Location of FoxP3⁺ Tregs in rats receiving CsA treatment.

<p>(a) Representative dot plots of donor-specific FoxP3⁺ Tregs in peripheral blood and spleen from allograft recipients in groups 5 and 6 at the end of the observation period. Mononuclear cells were obtained by Ficoll solution centrifugation, followed by magnetic isolation of CD4+ T cells. Isolated CD4+ T cells were then co-stained with anti-rat mAb RT1ⁿ-FITC and intracellularly stained with anti-mouse/rat Foxp3-PE. All cells were analyzed by flow cytometry. The results demonstrated that donor-specific FoxP3⁺ Tregs were exclusively obtained in both the peripheral blood and spleens of long-term allograft recipients in group 5. (b) Further analysis indicated an average proportion of 10.7%±1.1% versus 7.6%±1.6% and 12.6%±0.7% versus 8.3%±1.1 FoxP3⁺ Tregs located in the peripheral blood and spleen in group 5 and 6, respectively, with significant differences (n = 6, <i>P</i><0.05). (c) Expression of FoxP3 mRNA in the allografts was analyzed by real-time PCR normalized to GAPDH. Significantly higher levels of FoxP3 were observed in group 5 (n = 6, <i>P</i><0.05).