Gene expression analysis of adult lung ILC2s

To fully characterize the heterogeneity and gene expression profile of mouse lung ILC2s, we generated RORα lineage tracer mice by crossing RoraIRES-Cre (Chou et al., 2013) and R26R-EYFP mice, which have a loxP-flanked stop sequence followed by the gene encoding yellow fluorescent protein (YFP) in the ROSA26 locus. In these RORα-YFP mice, cells expressing Rorα during their development should be irreversibly labeled by YFP. Flow cytometric analyses of lymphocytes in RORα-YFP mice showed that innate lymphoid cells type 2 (ILC2s) were marked by YFP expression. However, both adult and neonatal lung Lin−YFP+ cells were heterogeneous and included not only ILC2s (GATA-3+Thy1+) but also GATA-3−Thy1− and GATA-3lowThy1+ cells. In order to fully assess the lung ILC2 heterogeneity, we analyzed all CD45low/+Linlow cells from adult (6-week-old) and neonatal (12-day-old) RORα-YFP mouse lungs by droplet-based scRNA-seq. Unsupervised clustering of the cells revealed distinct clusters of stromal, B, T, NK cell, DC and ILC2 based on their expression of well-characterized marker genes. We then isolated the ILC2 cluster and performed a second round of unsupervised clustering to examine the existence of possible ILC2 subsets. In both adult and neonatal datasets, ILC2s were divided into subsets with each subset expressing a distinct set of ILC2-associated genes. We validated the existence of distinct ILC2 subsets in adult and neonatal lungs using flow cytometry. We also comprehensively assessed the genes that were specifically and/or differentially expressed by ILC2s compared to other immune and non-immune cell populations in our datasets. This analysis revealed the expression of well-characterized ILC2 genes such as Gata3, Rora, Areg and Il7r as well as other genes with potential immune regulatory functions. Examination of adult lung ILC2 heterogeneity specific gene expression profile.