T-cell expansion protocol: FACS-isolated cells were plated at ~2.5 × 105 Tregs per well inmultiplewells of a 24-well plate (Nunc) and activatedwithDynabeads CD3/
CD28 CTS anti-CD3/anti-CD28–coated microbeads at a 1:1 bead/cell ratio. Cells were cultured either in X-VIVO 15 or in X-VIVO 15 customized by Lonza by substituting 100% of the glucose in the basemediumwith D-glucose. supplemented with 10% human heat-inactivated pooled AB serum. At day 2, the culture volume
was doubled and IL-2 was added (Proleukin, 300 IU/ml; Prometheus). Cells were resuspended, fresh medium and IL-2 were added at days 5, 7, 9, and 12, and the cellswere transferred to cell culture plates and flasks (Nunc), and/or bags (Saint-Gobain) of increasing size to maintain a seeding density of ~2 × 105 to 3 × 105 cells/ml in plates or flasks and a concentration of 500,000/ml in bags.On day 9, cells were restimulated with fresh anti-CD3/anti-CD28–coated beads at a 1:1 ratio.