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

采用新型体外分化平台表征人 ILC3 的分子身份和功能

• 批准号: 470195722
• 负责人: Professor Dr. Markus G. Uhrberg
• 金额: --
• 依托单位: Institut für Transplantationsdiagnostik und Zelltherapeutika (ITZ)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/470195722?language=en
• 关键词: Characterization; molecular; identity; function; human

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.

第 3 组先天淋巴细胞 (ILC) 是组织驻留淋巴细胞的异质家族，具有多效性和有时相反的功能。 ILC3 可以产生大量的 IL-22，并在粘膜屏障保护和共生微生物群的稳态中发挥重要作用。产生 IL-22 的 ILC3 通过调节 3 型免疫和受损内皮屏障的再生来控制炎症。另一方面，ILC3 还能够产生炎症细胞因子，例如 TNFα 和 IL-17，促进粘膜炎症并与炎症性肠病有关。第三类 3 组 ILC 是参与淋巴结形成的淋巴组织诱导细胞 (LTi)。目前，人类 ILC3 的研究受到严重阻碍，因为它们主要存在于组织中，并且在外周血 (PB) 中几乎不存在。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，能够与人间充质干细胞 (MSC) 共培养，产生产生 IL-22 的 ILC3、LTi 样细胞和 NK 细胞。从脐带血来源的造血干细胞/祖细胞 (HSPC) 中有效生成 IL-22+ ILC3 是基于 MSC 的存在，并且不能仅用小鼠基质细胞或细胞因子替代。本申请具有三个主要目标： 1. 使用 scRNAseq 在单细胞水平上通过表型和分子分析来彻底表征体外生成的 ILC3。单细胞分子数据将与扁桃体和肠道中 ILC3 的类似 scRNAseq 数据进行比较。 2. 体外生成的 ILC 可塑性的功能和表观遗传学分析。为此，ILC3 和 LTi 样细胞的表观遗传印记将通过 ATACseq 进行评估，从而能够在全球范围内分析染色质可及性。此外，ILC3和LTi样细胞将受到先前描述的合适的刺激，以诱导转化为ILC1、NK细胞，或促进从IL-22生产转变为IL-17生产。表观遗传印记和功能分析的结合分析将有助于评估体外生成的 ILC 子集的可塑性程度。 3. 最后，该研究旨在确定调节 ILC3 活性的微生物群和饮食来源的线索，这是了解 ILC3 在肠道中的作用的关键先决条件。在这方面，新型 MSC/HSPC 平台能够评估选定的饮食代谢物及其各自受体对人类 ILC3 的作用，该领域迄今为止主要仅限于小鼠模型。拟议的项目将建立一个在全人类系统中生成特征良好的效应器 ILC3 的协议。这可能为肠道屏障破坏情况下的细胞治疗开辟新途径，肠道屏障破坏是同种异体干细胞移植、炎症性肠病和艾滋病毒感染后移植物抗宿主病的主要并发症。