Analyzing the Heterogeneity of Innate Lymphoid Cells and the Relationship with their Microenvironments in situ and in vivo

天然淋巴样细胞(ILCs)被认为是各种器官中的感受器，以协调组织特异性免疫反应。在细胞因子的刺激下，ILCs是炎症过程中非常敏感的组织感受器，但也对维持组织内环境稳定至关重要。ILC在骨髓中发育。成熟的ILC通过其关键转录因子和细胞因子的表达而归类于ILC1-3，存在于全身各处，尤其是在屏障表面。然而，不同的ILC亚群在某些组织和器官上表现出明显的趋向性。ILC最初被认为主要是组织驻留的免疫细胞群，然而，这些发现最近受到了质疑。这就提出了这样的问题：某些“生态位”是否定义了ILC在不同器官中的微环境；ILC亚群的这些生态位在多大程度上不同；这些生态位的细胞和分子特征是否在动态平衡和炎症条件之间变化；ILC在多大程度上能够适应“新的”组织微环境；以及哪些机制适用于吸引和维持不同组织中的ILC亚群。反过来，所有这些问题都需要在其微环境中对国际法委员会进行现场分析。然而，ILCs与其他免疫细胞，特别是T细胞有许多共同的标志，再加上它们的低丰度，这使得对这些细胞的组织学分析变得非常困难。在之前的资助期间，我们建立了多表位配基图谱，允许在单个组织切片上对数十个标记进行染色，并将该方法与自动图像分析相结合，使我们能够识别ILC亚群，并在小鼠和人类组织中发展ILC。在第二个资助期，我们计划分析发育中和成熟的ILC在小鼠骨髓中的定位、造血细胞和基质微环境，以评估发育中的ILC和各种成熟的ILC亚群是否占据了不同的细胞特异性生态位。这些分析将扩展到其他组织，以便在细胞和分子基础上比较ILC生态位组成。我们将比较动态平衡情况和局部动态平衡状态，并通过活体显微镜分析ILC动力学。此外，我们将确定在体内平衡的系统性扰动下，组织特异性微环境如何塑造ILCs的表型和功能特征。最后，我们将把我们关于ILC生态位组成的发现转化到人类组织中，我们用多重组织学对其进行分析。

Innate lymphoid cells (ILCs) are thought to function as sensors within various organs in order to orchestrate tissue-specific immune responses. With cytokines as stimuli, ILCs are very sensitive tissue sensors during inflammatory processes, but also crucial for the maintenance of tissue homeostasis. ILCs develop in the bone marrow. Mature ILCs, grouped in ILC1-3 by their expression of key transcription factors and cytokines, are present throughout the body, and are especially enriched at barrier surfaces. However, different ILC subgroups show a clear tropism regarding certain tissues and organs. ILCs have initially been regarded to be a mainly tissue-resident immune cell population, however, these findings have been challenged recently. This raises the questions whether certain “niches” define the microenvironment of ILCs in various organs, to what extent these niches differ for ILC subgroups, whether the cellular and molecular characteristics of these niches change between homeostatic and inflammatory conditions, to what extent ILCs can adapt to “new” tissue microenvironments and which mechanisms apply for the attraction to and for the maintenance of the ILC subgroups in various tissues. In turn, all these questions call for an analysis of ILCs in situ, within their microenvironment. However, the fact that ILCs share many markers with other immune cells, especially T cells, in combination with their low abundance, has made histological analyses of these cells very difficult. In the previous funding period, we have established multi-epitope ligand cartography, which allows to stain for dozens of markers on one single tissue section, and combined this method with automated image analysis, which allows us to identify ILC subgroups and also developing ILCs in murine as well as human tissues. In the second funding period, we plan to analyze the localization, hematopoietic neighbour cells and the stromal microenvironment of developing and mature ILCs in the murine bone marrow of mice, to assess whether developing ILCs and various mature ILC subgroups occupy distinct cell-specific niches. These analyses will be extended to other tissues, in order to compare ILC niche composition on a cellular and molecular basis. We will compare the homeostatic situation to conditions of locally disturbed homeostasis, and analyze ILC dynamics by intravital microscopy. Furthermore, we will determine how tissue-specific microenvironments shape the phenotypic and functional characteristics of ILCs under systemic perturbations of homeostasis. Finally, we will translate our findings on ILC niche composition to human tissues, which we analyze by mutliplexed histology.