Molecular and functional analysis of human B-1 cell subsets and natural IgM

在人类中，是否存在单独的B-1细胞谱系一直存在争议。我们在第一个资助期的主要发现表明，根据分子、发育和功能特性，成熟的CD5+ B细胞代表人类B-1a细胞的对应物。我们发现，人B-1a细胞在人源化小鼠的腹腔（PerC）中从不同的造血前体发育到个体发育早期，并在外周血（PB）中产生天然的IgM滴度。此外，人B-1a细胞代表了人PerC的主要群体。人B-1a细胞的DNA甲基组分析揭示了由不同转录因子活性引起的特异性表观遗传标记，并支持其个体遗传和功能独特性。脐带血（UCB）来源的成熟CD5+ B细胞的IgH V基因库是有偏差的，通常在ighd - ighj关节处缺乏n -核苷酸。UCB中生发中心不依赖克隆的扩增表明这些细胞具有自我补充的潜力。最后，功能分析显示成熟CD5+ B细胞（人B-1a谱系）具有先天样的反应性和自发分泌Ig的潜力。在进一步的实验中，我们在UCB中鉴定了真正的人B-1b细胞，根据细胞大小和表面标记表达，它与B-1a细胞和传统naïve B细胞不同。此外，这些细胞在其Ig重排功能方面表现出与B-1a细胞相似的限制。在后续的应用中，我们计划加深我们对人类B-1a细胞功能作用的理解，并计划在分子和功能基础上定义和表征它们的B-1b姐妹细胞。为此，我们的目标是通过Ig库、rna测序和dna甲基化分析来比较两个亚群的分子特征。随后的功能分析将重点关注这两个亚群与常规B细胞相比的迁移、分化和分泌能力。此外，我们计划通过BCR克隆和蛋白质芯片分析来确定人B-1a、B-1b和人记忆B细胞的BCR特异性，以揭示自反应性/多反应性对B-1细胞亚群发育和选择的影响。此外，该策略将揭示人类天然（B-1a来源）与特异性（记忆B细胞来源）IgM的特异性。最后，我们计划通过质谱法对体外和体内产生的人类IgM分子进行结构分析，并对人类天然IgM的效应函数进行功能表征。综上所述，这项后续应用旨在对人B-1细胞亚群和天然IgM的精确定义和功能表征。

In humans, the existence of a separate B-1 cell lineage is debated. Our major findings from the first funding period revealed that mature CD5+ B cells represent the human B-1a cell counterpart, according to molecular, developmental and functional properties. We showed that human B-1a cells develop ontogenetically early from distinct hematopoietic precursors in the peritoneal cavity (PerC) of humanized mice and give rise to natural IgM titers in peripheral blood (PB). Moreover, human B-1a cells represent a major population in human PerC. The DNA methylome analysis of human B-1a cells revealed specific epigenetic marks resulting from divergent transcription factor activity and support their ontogenetical and functional distinctness. The IgH V gene repertoire of umbilical cord blood (UCB)-derived mature CD5+ B cells is biased, often lacking N-nucleotides at IGHD-IGHJ-joints. Germinal center-independent clonal expansions in UCB indicate a self-replenishing potential of these cells. Finally, functional analyses revealed an innate-like responsiveness and spontaneous Ig secretion potential of mature CD5+ B cells, the human B-1a lineage.In further experiments, we identified bona fide human B-1b cells in UCB, which differ from B-1a cells and conventional naïve B cells according to cell size and surface marker expression. Moreover, these cells show similar restrictions in their Ig rearrangement repertoire as B-1a cells.In this follow-up application, we plan to deepen our understanding of the functional role of human B-1a cells and plan to define and characterize their B-1b sister cells on a molecular and functional basis. For this, we aim at a comparative molecular characterization by Ig repertoire, RNA-sequencing and DNA-methylation analysis of both subsets. Subsequent functional analyses shall focus on migration, differentiation and secretion capacities of both subsets in comparison to conventional B cells. Furthermore, we plan to determine the BCR specificities of human B-1a, B-1b and human memory B cells by BCR cloning and protein microarray analysis to reveal an influence of auto-/ polyreactivity for the development and selection of B-1 cell subsets. Moreover, this strategy will reveal the specificity of human natural (B-1a-derived) versus specific (memory B cell-derived) IgM. Finally, we plan to perform a structural analysis of human IgM molecules ex vivo and generated in vivo by mass spectometry, and a functional characterization of the effector functions of human natural IgM.Taken together, this follow-up application aims at a refined definition and functional characterization of human B-1 cell subsets and natural IgM.