Flow cytometry assay
PBMCs were suspended at a density of 1 × 106 cells/mL and surface-stained with 5 μL of phycoerythrin (PE)-anti-hCD4, allophycocyanin (APC)-anti-hCD56, fluorescein isothiocyanate (FITC)-anti-hNKG2D and isotype control at 4°C in PBS containing 2% FBS in the dark. Each samples was then washed twice for the CD4+NKG2D+ T cell assay. After co-culture, 1 × 106 CD4+ T cells/mL were surface-stained with APC-anti-hCD25 for 30 min at 4°C, fixed, permeabilized with Cytofix/Cytoperm (BD Pharmingen, USA), stained with 20 μL anti-h Foxp3 PE for 30 min in the dark at 4°C, then washed for CD25+ Foxp3+ T cell assay.
To assay intracellular cytokines, 1×106CD4+ T cells/mL were stimulated with phorbol myristate acetate (50 ng/mL) and ionomycin (1 μM) in the presence of monensin (500 ng/mL; all from Sigma-Aldrich, USA) at 37°C in an atmosphere of 5% CO2 for 5 h, then washed, fixed, and permeabilized with Cytofix/Cytoperm and stained with 20 μL anti-hIFN-γ-PE, anti-hIL4-PE, and anti-hIL17-PE for 30 min in the dark at 4°C. The isotype controls were used to enable correct compensation and to confirm antibody specificity.
The expressions of the NKG2D ligands, major histocompatibility complex (MHC) class I-chain-related (MIC)A and MICB, in DCs were analyzed by flow cytometry. DCs were stimulated for 24 h using 20 ng/mL hIL-15 (Peprotech). Five microliters of PE-anti-hMICB or PE-anti-hMICB antibody and appropriately matched isotype controls were added to 1 × 106 DCs for 30 min at 4°C. The cells were washed and analyzed by flow cytometry as describe above. All the antibody and isotype controls were purchased from eBioscience. Fluorescence activated cell sorting was performed using a model LSR-II flow cytometer (BD Biosciences, USA) prior to data analysis with FlowJo software v10 (Tree Star, USA).