Transcriptional Profiling of CD200R-Dependent ILC2 Activation

Freshly sorted ILC2s after three intranasal administrations of rmIL-33 (0.5 μg per mouse), defined in this study as IL-33-activated ILC2s (aILC2s), were incubated with rmIL-2 (10 ng/mL) and rmIL-7 (10 ng/mL) with CD200-Fc or the isotype control for 24 h (5 × 10⁴ mL⁻¹). Total RNA was isolated using MicroRNAeasy (Qiagen). scRNA sequencing data [GSE102299] for mouse and human samples were obtained as described before^{17 (link),44 (link)}. In total, 10 ng of input RNA was used to produce cDNA for downstream library preparation. Samples were sequenced on a NextSeq 500 (Illumina) system. Raw reads were aligned, normalized, and further analyzed using Partek Genomics Suite software, version 7.0 Copyright; Partek Inc. Normalized read counts were tested for differential expression using Partek’s gene-specific analysis (GSA) algorithm leveraging lognormal with shrinkage^{45 (link)}. To explore expression gradient and narrow z-score range, we separately analyzed CD200R⁺ and CD200R^- ILC2s. During the analysis of CD200R⁻ ILC2s, we assigned z-scores of zero (black) to all ILC2s without any CD200R mRNA. In a second parallel analysis of CD200R⁺ ILC2s, we assigned z-scores of zero (black) to ILC2s with the lowest non-zero CD200R expression level (z-score of 5.22 in our data set) using Partek Flow.