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

先天性淋巴样细胞（ILC）是早期作用的淋巴细胞，其缺乏抗原特异性受体并且发挥T辅助细胞样（ILC 1 -3）或细胞毒性效应子功能（NK细胞）。在人类中，ILC祖细胞和导致第1、2和3组ILC特化的分子事件还没有很好地理解。除ILC 3 p外，其他ILC亚群的祖细胞群迄今仅在小鼠中描述。忠实鉴定人ILC祖细胞的一个主要挑战是提供合适的发育小生境，其模拟体内环境并提供必要的细胞接触依赖性和独立的外源性信号。在这方面，迄今为止，人类ILC分化的研究是基于鼠干细胞小生境或无基质培养系统，其可能无法提供存在于相应位置如人骨髓（BM）中的足够的物种特异性环境信号。在这个项目中，作为DFG优先计划先天类造血细胞的一部分，我们希望在BM和脐带血中鉴定早期人类ILC祖细胞，并使用新型人类共培养模型探索其分化潜力。在这方面，我们最近开发了一种采用人间充质干细胞（MSC）的体外分化方案，以支持NK细胞在全人类系统中的发育。因此，我们想从骨髓，脐带血，扁桃体MSC比较ILC的发展从早期HPC是如何受到不同的生态位条件的影响。通过分化扁桃体MSC上的早期BM祖细胞，我们希望模拟体内发现的条件，假设BM祖细胞被运输到外周淋巴器官，在那里它们将获得向ILC效应子亚群分化的必要信号。我们希望通过RNAseq监测这些培养物中的早期转录变化，并确定由小生境条件变化诱导的调控模块。此外，我们想解决两个转录因子E4 BP 4/NFIL 3和ID 2的作用，这两个转录因子通过调节它们在该系统中的表达在ILC发展中起关键作用。除了增加有关人ILC发育调控的基础知识外，该研究还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提供确定的条件。通过这种方式，该项目将有助于开发用于生产临床级产品的合适方案，例如从脐带血中生产，以便将来将ILC用作细胞治疗剂。

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.