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).