Both CD4⁺CD25⁺CD62L⁺ and CD4⁺CD25⁺CD62L^- T cells show Treg cell characteristics in vitro. (A) Alloresponse of C57BL/6-derived CD4⁺CD25^- T cells and CD4⁺CD25⁺CD62L⁺ and CD62L^- T cells toward BALB/c APC in vitro. Cultures were set up with 100 000 BALB/c-derived APC and 100 000 sorted CD4⁺CD25^- T cells from C57BL/6 mice plus variable numbers of C57BL/6-derived CD4⁺CD25⁺CD62L⁺ (▨) or CD62L^- T cells (▪) to obtain the indicated ratios. CD4⁺CD25⁺CD62L⁺ and CD62L^- T cells were also stimulated alone. Proliferation was assessed by labeling the cultures with ³H-thymidine for the final 16 hours of the 5-day incubation period. Data represent mean + SD of triplicate cultures. Shown is 1 of 5 experiments with similar results. (B) Sorted CD4⁺CD25⁺CD62L^- (▪), CD4⁺CD25⁺CD62L⁺ (▨), and CD4⁺CD25^- T cells (□; 25 000) were stimulated for 24 hours with 50 ng/mL PMA and 1 μM ionomycin. IL-2 in the supernatant was determined by ELISA. The results represent mean + SD of triplicate cultures. (C) cDNA was prepared from cell populations sorted as in panel B and analyzed for expression of FoxP3 by real-time quantitative PCR using β-actin as normalizing gene.