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挑战了多个实验室。多光子显微镜的发展揭示了次级淋巴器官中淋巴细胞运动的动力学。与此相反，很少有人了解的动力学和细胞相互作用，指导造血细胞分化的骨髓（BM）。B淋巴细胞从基本的但特征不明显的BM基质细胞小生境中的造血干细胞和祖细胞发育而来。一个重要的机构的工作集中在淋巴细胞的内在机制，淋巴细胞的发展至关重要。尽管这些研究很重要，但它们导致了以淋巴为中心的工作模型，这些模型不足以理解造血前体细胞和指导细胞谱系决策的BM基质细胞之间复杂的相互作用。此外，目前还没有概念或机制的框架来解释骨髓中淋巴支持性基质细胞龛的发展和维持。在这项研究中，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系支持性基质细胞小生境的发展起着关键作用。在目标1中，我们建议完全表征CXCR4缺陷（仅在BM基质细胞中）对野生型造血干细胞的维持和分化的影响。我们还将确定CXCR 4信号传导如何指导基质小生境支持BM中的B细胞发育。在目标2中，我们提供了令人信服的证据，造血前体定位在淋巴支持的基质龛是一个高度调节的过程，对B淋巴细胞的发展至关重要。我们将描述基质细胞的小生境是至关重要的B淋巴细胞谱系的发展。此外，我们将定义引导机制，吸引淋巴祖细胞，淋巴支持的基质壁龛。在B细胞发育的晚期阶段，未成熟的B淋巴细胞在BM小生境中经历严格的中心耐受检查点，其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观察B淋巴细胞克隆缺失和受体编辑的策略。我们将使用这种方法来测试的假设，即B淋巴细胞迁移和与基质细胞龛的相互作用，使B细胞的中枢耐受性，使用现有的B细胞受体转基因和敲入小鼠品系，以及建立的小鼠模型的人类自身免疫性疾病系统性红斑狼疮。总之，提出的目标将提供一个广泛的概念和机制框架，了解如何引导线索形状的骨髓基质细胞室和造血细胞分化过程中影响细胞谱系的决定。

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.