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

人类 B-1 细胞亚群和天然 IgM 的分子和功能分析

• 批准号: 251227433
• 负责人: Privatdozent Dr. Marc Seifert
• 金额: --
• 依托单位: Institut für Zellbiologie (Tumorforschung)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251227433?language=en
• 关键词: Molecular; functional; analysis; human; cell

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.

在人类中，是否存在单独的B-1细胞谱系还存在争议。我们在第一个资助期的主要发现显示，根据分子、发育和功能特性，成熟的CD5+B细胞代表了人类B-1a细胞的对应物。我们发现人类B-1a细胞是由人源化小鼠腹膜腔内不同的造血祖细胞在个体发育早期发育而来的，并在外周血中产生自然的IgM滴度。此外，人类B-1a细胞是人类PERC的主要群体。人类B-1a细胞的DNA甲基组分析揭示了转录因子活性分化导致的特殊表观遗传标记，并支持它们在个体发育和功能上的差异。脐带血(UCB)来源的成熟CD5+B细胞的IgH V基因谱系是有偏见的，通常在IGHD-IGHJ-关节处缺乏N-核苷酸。脐血中不依赖生发中心的克隆性扩增表明这些细胞具有自我补充的潜力。在进一步的实验中，我们在脐带血中鉴定了真正的人B-1b细胞，这些细胞在细胞大小和表面标志的表达上与B-1a细胞和传统的幼稚B细胞不同。此外，这些细胞在其Ig重排谱系中表现出与B-1a细胞相似的限制。在这一后续应用中，我们计划加深对人类B-1a细胞功能角色的理解，并计划在分子和功能的基础上定义和表征它们的B-1b姐妹细胞。为此，我们的目标是通过免疫球蛋白谱系、RNA测序和DNA甲基化分析来比较分子特征。随后的功能分析将重点放在这两个亚群与传统B细胞相比的迁移、分化和分泌能力上。此外，我们计划通过BCR克隆和蛋白质芯片分析来确定人类B-1a、B-1b和人类记忆B细胞的BCR特异性，以揭示自身/多反应对B-1细胞亚群发育和选择的影响。此外，这一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，我们计划通过质谱学对体外和体内产生的人IgM分子进行结构分析，并对人天然IgM的效应功能进行功能表征。总之，这一后续应用旨在完善人B-1细胞亚群和天然IgM的定义和功能表征。