FIGURE 1.

Both CD25⁺ and PI-T_Reg cells maintain their anergic phenotype following expansion in IL-2 in vitro. CD25⁺ and PI-T_Reg cells were cultured with IL-2 and Ac1-9 (10 μg/ml) for 5 days and CD4⁺ cells were purified using magnetic beads. Purified CD25⁺ (A) or PI-T_Reg cells (B) were cultured alone (1), with Ac1-9 peptide (2), with irradiated CD4-depleted splenocytes as APC (3), or with irradiated APC and Ac1-9 peptide for 72 h before addition of tritiated thymidine. Proliferation of T_Reg cells was compared with the response of naive Tg4 cells to Ac1-9 and APC (5). CD4⁺ T cells were cultured at 5 × 10⁴ cells per well with 1 × 10⁵ cells per well of APC in vitro with 100 μg/ml Ac1-9. Results are depicted as mean thymidine incorporation from triplicate samples ± SEM. The experiment has been repeated three times with similar results.