The human innate B cell niche and role in efferocytosis

描述(由申请人提供)：B-1细胞是小鼠体内一种独特的B细胞亚群，其特征是表达CD19HighCD43+IgMHighIgDlowCD5+/-，并分泌多反应性IgM天然抗体(NAB)。在稳态条件下，IgM NAB通过促进细胞吞噬和抑制炎症反应来应对凋亡细胞，发挥关键的体内平衡功能。由于这种先天免疫功能，这些细胞也被称为“先天”B细胞。相比之下，对于如何定义人类的先天B细胞，目前还没有达成多少共识。在初步实验中，我们产生了人类先天B细胞克隆，分泌多反应的NAB，对人类血液样本中的凋亡细胞起反应。值得注意的是，我们发现这些细胞中的一部分是记忆B细胞，它们在体内经历了体细胞的超突变。因为它们在外周血中的频率相对较低(~1/20-100,000)，我们假设先天B细胞主要存在于组织中，不经常再循环。我们提出的研究将利用从脑死亡的实体器官捐赠者收集的人体组织标本来调查先天B细胞在人体内的分布。我们还将在这些细胞的解剖利基中进行表型分析。我们的实验将通过对其免疫球蛋白谱系的深度测序分析来特别检查先天B细胞的克隆组成。在功能上，先天B细胞通过产生IgM NAB参与胞吐作用。我们认为，在激活后，这些细胞进行了类别切换重组，导致分泌Ig G和Ig A-NAB。我们的研究将解决比较不同类别的NaB对泡腾细胞吞噬的调节作用。实验将在两个具体目标下进行：AIM-1。以确定人类先天B细胞的特征并绘制其解剖生态位图。对凋亡细胞有反应的先天B细胞克隆将在体外产生 从脑死亡的固体器官捐赠者身上收集的各种人体组织标本。然后将识别它们的免疫球蛋白重链(IgH)重排的CDR3序列，并使用深度测序策略来评估它们在不同组织中的频率。IgH序列还将表明这些克隆是否在不同的位置积累了不同的体细胞突变，并进行了原位类切换重组(CSR)。最后，我们使用流式细胞术对不同组织和血液中的固有B细胞表面标志物进行了分析。AIM-2。目的：探讨先天B细胞产生的NAB在胞吐中的作用。我们将首先确定由来自不同组织标本的先天B细胞克隆产生的NAB的类别和亚类。关于它们结合凋亡细胞的能力，然后我们将检查它们的能力 在不同类型(IgM、Ig G和Ig A)和亚类(Ig G1-4)的NaBS中，1)激活补体，导致补体调理素沉积在靶细胞上；2)大多数常见吞噬细胞增强或阻断吞噬作用；3)清除细胞吞噬时触发不同的细胞因子分泌模式。

DESCRIPTION (provided by applicant): B-1 cells are a distinct B cell subset in mice characterized by expression of CD19highCD43+IgMhighIgDlowCD5+/-, and by secretion of polyreactive IgM natural antibodies (NAb). Under steady state conditions, IgM NAb play a critical homeostatic function by enhancing efferocytosis and suppressing inflammation in response to apoptotic cells. Due to this innate immune function, these cells are also referred to as "innate" B cells. In contrast, there is still little consensus on how to define innate B cells i humans. In preliminary experiments, we generated human innate B cell clones secreting polyreactive NAb that react to apoptotic cells from human blood samples. Remarkably, we found that a fraction of these cells were memory B cells, which had undergone somatic hypermutation in vivo. Because their frequency in peripheral blood was relatively low (~1/20-100,000), we hypothesized that innate B cells reside primarily in tissue and do not often recirculate. Our proposed studies will make use of a collection of human tissue specimens collected from brain dead solid organ donors to investigate the distribution of innate B cells in the human body. We will also phenotype these cells in their anatomical niches. Our experiments will particularly examine the clonal composition of innate B cells through a deep sequencing based analysis of their IgH repertoire. Functionally, innate B cells participate in efferocytosis through the production of IgM Nab. We propose that upon activation, these cells undergo class-switch recombination resulting in the secretion of IgG and IgA Nab. Our studies will address compare the modulatory effect of different classes of Nabs on efferocytosis. Experiments will be carried out in 2 specific aims: Aim-1. To characterize human innate B cells and map their anatomical niches. Innate B cell clones reactive to apoptotic cells will be generated in vitro from a variety of human tissue specimens collected from brain dead solid organ donors. Their Ig heavy chain (IgH) rearranged CDR3 sequences will then be identified and used to assess their frequency in the various tissues using a deep sequencing strategy. The IgH sequences will also indicate whether these clones have accumulated different somatic mutations at different sites and have undergone class switch recombination (CSR) in situ. Lastly, we use flow cytometry to profile innate B cell surface markers in various tissues and blood. Aim-2. To determine the function of NAb produced by innate B cells in efferocytosis. We will first determine the classes and subclasses of Nabs produced by innate B cell clones derived from the various tissue specimens. In relation to their capacity to bind apoptotic cells, we will then examine the capacity of the different types classes (IgM, IgG and IgA) and subclasses (IgG1-4) of Nabs to 1) activate complement leading to the deposition complement opsonins on target apoptotic cells, 2) enhance or block efferocytosis by most common phagocytes and 3) trigger different cytokine secretion pattern by scavenger cells upon phagocytosis.