Fig. 2.

Assessment of BM−DC maturation and OVA presentation induced by treatment with (OVA)S^−/+(CpG) or S^−/+(OVA−CpG) in vitro. (A) Effect of (OVA)S^−/+(CpG) or S^−/+(OVA−CpG) on BM−DC maturation. BM−DCs were incubated for 24 h with 5 μg/ml CpG, OVA, (OVA)S^−/+(CpG) or S^−/+(OVA−CpG), each contained 5 μg/ml OVA. BM−DCs were stained with fluorescently labelled specific antibodies against MHC I, MHC II, CD40, CD80 or CD86, and cell analysis was performed using flow cytometry. The mean fluorescence intensity (MFI) of the positive CD11c−expressing BM−DCs was measured to assess the fold change in the expression of each marker with respect to the naïve BM−DCs, results represent the mean ± S.D. (B, C) OVA presentation by (OVA)S^−/+(CpG) or S^−/+(OVA−CpG) treated BM−DCs. BM−DCs were incubated for 24 h with OVA + CpG, OVA−CpG, (OVA)S^−/+(CpG) or S^−/+(OVA−CpG), each contained 5 μg/ml OVA. Treated BM−DCs were co−cultured with CD4⁺ or CD8⁺ T cells isolated from the spleen of OT−2 or OT−1 C57BL/6 mice, respectively, at 1:4 ratio for 3 days. On the last 18 h of incubation, CD4⁺ T cells (B, left) or CD8⁺ T cells (B, right) were pulsed with 1 μCi of ³H−thymidine and the proliferation was measured using ³H−thymidine incorporation assay. The content of IFN−γ in the supernatants of the proliferating CD4⁺ T cells (C, left) or CD8⁺ T cells (C, right) was quantified using ELISA. Measurements were performed in triplicates for each condition, results represent the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001.