Neutralization by anti-HMGB1 inhibits Treg differentiation in a dose-dependent fashion. (a) Schematic diagram represents HMGB1 neutralization in the coculture (left panel) and the supernatant treatment (right panel) models. (b) The frequency of CD4+CD25+Foxp3+Tregs of CD4+T cells after PBMCs and SK-N-SH cell coculture in the presence of anti-HMGB1 mAb (1, 2, or 3 μg/mL). (c) The frequency of CD4+CD25+Foxp3+Tregs of CD4+T cells after the SK-N-SH supernatant treatment to PBMCs in the presence of anti-HMGB1 mAb (1, 2, or 3 μg/mL). Cells were collected on day 4 for Treg enumeration. Baseline Treg levels were measured in PBMCs culture using complete media at day 4. Goat IgG (1 μg/mL) was served as the isotype control. PBMCs were obtained from 3 healthy individuals. Values represent mean±SD of 3 independent experiments (∗p<0.05).