Fig 4. Effect of Everolimus plus Tacrolimus on allogeneic Lymphoproliferation and the generation of FOXP3⁺ Tregs in the Treg-MLR.

A. Lymphoproliferation assays (left) and CFSE Treg-MLRs (right) as shown in Figs 1 and 2 respectively were performed, but in the presence of indicated concentrations of TAC (instead of EVL). TAC inhibited the proliferative responses and the generation of new Tregs in the responding cells (unlike other agents) in a dose dependent manner (n = 7). B. EVL at indicated concentrations without or with 1 and 5 ng/ml TAC were tested in standard 7-day ³H-TdR assays lymphoproliferation assays (left) and in the CFSE Treg-MLRs (right). As in other experiments, the data were calculated as percentage of medium control (i.e. 100%) (n = 4). (Left): Note that both EVL and TAC inhibited the MLRs in a dose dependent manner and that their combinations had an additive inhibitory effect on lymphoproliferation. Statistical significance was observed below the horizontal dashed line at 67% (p<0.05). (Right): TAC inhibited the generation of new Tregs (comparing the 3 lines at 0 EVL) confirming results in Fig 4A, and unlike by EVL (solid line). When combinations of EVL + TAC were tested, EVL failed to abrogate the inhibition mediated by TAC.