CD4⁺CD25⁺ cells can be expanded in vitro but retain their suppressive phenotype.

(A) CD4⁺CD25⁺ or CD4⁺CD25⁻ cells were stimulated with immobilized anti-CD3 (1 μg/mL) and anti-CD28 (10 μg/mL) for 3 days, and the cells were washed and rested in medium containing 30 U/mL rh–IL-2 for 2 days. The cells were then stained with FITC- or PE-conjugated monoclonal antibodies as indicated. (B) CD4⁺CD25⁺ or CD4⁺CD25⁻(1 × 10⁴) cells generated as in panel A were cocultured with autologous unstimulated CD4⁺CD25⁻(1 × 10⁴) cells in the presence of autologus ACs (1 × 10⁴) and PHA (2 μg/mL) for 3 days.