Suppression at 7 hours by ex vivo expanded Tregs predicts suppression of proliferation at 96 hours. (A) Representative example of a CFSE dilution assay showing Tregs cultured alone (left) or in coculture with Tregs at 1:1 Tresp:Treg ratio (right). (B-C) Suppression of proliferation at 96 hours at increasing Tresp:Treg ratios, showing a representative experiment (B) and pooled data from 8 independent experiments (C). (D-E) Comparison of suppression of CD69 (D) and CD154 (E) expression at 7 hours with paired suppression of Tresp proliferation at 96 hours, with regression lines. (F-G) ROC curves illustrating performance of CD69 (F) and CD154 suppression (G) at correctly identifying suppression of Tresp proliferation at 96 hours, for 3 critical values of the CFSE dilution assay. (H-I) Graphs illustrating the performance of CD69 (H) and CD154 suppression (I) at correctly identifying median suppression of proliferation. Median and distribution statistics for each marker are also given. The left and right y-axes illustrate the Youden index for threshold values and arbitrary cost of threshold values, penalizing for false positives, respectively. (C-I) Pooled data from 8 independent experiments from Tresp:Treg ratios 8:1 to 1:1 for CD69 and 16:1 to 1:1 for CD154. *P < .05, ††P < .0001. #P < .05, ##P < .01, ###P < .0001 with respect to Tresp alone cultures (Tresp:Treg ratio of 1:0).