Early infusion of lower Treg numbers is sufficient for GvHD control and suppresses Tcon proliferation. (A) Induction of GVHD was complete and lethal when allogeneic FVB donor Tcons were infused on day 0 (○) or 2 (▪) following transplantation of TCD-BMs into lethally-irradiated Balb/c hosts (○ versus ▪, NS) compared with complete survival of animals that received Tregs on day 0 (X) or 2 (•) (▪ versus X or •, P < .001) (combined data of 2 independent experiments, n = 7). (B) When Tregs were infused on day 0 or concurrently with luc⁺ Tcons on day 2 at a 1:1 ratio, a reduction of Tcon proliferation was observed and was more significant with earlier infusion of Tregs on day 0 (▴, n = 4) compared with day 2 (○, n = 4) (▪ versus ▴, P = .001; ▪ versus ○, P = .03). Animals that received a transplant with only TCD-BMs (□, n = 4) were controls for background bioluminescence. Data represent 2 independent experiments. Dose ratio titrations of Tregs/Tcons of 1:1, 1:2 (data not shown), and 1:10 were evaluated (C-D). A 10-fold dose reduction protected transplant recipients from GvHD as demonstrated by average weight change (C) and GvHD-related survival (D) (P = .02 for ▴ versus ○, n = 7). Data were combined from 2 independent experiments. Error bars represent standard error of the mean.