Generation of Novel Osteolineage VHL Conditional Knockout Mice to Study B Cell Microenvironments

项目总结 我们的长期目标是确定影响B淋巴细胞发育的微环境的特征。 骨骼动态平衡。B淋巴细胞是产生抗体所必需的，而抗体对强健的适应性免疫至关重要。 回应。来自多个群体的研究确定了B细胞抗原的成熟阶段和分子机制 受体基因重排和受体多样性的产生及其自我耐受机制 细胞。骨髓中支持B细胞祖细胞和成熟B细胞的微环境一直是 描述(2-7)，并且似乎来自B细胞的强健抗体反应的发展受到相对 脾和淋巴结生发中心含氧量低(11)。然而，人们对此知之甚少。 氧水平调节骨髓中B细胞发育的机制。我们的目标是填补科学 关于影响成人B细胞亚群维持和功能的微环境的知识差距。在……里面 在我们研究骨骼稳态改变对免疫细胞发育的影响的过程中，我们发现 在缺氧反应途径基因von-Hippel Lindau的小鼠中，B细胞的发育受到严重损害 (VHL)，在骨细胞(12)中有条件地删除。我们假设，骨细胞中VHL缺乏导致结构性 以及BM微环境中血管结构的分子变化，这可能会将氧分压改变到 支持异常的B基因发育。为了支持这一点，我们的初步分析表明，骨细胞中VHL的缺失 导致血管系统通透性增加。然而，Dmp1-Cre转基因的非靶向表达 成骨细胞掩盖了这种解释。传统上用于研究骨骼系统显示的其他组织特异性Cre小鼠 偏离靶点或比预期更广泛的表达，降低了它们在确定特定细胞中基因缺失作用方面的作用 类型。此外，目前的小鼠模型不允许研究VHL缺陷对出生后免疫细胞发育的影响 和成年的BM微环境。MSCs是骨细胞的前体，可以支持骨髓中B细胞的发育(13)，但 MSCs、OBS和OCys对B细胞成熟的具体和独特的贡献尚不清楚。因此，我们建议 建立一种新的可诱导的VHL条件性基因敲除小鼠模型，以评估VHL基因缺失的细胞外在效应 MSCs对B细胞发育的影响。我们的目标是将移植策略与高分辨率活体(Live)相结合 颅骨显微镜和体外长骨显像对VHL缺陷性骨髓的鉴别 微环境，以确定可能影响B细胞发育的变化，如氧分压、血管 类型和结构，基质细胞或造血细胞的存在和位置，以及骨骼形态的变化。 最后，我们的目标是随着时间的推移纵向分析生态位，以确定不同的物理属性如何在 VHL缺陷性骨骼中的骨骼改变和影响B细胞发育缺陷的进展是时间的函数。 这一信息可用于未来对辐射、清髓性调节或骨形成影响的研究。 关于骨髓利基和免疫细胞发育的药物。

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. The microenvironments in the bone marrow (BM) that support B cell progenitors and mature B cells have been described (2-7), and it appears that development of robust antibody responses from B 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 hypothesized 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 B development. In support of this, our preliminary analyses suggest that deletion of Vhl in osteocytes results in increased permeability in the vasculature. However, off-target expression of the Dmp1-Cre transgene in osteoblasts clouds this interpretation. Other tissue-specific Cre mice traditionally used to study the skeletal system display off-target or broader expression than expected, diminishing their utility in defining the role of gene deletion in specific cell types. In addition, current mouse models do not permit the study of Vhl-deficiency on immune cell development in postnatal and adult BM microenvironments. MSCs are osteocyte precursors, can support B cell development in the BM (13) but the specific and unique contributions of the MSCs, OBs and OCYs to B cell maturation is unclear. Therefore, we propose to generate a novel inducible Vhl-conditional knockout mouse model, to assess the cell-extrinsic effects of Vhl deletion in MSCs on B cell development. We aim to combine transplantation strategies with high-resolution intravital (live) microscopy of the calvaria and ex vivo long bone imaging 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. 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 Vhl-deficient 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.