Abstract
Innate lymphoid cells (ILCs) emerge in the last few years as important regulators of immune responses and biological processes. Although ILCs are mainly known as tissue-resident cells, their precise localization and interactions with the microenvironment are still unclear. Here we combine a multiplexed immunofluorescence technique and a customized computational, open-source analysis pipeline to unambiguously identify CD127+ ILCs in situ and characterize these cells and their microenvironments. Moreover, we reveal the transcription factor IRF4 as a marker for tonsillar ILC3, and identify conserved stromal landmarks characteristic for ILC localization. We also show that CD127+ ILCs share tissue niches with plasma cells in the tonsil. Our works thus provide a platform for multiparametric histological analysis of ILCs to improve our understanding of ILC biology.
Highlights
Innate lymphoid cells (ILCs) emerge in the last few years as important regulators of immune responses and biological processes
Repetitive incubation— imaging—bleaching cycles with directly coupled fluorescence antibodies were performed by a pipetting robot, in order to generate a stack of 53 images from the same field of view (FOV)
While B cells, T cells, and myeloid cells represented rather large cell populations, each accounting for 10–30% of total cell numbers, plasma cells and, especially, CD127+ ILCs were found in very low numbers (Figs. 3c and 4c)
Summary
Innate lymphoid cells (ILCs) emerge in the last few years as important regulators of immune responses and biological processes. Helper ILCs are mainly known to be tissue-resident cells[8], to be abundant in barrier sites, and to act as sensors for tissue integrity, both promoting inflammation or tissue regeneration and wound healing[1,9,10,11] In line with these functional characteristics, their microanatomical localization within and across tissues should fulfill particular needs and, histological approaches are needed to further understand ILC biology in context, as recent reports have pointed out[12,13]. Image data acquisition is combined with a customized computational analysis pipeline This allows us to unambiguously identify CD127+ ILCs in situ, while further characterizing these cells and their microenvironments, and defining potential microanatomical and molecular fingerprints characteristic for ILC localization and function, in various chronically inflamed tissues. The workflow used for the analysis of high-dimensional image data can be adapted for the in situ identification and in-depth characterization of several immune cell types, as well as for mapping particular tissue niches
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