Cellular and molecular analysis of B lymphocyte development and selection

描述(由申请者提供)：对淋巴细胞迁移的探索挑战了多个实验室几十年。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。相比之下，对指导骨髓造血细胞分化的动力学和细胞相互作用知之甚少。B淋巴细胞来自造血干细胞和祖细胞，在基本的，但特征不佳的骨髓基质细胞利基内。大量的工作集中在淋巴细胞的内在机制上，这些内在机制对淋巴细胞的发育至关重要。尽管这些研究很重要，但这些研究导致了以淋巴为中心的工作模型，不足以理解造血前体和指导细胞谱系决定的骨髓基质细胞之间的复杂相互作用。此外，目前还没有概念性或机械性的框架来解释骨髓中支持淋巴的基质细胞的发展和维持。在这项资助中，我们提供了突破性的初步证据，证明趋化因子受体CXCR4在骨髓基质细胞中对B系支持性基质细胞生态位的发展起着关键作用。在目标1中，我们建议完全表征CXCR4缺乏对野生型造血干细胞维持和分化的影响，仅限于骨髓基质细胞。我们还将确定CXCR4信号如何指导基质利基支持骨髓中B细胞的发展。在目标2中，我们提供了令人信服的证据，证明在支持淋巴的基质中定位造血祖细胞是一个对B淋巴细胞发育至关重要的高度调控的过程。我们将描述对于B淋巴细胞系的发展至关重要的基质细胞的特点。此外，我们还将确定引导机制，将淋巴祖细胞吸引到支持淋巴的基质中。在B细胞发育的后期，未成熟的B淋巴细胞在骨髓中经历严格的中心耐受检查点，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(称为克隆性删除的过程)来消除自我反应性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淋巴细胞克隆性缺失和受体编辑的策略。我们将使用这种方法，使用现有的B细胞受体转基因和敲入小鼠品系，以及已建立的人类自身免疫性疾病系统性红斑狼疮的小鼠模型，验证B淋巴细胞迁移和与基质细胞利基相互作用使B细胞中枢耐受的假设。总之，建议的目标将提供一个广泛的概念和机制框架，以了解指导线索如何塑造骨髓基质细胞室，并在造血细胞分化过程中影响细胞谱系决定。

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.