Functional properties of Treg after expansion with GM-CSF. After 3 days of culture of CD4⁺CD25⁺ T cells with CD3/CD28-coated beads and 2 ng/mL GM-CSF (Δ) or PBS (□), cells were washed and cotransferred (10⁶) with diabetogenic NOD splenocytes (3 × 10⁶) into 4-week-old NOD-Rag2^−/− recipients. A control transfer group was injected with diabetogenic NOD splenocytes (3 × 10⁶) alone (●). Mice (n = 8-10 per group) were screened for glycosuria (Glucotest, Boehringer-Mannheim, Mannheim, Germany) twice a week and/or glycemia (Haemoglukotest and Reflolux F, Boehringer-Mannheim) and considered diabetic when nonfasting blood glucose levels were more than 250 mg/dL on 2 consecutive readings. P < .001, using Kaplan-Meier estimates and log-rank analysis, comparing diabetes incidence measured in mice cotransferred with Treg and diabetogenic splenocytes versus injected with diabetogenic NOD splenocytes (3 × 10⁶) alone. P = .047, comparing diabetes incidence in mice cotransferred with GM-CSF– versus PBS-expanded CD4⁺CD25⁺ cells. One representative experiment of 2 is shown.