Mixed Leukocyte Reaction (MLR) Analysis

The stimulatory capacity of DCs was tested by mixed leukocyte reaction (MLR) analysis. DCs pretreated with mitomycin C (25 μg/mL, Amresco, USA) were co-cultured with splenic naive T cells from Balb/c mice at DC: T-cell ratios of 10:1, 20:1 and 40:1 for 72 h in RPMI 1640 with 10% FBS at 37°C. The total volume was adjusted to 200 μL/well. Then, Cell Counting Kit (CCK-8; Tongren, Japan) was added to the medium for 4 h before the end of culture, and T cell group (without dendritic cells incubated together) and only RPMI 1640 with 10% FBS medium group were established as negative control group and background group. Absorbance (A) at a wavelength of 450 nm was measured using a Microplate reader (Bio-Rad, iMark™). The results were expressed as the stimulation index (SI), calculated by the following formula: stimulation index = (absorbance of the sample − the background absorbance)/(absorbance of the negative control − the background absorbance).

To evaluate secretion of cytokines (IL‐10, IL‐17, IFN‐γ) by DCs, the culture supernatants were collected after 72 h and cytokine concentrations were measured using ELISA kits according to the manufacturer’s protocols.