In vitro expansion protocols
Sorted naïve Treg were plated at 5x104/ml in 96-well U
bottom plate (Costar) with X-VIVO 15 medium (Lonza, catalog
no. 04-418Q) containing 5% heat inactivated pooled
bovine serum (Sigma-Aldrich). In the standard method (StM) culture
medium was supplemented with Dynabeads human T activator CD3/CD28
(Thermofisher) at a 1:1 bead/cell ratio, and 100 UI/ml rhIL-2
(Bio-Techne). In the IL-7 method (IL-7M), culture medium of the StM was
supplemented also with rhIL-7 (R&D Systems) at 10ng/ml. Treg were
cultured for 14 days and the culture medium and supplements were
replaced at day 7 and 11.