Prolonged survival of Tregs in vivo correlates with persistent protection. (A) Bioluminescent imaging of donor allogeneic luc⁺ Tregs in lethally irradiated Balb/c host (▪, n = 4) showed persistent and stable signal over a 3-month period following transplantation compared with control animals that received only TCD-BMs and no luc⁺ cells (▵, n = 4) (P < .001, d91). Data are representative of 2 separate experiments. (B) Lethally-irradiated Balb/c recipients were challenged with Tcons at various time points to induce GvHD (d7 ▾, n = 5; d14 •, n = 5; d21 □, n = 6; d30 ○, n = 6). By day 30, no evidence of GvHD was noted. Data represent 1 of 2 independent experiments. (C-D) When transplant recipients received Tregs on day 0, they were protected from GvHD with the infusion of Tcons on days 7, 14, and 21 (data shown for day 21). Average weight change (indicator of GvHD; *2 animals remain alive after day 39) (C) and survival (D) shown for animals that underwent transplantation with (▪, n = 6) or without transferred Tregs on day 0 (○, n = 5) and challenged with Tcons on day 21 (P < .05). Animals that received Tcons with (▵, n = 6) or without (▴, n = 6) Tregs on day 0 were controls (P < .001). Data were combined from 2 independent experiments. (E) Protection from delayed GvHD induction was associated with the reduction of Tcon proliferation by Tregs infused on day 0 of transplantation (▪, n = 3) compared with animals that received only Tcons on day 21 (○, n = 5) (P = .001, day 29; P < .001, day 35). Data are representative of 2 independent experiments. Error bars represent standard error of the mean.