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.
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.