The human innate B cell niche and role in efferocytosis

描述（由申请方提供）：B-1细胞是小鼠中的一种独特的B细胞亚群，其特征在于表达CD 19高CD 43 + IgM高IgD低CD 5 +/-，并分泌多反应性IgM天然抗体（NA B）。在稳态条件下，IgM NAb通过响应于凋亡细胞而增强红细胞增多和抑制炎症而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，关于如何定义人类先天性B细胞的共识仍然很少。在初步实验中，我们产生了分泌多反应性NA B的人先天性B细胞克隆，其与来自人血液样品的凋亡细胞反应。值得注意的是，我们发现这些细胞中有一部分是记忆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（~1/20- 100，000），我们假设先天性B细胞主要存在于组织中，并且不经常再循环。我们建议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研究先天性B细胞在人体中的分布。我们还将在它们的解剖学小生境中对这些细胞进行表型。我们的实验将通过基于IgH库的深度测序分析来特别检查先天性B细胞的克隆组成。在功能上，先天性B细胞通过产生IgM Na B参与红细胞增多。我们提出，在激活后，这些细胞进行类别转换重组，导致IgG和伊加Nab的分泌。我们的研究将致力于比较不同类别的Nab对红细胞增多症的调节作用。实验将在2个特定目标中进行：目标1。描述人类先天性B细胞的特征并绘制其解剖学小生境。与凋亡细胞反应的先天性B细胞克隆将在体外从 从脑死亡的实体器官捐赠者身上收集的各种人体组织标本。然后将鉴定其IG重链（IgH）重排的CDR 3序列，并使用深度测序策略评估其在各种组织中的频率。IgH序列还将指示这些克隆是否在不同位点积累了不同的体细胞突变，并在原位经历了类别转换重组（CSR）。最后，我们使用流式细胞术来分析各种组织和血液中的先天性B细胞表面标志物。目标二探讨先天性B细胞产生的NA B b在红细胞增多症中的作用。我们将首先确定来源于各种组织标本的先天性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，我们将检查它们结合凋亡细胞的能力。 Nab的不同类型类别（IgM、IgG和伊加）和亚类（IgG 1 -4）的功能，以1）激活补体，导致补体调理素沉积在靶凋亡细胞上，2）增强或阻断最常见的吞噬细胞的吞噬作用，和3）在吞噬作用后触发清道夫细胞的不同细胞因子分泌模式。

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.