Anatomical and Molecular Investigation of Microenvironments that Influence B Lymphocytes

项目摘要 我们的长期目标是表征在改变的免疫应答过程中影响B淋巴细胞发育的微环境。 骨稳态B淋巴细胞是产生抗体所必需的，而抗体对强大的适应性免疫至关重要 反应许多研究小组已经确定了成熟阶段，B细胞抗原的分子机制 受体基因重排和受体多样性的产生，以及常规B细胞中自身耐受的机制 (also称为“B2”细胞）。骨髓（BM）中支持B2祖细胞和成熟B2细胞的微环境 已经描述了（2-7），并且似乎来自B2细胞的稳健的抗体应答的发展受到以下因素的影响： 脾脏和淋巴结的生发中心的氧气水平相对较低（11）。然而，相对而言， 关于氧气水平调节骨髓中B细胞发育的机制。我们的目标是填补科学 对影响成人B细胞亚群的维持和功能的微环境生态位的知识差距。在 在我们研究骨稳态改变对免疫细胞发育的影响的过程中，我们发现 缺氧反应途径基因von-Hippel Lindau (Vhl)在骨细胞中有条件地缺失（12）。我们假设骨细胞中的Vhl缺陷导致结构性骨损伤。 以及BM微环境中血管结构的分子变化，这可能会改变氧张力， 支持异常的B2发育我们进一步假设，骨细胞的分子变化导致了骨细胞的凋亡。 在某些实施方案中，B细胞是不足以产生细胞因子以适当地支持B细胞成熟的微环境。为支持这些 假设，我们的初步分析表明，在骨细胞中Vhl的缺失导致骨细胞的渗透性增加， 脉管系统我们的项目目标是进一步表征骨细胞调节的微环境对骨形成的影响。 由于Vhl缺乏，B淋巴细胞的发育和维持。我们的目标是利用高分辨率活体 （活）和离体器官成像和移植策略以表征Vhl缺陷骨髓 微环境，以便识别可能影响B细胞发育的变化，如氧张力、血管张力 类型和结构，基质细胞或造血细胞的存在和位置，以及骨形态的变化。在 同时，我们还将使用细胞因子免疫测定和共培养方法来鉴定VhlcKO中的分子变化。 骨髓基质细胞龛。最后，我们的目标是随着时间的推移纵向分析生态位，以确定如何 骨中不同的物理性质改变并影响VhlcKO骨中B细胞发育缺陷的进展 作为时间的函数。这一信息可以应用于未来的研究辐射的影响，骨髓清除， 调理，或骨建设药物对骨髓龛和免疫细胞的发展。

PROJECT SUMMARY Our long-term objective is to characterize the microenvironments that influence B lymphocyte development during altered bone homeostasis. B lymphocytes are required for the production of antibodies that are crucial for a robust adaptive immune response. Studies from many groups have determined the stages of maturation, the molecular mechanisms of B cell antigen receptor gene rearrangements and generation of receptor diversity, and mechanisms of self-tolerance in conventional B cells (also called “B2” cells). The microenvironments in the bone marrow (BM) that support B2 progenitors and mature B2 cells have been described (2-7), and it appears that development of robust antibody responses from B2 cells is influenced by the relatively low oxygen levels in the germinal centers of the spleen and lymph nodes (11). However, relatively little is known about the mechanisms by which oxygen levels regulate B cell development in the bone marrow. We aim to fill the scientific knowledge gap on the microenvironmental niches that influence the maintenance and function of adult B cell subsets. In the course of our studies to examine the effects of altered bone homeostasis on immune cell development, we discovered that B cell development was severely impaired in mice in which a hypoxia response pathway gene, von-Hippel Lindau (Vhl), is conditionally deleted in osteocytes (12). We hypothesize that Vhl-deficiency in osteocytes results in structural and molecular changes in the vascular architecture in the BM microenvironments, which may alter oxygen tension to levels that support aberrant B2 development. We further hypothesize that molecular changes in the osteocytes results in a microenvironment that insufficiently produces cytokines to appropriately support B cell maturation. In support of these hypotheses, our preliminary analyses suggest that deletion of Vhl in osteocytes results in increased permeability in the vasculature. Our project objective is to further characterize the osteocyte-regulated microenvironmental influences on the development and maintenance of B lymphocytes because of Vhl deficiency. We aim to utilize high-resolution intravital (live) and ex vivo organ imaging and transplantation strategies to characterize the Vhl-deficient bone marrow microenvironment in order to identify changes that may influence B cell development, such as oxygen tension, blood vessel type and structure, the presence and location of stromal cells or hematopoietic cells, and changes in bone morphology. In parallel, we will also use cytokine immunoassays and co-culture methods to identify the molecular changes in the VhlcKO BM stromal cell niches. Finally, we aim to analyze the niches longitudinally over time, in order to determine how the distinct physical properties in the bone change and affects the progression of B cell developmental defects in VhlcKO bones as a function of time. This information could be applied to future studies of the effects of irradiation, myeloablative conditioning, or bone-building drugs on bone marrow niches and immune cell development.