iTEC as a new experimental system for TEC biology

项目概要/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细胞，是所有胸腺功能所必需的， 协调胸腺内所有其他细胞类型的组装和分化。遗传途径 这些胸腺上皮细胞（TEC）的特化和分化的基础仍然知之甚少。 然而，已知单个转录因子FOXN 1控制TEC增殖的多个关键方面， 分化和维持在胎儿和出生后胸腺。我们实验室和其他机构的研究表明 Foxn 1在TEC亚群中的作用不同，并且具有令人难以置信的剂量敏感性， 祖细胞足以驱动大多数（如果不是全部的话）TEC分化程序。由于这一核心作用， Foxn 1是通过诱导多能造血干细胞定向分化产生TEC的持续努力中的关键靶点。 干细胞（iPSC）。然而，关于FOXN 1在TEC分化中的确切功能仍存在许多问题 和扩散。虽然TEC功能所需的至少一些FOXN 1目标是已知的，但关键问题是 仍然没有答案，包括建立TEC身份和启动Foxn 1表达的分子途径， 以及FOXN 1在不同TEC亚群中的不同水平如何不同地控制TEC生物学。我们是 一个合作小组的研究表明，FOXN 1在鼠胚胎成纤维细胞（MEFs）中的强制表达， 足以将MEF转化为功能性TEC。这些“诱导的TEC”（iTEC）可以在移植后直接 在体内支持T细胞发育的功能齐全的胸腺器官的组装。iTEC还显示， 有望在二维培养中促进未成熟胸腺细胞分化为单阳性T细胞。 因此，iTEC可以为体外产生自体TEC的长期目标提供一种新的工具， 以产生用于移植的类器官，或用于治疗目的的T细胞的体外产生。更广泛地说， 胸腺生物学领域缺乏用于研究TEC的分子需求的可行的体外培养系统 生物学和分化，或指导T细胞发育和选择的TEC-胸腺细胞相互作用。iTECs 因此，可以提供一个有用的体外系统，用于研究FOXN 1的功能和遗传途径， 控制TEC分化和功能。该提案旨在解决iTEC生成的关键问题 这限制了它作为实验系统的广泛采用。我们提出了三个具体目标，重点是改善 iTEC分化和增殖的控制将使我们能够开发这种方法用于广泛的实验 应用：1）iTEC生成期间的Foxn 1剂量敏感性; 2）促进mTEC的机制 iTEC培养物中的分化;和3）MHCII表达和iTEC增殖。成功完成 拟议的实验将大大改善iTEC的生成和功能，目标是建立 iTECs作为一个急需的体外实验系统，具有广泛的实用性，用于研究FOXN 1功能， TEC生物学、T细胞分化和TEC-胸腺细胞相互作用。

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.