Tregs expanded with cell-based aAPCs have increased survival and accumulation in in vitro suppression assays. Cord blood Tregs were expanded in vitro using bead-or cell-based aAPCs with or without 4.1BBL or OX40L costimulation, then CFSE-labeled and incubated with allogeneic PBMNCs and anti-CD3 beads for 4 days and Treg cell division monitored by CFSE-dye dilution. (A) Representative example of CFSE dilution in Tregs during bead-based suppression assays. (B) Quantitation of Treg cell number (CD4⁺, CFSE⁺) on day 4 of suppression assay at Treg-to-PBMNC ratio of 1:2 as assessed by flow cytometry using counting beads and CFSE-dilution. (C) Pro- and antiapoptotic gene expression in UCB Tregs expanded with CD3/28 beads versus KT32/4.1BBL. Data are mean plus or minus SEM for 5 independent cultures. (E) Data in panel A is representative of a single experiment, while data in panels B and C represent the mean plus or minus SEM for 3 and 5 independent experiments, respectively. *P < .05.