Multiplexed histology analyses for the phenotypic and spatial characterization of human innate lymphoid cells.

Pascual-Reguant, Anna; Köhler, Ralf; Mothes, Ronja; Bauherr, Sandy; Hernández, Daniela C; Uecker, Ralf; Holzwarth, Karolin; Kotsch, Katja; Seidl, Maximilian; Philipsen, Lars; Müller, Werner; Romagnani, Chiara; Niesner, Raluca; Hauser, Anja E

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.