Il 4 pe

T-lymphocytes were studied in PBMCs by nine-color flow cytometry. PBMCs were incubated with the next surface-labeled monoclonal-antibodies, CD3-PercP, CCR7-PECY7 (Becton-Dickinson, BD, CA, USA), CD8-Alexa405, CD45RA-APC (Caltag, Carlsbad, CA, USA) and CD27-APCAlexa780 (eBioscience, San Diego, CA, USA).
For intracytoplasmic staining, cells were fixed and permeabilized (Fix and Perm, Caltag, Carlsbad, CA, USA), and cytokines were stained with IL-4-PE, IFNγ-Alexa700 and IL-17A-FITC (Becton-Dickinson, BD, CA, USA). All samples were stained with a dead cell-discriminator simultaneously with antibody addition (Fixable aqua dead cell stain kit for 405 nm excitation; Molecular Probes, Eugene, OR, USA).
Samples were acquired in a FacsAria-II flow cytometer and were analyzed using FacsDiva 5.0 and Flow-Jo 7.0 software (Becton-Dickinson, BD, San Jose, CA, USA).

PBMCs and other cells were stained with anti-human mAbs: anti-CD3 FITC, anti-CD4 BV605, anti-CD8 BV421, anti-CD14 BV711, anti-CD19 BV605, anti-CD1d APC, anti-HLA-DR BV421, IFN-γ PE, and IL-4 PE (all from BD Biosciences, Vienna, Austria). APC-labeled human CD1d tetramers loaded with the α-GalCer analogue PBS-57 were obtained from the MHC Tetramer Core Facility (Emory University Vaccine Center, Atlanta, GA). Anti-human ADRB2 (AbD Serotec, Oxford, UK) was coupled with alexa fluor647 fluorochrome by using a protein labeling kit (Life Technologies, Paisley, UK). Viability was assessed with Zombie Nir viability dye (BioLegend, London, UK) or with fixable viability dye eFluor506 (eBiosciences, Vienna, Austria). The corresponding isotype control mAbs were used to assess staining specificity.

Monocyte-derived DCs were stimulated with plate-bound FhTE in the presence or absence of LPS (10 ng/ml). After 48 h, expression of costimulatory molecules was measured by flow cytometry using the following antibodies: anti-CD86 (BU63), -CD83 (HB15e), -HLA-DR (L203), -CD40 (5C3), and -OX40L (Ik-1).
After stimulation, cells were washed and cocultured with allogenic naïve CD4 T cells (CD4⁺ CD45RA⁺, ratio 1:10), purified by MACS Beads (Miltenyi), in the presence of Staphylococcal Enterotoxin B (10 pg/ml, Sigma). On day 5, supernatants were harvested (for evaluation of IFNγ) and replaced with rhuIL-2 (100 U/ml, immunotools). Primed CD4⁺ T cells were stimulated with a cocktail containing 100 ng/ml Phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin, and 10 μg/ml brefeldin A for 5–6 h. The cells were washed, fixed, and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) and subsequently stained with a combination of IL-4-PE and IFN-γ-FITC antibodies (BD Biosciences).

An additional experiment was designed and performed to evaluate CD4+ T‐cell phenotypes. The mouse spleen was acquired under sterile condition, cut into small pieces and filtered with 70‐μm strainer to remove the capsule and connective tissue. The cell supernatant was collected and centrifuged at 1500 rpm for 10 min., with thrice washes atTris‐NH4 cl. FACSAria II flow cytometry (BD Biosciences, San Diego, CA, USA) was used to test spleen CD4+ T‐cell subgroups in animals mentioned above. The CD4+ T cells were isolated by flow cytometry and then were labelled with IFN‐γ‐PE, IL‐4‐PE and Foxp3‐PE antibodies (BD). The proportion of CD4 + IFN‐γ+ T cells, CD4 + IL‐4+ T cells and CD4 + Foxp3+ T cells was accounted. The mRNA expression of T‐bet, GATA‐3 and Foxp3 in lung tissues harvested from various groups was measured on basis of gene probes as listed in Table 1, using Rotor‐Gene 3000 fluorescence ration PCR instrument (Corbett Research, Sydney, Australia).

Intracellular flow cytometry was utilized to determine the percentage of IFN-γ, IL-4, IL-17 and IL-10-producing lymphocytes within the CD4⁺subset in 10 patients, in both IC and PB samples. Briefly, heparinized whole blood was diluted 1:1 with RPMI medium (Sigma-Aldrich Co., St Louis, MO, USA), and cells were stimulated for 5 h at 37 °C, in 5 % CO2 atmosphere with 0.5 ng/ml ionomycine (Sigma-Aldrich) and 1 γg/ml phorbol myristate acetate (PMA; Sigma-Aldrich) in the presence of 5 mg/ml Brefeldin A (Sigma-Aldrich) to induce cytokine production (Grille et al. 2010 (link)). Lymphocytes were then stained with anti-human CD4 and anti-human CD3 (BD Pharmingen, San Diego, USA). Red blood cells were lysed with FACS Lysing Solution (BD Biosciences), and lymphocytes were permeabilized with FACS Permeabilizing Solution (BD Biosciences). To detect intracellular cytokines, cells were stained with the following cytokine-specific antibodies: anti-human IL-17-PE, IFN-γ-APC, IL-4-PE and IL-10-APC (BD Pharmingen, San Diego, USA), washed, and fixed with 1 % paraformaldehyde. Data acquisition and analysis was performed as previously described. A gate was set on CD4⁺CD3⁺ lymphocytes and at least 5000 cells were counted and evaluated for the expression of IFN-γ, IL-17, IL-4 and IL-10.