Mixed Lymphocyte Reaction (MLR) Assay

Following the established methodology, single-cell suspensions of splenocytes were obtained from 7-week-old BALB/c mice (Orient Bio, Inc.) [17]. CD4⁺ and CD8⁺ T cells were separated from splenocytes using anti-CD4 or anti-CD8-coated magnetic microbeads (Miltenyi Biotec) and LS columns (Miltenyi Biotec) according to the manufacturer’s protocol. The isolated CD4⁺ and CD8⁺ T cells were stained with CellTrace Violet Cell Proliferation Kit for 20 min at 37°C and washed with 2% FBS in PBS for 10 min. The CellTrace-labeled CD4⁺ and CD8⁺ T cells were cocultured with each group of BMDCs (C57BL/6 background) for 48 h on 1 μg/ml anti-CD3-coated round-bottom 96-well plates in the presence of 1 μg/ml anti-CD28 (DC:T cell ratio 1:5). After coculturing, the cells were stained with FITC-conjugated anti-CD4 and PE-Cy7-conjugated anti-CD8 mAbs for 20 min at 25°C. The proliferation of CD4⁺ and CD8⁺ T cells was detected using flow cytometry. The levels of IFN-γ, IL-2, and IL-5 in the culture supernatants were measured using commercial ELISA kits.