Phenotype of in vitro–expanded CD4⁺CD25⁺ and CD4⁺CD25⁻ T-cell lines. CD4⁺ T cells were isolated from spleens and pooled LNs of nonmanipulated CBA mice and separated in CD25⁺ and CD25⁻ subsets. (A) Representative flow cytometry histograms showing the level of surface expression of CD25, CD45RB, and CD62L on the selected cells used to generate the T-cell lines. (B) Surface expression of CD25, CD69, CD44, GITR, CD62L, CCR7, CD103, and intracellular expression of CTLA-4 was compared on the in vitro–expanded CD25⁻ (gray line) and CD25⁺ line (black line) with the respective isotype controls (gray area). (C) Foxp3 mRNA was detected by RT-PCR in freshly isolated CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells and in the expanded CD25⁻ and CD25⁺ line. The amount of β-actin mRNA was used as control. The phenotypic characterization of the T-cell lines was carried out more than 6 days after restimulation (ie, in the resting phase). Results are representative of 4 independent experiments.