Il 13

Cells suspensions were prepared from lymph nodes and pancreatic islets. Single-cell suspensions were labeled with fluorochrome-conjugated monoclonal antibodies: anti-mouse CD3, CD4, CD8, ST2, and CXCR3 (BD Biosciences), CD11c and CD11b antibodies (BioLegend, San Diego, CA) or with isotype-matched control and analyzed on a FACSCalibur (BD) using CELLQUEST software (BD). The intracellular staining was performed with lymph node cells incubated for 6 h in the presence of Phorbol 12-myristate13-acetate (50 ng/ml) (Sigma, USA), Ionomycin (Sigma, USA) (500 ng/ml), and GolgyStop (BD Pharmingen) at 37°C, 5% CO2, stained with anti-CD4 monoclonal antibodies or appropriate isotype controls, fixed and permeabilized with a Cytofix/Cytoperm solution. Intracellular staining was performed using monoclonal antibodies: IFN-γ, IL-17, IL-10, IL-5, IL-13, IL-2, and Foxp3 (BD Biosciences) or appropriate negative controls. Cells were analyzed with the FACSCalibur Flow Cytometer (BD Biosciences), and analysis was conducted with FlowJo (Tree Star).

The mesenteric lymph node (mLN) from each mouse was removed aseptically. The mLNs were then homogenized into single-celled suspensions; 5 × 10⁶ cells/mL were added to RPMI medium, and cell viability was determined using trypan blue dye exclusion. Cells (200 μL/well) were cultured in the presence or absence of OVA (100 μg/mL) at 37°C for 72 h in a humidified incubator with 5% CO₂ and 95% air. Cytokine assay kits (interferon γ [IFN-γ], IL-4, IL-5, IL-10, and IL-13, BD Pharmingen, San Diego, CA, and IL-17 and TGF-β, R&D Systems, Minneapolis, MN) were used to quantify cytokines following the manufacturer's instructions. The absorbance at 450 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA).

The harvested BMDMs were plated in 6-well plates for overnight incubation. Following incubation cells were either left untreated or stimulated with IFNγ (100 unit/ml, BD Biosciences, San Jose, CA, USA) or IL-4/IL-13 (100 units/ml each, BD Biosciences, San Jose, CA), IL-4 (100 units/ml, BD Biosciences, San Jose, CA, USA), IL-13 (100 units/ml, BD Biosciences, San Jose, CA, USA) and incubated at 37°C under 5% CO_2. At 0, 2, 4, 6, 12, and 24 hours post stimulation, BMDMs were lyzed with 700 μl of Qiazol (Qiagen, Valencia, CA, USA) and stored at minus 80°C for RNA extraction. Total RNA was prepared using miRNAeasy kit (Qiagen, Valencia, CA, USA) and its concentration and quality was measured using nanodrop and bioanalyser, respectively. Total RNA was used for CAGE library preparation.

The culture medium used was X-VIVO media (Lonza, USA) supplemented with 10% human AB serum, 1% GlutaMAX (Invitrogen, USA), 1% sodium pyruvate (Invitrogen, USA), and 1% penicillin/streptomycin (Invitrogen, USA). Fluorochrome-conjugated anti-CD4 was from BioLegend (USA). Fluorochrome-conjugated anti-Ki67, CTLA4, and GITR were from eBioscience (USA). Fluorochrome-conjugated anti-CD45RA, TGF-β, IL-10, IFN-γ, IL-4, IL-5, IL-13, FOXP3, and GATA3 were from BD (USA). PE Annexin V Apoptosis Detection Kit I, Cytofix/Cytoperm Kit, Pharmingen™ Leukocyte Activation Cocktail with BD GolgiPlug™, and Leukocyte Activation Cocktail with BD GolgiPlug were also from BD (USA). Anti-Human CD25 PerCP-Cyanine5.5 was from eBioscience (USA), and anti-Human CD25-PE was from BD (USA). Human Neuropilin-1 PerCP MAb was from R&D (USA). Anti-Human USP21 was purchased from Sigma-Aldrich Co. (USA), and anti-Human PIM2 was from Santa Cruz (USA); the secondary antibodies were from Sigma-Aldrich Co. (USA). Recombinant human cytokine IL-2 was purchased from R&D, and rIL-4 and TGF-β were from PeproTech (USA). Ficoll-Paque PLUS was purchased from GE Healthcare (UK). Human IL-10 ELISA Kit and Human TGF-β1 ELISA Kit were from RayBiotech (USA). Anti-CD3/CD8 Dynabeads were purchased from Invitrogen (USA).

Antibodies used for flow cytometry analysis were as follows: CD3ϵ, CD4, IL-4, IL-5, IL-6, IL-10, IL-13, CD45, CD11b, F4/80, and MHC II purchased from BD Biosciences (Franklin Lakes, New Jersey) and eBioscience (San Diego, California). For staining of cell surface markers, cells (1 x 10⁶) were labelled and washed in PBS containing 1% BSA (Roche, Switzerland) and 0.1% NaN₃ (FACS buffer). For detection of intracellular cytokines, cells were seeded at a density of 2 x 10⁶ cells/well in a complete RPMI culture medium and stimulated with 50 ng/ml phorbol myristate acetate (PMA), 250 ng/ml ionomycin, and 200 µM monensin (all from Sigma) for 6 hr at 37°C in a humidified atmosphere containing 5% CO₂. After the incubation period, cells were harvested, washed, fixed in 2% (w/v) paraformaldehyde, permeabilized with 0.5% saponin buffer, and then stained for cytokine production as previously described (23 (link), 24 (link)). Fluorescence minus one (FMO) was used as a control for intracellular staining whereby all fluorophores were added except one, i.e. cytokine fluorophore, to detect positive signal. Acquisition was performed using BD LSRFortessa (BD Biosciences) and data were analyzed using FlowJo software (Treestar, Ashland, Oregon).