Objective    Intranuclear forkhead/winged helix transcription factor p3(Foxp3) plays a key role in T cell-mediated immunosuppression. The present study was performed to investigate the effects of high mobility group box-1 protein (HMGB1) on Foxp3 gene as well as protein expressions in splenic regulatory T cells (Tregs) and their potential regulating mechanisms in mice.     Methods    CD4

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CD25

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Tregs isolated from the spleens of male BABL/c mice by magnetic beads were seeded on 96-we

ll (1×10

5

cells/well) cell culture plates coated with anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml)

and the cells were stimulated with HMGB1 at various intervals or at different concentrations. After being stimulated

the Foxp3 mRNA/protein expressions in the Tregs were determined. The time-dependent and dose-dependent responses between HMGB1 and intranuclear Foxp3 expression were analyzed by flow cytometry

and the expressions of Foxp3 mRNA of Tregs were analyzed by quantitative PCR of SYBR GREEN.     Results    After stimulation with HMGB1

the intranuclear Foxp3 protein and mRNA expressions of splenic Tregs in mice were markedly down-regulated in 24 h to 72 h (P0.05 or 0.01)

and the expression levels of Foxp3 protein and Foxp3 mRNA were lowest at 72 h (P0.01). When Tregs were cultured in the presence of 10

100

and 1 000 ng/ml HMGB1 for 72 h

the expressions of Foxp3 protein and mRNA were significantly down-regulated (P0.05 or 0.01)

and values of this molecules were lowest in 1 000 ng/ml HMGB1-treated group (P0.01).      Conclusion    These data indicate that HMGB1 stimulation can result in significant down-regulation of the Foxp3 mRNA and protein expressions in mouse splenic Tregs. HMGB1 appears to be a potential immunoregulatory signal that influences the function of Tregs by inhibiting CD4

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CD25

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Treg activity.