Anatomical and Molecular Investigation of Microenvironments that Influence B Lymphocytes

影响 B 淋巴细胞的微环境的解剖学和分子研究

• 批准号: 10046904
• 负责人: JENNIFER O MANILAY
• 金额: $ 45.02万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046904
• 关键词: Adoptive Transfer; Adult; Affect; Aging; Anatomy; Antibody Formation; Antibody Response; Apoptosis; Architecture; Autoimmunity; B Cell Proliferation; B cell differentiation; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; Biological Assay; Biomedical Engineering; Blood Vessels; Blood flow; Bone Marrow; Bone Matrix; Cell Cycle; Cell Maturation; Cell physiology; Cells; Chemicals; Coculture Techniques; Confocal Microscopy; Data; Defect; Development; Endothelium; Engraftment; Flow Cytometry; Fluorouracil; Future; Gene Rearrangement; Generations; Genes; Hematopoietic; Hematopoietic stem cells; Homeostasis; Hypoxia; Image; Immune; Immunoassay; Immunology; Impairment; Investigation; Knowledge; Location; Longevity; Lymphocyte; Lymphocyte Biology; Maintenance; Measurement; Measures; Mesenchymal Stem Cells; Methods; Molecular; Monitor; Morphology; Movement; Mus; Organ; Osteoblasts; Osteocytes; Output; Oxygen; Pathway interactions; Permeability; Pharmaceutical Preparations; Proteins; Receptors, Antigen, B-Cell; Recovery; Research Assistant; Resolution; Role; Self Tolerance; Spleen; Stem cell transplant; Stromal Cells; Structure; Structure of germinal center of lymph node; Supporting Cell; System; Techniques; Time; Transgenic Mice; Transplantation; VHL gene; Work; adaptive immune response; applied biomedical research; bone; bone cell; bone imaging; cell type; conditioning; cytokine; high resolution imaging; in vivo; intravital microscopy; irradiation; lymph nodes; novel; physical property; progenitor; receptor; response; stem cells; two-photon

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.

项目概要 我们的长期目标是表征改变过程中影响 B 淋巴细胞发育的微环境。 B 淋巴细胞是产生抗体所必需的，而抗体对于强大的适应性免疫至关重要。 许多小组的研究已经确定了 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以及传统 B 细胞的自我耐受机制 （也称为“B2”细胞）骨髓 (BM) 中支持 B2 祖细胞和成熟 B2 细胞的微环境。 已被描述 (2-7)，并且 B2 细胞产生的强烈抗体反应似乎受到以下因素的影响： 脾脏和淋巴结生发中心的氧含量相对较低 (11) 然而，人们对此知之甚少。 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空白。 影响成体 B 细胞亚群维持和功能的微环境生态位的知识差距。 在我们研究改变骨稳态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 的小鼠 B 细胞发育严重受损 (Vhl) 在骨细胞中被条件性删除 (12)，我们发现骨细胞中的 Vhl 缺陷会导致结构性损伤。 以及骨髓微环境中血管结构的分子变化，这可能会改变氧张力的水平 我们进一步发现骨细胞中的分子变化会导致 B2 发育异常。 不足以产生细胞因子以适当支持 B 细胞成熟的微环境。 根据假设，我们的初步分析表明，骨细胞中 Vhl 的缺失导致骨细胞通透性增加。 我们的项目目标是进一步表征骨细胞调节的微环境对血管系统的影响。 由于 Vhl 缺陷，B 淋巴细胞的发育和维持我们的目标是利用高分辨率的活体细胞。 （活体）和离体器官成像和移植策略来表征 Vhl 缺陷骨髓 微环境，以确定可能影响 B 细胞发育的变化，例如氧压、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形态的变化。 同时，我们还将使用细胞因子免疫分析和共培养方法来鉴定VhlcKO的分子变化 最后，我们的目标是随着时间的推移纵向分析这些生态位，以确定如何 骨骼中独特的物理特性发生变化，并影响 VhlcKO 骨骼中 B 细胞发育缺陷的进展 作为时间的函数，该信息可应用于辐射、清髓作用的未来研究。 调节或造骨药物对骨髓生态位和免疫细胞发育的影响。

项目成果

Intravital fluorescence microscopy with negative contrast.

具有负对比度的活体荧光显微镜。

• 发表时间: 2021
• 影响因子: 3.7
• 作者: Wu, Juwell W;Jung, Yookyung;Yeh, Shu;Seo, Yongwan;Runnels, Judith M;Burns, Christian S;Mizoguchi, Toshihide;Ito, Keisuke;Spencer, Joel A;Lin, Charles P
• 通讯作者: Lin, Charles P

Deletion of Vhl in Dmp1-Expressing Cells Causes Microenvironmental Impairment of B Cell Lymphopoiesis.

Dmp1 表达细胞中 Vhl 的缺失会导致 B 细胞淋巴细胞生成的微环境受损。

• 发表时间: 2022
• 作者: Chicana, Betsabel;Abbasizadeh, Nastaran;Burns, Christian;Taglinao, Hanna;Spencer, Joel A;Manilay, Jennifer O
• 通讯作者: Manilay, Jennifer O