ILC2s are meningeal-resident cells concentrated around the dural sinus. (A) Representative gating of meningeal ILC2s. ILC2s were identified by flow cytometry as CD45⁺/singlets/lineage⁻/viable/ST2⁺/CD25⁺ cells (Lineage cocktail = CD11b, GR1, CD3, B220, FcεRα, Ter-119; representative of three biological replicates). (B) Flow cytometry characterization of meningeal ILC2s (representative of three biological replicates). (C) Counts of mouse ILC2s and ILC3s (CD45⁺/singlets/lineage⁻/viable/RORγt⁺ cells) in healthy mouse brain meninges (P = 0.007; left y axis), spinal meninges (P = 0.015), and spinal cord parenchyma (P = 0.002; right y axis; n = 3; multiple Student’s t tests). (D–G) Characterization of the IL-13^tdt mouse by flow cytometry. (D) Representative flow plots of ILC2 tdTomato expression. Lineage cocktail omitted anti-FcεRα in D and F to allow separate visualization. (E) >95% of ILC2s are tdTomato⁺ in IL-13^tdt mouse brain and spinal cord meninges and lung (n = 3; representative of three experiments). (F) Representative flow plots of tdTomato expression of other meningeal cell types. (G) ILC2s, T cells, and mast cells account for the majority of total tdTomato⁺ cells in brain and spinal cord meninges and lung (n = 3; representative of two experiments). (H–J) Representative images of ILC2s identified as IL-13^tdt+/CD3⁻/toluidine blue⁻ cells in dural sinus (H), dura/arachnoid mater (I), or spinal cord meninges (J) whole mounts (blue arrows represent IL-13^tdt+/CD3⁻/toluidine blue⁻ ILC2s, green arrows represent IL-13^tdt+ or −/CD3⁻/toluidine blue⁻ T cells, and yellow arrows represent IL-13^tdt+/CD3⁻/toluidine blue⁺ mast cells). (K) Quantification of ILC2 localization demonstrates a concentration in the dural sinus versus other meninges areas (P < 0.0001; n = 3–4; one-way ANOVA with Tukey’s multiple comparisons test). (L–N) Mice were treated with IL-33 every other day for 6 d i.p., and on the eighth day, ILC2 numbers were analyzed by flow cytometry. (L) Representative plots demonstrating expansion of ILC2s in IL-33–treated mice relative to PBS treatment (gated on live/CD45⁺/singlet/Lineage⁻ cells). (M and N) Counts demonstrating expansion in brain meninges (M; P = 0.009) and spinal cord meninges (N; P = 0.001; n = 3, representative of three experiments; Student’s t test). Error bars represent mean ± SEM; **, P ≤ 0.01; ***, P ≤ 0.001.