Suppression at 7 hours predicts suppression of proliferation at 96 hours. (A) Representative example of a 96-hour CFSE dilution assay, gated on live Tresps, cultured alone (left) or at a 1:1 Tresp:Treg ratio (right). (B-C) Suppression of proliferation at 96 hours at increasing Tresp/Treg ratios from a representative experiment (B) and pooled data from 10 independent experiments (C). (D-E) Comparison of suppression of CD69 (D) and CD154 (E) expression at 7 hours with paired suppression of autologous Tresp proliferation at 96 hours, with regression lines. (F-G) ROC curves illustrating the performance of CD69 suppression (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) 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. (D-I) Pooled data from Tresp/Treg ratios 4:1 to 1:1 from 10 independent experiments. *P < .05, **P < .01, ***P < .001, †P < .005, ††P < .0001. UQ indicates upper quartile; and LQ, lower quartile. #P < .005, ##P < .0001 with respect to Tresp alone cultures (Tresp:Treg ratio of 1:0).