Flow cytometric analyses were performed using the following antibodies: anti-CD4, anti-CD11b, anti-CD33, anti-CD83, anti-CD86, anti-CD206 (BD Biosciences), anti-CD14, anti-CD58, anti-CD80, anti-HLA-DR (ImmunoTools), anti-CD16 (Thermo Scientific), anti-CD25, anti-GARP (eBioscience) and anti-Rab-32 (Abnova).
For intracellular staining of Foxp3, cells were fixed and permeabilized using a Fix/Permeabilization kit (eBioscience) and stained with anti-Foxp3 mAb (BD Biosciences). Cytokine expression was analyzed in T cells re-stimulated with 50 ng/ml PMA plus 1 μg/ml Ionomycin for 5 h in the presence of Monensin (1.3 μM) 7 days after in vitro primary stimulation (day 0). Cells were then permeabilized as above and stained with anti-IL-2, anti-IFN-γ or anti-granzyme B mAb (BD Biosciences). Flow cytometry was performed on an LSRII FACS and FACSCalibur (BD Biosciences), using FlowJo software (Tree star).
For intracellular staining of Foxp3, cells were fixed and permeabilized using a Fix/Permeabilization kit (eBioscience) and stained with anti-Foxp3 mAb (BD Biosciences). Cytokine expression was analyzed in T cells re-stimulated with 50 ng/ml PMA plus 1 μg/ml Ionomycin for 5 h in the presence of Monensin (1.3 μM) 7 days after in vitro primary stimulation (day 0). Cells were then permeabilized as above and stained with anti-IL-2, anti-IFN-γ or anti-granzyme B mAb (BD Biosciences). Flow cytometry was performed on an LSRII FACS and FACSCalibur (BD Biosciences), using FlowJo software (Tree star).