(A) Cryosections from affected Cd18^hypo mice were double stained with CD25-FITC and TGF-β1–Cy3 mAbs. Original magnification, ×20. (B) FACS analysis of pooled DLNs from affected Cd18^hypo mice using TGF-β1 and CD25 mAbs. (C and D) Seven days after adoptive transfer of MACS-sorted Cd18^wt Tregs into affected Cd18^hypo mice, skin sections were stained with antibody against TGF-β1 and CD18. The overlay (yellow) of CD18-positive Cd18^wt Tregs (green) and TGF-β1–expressing cells (red) indicate that most of the Cd18^wt Tregs express TGF-β1 in the skin (C) and skin DLNs (D) after transfer into Cd18^hypo mice. The dotted line indicates the border between epidermis and dermis. Three independent experiments were performed in total. (E) A total of 1 × 10⁶ Tregs from either Cd18^wt or Cd18^hypo mice were labeled with CFSE and adoptively transferred into affected Cd18^hypo mice. At day 4 after adoptive Tregs transfer, FACS analysis was performed to measure TGF-β1 expression of CFSE-labeled Cd18^wt Tregs (left panel) or Cd18^hypo Tregs (right panel) from skin DLNs of affected recipients. Gray region, TGF-β1 expression; white region, normal goat IgG control for TGF-β1 staining. Numbers on the top of B and E indicate the percentage of CFSE-labeled proliferating cells. (F) Following adaptive transfer of 1 × 10⁶ Cd18^wt Tregs, 250-μg TGF-β1–neutralizing mAb (left panel) or isotype control IgG (right panel) were injected intraperitoneally into affected Cd18^hypo recipients. Repetitive injection of TGF-β neutralizing antibody or isotype control IgG after adoptive transfer of Cd18^wt Tregs was performed till the end of treatment (21 days). Original magnification, ×40 (C and D). **P = 0.002, using Student’s t test.