Novel regulation of Notch-induced HSPC expansion

项目摘要 我们的实验室开发了新的培养方法，使用工程Notch配体来增加细胞数量 CD34+前体细胞，包括那些能够提供快速造血植入的患者 脐带血移植。临床试验表明，需要获得更大的造血干细胞和 祖细胞(HSPC)的数量为一种更有效、经济可行的治疗方法。因此，这笔赠款 旨在阐明调节Notch诱导的信号强度以最大化HSPC生长的新策略 利用1)HSPC亚群与2)Notch受体激活敏感性的差异 促进HSPC扩增与T细胞分化所需的信号强度。为了实现这个目标，我们 已经确定针对Notch胞外区氨基末端产生的抗体比 比Notch配体更有效地激活原始HSPC上的受体，导致In的生成增加 体内重新繁殖细胞；初步研究表明，这是由于抗体介导的颠覆 内源性表达的配体对原始HSPC的抑制作用(顺式抑制)。因此，在AIM中 1，我们验证了我们的假设，即不同的激动剂在促进Notch激活和HSPC生成方面的效力 结果由配体介导的顺式抑制。我们将研究抑制性配体在CB中的表达和功能 HSPC采用shRNA敲除和/或阻断抗体治疗，同时进行移植检测。至 阐明抗体颠覆顺式抑制的机制，指导改良的未来发展 激动剂，我们将确定目标表位位置的贡献和激动剂亲和力的增加。我们的 以前的研究也确定了对不同水平的Notch信号产生的要求 HSPC对T细胞分化的促进作用。因此，在目标2中，我们测试了我们的假设，即使用Paralog- 特定的Notch抗体激动剂将微调Notch的激活水平，以同时增强 骨髓和胸腺再生细胞的生成。我们将研究动态使用低密度的 缺口类对数特异性抗体激动剂通过诱导进一步增加骨髓再生细胞生成 在顺式抑制的原始前体中的缺口激活，但保持低水平的缺口信号强度以 防止这些细胞向T细胞转移。我们进一步探讨了Paralog特异性抗体激动剂 通过诱导不同水平的Notch同时产生骨髓和胸腺再生细胞 在不同的造血亚群中的激活。总体而言，这些研究将提供对细胞的洞察- 自主调节Notch受体功能的机制--一个具有概念和实践意义的成果 对于那些专注于操纵Notch依赖的干细胞类型的细胞命运决定的人来说，这里最相关的是， HSPC和前胸腺细胞前体细胞的扩增，以开发可行的临床治疗方法。

Project Summary Our laboratory has developed novel culture methods using engineered Notch ligands to increase the number of CD34+ precursors, including those able to provide rapid hematopoietic engraftment in patients undergoing cord blood (CB) transplantation. Clinical trials indicate the need to derive greater hematopoietic stem and progenitor cell (HSPC) numbers for a more potent, economically feasible therapy. Consequently, this grant aims to elucidate new strategies to regulate Notch-induced signal strength to maximize HSPC growth, focusing on exploiting differences in 1) Notch receptor susceptibility to activation among HSPC subsets and 2) Notch signal strength necessary to promote HSPC expansion versus T cell differentiation. Towards this goal, we have determined that antibodies raised against the amino-terminus of the Notch extracellular domain are more effective than Notch ligands in activating receptors on primitive HSPC, leading to increased generation of in vivo repopulating cells; preliminary studies have suggested this results from antibody-mediated subversion of inhibitory effects imposed by endogenously expressed ligands in primitive HSPC (cis-inhibition). Thus, in Aim 1, we test our hypothesis that differential agonist potency in promoting Notch activation and HSPC generation results from ligand-mediated cis-inhibition. We will investigate inhibitory ligand expression and function in CB HSPC using shRNA knockdown and/or blocking antibody treatment together with transplantation assays. To elucidate the mechanism whereby antibody subverts cis-inhibition, guiding the future development of improved agonists, we will determine the contribution of target epitope location and increased agonist affinity. Our previous studies have also determined requirements for distinct levels of Notch signaling for generation of HSPC versus promotion of T cell differentiation. Thus, in Aim 2, we test our hypothesis that the use of paralog- specific Notch antibody agonists will fine-tune the level of Notch activation to concurrently enhance the generation of marrow and thymic repopulating cells. We will investigate the dynamic use of low densities of Notch paralog-specific antibody agonists to further increase marrow repopulating cell generation by inducing Notch activation in cis-inhibited primitive precursors, yet maintain a low level of Notch signal strength to prevent diversion of these cells to the T-lineage. We further explore whether paralog-specific antibody agonists can simultaneously generate marrow and thymic repopulating cells by inducing different levels of Notch activation in different hematopoietic subsets. Overall, these studies will provide insight into the cell- autonomous mechanisms regulating Notch receptor function, an outcome of conceptual and practical interest to those focused on manipulating cell-fate decisions in Notch-dependent stem cell types, most relevant here, the expansion of HSPC and prothymocyte precursors for the development of viable clinical therapeutics.