胞。结果将使用各种互补的统计技术和降维算法进行分析。一旦这项分析确定了与杀死感染病毒的细胞有关的特定NK细胞亚群，我们将 使用体外功能分析来补充这些发现，以准确地定义NK细胞如何识别和杀死受感染的细胞。这些分析方法经典地检测单个受体或功能，但将被调整为以组合方式同时询问多个受体或功能。我们的创新方法旨在了解哪些NK细胞亚群需要启动，哪些功能需要刺激，以便有目的地在疫苗或治疗环境中引发抗病毒反应。因此，我们的发现将推动疫苗接种创新方法的发展。

DESCRIPTION (provided by applicant): Vaccination is one of the most effective methods to prevent morbidity and mortality related to infectious diseases, yet there are many viral infections, such as Human Immunodeficiency Virus (HIV) and cytomegalovirus (CMV), for which durable, broadly cross-protective vaccines remain desperately needed. Although most of the focus in vaccine development has been on adaptive immune responses, natural killer (NK) cells are innate lymphocyte responders that rapidly and robustly kill virus-infected cells. Recent data has demonstrated that NK cells share features of adaptive immune responses-including the ability to respond more quickly and robustly upon a second challenge with the same antigen. Thus, they show great promise in establishing early control of infection in a vaccine or therapeutic setting where adaptive immune responses have proven to be too little and too late. Before this response can be elicited successfully, NK cell recognition of virus-infected cells must be understood at a detailed level. However, NK cells are a highly heterogeneous cell population, capable of a vast variety of functions and possessing a complex mosaic of surface receptors. The limits of fluorescence cytometry have prevented the simultaneous examination of all of these traits. To address this question, we propose the use of a novel technology, Cytometry by Time-Of-Flight (CyTOF). CyTOF can detect up to 40 cellular markers simultaneously, a considerable advance from the traditional fluorescence cytometry limit of 12-18. Thus, we will use CyTOF to comprehensively measure NK cell phenotype and functions during killing of virus-infected cells. We will define the specific populations of effector NK cells critical for responsesto HIV, influenza, and CMV by developing in vitro viral suppression assays in which NK cells from healthy donors recognize and kill autologous cells infected with these viruses. The highly parametric, deep profiling capabilities of CyTOF, applied to this specific, controlled interrogatio of antiviral NK cell function, will allow us to gain profound insight into which NK cells are activly recognizing and killing virus- infected cells. Results will be analyzed using a variety of complementary statistical techniques and dimensionality reduction algorithms. Once this analysis has defined specific subsets of NK cells involved in killing virus-infected cells, we will complement these findings using in vitro functional assays to define precisely how the NK cells are recognizing and killing the infected cells. These assays classically examine single receptors or functions, but will be adapted to simultaneously interrogate multiple receptors or functions in a combinatorial manner. Our innovative approach aims to understand which NK cell subsets to prime and which functions to stimulate in order to purposefully elicit an antiviral response in a vaccine or therapeutic setting. Our findings will therefore drive the development of innovative new approaches to vaccination.