Treg lines expanded with cell-based aAPCs have equivalent purity and expansion, with increased suppressive function. (A) Representative example (i) and summary (ii) of the CD4, CD25 versus Foxp3 (CD4-gated), and CD127 versus Foxp3 (CD4-gated) profiles for purified cord blood cells expanded 21 days in vitro with bead- or cell-based aAPCs. (B) Representative example (i) and summary (ii) of in vitro Treg expansion demonstrating the increased effectiveness of 4-1BBL or OX40L costimulation. (C) Representative example (i) and summary (ii; average values for Treg-to-Tresponder ratio of 1:4) demonstrating cord blood Tregs expanded with aAPCs potently suppress an in vitro MLR assay, and cell-based aAPCs are even more effective than bead-based aAPCs. Tregs were cultured for 18 to 21 days, and each summary is presented as the mean plus or minus SEM, with n = 6. TGF-β (D) or IFN-γ (E) present in day 6 or 17 supernatants from UCB Treg cultures expanded with anti-CD3/28 beads or KT32/4.1BBL. Note that the limit of detection for IFN-γ was 25 ng/mL. *P ≤ .05 for comparison with CD3/28 beads; #P ≤ .05 for comparison with KT32.