Molecular and functional analysis of human splenic marginal zone B cells

在小鼠中，B淋巴细胞池分为三个主要的B细胞谱系，即B-1、滤泡（B-2）和边缘区（MZ）B细胞。MZ B细胞分布于专门的组织学结构，如脾边缘区（sMZ），在针对血源性病原体的免疫保护中发挥重要作用，并参与凋亡细胞清除。然而，sMZ B细胞对先天性刺激快速有效地应答，也能够使生发中心（GC）成熟并产生高亲和力抗体。基于先天和适应性倾向的这种组合，sMZ B细胞被认为具有跨越先天和适应性免疫系统之间的边界的淋巴细胞的功能。目前关于sMZ B细胞功能多样性的分子机制的观点主要基于小鼠模型，表明sMZ B细胞的发育和功能需要独特的遗传程序。此外，它们的组织学呈递、先天细胞的引发和受限的B细胞受体（BCR）库是必需的。与小鼠模型相反，对人sMZ B细胞的产生和功能了解甚少。人sMZ B细胞显示出与记忆B细胞的高度相似性，因此，目前争论的是这些细胞是否是GC经历的或代表与小鼠中不同的谱系，或者人sMZ B细胞是否由一个以上的亚群组成。去年，我们收集了一组超过80例不同年龄的人脾活检标本，其中包括100例正常人脾活检标本。我们对人sMZ B细胞的单细胞转录谱的初步结果支持它们由不同的B细胞亚群组成的想法，重要的是，这种情况随着年龄的变化而变化。在这个建议中，我们的目的是深化这一分析，揭示这些子集之间的推定分子或功能的独特性。此外，我们计划在体外跟踪婴儿和成人sMZ B细胞的功能特征，包括BCR库分析、包封细菌菌株的特异性以及sMZ B细胞对先天性刺激与适应性刺激的反应性。使用我们建立的方法来测量细胞因子分泌、迁移和细胞粘附，我们的目标是识别控制sMZ归巢和保留的信号。此外，我们希望深化我们的初步研究结果，表明激活的sMZ B细胞与红髓巨噬细胞和TH 1细胞的密切相互作用。最后，我们计划量化我们收集的配对脾和外周血（PB）B细胞样本中的克隆相关BCR重排，以确定人sMZ和PB IgM记忆B细胞库的连接程度。

In mice, the B lymphocyte pool is divided into three main B cell lineages, namely B-1, follicular (B-2) and marginal zone (MZ) B cells. MZ B cells populate specialized histological structures like the splenic marginal zone (sMZ), fulfill an important role in immune protection from blood-borne pathogens and participate in apoptotic cell clearance. sMZ B cells respond fast and efficiently to innate stimulation, however, are also capable of germinal center (GC) maturation and production of high affinity antibodies. Based on this combination of innate and adaptive propensities, sMZ B cells are thought to function as lymphocytes that cross over the boundaries between innate and adaptive immune system. The current view on the molecular mechanisms underlying the functional versatility of sMZ B cells is primarily based on mouse models, showing that sMZ B cells require a distinct genetic program for development and function. Moreover, their histological presentation, priming by innate cells and a restricted B cell receptor (BCR) repertoire are mandatory. In contrast to mouse models, the generation and function of human sMZ B cells are poorly understood. Human sMZ B cells show a high similarity to memory B cells and hence, it is currently debated whether these cells are GC-experienced or represent a distinct lineage as in mice or if human sMZ B cells are composed of more than one subset. In line with their unresolved development, the molecular mechanisms guiding human sMZ B cell functions are largely unexplored.Last year, we collected a cohort of > 80 human spleen biopsies of different ages. Our preliminary results on single cell transcription profiling of human sMZ B cells support the idea that they are composed of distinct B cell subsets and importantly, this picture changes with age. In this proposal, we aim at deepening this analysis to reveal a putative molecular or functional distinctness among these subsets. In addition, we plan to follow up our functional characterization of infant and adult human sMZ B cells in vitro, including the analysis of BCR-repertoires, the specificity for encapsulated bacteria strains and the responsiveness of sMZ B cells towards innate versus adaptive stimulation. Using our established methods to measure cytokine secretion, migration and cell adhesion, we aim at identifying the signals that govern sMZ homing and retention. Moreover, we want to deepen our preliminary findings that suggest a close interaction of activated sMZ B cells with red-pulp-macrophages and TH1 cells. Finally, we plan to quantify clonally related BCR-rearrangements among our collection of paired splenic and peripheral blood (PB) B cell samples to determine, how much the human sMZ and PB IgM memory B cell pools are connected.