(A) CFSE-labeled CD4⁺CD25^– T cells were transferred into Ccl17^+/+ or Ccl17^E/E mice, and frequencies of CFSE⁺Foxp3⁺ Tregs and CFSE⁺CD4⁺ cells among T cells in LNs were analyzed by flow cytometry after 10 days. Representative dot plots and percentages within gates are shown (n = 10 per group). (B) Sorted unpulsed or OVA-2–pulsed EGFP^– or EGFP⁺Ccl17^E/+ or Ccl17^E/E BMDCs were incubated with OT-II T cells in vitro (n = 5 independent experiments). T cell proliferation was quantified by CSFE dilution and FACS analysis after 3 days. OT-II T cells were back-sorted, and Foxp3 mRNA expression was analyzed by real-time PCR. Frequencies of apoptotic annexin V⁺ cells were quantified by FACS analysis. *P < 0.05 versus unpulsed; ^#P < 0.05 versus OVA-pulsed EGFP⁺Ccl17^E/+ BMDCs. Phosphorylation of Stat5 (pStat5) was assessed by flow cytometry; representative histograms are shown. Fluorescence-minus-one measurements served as control. (C) OVA-2–pulsed Ccl17^+/+ or Ccl17^E/E BMDCs were incubated with OT-II Tregs (n = 3 independent experiments). Frequencies of Foxp3⁺CD25⁺CD4⁺ and of annexin V⁺ Tregs among Foxp3⁺CD25⁺CD4⁺ Tregs were quantified by FACS analysis after 3 days; representative dot plots and percentage of Foxp3⁺CD25⁺ Tregs among CD4⁺ T cells within gates are shown. *P < 0.05 versus Ccl17^+/+ BMDCs. pStat5 was assessed by flow cytometry in Tregs; representative histograms are shown. (D) FACS analysis of annexin V⁺CD4⁺Foxp3⁺ Tregs in LNs of Ccl17^+/+ and Ccl17^E/E mice. Data points represent frequencies of cells in individual mice; horizontal bars denote mean of all mice. *P < 0.05.