Genome-wide methylation analysis of LN ILCs. (A) Characterization of ILC2 in different LNs. Cells were isolated from inguinal (ingLN) or mesenteric (mLN) lymph nodes of Gata3 reporter mice (Gatir mice; Rao et al., 2020). ILC2 were gated as Lin⁻CD127⁺Gata3-YFP^high cells and further discriminated by expression of ST2, IL17RB, and Klrg1. Data pooled from n = 2 independent experiments with n = 2 mice per group. Statistical significance was analyzed using unpaired two-tailed Student’s t test with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B) Sorting strategy for ILC WGBS. ILCs from pooled murine pLNs of WT C57BL/6J mice were sorted by indicated marker. Lineage markers (Lin) included CD3 and CD19. NK cells (Lin⁻CD127⁻NK1.1⁺Tbet⁺), ILC1 (Lin⁻CD127⁺Tbet⁺Gata3⁻RORgt⁻), ILC2 (Lin⁻CD127⁺Tbet⁻Gata3⁺RORgt⁻), ILC3 (Lin⁻CD127⁺CCR6⁻Tbet⁻Gata3⁻RORgt⁺), and LTi cells (Lin⁻CD127⁺CCR6⁺Tbet⁻Gata3⁻RORgt⁺). (C) Purity reanalysis of sorted ILCs. (D) Unsupervised hierarchical clustering of the top 1,000 DMRs among ILC subsets. The color represents the degree of mean methylation value, ranging from yellow (methylation level = 0) to blue (methylation level = 100%).