iTEC as a new experimental system for TEC biology

项目摘要/摘要 胸腺是T细胞生成和形成适应性免疫反应所需的关键器官。 一种关键的细胞类型，内胚层来源的胸腺上皮，是所有胸腺功能和 协调胸腺内所有其他类型细胞的组装和分化。遗传途径 这些胸腺上皮细胞(TECs)的规范和分化的基础仍然知之甚少。 然而，已知单个转录因子FOXN1控制TEC增殖的多个关键方面， 胎儿和出生后胸腺的分化和维持。我们实验室和其他实验室的研究表明 Foxn1在TEC亚群中的作用是不同的，并且对剂量非常敏感，它在TEC中的表达 祖细胞足以推动TEC差异化计划的大部分(如果不是全部)。因为这个中心角色， FOXN1是通过诱导多能性定向分化产生TEC的持续努力中的一个关键靶点 干细胞(IPSCs)。然而，关于FOXN1在TEC分化中的精确功能仍然存在许多问题 和扩散。虽然TEC功能所需的至少一些FOXN1目标是已知的，但关键问题 仍然没有答案，包括建立TEC身份和启动Foxn1表达的分子途径， 以及不同TEC亚群中不同水平的FOXN1如何不同地控制TEC生物学。我们是一个 合作小组发现FOXN1在小鼠胚胎成纤维细胞(MEF)中的强制表达是 足以将MEF转化为功能性TEC。这些“诱导的TECs”(ITECs)在移植后可以直接 支持体内T细胞发育的功能齐全的胸腺器官的组装。ITEC还展示了 在二维培养中促进未成熟胸腺细胞分化为单一阳性T细胞的前景。 因此，iTEC可能为在体外产生可使用的自体TEC的长期目标提供一种新的工具 用于移植或用于治疗目的的T细胞的体外生成。更广泛地说， 胸腺生物学领域缺乏一个可行的体外培养系统来研究TEC的分子需求。 生物学和分化，或TEC与胸腺细胞的相互作用，指导T细胞的发展和选择。ITEC 因此，可以为研究FOXN1的功能和遗传途径提供一个有用的体外系统 控制TEC的分化和功能。本提案旨在解决iTEC生成的关键方面 这限制了它作为实验系统的更广泛采用。我们提出了三个专注于改进的具体目标 对iTEC分化和增殖的控制，将使我们能够发展这种方法进行广泛的实验 应用：1)Foxn1在iTEC生成过程中的剂量敏感性；2)促进mTEC的机制 (3)MHCII表达与iTEC增殖。圆满完成 拟议的实验将大大改善iTEC的生成和功能，目标是建立 ITECs AS为研究FOXN1功能和功能的急需的体外实验系统 TEC生物学，T细胞分化，以及TEC与胸腺细胞的相互作用。

Project Summary/Abstract The thymus is the key organ required for T cell generation and the formation of an adaptive immune response. One key cell type, endoderm-derived thymic epithelium, is required both for all thymus functions and to orchestrate the assembly and differentiation of all other cell types within the thymus. The genetic pathways underlying the specification and differentiation of these thymic epithelial cells (TECs) are still poorly understood. However, a single transcription factor, FOXN1, is known to control multiple key aspects of TEC proliferation, differentiation, and maintenance in both the fetal and postnatal thymus. Work from our lab and others has shown that Foxn1 acts differentially in TEC subsets and is incredibly dosage-sensitive, and that its expression in TEC progenitors is sufficient to drive most if not all of the TEC differentiation program. Because of this central role, Foxn1 is a key target in ongoing efforts to generate TEC by the directed differentiation of induced pluripotent stem cells (iPSCs). However, many questions remain about the precise FOXN1 functions in TEC differentiation and proliferation. While at least some of the FOXN1 targets required for TEC function are known, key questions remain unanswered, including the molecular pathways that establish TEC identity and initiate Foxn1 expression, and how diverse levels of FOXN1 in different TEC subsets differentially control TEC biology. We were part of a collaborative team that showed that enforced expression of FOXN1 in murine embryonic fibroblasts (MEFs) is sufficient to convert MEFs into functional TEC. These “induced TECs” (iTECs) can, upon transplantation, direct the assembly of a fully functional thymus organ that supports development of T cells in vivo. iTECs also show promise in promoting differentiation of immature thymocytes into single positive T cells in 2-dimensional culture. iTECs may thus provide a novel tool for long-term goals of generating autologous TEC in vitro that could be used to generate organoids for transplant, or for in vitro generation of T cells for therapeutic purposes. More broadly, the field of thymus biology lacks a viable in vitro culture system for studying the molecular requirements for TEC biology and differentiation, or TEC-thymocyte interactions that direct T cell development and selection. iTECs thus could provide a useful in vitro system for studying both FOXN1 function and the genetic pathways that control TEC differentiation and function. This proposal is designed to address key aspects of iTEC generation that limit its broader adoption as an experimental system. We propose three specific aims focused on improving the control of iTEC differentiation and proliferation that will allow us to develop this method for broad experimental applications: 1) Foxn1 dosage sensitivity during iTEC generation; 2) mechanisms to promote mTEC differentiation in iTEC cultures; and 3) MHCII expression and iTEC proliferation. Successful completion of the proposed experiments will substantially improve iTEC generation and function, with the goal of establishing iTECs as into a much-needed in vitro experimental system with broad utility for studying FOXN1 function and TEC biology, T cell differentiation, and TEC-thymocyte interactions.