Tissue-niches and cellular interactions of mouse and human ILCs at single-cell resolution

新出现的证据表明，驻留在特定组织中的ILC原则上是由不同的细胞来源(即本地居民细胞与招募细胞)在发育过程中的不同时间点(例如胚胎、新生儿、成人)产生的。ILC的局部池在炎症和感染期间会发生实质性的变化，我们才刚刚开始了解不同亚型的组织ILC之间的关系、它们的“分工”以及它们的局部相互作用。随着单细胞技术的出现，我们已经开始破译ILC的异质性。然而，由于缺乏能够在其组织背景下高分辨率描绘细胞类型的实验方法，ILC的组织微环境在很大程度上仍未被探索。组织壁龛的分子调控，例如通过旁分泌信号，可能影响祖细胞的维持和局部分化，并控制ILC的组织适应以促进特定的功能。揭示驻留在给定组织中的不同ILC亚型的微环境的细胞类型组成，将有助于纠正亚型或分化阶段特异性的生态位，并测试参与这些细胞相互作用的分子。这些依赖于环境的机制的研究具有挑战性，尤其是非人类组织，迫切需要新的方法来验证小鼠模型的发现并在患者样本中解决这些问题。我们在第一个资助期间的发现强调，通过结合单细胞RNA-Seq、分化轨迹的预测和疾病模型的体内验证，我们能够推断ILCs的早期分化阶段，并揭示这些细胞的发育和功能异质性以及在免疫挑战过程中发生的动态变化。我们现在已经开发出新的方法，将单细胞RNA-Seq在细胞类型鉴定中的能力与单分子FISH的空间分辨率相结合，跨&gt；100基因多路复用，以便原位绘制细胞类型图。在此基础上，我们建议研究人和小鼠ILC及其在肝脏内动态平衡、组织损伤和修复过程中的原位组织背景。该项目的基本目标是揭示功能上不同的亚型及其分化动力学，并确定细胞间串扰在健康和疾病中的出现和调节中所涉及的机制。为此，我们将在肝组织损伤和再生模型中ILCs的体内实验研究、scRNA-seq经验、深入的生物信息学分析和肝脏微环境的空间重建方面建立我们的联合已有专业知识。

Emerging evidence suggests that ILCs residing in a given tissue canin principle be generated from different cellular sources (i.e. localresident versus recruited cells) and at different timepoints duringontogeny (e.g. embryonic, neonatal, adult). The local pools of ILCscan undergo substantial changes during inflammation and infection.We are only beginning to understand the relationship ofheterogeneous subtypes of tissue-ILCs, their “division of labor” aswell as their local interactions. With the advent of single-celltechnologies we have started to decipher the heterogeneity of ILCs.However, the tissue microenvironment of ILCs remains largelyunexplored due to the lack of experimental methods enabling thehigh-resolution profiling of cell types in their tissue context. Molecularcontrol exerted by tissue-niches, e.g. via paracrine signaling, likelyaffects the maintenance and local differentiation of progenitor cells,and controls tissue-adaptation of ILCs to facilitate particular functions.Revealing the cell type composition of the microenvironment fordistinct ILC sub-types residing in a given tissue context would allow topredict sub-type- or differentiation stage-specific niches and to testmolecules involved in these cellular interactions. These contextdependentmechanisms are challenging to study, particularly inhuman tissues, and novel approaches to validate findings from mousemodels and to address these questions in patient samples areurgently needed. Our findings obtained during the first funding periodunderscore that by combining single-cell RNA-Seq, the in silicoprediction of differentiation trajectories and the in vivo validation indisease models, we are able to infer early differentiation stages ofILCs and to reveal the developmental and functional heterogeneity ofthese cells as well as the dynamic changes occurring during immunechallenge. We have now developed novel approaches to combine thepower of single-cell RNA-Seq in cell type identification with the spatialresolution of single-molecule FISH, multiplexed across >100 genes, inorder to map cell types in situ. On this basis, we propose toinvestigate human and mouse ILCs and their in situ tissue-context inthe liver during homeostasis, tissue-damage and repair. The essentialgoal of the project is to reveal functionally distinct sub-types and theirdifferentiation dynamics and to identify the mechanisms of intercellularcross-talk involved in the emergence and regulation of thesepopulations in health and disease. To this end, we will build on ourcombined established expertise in the experimental in vivo study ofILCs in models of hepatic tissue damage and regeneration and ourexperience in scRNA-seq, in-depth bioinformatical analyses andspatial reconstruction of the liver microenvironment.