Non-cell-autonomous hedgehog signaling in B lymphopoiesis

描述（由申请人提供）：B淋巴细胞稳健生产和工程化的一个障碍是我们对支持B淋巴细胞生成的环境线索的不完全理解。促进离体B淋巴细胞生成的可溶性因子的鉴定受到了很大的关注，但对基质细胞的需求仍然不太清楚。在初步工作中，我们已经取得了意想不到的观察，即基质细胞中的非细胞自主Hh信号传导促进造血祖细胞的B淋巴细胞生成。我们的推论基于以下几个观察：（1）Hh信号有助于维持B祖细胞;（2）细胞自主Hh信号对B细胞发育和功能起重要作用;（3）基质细胞中Hh信号的消失会损害B淋巴细胞生成，但在造血祖细胞中不会。间质细胞Smo的耗竭与成骨细胞特性和B淋巴细胞生成活性相关基因的协同下调有关，这使我们提出Hh通路活性维持促进早期B淋巴细胞生成的间质表型。根据该提议提出的工作旨在测试这一假设，并在这样做的过程中，发展对B细胞发育中非细胞自主信号的更深入的机制理解。在第一个目标下，我们将定义在基质细胞系和原代基质中Hh信号消失后对B淋巴细胞生成的发育阻断。在第二个目标中，我们将利用生物化学和计算机模拟方法、广泛的基因组资源和强大的生物系统来鉴定和验证促进小鼠和人类B淋巴细胞生成的新型Hh依赖性基质因子。这一目标特别重要，因为它有望揭示基质细胞指导早期造血的新机制和免疫应答工程的新工具。第三个目标将定义非细胞自主的Hh信号对淋巴发育的影响，在体内通过使用小鼠模型在成骨细胞样基质中操纵Hh信号。因此，该提案的首要主题是确定Hh形态发生途径对B细胞发育的贡献。这些目标的实现将使得能够从造血祖细胞构建用于B淋巴分化的稳健系统，从而克服体液免疫疗法工程化中的关键障碍。

DESCRIPTION (provided by applicant): One obstacle to the robust production and engineering of B lymphocytes is our incomplete understanding of the environmental cues that support B lymphopoiesis. Much attention has been afforded the identification of soluble factors that promote B lymphopoiesis ex vivo, but the requirement for stromal cells remains less well understood. In preliminary work we have made the unexpected observation that non-cell-autonomous Hh signaling in stromal cells promotes B lymphopoiesis from hematopoietic progenitors. Our inference rests on several observations: (1) Hh signaling contributes to the maintenance of B progenitors; (2) cell-autonomous Hh signaling is dispensable for B cell development and function; and (3) B lymphopoiesis is impaired by extinction of Hh signaling in stromal cells, but not in hematopoietic progenitors. Depletion of Smo from stromal cells is associated with coordinate downregulation of genes associated with osteoblastoid identity and B lymphopoietic activity, leading us to propose that Hh pathway activity maintains a stromal phenotype that promotes early B lymphopoiesis. The work proposed under this proposal aims to test this hypothesis, and in so doing to develop deeper mechanistic understanding of non-cell-autonomous signals in B cell development. Under the first aim we will define the developmental block to B lymphopoiesis upon extinction of Hh signaling in stromal cell lines and in primary stroma. In the second aim we will exploit biochemical and in silico methods, extensive genomic resources and robust biological systems to identify and validate novel, Hh-dependent stromal factors that pro- mote B lymphopoiesis in the mouse and human. This goal is of particular importance as it is expected to un- cover new mechanisms by which stromal cells instruct early hematopoiesis and new tools for the engineering of immune responses. The third aim will define the effects of non-cell-autonomous Hh signaling on lymphoid development in vivo through the use of mouse models for manipulation of Hh signaling in osteoblastoid stroma. Thus the overarching theme of this proposal is to define the contribution of the Hh morphogenetic pathway to B cell development. Accomplishment of these aims would enable the construction of robust systems for B lymphoid differentiation from hematopoietic progenitors, thereby overcoming a critical roadblock in the engineering of humoral immunotherapies.