Figure 1

HMGB1 induced DC maturation and activation. Immature splenic DCs were cultured with HMGB1 at the indicated doses (100 and 1000 ng/ml) or without HMGB1 (0 ng/ml, as control) for 48 hours. (a1) Flow cytometry gating strategy for CD11c⁺DC cells with the purity of 95.58% after twice MS colume separations. (a2) Data shown are representative histograms of CD80, CD86 and MHC II, using FACS with APC-, PE- or FITC-conjugated antibodies, respectively. The gate was set according to the data of unstained negative group (Isotype control). (a3) The percentage of DCs expressing CD80, CD86, and MHC II was significant elevated after 100 or 1000 ng/ml HMGB1 stimulating and peaked in the concentration of 100 ng/ml. Results of 3 independent experiments were shown as the mean ± SEM. (b) The levels of IL-12 and TNF-α secreted by DCs stimulated with HMGB1 were measured via ELISA. Results of 3 independent experiments were shown as the mean ± SEM. T cells were incubated with ConA (5 μg/ml) for 24 hours, and co-cultured with stimulated DCs at a ratio of 1:100. (c1) A CCK-8 cell proliferation assay was used to assess T-cell proliferative activity after co-culture for 68 hours. (c2) Meanwhile, using ELISAs, the levels of IFN-γ in culture medium were determined to evaluate Th1 and Th2 polarization. (c3) The levels of IL-2 in cell culture supernatants were also measured. Results of 3 independent experiments were shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 ng/ml group.