Peripheral accumulation of human suppressive CD4⁺CD25⁺icFoxP3⁺T cells in treated HIS (BALB-Rag/γ) mice. (A) The percentage of CD25⁺ cells among CD3⁺CD4⁺ T cells (top) and their absolute number (bottom) was monitored in the blood of IgG1 control (▪) and SA anti-CD28-treated mice (○) over a 2-month period (mean ± SEM). Each point shows the mean value obtained from 4 to 9 mice, and this experiment is a representative out of a total of 3. (B) The expression of FOXP3 mRNA was analyzed at day 6 by RT-PCR in sorted human CD4⁺CD25⁺ thymocytes from both control (left) and treated (right) animals. The corresponding numeric results (FoxP3/actin ratio) are shown in the bottom graphs. Representative results from 1 experiment out of 3 are shown, and similar results were obtained from spleens. (C) Thymocytes of control (left) and treated (right) animals were stained for intracellular FoxP3 (icFoxP3) expression, and results show representative staining in gated CD4⁺ cells. (D) CD4⁺CD25high and CD4⁺CD25-thymocytes were sorted from 6-day-treated HIS (BALB-Rag/γ) mice and cultured for suppression assay, as described in “Materials and methods.” Values are representative of 3 independent experiments.