The suppression of responder T cells mediated by CD4⁺CD25^hiFoxP3⁺ Treg cells is due to the induction of G₀/G₁ cell-cycle arrest. (A) Isolation of CD4⁺CD25^hiFoxP3⁺ Treg cells and CD4⁺CD25⁻ effector T cells from PBMCs of healthy donors by FACS sorting. FoxP3 expression in the isolated T cells was further confirmed by FACS analyses. (B) Relative FoxP3 methylation levels of different T cells were determined by real-time quantitative PCR with methylation-specific primers, normalized to β-actin expression, and compared with the expression level of methylated FoxP3 in CD4⁺CD25⁻ T cells. CD4⁺CD25⁻ and anti-CD3–activated CD4⁺ T cells were included as controls. All experiments were performed in triplicate. (C-D) Suppression of naive T-cell proliferation by CD4⁺CD25^hiFoxp3⁺ Treg cells. CD4⁺CD25⁻ effector T cells served as a negative control displaying no suppressive activity. Naive CD4⁺ T cells were cocultured with Treg cells or control T cells at a ratio of 10:1. The proliferation of naive CD4⁺ T cells in the presence of anti-CD3 Ab was determined by [³H]-thymidine incorporation assays (C) or CFSE dilution assays (D). (E) Suppression of naive CD4⁺ T-cell proliferation mediated by CD4⁺CD25^hiFoxp3⁺ Treg cells is not due to the induction of apoptosis. Naive CD4⁺ T cells were cocultured with CFSE-labeled Treg cells or CD4⁺CD25⁻ cells in the presence of plate-bound anti-CD3 Ab. Apoptosis in naive CD4⁺ T cells was analyzed after staining with PE-labeled annexin V and 7-amino-actinomycin D gating on CFSE⁻ cell populations. (F) CD4⁺CD25^hiFoxP3⁺ Treg cells promoted the accumulation of naive CD4⁺ T cells in G₀/G₁ cell-cycle arrest. Cell treatment was the same as in panel E. Cell-cycle distribution in naive CD4⁺ T cells was analyzed after incubation with 1 μg/mL of propidium iodide and 100 μg/mL of RNase A. Naive CD4⁺ T cells cocultured with or without CD4⁺CD25⁻ T cells served as controls. Data are representative of 3 independent experiments with similar results.