The human innate B cell niche and role in efferocytosis

描述（由申请人提供）：B-1细胞是小鼠中一个独特的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通过增强efferocytosis和抑制凋亡细胞的炎症反应发挥关键的稳态功能。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，对于如何定义人体内的先天B细胞，目前还没有多少共识。在初步实验中，我们生成了人类先天B细胞克隆，分泌多反应性NAb，对来自人类血液样本的凋亡细胞起反应。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超突变。由于它们在外周血中的频率相对较低（约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实体器官供体收集的人体组织标本来研究先天B细胞在人体内的分布。我们还将在这些细胞的解剖壁龛中对它们进行表型分析。我们的实验将通过对先天B细胞的IgH库进行深度测序分析，特别检查其克隆组成。功能上，先天B细胞通过产生IgM抗体参与efferocytosis。我们认为，在激活后，这些细胞进行类转换重组，导致IgG和IgA Nab的分泌。我们的研究将比较不同种类的nab对efferocytosis的调节作用。实验将在两个具体目标下进行：Aim-1。表征人类先天B细胞并绘制其解剖壁龛图。对凋亡细胞有反应的先天B细胞克隆将在体外产生

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.