Characterizing human ILC development from early hematopoietic progenitors: regulation by intrinsic and extrinsic signals

先天淋巴样细胞（ILC）是缺乏抗原特异性受体的早期淋巴细胞，可以发挥T辅助细胞样（ILC1-3）或细胞毒性效应细胞（NK细胞）的功能。在人类中，ILC祖细胞和导致1、2和3组ILC特异性的分子事件尚不清楚。除了ILC3p外，其他ILC亚群的祖群体到目前为止只在小鼠中被描述。准确鉴定人类ILC祖细胞的一个主要挑战是提供合适的发育壁龛，以模拟体内环境，并提供必要的细胞接触依赖和独立的外部信号。在这方面，人类ILC分化的研究迄今为止是基于小鼠干细胞壁龛或无基质培养系统，这可能无法提供存在于各自位置（如人类骨髓）的足够的物种特异性环境信号。在这个项目中，作为DFG优先计划先天淋巴样细胞的一部分，我们希望在骨髓和脐带血中识别早期人类ILC祖细胞，并利用新的人类共培养模型探索它们的分化潜力。在这方面，我们最近开发了一种体外分化方案，利用人间充质干细胞（MSC）来支持NK细胞在完全人类系统中的发育。因此，我们希望利用来自骨髓、脐带血和扁桃体的MSC来比较不同生态位条件对早期HPC的ILC发育的影响。通过在扁桃体间充质干细胞上分化早期BM祖细胞，我们希望模拟体内发现的条件，假设BM祖细胞被转运到外周淋巴器官，在那里它们将获得向ILC效应亚群分化所需的信号。我们希望通过RNAseq监测这些培养物的早期转录变化，并确定由生态位条件变化诱导的调控模块。此外，我们希望解决两个转录因子E4BP4/NFIL3和ID2的作用，这两个转录因子通过调节其在该系统中的表达在ILC的发展中起关键作用。除了增加对人类ILC发育调控的基础知识外，该研究将为在没有异种饲养细胞的情况下从造血祖细胞体外分化和扩增人ILC提供明确的条件。通过这种方式，该项目将有助于制定适当的方案，用于生产临床级产品，例如从脐带血中生产，以便将来将ILC用作细胞疗法。

Innate lymphoid cells (ILC) are early acting lymphocytes that lack antigen-specific receptors and either exert T helper-like (ILC1-3) or cytotoxic effector functions (NK cells). In humans, ILC progenitors and the molecular events leading to the specification of group 1, 2, and 3 ILCs are not well understood. Except the ILC3p, progenitor populations for other ILC subsets were so far only described in the mouse. One major challenge for the faithful identification of human ILC progenitors is to provide suitable developmental niches, which mimic the in vivo environment and provide the necessary cell-contact dependent and independent extrinsic signals. In this regard, studies of human ILC differentiation are so far based on murine stem cell niches or stroma-free culture systems, which might not provide the adequate species-specific environmental signals that are present in the respective locations such as human bone marrow (BM). In this project, as part of the DFG Priority Program Innate Lymphoid Cells, we would like to identify early human ILC progenitors in BM and cord blood and explore their differentiation potential using novel human co-culture models. In this regard, we have recently developed an in vitro differentiation protocol employing human mesenchymal stem cells (MSC) to support NK cell development in a fully human system. We would thus like to employ MSC from bone marrow, cord blood, and tonsils to compare how ILC development from early HPC is influenced by different niche conditions. By differentiating early BM progenitors on tonsil MSC we hope to mimic the conditions found in vivo assuming that BM progenitors are trafficking to peripheral lymphoid organs where they would get the necessary signals for differentiation towards ILC effector subsets. We would like to monitor early transcriptional changes in these cultures by RNAseq and identify regulatory modules that are induced by variation in niche conditions. Furthermore, we would like to address the role of the two transcription factors E4BP4/NFIL3 and ID2, which play key roles in ILC development by modulating their expression in this system. Besides increasing basic knowledge about the regulation of human ILC development, the study will provide defined conditions for in vitro differentiation and expansion of human ILC from hematopoietic progenitors without xenogeneic feeder cells. In this way, the project will help to develop suitable protocols for production of clinical-grade products, for example from cord blood, for the future use of ILC as cellular therapeutics.