Characterization of the molecular identity and function of human ILC3 employing a novel in vitro differentiation platform

第三组先天淋巴样细胞(ILC)是一个异质性的组织内淋巴细胞家族，具有多向性，有时还具有相反的功能。ILC3可以产生大量的IL-22，在粘膜屏障保护和共生微生物群的动态平衡中发挥重要作用。产生IL-22的ILC3通过调节3型免疫和受损内皮屏障的再生而强烈参与控制炎症。另一方面，ILC3也能产生炎性细胞因子，如TNFpha和IL-17，促进粘膜炎症，并与炎症性肠病有关。第三类ILCs为淋巴组织诱导细胞(LTI)，参与淋巴结的形成。目前，对人类ILC3的研究受到以下事实的严重阻碍：它们主要是组织驻留的，几乎不存在于外周血(PB)中。也没有合适的细胞系。在这方面，我们最近开发了一个体外平台，可以在与人间充质干细胞(MSC)共培养时产生产生IL-22的ILC3、LTI样细胞和NK细胞。脐带血来源的造血干/祖细胞(HSPC)高效地产生IL-22+ILC3是基于MSC的存在，不能仅被小鼠基质细胞或细胞因子所替代。本申请有三个主要目标：1.通过使用scRNAseq在单细胞水平上的表型和分子分析来彻底表征体外产生的ILC3。单细胞分子数据将与ILC3在扁桃体和肠道中的类似scRNAseq数据进行比较。2.体外培养ILCs可塑性的功能和表观遗传学分析为此，ATACseq将评估ILC3和LTI样细胞的表观遗传印记，从而能够在全球范围内分析染色质的可及性。此外，ILC3和LTI样细胞将受到适当的刺激，以诱导转化为ILC1，NK细胞，或促进从IL-22到IL-17的产生。联合分析表观遗传印迹和功能分析将有助于评估体外产生的ILC亚群的可塑性程度。3.最后，这项研究旨在确定调节ILC3活性的微生物区系和饮食来源的线索，这是了解ILC3在肠道中的作用的关键前提。在这方面，新的MSC/HSPC平台能够评估选定的饮食代谢物及其各自的受体在人类ILC3上的作用，这一领域迄今主要局限于小鼠模型。拟议的项目将建立一个在完全人类系统中生成特征良好的效应器ILC3的协议。这可能为肠屏障破坏情况下的基于细胞的治疗开辟新的途径，肠屏障破坏是异基因干细胞移植后移植物抗宿主病、炎症性肠病和艾滋病毒感染的主要并发症。

Group 3 innate lymphoid cells (ILC) are a heterogenous family of tissue-resident lymphocytes with pleiotropic and sometimes opposing functions. ILC3 can produce large amounts of IL-22 and serve important functions in mucosal barrier protection and homeostasis of the commensal microbiome. IL-22-producing ILC3 are strongly implicated in the control of inflammation by regulating type 3 immunity and regeneration of damaged endothelial barriers. On the other hand, ILC3 are also able to produce inflammatory cytokines such as TNFalpha and IL-17 that promote mucosal inflammation and are implicated in inflammatory bowel disease. The third kind of group 3 ILCs are lymphoid tissue inducer cells (LTi) that are involved in formation of lymph nodes. Currently, research on human ILC3 is seriously hampered by the fact that they are primarily tissue-resident and virtually absent from peripheral blood (PB). Suitable cell lines are also not available. In this regard, we have recently developed an in vitro platform enabling generation of IL-22-producing ILC3, LTi-like, and NK cells in coculture with human mesenchymal stem cells (MSC). The efficient generation of IL-22+ ILC3 from cord blood-derived hematopoietic stem/progenitor cells (HSPCs) was based on the presence of MSC and could not be replaced by murine stromal cells or cytokines only. The present application has three primary goals: 1. Thorough characterization of in vitro-generated ILC3 by phenotypic and molecular analysis on the single cell level employing scRNAseq. Single cell molecular data will be compared with similar scRNAseq data from ILC3 in tonsils and intestine. 2. Functional and epigenetic analysis of the plasticity of in vitro-generated ILCs. To this end, epigenetic imprinting of ILC3 and LTi-like cells will be assessed by ATACseq, enabling the analysis of chromatin accessibility on a global scale. Furthermore, ILC3 and LTi-like cells will be subjected to suitable stimuli previously described to induce conversion to ILC1, NK cells, or to promote a switch from IL-22 to IL-17 production. Combined analysis of epigenetic imprinting and functional analysis will help to assess the degree of plasticity of in vitro generated ILC subsets. 3. Finally, the study aims at identifying microbiota- and dietary-derived cues regulating ILC3 activity, a crucial prerequisite for understanding the role of ILC3 in the gut. In this regard the novel MSC/HSPC platform enables to assess the role of selected dietary metabolites and their respective receptors on human ILC3, a field that is so far largely restricted to murine models. The proposed project will establish a protocol for generation of well-characterized effector ILC3 in a fully human system. This could open novel avenues for cell-based therapy in conditions of intestinal barrier breakdown, which is a major complication in graft-versus-host disease following allogeneic stem cell transplantation, inflammatory bowel disease, and HIV infection.