Anti il 9

Cryosections of colonic tissue were stained with H&E [haematoxylin [DAKO] and eosin [Sigma-Aldrich]]. For immunofluorescence staining, tissue sections were fixed in 4% paraformaldehyde/PBS or methanol, followed by Protein Block [DAKO]. For intracellular markers, sections were permeabilised with perm buffer [Ebioscience] for 20 min at 18°C. Staining was performed with the following primary antibodies at 4°C overnight: anti-CD3 [Ebioscience, 1:50], anti-IL-9R [Acris, 1:50], anti-Ki67 [DAKO, 1:50], anti-PU.1 [ThermoScientific, 1:50], anti-IL-9 [Biolegend, 1:50], anti-CD4 [Biolegend, 1:200], anti-IL-6 [Biolegend, 1:50], anti-claudin2 [1:200, Invitrogen], anti-claudin3 [1:100, Invitrogen], anti-SOCS3 [Invitrogen, 1:50], and anti-pSTAT3 [Cell Signaling, 1:50]. For staining of human cryosections the following antibodies were used: anti-CD3 [Ebioscience, 1.50], anti-IL-9 [BioLegend, 1:50], anti –PU.1 [ThermoScientific, 1:50], and anti-CD4 [Biolegend, 1:50]. Sections were then incubated with the secondary antibodies anti-rabbit ^Alexa488, anti-rabbit ^Alexa594, anti-rat ^Alexa488, or anti-rat ^Alexa555 [Invitrogen] and counterstained with DAPI [Vektor]. Image acquisition was performed on a confocal microscope [Leica]. Positive cells in high-power fields were counted in all samples per condition. Antibodies used are registered in Supplementary Figure 2].

We used flow cytometry analysis to assess the production of FOXP3, Helios, GATA3, IL-9, IL-17A, and IL-10 by CXCR6+ and CD4+ T cells. Briefly, splenocytes were incubated with phorbol 12-myristate 13-acetate (PMA)/ionomycin (Sigma-Aldrich) for 4 h in the presence of brefeldin-A (GolgiPlug, BD Biosciences), which prevents the transport of cytokines and transcription factors out of the cell [12 (link),33 (link),41 (link)]. Cells were washed and surface stained for CD4, and CXCR6 surface receptors (BioLegend, San Diego, CA, USA). After permeabilization and fixation (BioLegend), the cells were stained with intracellular cytokines (anti-IL-9, anti-IL-10, and anti-IL-17A; BioLegend) and transcription factors (anti-FOXP3, anti-GATA3, and anti-Helios; BioLegend). The proportions of CXCR6+FOXP3+, CXCR6+Helios+, CD4+GATA3+, CD4+IL-9+, CXCR6+IL-10+, and CD4+IL-17A+ cells were acquired via a FC 500 flow cytometer and analyzed using CXP software (Beckman Coulter, Indianapolis, IN, USA).

We used rabbit anti-ESAT-6 antibody (Abcam, Cambridge, MA), mouse anti-His_6X antibody (Sigma-Aldrich, St. Louis MA), anti-FLAG antibody (Sigma-Aldrich, St. Louis MA), HRP conjugated anti-mouse IgG (Bio-rad, Hercules, CA), anti-rabbit FITC conjugated secondary antibody (Jackson-ImmunoResearch, West Grove, PA) and anti-mouse Texas Red secondary antibody (Jackson-ImmunoResearch, West Grove, PA) for in vitro studies. For in vivo studies, anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5 or -APC, anti-CD8 (clone: 53–6.7)-FITC, -PerCP-Cy5 or -APC, anti-CD44 (clone: IM7)-APC, anti-Brdu (clone: Bu20a)-PE, anti-CD11b (clone: M1/70)-APC, anti-CD11c (clone: N418)-APC, 7AAD, anti-IFN-γ (clone: XMG1.2)-APC, anti-IL-17 (clone: TC11-18H10.1)-PE, anti-IL-4 (clone: 11B11)-PE, anti-IL-6 (clone: MPS-20 F3)-PE, anti-IL-12 (clone: C15.6)-PE, anti-IL-22 (clone: Poly5164)-PE, anti-IL-10 (clone: JES5-16E3)-PE, anti-IL-9 (clone: MH9A4)-PE, anti-TNF-α (clone: MP6-XT22)-PE, (all from Biolegend, USA) and anti-CD69 (clone: H1.2 F3)-PE (from eBiosciences, USA) were used.