Source and homeostatic functions of anti-adduct IgM in humans

项目摘要 血清免疫球蛋白M具有重要的“看家”功能，如清除凋亡细胞和细胞碎片， 防止它们在组织中积聚。然而，尽管它们扮演着重要的角色，但IgM在很大程度上 与其他免疫球蛋白同种类型，特别是免疫球蛋白免疫球蛋白相比，研究不足。血清的一个中心特征 免疫球蛋白与其生物学功能有关的是它们对暴露在细胞膜上的简单化学部分的反应能力 和大分子。例如，IgM与凋亡细胞上的磷胆碱结合以促进其消除。 当这些部分与蛋白质或其他大分子结合时，会形成加合物。值得注意的是，只有少数 到目前为止，已鉴定出由血清IgM识别的化学加合物。这些是不是 以血清IgM为靶点的更大一组化学自由基尚不清楚。为了解决这一知识差距，我们 开发了一个高维平台来评估单抗和血清对87个无处不在的IgM的反应性 加合物。结果表明：1)从记忆血B细胞中克隆出的单抗具有多反应性并与其结合 对于凋亡的细胞，因此表现出典型的“天然抗体”特征，事实上对特定的加合物起反应；2) 抗加合物IgM谱系在新生儿中高度受限，仅包括有限数量的 加合物；3)IgM反应性在6个月左右突然多样化，标志着从限制性的 新生儿曲目广泛的成人曲目和4)这一转变似乎与接触 环境抗原。总体而言，这些研究揭示了一种更复杂的抗加合物IgM免疫 最初是预料到的。然而，有几个重要的问题仍然没有得到回答： 抗加合物B细胞/PC？抗加合物B细胞是否构成具有特定表型的不同亚群 特征是什么？抗加合物IgM在吞噬和调节炎症中的作用是什么？是这些吗？ 依赖于单个加合物的IgM特异性的功能？我们建议的研究将解决这些突出的问题 问题通过在单细胞水平上表征抗加合物B细胞和PC。 具体目标--1.抗加合物记忆B细胞和浆细胞的特征及其生态位图谱 我们将首先检测骨髓、脾、胸腺中的反加合物记忆IgM+B细胞和IgM分泌细胞 和肠道相关淋巴组织，以确定它们的主要生态位。然后我们将确定表型的特征 应用流式细胞术和单细胞RNA-SEQ联合BCR-SEQ检测抗加合物记忆IgM+B细胞 描述它们的转录组，并在体内评估它们的克隆组成。 具体目标--2.确定抗加合物IgM在泡腾和炎症调节中的作用 在特定目标2中，我们将检测针对单个加合物的IgM单抗调理细胞凋亡的能力。 并通过不同类型的吞噬细胞提高它们的清除能力。我们还将调查 此函数中的补充符。在第二部分，我们将检查免疫球蛋白M反应是否调理了凋亡细胞。 不同的加合物在被吞噬细胞摄取时会触发不同的细胞因子分泌。

Project Summary Serum IgM have crucial “housekeeping” functions such as the clearance of apoptotic cells and cellular debris, preventing their accumulation in tissue. However, despite their important role, IgM have been largely understudied in comparison to other immunoglobulin isotypes, especially IgG. A central characteristic of serum IgM linked to their biological function is their reactivity to simple chemical moieties exposed on cell membranes and macromolecules. For instance, IgM bind to phosphorylcholine on apoptotic cells to promote their elimination. These moieties form adducts when attached to proteins or other macromolecules. Remarkably, only a few chemical adducts recognized by serum IgM have been identified thus far. Whether these are examples of a much larger group of chemical radicals targeted by serum IgM is unknown. To address this knowledge gap, we developed a high-dimensional platform to assess monoclonal IgM and serum IgM reactivity to 87 ubiquitous adducts. Results showed that: 1) monoclonal “polyreactive” IgM cloned from memory blood B cells and binding to apoptotic cells, hence displaying a typical “natural antibody” profile, react in fact to specific adducts; 2) the anti-adduct IgM repertoire is highly restricted in newborns and only includes reactivity to a limited number of adducts; 3) IgM reactivity diversifies abruptly around 6 months of age, marking a transition from a restricted neonatal repertoire to a broad adult repertoire and 4) this transition appears to coincide with exposure to environmental antigens. Overall, these studies uncovered a much more complex anti-adduct IgM immunity than was initially anticipated. Yet, several important questions remain unanswered: What are the functional niches of anti-adduct B cells/PC? Do anti-adduct B cells constitute a distinct subset with specific phenotypic characteristics? What is the role of anti-adduct IgM in efferocytosis and regulation of inflammation? Are these functions dependent on the IgM specificity to individual adducts? Our proposed studies will address these salient questions through the characterization of anti-adduct B cell and PC at the single-cell level. Specific aim-1. To characterize anti-adduct memory B cells and plasma cells and map their niches We will first detect anti-adduct memory IgM+ B cells and IgM-secreting cells in the bone marrow, spleen, thymus and gut-associated lymphoid tissue in order to identify their main niches. We will then characterize the phenotype of anti-adduct memory IgM+ B cells using flow cytometry and single-cell-RNA-seq combined with BCR-seq to profile their transcriptome and evaluate their clonal composition in vivo. Specific aim-2. To determine the role of anti-adduct IgM in regulation of efferocytosis and inflammation In specific aim 2 we will examine the capacity of IgM mab specific to individual adducts to opsonize apoptotic cells and enhance their clearance by different types of phagocytes. We will also investigate the role of complement in this function. In a second part, we will examine whether apoptotic cells opsonized by IgM reactive to different adducts trigger distinct cytokine secretion upon their uptake by phagocytes.