(A) CD25^–CD4⁺ T cells were sorted from wild-type C57BL/6 mice and labeled with violet dye. Labeled CD4⁺ T cells were then activated by anti-CD3 antibodies plus irradiated APCs in the presence of various ratios of Treg cells sorted from Itch^fl/flFoxp3^Cre and Itch^+/+Foxp3^Cre mice. Teff, effector T cells. (B–F) Rag1^–/– mice were given sorted wild-type or Itch-deficient (CD45.2⁺) Treg cells, together with CD4⁺CD45RB^hi (CD45.1⁺) naive T cells, or CD4⁺CD45RB^hi (CD45.1⁺) naive T cells alone. (B) Weight loss of individual mice was monitored every week for 2 months. Data are compiled from three independent experiments with four mice each. Error bars indicate the mean (± SD). (C) Photograph of spleen (Sp) and mesenteric lymph nodes (mLN). (D) H&E staining of colon sections of recipient mice at 8 weeks after adoptive transfer. (E) Absolute number of CD4⁺CD45.1⁺ and CD4⁺CD45.2⁺ T cells in the spleens of the recipient mice at 8 weeks after adoptive transfer. Error bars indicate the mean (± SD). (F) Foxp3 expression by CD4⁺CD45.2⁺ cells from Rag1^–/– recipient mice. Data are representative of three independent experiments. (G) Flow cytometric analysis of YFP and RFP expression in CD4⁺ T cells of 4-month-old WT^R26R and Itch^R26R mice (left panel). Percentages of ex-Treg cells (YFP^–RFP⁺) in total RFP⁺ cells (YFP⁺RFP⁺ and YFP^–RFP⁺) are indicated (middle panel). Absolute number of ex-Treg cells (YFP^–RFP⁺) cells in the spleen (n = 4) (right panel). *P < 0.05 (unpaired 2-tailed Student’s t test).