Fc block

For flow cytometry experiments, SI organoids were generated from crypts extracted from Lgr5-GFP mice. After 4 days of treatment, organoids were harvested and washed 5 times with cold PBS-BSA 0.1%. Organoids were disaggregated to single cells by incubating with TrypLE supplemented with DNase I for 5 min at 37 °C. Cells were incubated in Fc block (1:1000; Invitrogen, Cat. No. 14-0161-85) and Fixable Viability Dye eFluor 780 (1:1000; eBioscience, Cat. No. 65-0865-14) for 15 min at 4 °C, and subsequently stained with anti-Epcam PE-Cy7 (1:200; BioLegend, Cat. No. 118215), anti-CD24 BV421 (1:200; BioLegend, Cat. No. 101825) and WGA Alexa Fluor 555 (1:5000; Invitrogen) for 1 h at 4 °C. Flow cytometry was performed using an LSRFortessa flow cytometer (BD Biosciences, USA), and analysis was perfumed using FlowJo v10 (Treestar, USA).

Flow cytometry was performed as previously described^{80 (link)}. In short, tissues were cut into 1 mm pieces and digested in collagenase/dispase (Roche) and DNase I (Roche) in Ca2⁺- and Mg2⁺-free Hanks medium plus 5% FCS for 30 min at 37 °C with gentle shaking. Samples were then vortexed for 15 s, filtered and washed in PBS plus 5% FCS. Single cell suspensions were blocked with FC block (Invitrogen) for 20 min at 4 °C, before staining with fluorophore-conjugated primary antibodies (Supplementary Table 3) for 20 min at 4 °C in the dark. Cells were washed twice and re-suspended in PBS supplemented with 5% FCS prior to analysis with either an Aria III cell sorter or BD FACS Canto.
Isotype antibodies (Supplementary Table 3) and fluorescent-minus-one (FMO) controls were used to estimate background fluorescence in combination with either compensation beads and/or unstained controls. Dead cells were detected and excluded from analysis using Sytox Blue or Fixable Viability Dye, eF506. Tuft cells were identified as EpCAM⁺CD45^-/lowCD24⁺SiglecF⁺ (Supplementary Fig. 8a). Inflammatory ILC2s were identified as ST2⁻KLRG1⁺CD90.2⁺Lineage⁻(CD11b⁻CD11c⁻CD19⁻Ly-6G⁻NK1.1⁻CD3^-)CD45⁺. Natural ILCs were identified as ST2⁺KLRG1⁺CD90.2⁺Lineage^-CD45⁺(Supplementary Fig. 8b). All experiments were analyzed with FlowJo software (Version 10).

2.5 x 10⁵ lung cells were restimulated ex vivo in 250 µL Iscove’s modified Dulbeccos’s Medium (IMDM; Thermo Fisher Scientific Inc.) with Phorbol 12-myristate 13-acetate (PMA)/ionomycin (all Sigma-Aldrich Inc.; 50 ng/mL and 500 ng/mL) and 1x Brefeldin/Monensin (Sigma-Aldrich Inc.) for 4 h at 37°C. Cells were centrifuged (400g, 3 min) and the pellet was resuspended in PBS/bovine serum albumin (BSA; 0.5 %; Sigma-Aldrich Inc.). Nonspecific antibody binding was blocked using anti-CD16/CD32 Mouse BD Fc-Block™ (Becton, Dickinson Inc.) for 15 min at 37°C. Cells were stained with anti-CD90.2-BV605 (clone 53-2.1, BioLegend Inc.), anti-TCRβ-AF700/AF488 (clone H57-597, BioLegend Inc.) and anti-CD3ϵ-APC-Cy7 (clone eBio500A2, Invitrogen Inc.) for 30 min at 37°C. Cells were washed and BD Horizon Viability Dye V500 (Becton Dickinson Inc.) was added in PBS in darkness for 15 min at RT. Cells were washed and then fixed/permeabilized using Cytofix/Cytoperm™ (Becton Dickinson Inc) in darkness for 60 min at RT. After additional blocking with Fc-Block, intracellular staining with anti-IL-17A-PE (clone eBio17B7; Invitrogen Inc.) for 30 min at 4°C was performed. Flow cytometric measurements were performed using a BD™ LSR II flow cytometer (Becton Dickinson Inc.). IL-17A⁺ cells were analyzed as depicted in Supplementary Figure 1.