Upon depletion of administered Treg, host Treg are required for acceptance of skin allografts. (A-C) Sublethally irradiated WT B6 mice were grafted with DBA/2 BM under cover of 2.106 DEREG or WT Treg in vitro cultured in presence of (B6 × DBA/2)F1 antigen-presenting cells. Mice were left untreated or received diphtheria toxin (DTx) 4 or 10 weeks after reconstitution, as indicated. Skin allografts were placed 6 weeks after BM grafting. Control mice were not injected with Treg at the time of BM transplantation. (A) Skin allograft survival was monitored daily by assessment of macroscopic signs of rejection. Graft-survival curves for indicated conditions (4 independent experiments). Representative macroscopic (B) and histologic (C) features of skins 100 days after transplantation. (HE, hematoxylin and eosin; F4/80, immunohistocheministry with antibody to the macrophage marker F4/80; Luna, Luna’s eosinophil stain) Scales represent 200 µm for HE and F4/80 staining and 20 µm for Luna staining. (D-E) Sublethally irradiated DEREG mice were grafted with DBA/2 BM under cover of DEREG Treg in vitro cultured in presence of (B6 × DBA/2)F1 stimulator cells. Injected Treg were depleted or not by diphtheria toxin (DTx) injection 4 weeks after BM grafting. DBA/2 Skin transplantation was performed 2 weeks later. Skin allograft survival was monitored daily. (D, left panel) Skin allograft survival during the 100-day observation period. (D, right panels) In toxin-treated mice, rejection was macroscopically clearly visible (2 independent experiments). (E) Chimerism was analyzed in PBMC 2 days before skin transplantation and immediately after rejection in toxin-treated mice.
