PBMC activation.
The levels of IL-17A– and IL-22–producing T cells were evaluated by intracellular staining or by ELISA, as previously described (de Beaucoudrey et al., 2008). In brief, PBMCs were purified by centrifugation on a gradient (Ficoll-Paque PLUS; GE Healthcare) and resuspended in RPMI supplemented with 10% FBS (RPMI/10% FBS; Invitrogen). Adherent monocytes were removed from the PBMC preparation by incubation for 2 h at 37°C, under an atmosphere containing 5% CO2. For the ex vivo evaluation of IL-17– and IL-22–producing T cells by flow cytometry, we resuspended 5 × 106 nonadherent cells in 5 ml RPMI/10% FBS in 25-cm2 flasks and stimulated them by incubation with 40 ng/ml PMA (Sigma-Aldrich) and 10−5 M ionomycin (Sigma-Aldrich) in the presence of a secretion inhibitor (1 µl/ml GolgiPlug; BD) for 12 h. For the evaluation of the IL-17– and IL-22–producing T cell blasts after in vitro differentiation, the nonadherent PBMCs were dispensed into 24-well plates at a density of 2.5 × 106 cells/ml in RPMI/10% FBS and activated by incubation with 2 µg/ml of an antibody directed against CD3 (Orthoclone OKT3; Janssen-Cilag) either alone or together with 20 ng/ml IL-23 (R&D Systems), 50 ng/ml IL-6 (R&D Systems), 10 ng/ml IL-1β (R&D Systems), or combinations of these three cytokines. After incubation for 3 d, the cells were restimulated in the same activation conditions, except that the anti-CD3 antibody was replaced with 40 IU/ml IL-2 (Proleukin i.v.; Chiron). We added 1 ml of the appropriate medium, resuspended the cells by gentle pipetting, and then split the cell suspension from each well into two. Flow cytometry was performed on one of the duplicated wells 2 d later, after stimulation by incubation for 12 h with 40 ng/ml PMA and 10−5 M ionomycin in the presence of 1 µl/ml GolgiPlug. Cells were washed in cold PBS, and surface labeling was achieved by incubating the cells with VioBlue-anti–human CD3 (Miltenyi Biotec) or FITC-anti–human CD4 (BD) antibody in 2% FBS in PBS for 20 min on ice. The cells were then washed twice with 2% FBS in cold PBS, fixed by incubation with 100 µl Cytofix for 30 min on ice, and washed twice with Cytoperm (Cytofix/Cytoperm Plus fixation/permeabilization kit; BD). The cells were then incubated for 1 h on ice with Alexa Fluor 488–conjugated anti–human IL-17A (eBioscience), IL-17F PE-conjugated anti–human IL-22 (R&D Systems), or PE-conjugated anti–human IFN-γ (R&D Systems) antibodies, washed twice with Cytoperm, and analyzed with a FACSCanto II system (BD).The contents of the other duplicated well were split into two, with one half left unstimulated and the other stimulated by incubation with 40 ng/ml PMA and 10−5 M ionomycin for another 2 d. Supernatants were collected after 48 h of incubation for ELISA.