Flow cytometry sample preparation:

For cell sorting for single cell RNA sequencing, meningeal cell suspensions were incubated with Fc block (2.4G2) and stained with the following antibody solution in FWB (45 min at 4°C): CD45-BUV395, CD31-AF488, CD11b-PB, PDGFRa-APC, gp38-PECy7, DAPI (Antibody details in Key Resources Table). Cells were washed in FWB and fluorescence activated cell sorting was performed on a BD FACS Aria Fusion. Cells were gated for viable single cells and were enriched for lymphocyte and stromal populations by sorting CD45⁺CD11b- (non-myeloid immune cells) and gp38⁺CD31- (stromal cells) separately, and adding each of those populations to 25% of total cells (1:1:2 ratio of lymphocytes: stroma: total viable populations, Fig. S1A). Genotypes of mice sequenced were IL-33 deficient (Il33^{mcherry/mcherry}) or heterozygous controls (Il33^mcherry/+). Meninges from both sexes were pooled by genotype to obtain technical replicates and sufficient numbers of cells for sorting. Il33^wt/mCherry controls included 5 females and 7 males, and Il33^{mCherry/mCherry} knockouts included 7 females and 8 males.

For immunophenotyping over development, flow cytometric analyses were performed. For cytokine reporter tracking, cell suspensions were incubated with a viability dye (Zombie-NIR, 20 minutes), followed by Fc block (2.4G2) and stained with the following antibody solution in FWB (45 min at 4°C): CD45-BUV395, CD3e-AF700, CD4-BV711, CD8a-BV785, Thy1.2-BV421, CD11b-BV605, NK1.1-BV650, CD19-PEDazzle 594, CD11c-PeCy7, CD25-PerCPCy5.5, Hu-CD4-APC (Antibody details in Key Resources Table), followed by wash and resuspension in FWB prior to analysis. Intracellular stains were combined with intravenous labelling of blood circulating cells. Three minutes prior to euthanasia, mice were administered intravenous CD45.2- APCCy7 to label blood circulating cells. Cell suspensions were incubated with a viability dye (Live/Dead Aqua, 20 minutes), followed by Fc block (2.4G2) and surface marker staining with the following antibody solution in FWB (45 min at 4°C): CD45-BUV395, CD3e-AF700, CD4-BV711, CD8a-BV785, Thy1-BV421/PB, CD11b-BV605, TCR γ/δ-PerCPCy5.5, NK1.1-BV650, CD19-PEDazzle 594 (Antibody details in Key Resources Table). Cells were washed in FWB and fixed and permeabilized (60 min at RT) using the FoxP3/transcription factor staining buffer set (eBioscience), followed by intracellular staining with the following antibody solution in 1x PermBuffer (eBioscience) supplemented with 5% rat serum): Rorγt-APC, EOMES-FITC, Tbet-PeCy7, GATA3-PE (60 min at 4°C). Cells were washed two times in 1x PermBuffer and resuspended in FWB prior to analysis. Flow cytometric analysis was performed on a BD Fortessa and data analysis was performed using FlowJo^™ software (BD).