Cellular and molecular analysis of B lymphocyte development and selection

描述（由申请人提供）：几十年来，对理解淋巴细胞迁移的探索一直困扰着多个实验室。多光子显微镜的发展为研究淋巴细胞在次生淋巴器官中的运动动力学提供了新的途径。相比之下，对骨髓中指导造血细胞分化的动力学和细胞相互作用知之甚少。B淋巴细胞由造血干细胞和祖细胞在骨髓基质细胞龛中发育而来。大量的工作集中在淋巴细胞内在机制对淋巴细胞发育至关重要。尽管它们很重要，但这些研究导致了以淋巴细胞为中心的工作模型，不足以理解造血前体和骨髓基质细胞之间复杂的相互作用，这些相互作用指导细胞谱系的决定。此外，目前还没有概念或机制框架来解释骨髓性白血病中淋巴细胞支持基质细胞龛的发展和维持。在这项资助中，我们提供了开创性的初步证据，证明趋化因子受体CXCR4在BM基质细胞中对b系支持性基质细胞龛的发展起着关键作用。在目的1中，我们建议充分表征cxcr4缺乏（仅在BM基质细胞中）对野生型造血干细胞维持和分化的影响。我们还将确定CXCR4信号如何指导基质龛支持BM中B细胞的发育。在目标2中，我们提供了令人信服的证据，证明造血前体在淋巴细胞支持基质龛中的定位是一个高度调控的过程，对B淋巴细胞的发育至关重要。我们将描述对B淋巴细胞谱系发育至关重要的基质细胞龛。此外，我们将定义引导机制吸引淋巴祖细胞到这些淋巴细胞支持基质壁龛。在B细胞发育的后期阶段，未成熟的B淋巴细胞在BM壁龛中经历严格的中央耐受检查点，通过允许细胞改变其受体特异性（称为受体编辑的过程）或通过促进细胞死亡（称为克隆缺失的过程）来消除自反应性B细胞。在目标3中，我们描述了一种通过活体双光子显微镜可视化b淋巴细胞克隆缺失和受体编辑的策略。我们将使用现有的B细胞受体转基因和敲入小鼠菌株以及建立的人类自身免疫性疾病系统性红斑狼疮小鼠模型，使用这种方法来验证B淋巴细胞迁移和与基质细胞龛的相互作用能够使B细胞中枢耐受的假设。总之，提出的目标将提供一个广泛的概念和机制框架，以理解引导线索如何塑造骨髓基质细胞区室并影响造血细胞分化过程中的细胞谱系决定。

DESCRIPTION (provided by applicant): The quest for understanding lymphocyte migration challenged multiple laboratories for several decades now. The development of multi-photon microscopes has shed light on the dynamics of lymphocyte movement in secondary lymphoid organs. In contrast, very little is understood about the dynamics and cellular interactions instructing hematopoietic cell differentiation in bone marrow (BM). B lymphocytes develop from hematopoietic stem and progenitor cells within essential, yet poorly characterized, BM stromal cell niches. A significant body of work has focused on the lymphocyte-intrinsic mechanisms critical for lymphocyte development. Despite their importance, these studies led to lymphoid-centric working models that are insufficient for understanding the complex interactions between hematopoietic precursors and the BM stromal cells that instruct cell lineage decisions. Furthermore, there are currently no conceptual or mechanistic frameworks explaining the development and maintenance of lymphoid-supportive stromal cell niches in BM. In this grant we provide groundbreaking preliminary evidence for a critical role played by the chemokine receptor CXCR4 in BM stromal cells for the development of B-lineage supportive stromal cell niches. In aim 1, we propose to fully characterize the impact of CXCR4-deficiency, exclusively in BM stromal cells, on the maintenance and differentiation of wild-type hematopoietic stem cells. We will also determine how CXCR4 signaling instructs stromal niches to support B cell development in BM. In aim 2, we provide compelling evidence that hematopoietic precursor positioning in lymphoid-supportive stromal niches is a highly regulated process vital for B lymphocyte development. We will characterize the stromal cell niches that are critical for B lymphocyte lineage development. Furthermore, we will define guidance mechanisms attracting lymphoid progenitors to such lymphoid-supportive stromal niches. In late stages of B cell development, immature B lymphocytes undergo stringent central tolerance checkpoints in BM niches that eliminate self-reactive B cells by allowing cells to change their receptor specificity a process named receptor editing) or by promoting cell death (a process known as clonal deletion). In aim 3, we describe a strategy for visualizing B-lymphocyte clonal deletion and receptor editing by intravital 2-photon microscopy. We will use this approach to test the hypothesis that B lymphocyte migration and interactions with stromal cell niches enable B cell central tolerance, using available B cell receptor transgenic and knock-in mouse strains as well as established mouse models of the human autoimmune disease systemic lupus erythematosus. Together, the proposed aims will provide a broad conceptual and mechanistic framework for understanding how guidance cues shape the BM stromal cell compartment and influence cell lineage decisions during hematopoietic cell differentiation.