(A) PBMC preparations were obtained from 10 healthy individuals and cultured in the presence or absence of the indicated cytokines (25 ng/ml) for 24 hours. CD4⁺CD25⁺ Tregs were used as a reference. The resulting cells were analyzed for the expression of Foxp3 by real-time PCR and immunoblot. The real-time PCR histogram represents analysis of 10 individual specimens. Relative change in Foxp3 expression in immunoblot is presented in folds (intensity of experimental band/intensity of control band). (B) Purified CD4⁺CD25^– T cell preparations (n = 10) were cultured in the presence of IFN-γ at the indicated concentrations for 24 hours and measured for Foxp3 expression. (C) CD4⁺CD25^– T cells treated in the presence or absence of IFN-γ (25 ng/ml) were analyzed for intracellular Foxp3 expression by flow cytometry. CD4⁺CD25⁺ Tregs were used as a reference. (D) CD4⁺CD25^– T cells were cultured in the presence of IFN-γ and the indicated antibodies (10 μg/ml). The resulting T cells were analyzed for mRNA expression of Foxp3 by real-time PCR. Horizontal line represents the level of Foxp3 expression in untreated CD4⁺CD25^– T cells. (E) Purified CD4⁺CD25^– T cells were treated with IFN-γ under the experimental conditions described above. The resulting T cells were FACS sorted and assayed for inhibitory activity on the proliferation of autologous CD4⁺CD25^– T cells. Results are expressed as mean percentage inhibition ± SEM from 5 independent experiments. Asterisks indicate that differences between groups are statistically significant; *P < 0.05.