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组先天淋巴样细胞（ILC）是具有多效性，有时相反功能的组织居民淋巴细胞的异源家族。 ILC3可以产生大量的IL-22，并在共生微生物组的粘膜屏障保护和稳态中发挥重要作用。产生IL-22产生的ILC3与调节3型免疫力和受损内皮屏障的再生有关，与控制炎症有关。另一方面，ILC3还能够产生炎症细胞因子，例如TNFALPHA和IL-17，它们促进粘膜炎症，并与炎症性肠病有关。第三种第3组ILC是参与淋巴结形成的淋巴组织诱导细胞（LTI）。目前，对人ILC3的研究主要是居住在组织居民，几乎没有外周血（PB）的事实受到了严重阻碍。合适的细胞系也不可用。在这方面，我们最近开发了一个体外平台，从而使IL-22产生的ILC3，LTI样和NK细胞与人间充质干细胞（MSC）产生。从脐带血造血干/祖细胞（HSPC）中有效产生IL-22+ ILC3是基于MSC的存在，不能仅被鼠基质细胞或细胞因子所取代。本应用具有三个主要目标：1。通过SCRNASEQ对单细胞水平的表型和分子分析对体外生成的ILC3的彻底表征。将单细胞分子数据与扁桃体和肠中ILC3的类似SCRNASEQ数据进行比较。 2。对体外生成的ILC的可塑性的功能和表观遗传分析。为此，将通过ATACSEQ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样细胞将受到以前描述的合适刺激，以诱导转化为ILC1，NK细胞，或促进从IL-22到IL-17产生的转换。表观遗传印记和功能分析的结合分析将有助于评估体外产生的ILC子集的可塑性程度。 3。最后，该研究旨在鉴定调节ILC3活性的微生物群和饮食中的线索，这是了解ILC3在肠道中的作用的关键先决条件。在这方面，新型的MSC/HSPC平台可以评估所选饮食代谢产物及其各自受体在人ILC3上的作用，该领域迄今已限于鼠模型。拟议的项目将建立一种在完全人类系统中生成良好特征效应的ILC3方案。这可能在肠道屏障崩溃的情况下为基于细胞的疗法开辟了新的途径，这是同种异体干细胞移植，炎症性肠病和HIV感染后移植物抗宿主疾病的主要并发症。