B cell distribution, differentiation and diversity in tissues

项目2摘要 免疫系统显示出非凡的、分层的复杂性。多种细胞类型可以采用不同的效应子 程序-通常基于一个或多个定义转录因子的表达-导致不同的细胞 具有不同功能的亚型。新出现的数据表明，在极化效应阶段之后，异源基因- 记忆或类似记忆的人群也会发展出这种能力。虽然众所周知的T细胞，这种模式 也适用于B细胞。我们的小组已经确定了几个表型和转录不同的MBC亚群， 在小鼠体内具有不同的功能。在这个项目的第一阶段，我们已经定义了有意义的和重复的， 人类MBC区室中的可诱导细胞多样性。为此，我们使用了基于流的 筛选、RNA-seq、V区高通量测序（V-seq）和功能分析，以确定 在同一个体的多个组织中，然后在许多不同的个体中。功能 这些细胞的意义以及人类MBC上基因和表面标志物的异质性表达 大部分仍有待阐明。鉴于我们通过该PPG独特地进入次级淋巴和粘膜 组织，这两个项目的主要重点是组织中的常驻MBC（BRM）。在最近的研究中，我们发现 定义BRM的表面标记和转录谱。使用V区高通量测序（V- seq）我们进一步鉴定了MBC的出乎意料的大克隆，并追踪了它们的迁移模式 在组织中传播这些发现显示了我们方法的力量，并为更深入的分析奠定了基础， 确定这些新鉴定的细胞类型和克隆的功能，它们如何相互关联，以及它们之间的关系。 推动他们的扩张和差异化。考虑到这一点，我们的目标是：1）定义新的BRM子集， 单细胞RNA-seq;纯化群体的深度RNA-seq和表观遗传学分析;以及成像。2)到 确定MBC亚群在组织和组织中的功能反应，检验BRM在组织中的假设。 粘膜部位包括具有类似先天性质的细胞。3)确定大B细胞的抗体网络和特异性 不同组织中的克隆。我们将使用深度V-seq来鉴定不同MBC子集中的大克隆， 组织来阐明MBC亚群和组织间的克隆流和分化。我们将表达- 从这样的大克隆中选择mAb（来源于单细胞HL序列），并在自身和 微生物阵列来识别驱动抗原。如果我们能找到大克隆的驱动抗原，就像CMV 和EBV扩增CD 8细胞，这将对理解微生物组免疫间 行为和自身免疫。总的来说，这些研究将提供重要的，有用的和新颖的见解，人类如何 B细胞记忆的发展以及如何在粘膜和淋巴部位发挥作用。

Project 2 Summary The immune system displays remarkable, layered complexity. Multiple cell types can adopt different effector programs—often based on the expression of one or more defining transcription factors—leading to diverse cell subtypes that have distinct functions. Emerging data reveals that after the polarized effector phase, heterogene- ity also develops among memory or memory-like populations. Though well known for T cells, this paradigm applies to B cells as well. Our group has defined several phenotypically and transcriptionally distinct MBC sub- sets with distinct functions in mice. During the first period of this project we have defined meaningful and repro- ducible cellular diversity within human MBC compartments. To do so we have used a combination of flow-based screening, RNA-seq, V-region high throughput sequencing (V-seq), and functional analysis to define the nature of MBCs across multiple tissues of the same individual and then over many different individuals. The functional significance of these cells and the heterogeneous expression of genes and surface markers on human MBC largely remain to be elucidated. Given our unique access via this PPG to both secondary lymphoid and mucosal tissues, a major focus of both this Project is resident MBCs in tissues (BRM). In recent work we have discovered surface markers and transcriptional profiles that define BRM. Using V region high-throughput sequencing (V- seq) we have further identified unexpectedly large clones of MBC and have tracked their migration patterns across tissues. These discoveries show the power of our approach and set the stage for deeper analysis to determine the functions of these newly identified cell types and clones, how they relate to each other, and what drives their expansion and differentiation. With this in mind, our Aims are: 1) To define novel subsets of BRM by single cell RNA-seq; deep RNA-seq and epigenetic analysis of purified populations; and by imaging. 2) To determine the functional responses of MBC subsets in tissues and SLT, testing the hypothesis that BRMs in mucosal sites include cells with innate-like qualities. 3) To define Ab networks and specificities of large B cell clones in different tissues. We will use deep V-seq to identify large clones among different MBC subsets and tissues to elucidate clonal flow and differentiation across MBC subsets and tissues. We will then express se- lected mAbs (derived from single cell HL sequences) from such large clones and screen them on self- and microbial- arrays to identify driving antigens. If we can find driving antigens for large clones, akin to how CMV and EBV expand CD8 cells, it would have major implications for understanding both microbiome-immune inter- actions and autoimmunity. Overall, these studies will provide important, useful and novel insights into how human B cell memory develops and how if functions at mucosal and lymphoid sites.