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

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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.