Il 10 pe jes5 16e3

Collagenase/DNAse-treated DLN cell suspensions or mASC-cultured DCs were preincubated with anti-FcγRII/FcγRIII mAb at 2.5 μg/mL (2.4G2; BD Pharmingen) and 7-aminoactinomycin D (2 μg/mL; Sigma Aldrich) and then stained with the following antibodies: CD11c-APC (N481; eBioscience), CD40-PE (2/23), CD80-PE (16-10A1), and/or CD86-PE (GL-1) (BD Pharmingen). For intracellular cytokine staining cells were fixed, permeabilized, and stained for IFN-γ-FITC (XMG1.2), IL-17-PE (TC11-18H10), TNF-α-PE (MP6-XT22), IL-4-PE (BVD4-1D11), and IL-10-PE (JES5-16E3) (all from BD Biosciences, San Diego, CA) using the BD cytofix/cytoperm kit (BD Biosciences) according to the manufacturer's instructions. Each sample was stained with appropriate isotype controls. For the analysis of foxp3 expression cells were stained with CD4-FITC (GK1.5; BD Biosciences) and subsequently processed for foxp3 staining using the foxp3 staining kit (eBioscience) according to the manufacturer's instructions. Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences).

Cells in BALF were stained with CD45-FITC (clone 30-F11), CD11b-APC (clone M1/70), CD11c-PE (clone N418), F4/80-PE-CY7 (clone BM8), and Ly6G-BV421 (clone 1A8, BioLegend) in the presence of an Fc blocker (CD16/CD32, BD Biosciences). A total of 2 × 10⁶ single lung cells were stimulated with cell stimulation cocktail and protein transport inhibitor cocktail (Cat#00-4970-03 and #00-4980-93, eBioscience) in IMDM medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin for 4 h in a 37°C incubator with 5% CO₂. The cells were incubated with a Zombie NIR™ Fixable Viability Kit (Cat#423105, BioLegend) at room temperature for 15 min and then stained with antibodies to extracellular antigens in the presence of Fc blocker at 4°C for 25 min. Intracellular and nuclear staining was performed with the Foxp3/Transcription buffer set (Cat# 00-5523-00, eBiosciences). Data were acquired using LSRFortessa and FACSDiva software (BD Biosciences) or Attune NxT 3 L-BRV and Attune NxT software (Thermo Fisher Scientific) and analyzed using FlowJo 10.7.2 (TreeStar, Ashland). The following antibodies were used: CD4-FITC (clone GK1.5), TCR-BV421 (clone H57-597), Foxp3-AF647 (clone MF23), IL-10-PE (JES5-16E3, BD Biosciences), IFN-γ-PE-Cy7 (clone XMG1.2), IL-4-BV605 (clone 11B11), IL-17a-BV510 (clone TC11-18H10.1, Biolegend), and IL-13-PE-eFluor 610 (eBiosciences).