Anti il 4 pe

Anti-CD3 FITC, anti-CD3 APC, anti-CD4 PerCP-cy5.5, anti-CD8-PerCP-cy5.5, anti-CD8 FITC, anti-CD45RO FITC, anti-CD45RO PE, anti-IFN-γ FITC, anti-IFN-γ APC, anti-IL-4 PE and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-22 PE was purchased from R&D Systems (Abingdon, UK). Anti-IL-17 APC was purchased from eBioscience (San Diego, CA, USA). Phorbol myristate acetate (PMA), ionomycin, saponin and Brefeldin A (BFA) were purchased from Sigma-Aldrich (Fluka, Sigma, USA).

Anti-CD4 PerCP, Anti-CD4 PerCP-cy5.5, anti-CD3 APC, anti-CXCR5-Alexafluor 488, anti-CD45RO PE, anti-CCR7 PE, anti-CD28 PE, anti-CD69 PE, anti-HLA-DR PE, anti-IL-21 PE, anti-IL-4 PE, anti-IFN-γ APC and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-17 APC and ELISA for determining IL-21 was purchased from eBioscience (San Diego, CA, USA). Anti-IL-22 PE was purchased from R & D Systems (Abingdon, UK). PMA, ionomycin (INO), saponin and Brefeldin A were purchased from Sigma-Aldrich (Fluka, Sigma, USA).

For the ICS, T cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (2 μg/mL; Sigma-Aldrich) for 5 hrs and with Monensin (1 μl/ml; BD Pharmingen) added for the final 4 hrs. Cells were pre-incubated with mouse IgG (20 μg) to block non-specific binding. Each sample was then labeled with anti-CD4-PerCP-Cy5.5 (1:200 μl) and anti-CD8-PE-Cy7 (1:200 μl) for 30 min. Cells were fixed by addition of 2% paraformaldehyde (100 μl) for 10 min followed by staining buffer (5% BSA and 0.1% sodium azide in PBS). ICS was performed with Fix and Perm cell permeabilization kit (Invitrogen) according to the manufacturer’s instructions. Cells were suspended in permeabilization buffer along with mouse IgG (50 μl). For cytokine staining, cells were incubated with either anti-IL-4-PE or anti-IFN-γ-APC-Cy7 (1:100) for 30 min (BD Biosciences, San Diego, CA). The cells were incubated for 30 min and washed with staining solution (5% BSA and 0.1% sodium azide in PBS). Flow cytometry was performed as described with appropriate gating on the CD4⁺ and CD8⁺ populations.

The immune cell phenotyping and analysis of functional markers were performed by multiparametric flow cytometry analysis. Briefly, cells isolated from the spleen following RBC lysis were incubated at 37°C, 5% CO₂ for 3–4 h with brefeldin A (GolgiPlug). Cells were first blocked using mouse Fc-block (anti-CD16/32) followed by staining for surface markers. Next, cells were washed, fixed, and permeabilized using the Fix-Perm reagent kit (BD Biosciences, Franklin Lakes, NJ, USA) followed by staining for intracellular markers. The following fluorochrome-conjugated anti-mouse antibodies were used: anti-CD11c APC, anti-CD19 BV605, anti-CD44 FITC, anti-CD62L APC-Cy7, anti-granzyme B BV421, anti-IFN-γ BV711, anti-IL4 PE, anti-B220 BUV737, anti-CD3 BV650, anti-CD4 BV786, anti-CD8 BUV396, anti-Foxp3 PE-CF594, and anti-GL7 PerCp-Cy5.5 (BD Biosciences). FACS data acquisition was done on a five-laser Fortessa X-20 flow cytometer (BD Biosciences) and analyzed using FlowJo version 10 (FlowJo LLC, Ashland, OR). Forward and side scatter parameters were used to set singlets and leukocyte gates. Fixable viability stain BV510 included in the surface antibody cocktail was used to gate out dead cells and analyze only viable cells. Overall gating strategy is shown (Supplemental Figure S1).

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Plaque Plus density gradient centrifugation (Amersham Biosciences, NJ, USA). Fresh tissues were washed and cut into small fragments, prior to homogenization by tissue disaggregation vessels (BD Medimachine System). The cells in each sample were adjusted to a concentration of 2 × 10⁶ cells/mL, and 0.5 mL cell suspension stimulated with 2μL Leukocyte Activation Cocktail and BD GolgiplugTM (BD Pharmingen, San Diego, CA, USA) for 4 hours.
T cell subsets were phenotyped in isolated PBMCs or tissue cells by flow cytometry (FACSAria flow cytometer, BD Biosciences, NJ, USA) according to manufacturer’s instructions. The cells were labelled with specific monoclonal antibodies; including anti-CD4-PerCP, anti-CD25-PE, anti- Foxp3-FITC, anti-IL-4-PE and anti-IFN-γ-FITC (all from BD Biosciences, NJ, USA). T cell subsets were selected for detailed phenotypic analysis as follow: (1) Th1 cells: IFN-γ⁺IL-4^- CD4⁺ T cells; (2) Th2 cells: IFN-γ^-IL-4⁺ CD4⁺T cells; (3) Treg cells: CD4⁺CD25⁺Foxp3⁺ T cells. A minimum of 10⁶ cells per staining were assessed, with at least 10⁵ events being measured. To measure CCRs in peripheral Treg cells, anti-CCR3, anti-CCR4, anti-CCR5 and anti-CCR8 (R & D Systems, Mineapolis, MN, USA) were used to evaluate corresponding receptor expression in circulating Treg cells of 3 groups.

For the analysis of Th1, Th2, and Th17 cells, cells in 1 ml of heparin-anticoagulated venous blood were stimulated for 5 h with 10 μl of PMA, 10 μl of ionomycin (final concentration was 750 ng/ml), and 1 μl of GolgiStop, respectively, in a 37°C incubator. Stimulated cells (80 μl) were taken for surface staining, and the cells were fixed and permeabilized using fixation/permeabilization reagent (BD, USA) and then stained with anti-IFN-γ-APC (for Th1), anti-IL-4-PE (for Th2), and anti-IL-17A-PE (for Th17) monoclonal (Supplemental Table 2).
For the analysis of CD4 Tregs, cells in 80 μl of heparin-anticoagulated venous blood were aliquoted into tubes without PMA and ionomycin stimulation and surface-labeled with anti-CD4-FITC and anti-CD25-APC followed by fixation, permeabilization, and intracellular staining with anti-FoxP3-PE (all from BD, USA). The labeled cells were washed and analyzed with a FACSCalibur flow cytometer (Becton-Dickinson) using the CellQuest software (BD, USA). At least 5,000 to 10,000 cells were collected to calculate the percentage of these CD4⁺ T subsets, and the total number of CD4⁺ T cells was calculated using the internal microsphere counting standard.
In this study, we used an equation to calculate the absolute number of CD4⁺ T subsets: the absolute number of CD4⁺ T subsets = the percentage of each CD4⁺ T subset * the absolute number of total CD4⁺ T cells.