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

项目总结 像质量细胞术这样的新技术极大地扩展了我们深化我们的 了解淋巴细胞及相关人群的复杂性。斯坦福CCHI小组已经 我特别注意到了跟随诺兰实验室利用这一点的潜力 技术，对T细胞、NK细胞和癌症免疫学进行开创性的研究。但一个明显的需求 已经用于在各种情况下询问组织切片的高维方法。 这激发了诺兰博士和他的团队的几项互补努力，特别是MIBI，它使用 高分辨率格式的金属标记抗体和CODEX(通过索引检测CODEX)，多 适用于大多数标准三色荧光的基于参数荧光的成像技术 显微镜，目前能够灵敏和定量地测量60多个标记在一个 单一组织。Codex扩展了多参数流式细胞术的深度表型能力，同时 支持多种单元类型的关联空间上下文，包括 疾病机制。为了实现这种高参数能力，针对目标表位的抗体分别是 标记有独特的DNA寡核苷酸以及相应标记的成像和移除的迭代循环 执行以收集所有参数的单细胞蛋白质组测量。我们将部署Codex用于 扁桃体器官的2D和3D构型的深层表型。认识到日益国际化的 生物医学和药学在免疫学、疫苗和药物成像应用中的兴趣 开发，这一技术开发项目将把Codex的现有功能扩展到深度 暴露于流感疫苗前后扁桃体组织结构的表型图谱。 具体地说，戴维斯实验室已经开发出一种独特的扁桃体器官系统，可以接触到流感 疫苗随后在几天到一周后产生高亲和力抗体。的多功能性 这种有机系统为修改和测试流感疫苗结构提供了前所未有的机会。 和佐剂在完全人体系统中，并确定如何最好地触发产生广泛中和 流感抗体，这是产生通用疫苗的目标。我们将使用此选项提取要素数据 史无前例的深入数据，用于了解发生在 应对流感疫苗挑战--首次在单细胞上绘制“组织组学”图谱 流感疫苗反应的水平。我们还将利用各种操作 将在项目1中使用这个有机系统，以评估它们对组织的影响 这些细胞，并用它来阐述关于特定细胞分组的重要性的假设 这是我们在扁桃体中看到的，我们称之为“邻里关系”。

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”.