Expanded Treg suppressive capacity was assessed in 72 hours in vitro assays in the absence of TAC. Variable numbers of Treg were incubated with 10 × 104 autologous PBMC labelled with 1 μM VPD450 (BD) proliferation dye in triplicate wells containing 2 × 104 αCD3/αCD28 beads in 96-well plates. Labeled PBMC cultured alone in the presence of αCD3/αCD28 beads were used as a negative control for suppression. After a 72-hour incubation, cocultures were stained to distinguish CD4+ and CD8+ cells, and violet proliferation dye dilution was analyzed by flow cytometry. The percentage of PBMC suppression was calculated by using division index of cocultures containing Treg and PBMC compared with division index of PBMC alone, according to the following formula (1 − (div.index Treg + PBMC/div.index PBMC)). Data were analyzed with FlowJo software version 9.5.3. The program calculates division index as the average of cell divisions that a cell in the original population has undergone.