The Role of Cellular-Spatial Neighborhoods in Tonsil Organoid Immunology.

项目摘要 新技术，如质谱细胞仪，大大扩展了我们的能力，加深我们的认识， 了解淋巴细胞和相关群体的复杂性。斯坦福大学CCHI小组 我特别注意到了跟随诺兰实验室的领导来利用这一点的潜力 技术，突破性的研究T细胞，NK细胞和癌症免疫学。但显然需要 一直是用于在各种情况下询问组织切片的高维方法。 这启发了诺兰博士和他的团队，特别是MIBI， 高分辨率格式的金属标记抗体，以及CODEX（CODetection by inDEXing），一种多功能 适用于大多数标准三色荧光的参数荧光成像技术 显微镜，目前能够灵敏和定量地测量60多个标记， 单个组织。CODEX扩展了多参数流式细胞术的深度表型分析能力， 使众多细胞类型的相关空间环境成为可能，包括涉及 疾病机制。为了实现这种高参数能力，针对靶表位的抗体各自是 用独特的DNA寡核苷酸标记，并重复成像和去除相应标记的循环， 以收集所有参数的单细胞蛋白质组学测量。我们将部署CODEX， 扁桃体类器官的2D和3D结构的深度表型分析。认识到日益增长的国际 对免疫学、疫苗和药物的成像应用感兴趣 开发，该技术开发项目将扩展CODEX的当前功能，以深入 暴露于流感疫苗之前和之后扁桃体组织结构的表型分析。 具体来说，戴维斯实验室已经开发出一种独特的扁桃体类器官系统，可以接触到流感 随后在几天至一周后产生高亲和力抗体。的多功能性 这种类器官系统为修饰和测试流感疫苗构建体提供了前所未有的机会 和佐剂，并确定如何最好地触发广泛中和的生产。 流感抗体，一个目标是产生一种通用疫苗。我们将提取特征数据与此 前所未有的深入的数据，了解批发和轻微的组织变化，发生在 对流感疫苗挑战的反应--首次在单细胞上绘制“组织组学”图谱 流感疫苗反应水平。我们还将利用各种操纵， 将在项目1中使用这个类器官系统，以衡量它们对组织的影响。 这些细胞，并使用它来制定关于特定细胞分组的重要性的假设 我们称之为“邻里”

PROJECT SUMMARY New technologies such as mass cytometry have greatly expanded our ability to deepen our understanding of the complexity of lymphocytes and related populations. The Stanford CCHI group has been particularly attuned to the potential in following the lead of the Nolan lab in exploiting this technology, with ground-breaking studies of T cells, NK cells, and cancer immunology. But a clear need has been for high dimensional methods to interrogate tissue sections in a wide variety of circumstances. This inspired several complementary efforts by Dr. Nolan and his group, specifically MIBI, which uses metal labeled antibodies in a high-resolution format, and CODEX (CODetection by inDEXing), a multi- parameter fluorescence-based imaging technology adaptable to most standard three-color fluorescence microscopes, and currently capable of sensitively and quantitatively measuring more than 60 markers in a single tissue. CODEX extends the deep phenotyping capabilities of multi-parameter flow cytometry while enabling the associated spatial context of a multitude of cell types, including rare cell types implicated in disease mechanisms. To achieve this high-parameter capability, antibodies against target epitopes are each tagged with unique DNA oligonucleotides and iterative cycles of imaging and removal of corresponding tags is performed to collect single cell proteomic measurements across all parameters. We will deploy CODEX for deep phenotyping of the 2D and 3D architecture of tonsil organoids. Recognizing a growing international biomedical and pharmaceutical interest in imaging applications to immunology, vaccine and drug development, this Technology Development Project will extend the current features of CODEX to deep phenotypic profiling of tonsil tissue architecture before and after exposure to influenza vaccine. Specifically, the Davis lab has developed a unique tonsil organoid system that can be exposed to a flu vaccine with subsequent production of high affinity antibodies several days to a week later. The versatility of this organoid system provides an unprecedented opportunity to modify and test influenza vaccine constructs and adjuvants in a fully human system and determine how best to trigger production of broadly neutralizing influenza antibodies, a goal toward generating a universal vaccine. We will extract feature data with this unprecedentedly deep data for the understanding of wholesale and minor tissue alterations that occur in response to influenza vaccine challenge—enabling a first ever map of “tissue-omics” at the single cell level for the influenza vaccine response. We will also take advantage of the various manipulations that will be employed in Project 1 on this organoid system in order to gauge their effects on the organization of these cells and use this to formulate hypotheses regarding the significance of particular cellular groupings that we see in tonsils, which we refer to as “neighborhoods”.