In vitro–expanded cord blood Tregs decrease mortality in a xenogeneic model of GVHD. Tregs were purified from frozen cord blood and expanded with a single stimulation of bead- or cell-based aAPCs for 18 days, then were cotransferred with allogeneic PBMNCs into clodronate-treated, irradiated Rag2^−/−, γ_c^−/− mice. (A-C) Day-18 flow phenotype showing percent CD4⁺ and percent CD25⁺Foxp3⁺ (CD4-gated), fold expansion, and suppression index (respectively) of the cell lines used. (D) Kaplan-Meyer survival curve showing increased survival of clodronate-treated, irradiated Rag2^−/−, γ_c^−/− mice receiving human PBMNCs with or without groups of Tregs in a 1:1 ratio (ie, 30 × 10⁶ each). For groups PBMNCs, CD3/28 beads, KT32/4.1BBL, KT32/OX40L, and PBS, n = 8, 9, 8, 8, and 4, respectively. P ≤ .05 for each Treg-treated group compared with PBMNCs. (E) Average weight (% of initial) for mice surviving on given day for different groups of mice. *P ≤ .05 from days 8 to 73 for 4.1BBL and days 10 to 31 and 49 to 80 for OX40L. Data are representative of 4 experiments ameliorating XGVHD with Tregs.