Abstract

Group 3 innate lymphoid cells (ILC3s) are critical mediators of innate immune responses at mucosal barriers in both health and disease. ILC3s rapidly respond to environmental cues to reinforce barrier function and foster a mutualistic microbiota. ILC3s are defined by the expression of the master transcription factor RORγt, but can be further subdivided by the surface expression of the chemokine receptor CCR6 or the natural killer cell-associated receptor NKp46, as well as through the coexpression of the transcription factor T-bet. Importantly, while these subsets exhibit overlapping functions such as the secretion of the cytokines IL-17A and IL-22, they also differ significantly transcriptionally, functionally and by their localization within tissues. Thus, it is critical that studies investigating ILC3 biology consider the heterogeneity and tissue specificities of these subsets. Here, we describe common tools used to dissect and characterize ILC3s subset phenotypes and functions by flow cytometry and strategies for cell sorting of these cells in both the gastrointestinal tract and associated lymph nodes. Together, these approaches provide a tool kit for researchers aiming to dissect ILC3 subset responses at homeostasis, during infection, or in the context of inflammation.