4.2. Isolation and Stimulation of Human CD4+ T Cells and Treg

Buffy coats were obtained from healthy volunteers, with approval by the local ethical committee (Landesärztekammer Rhineland Palatine No. 837.019.10 (7028), approved on 4 March 2010). The CD4⁺ T cells were isolated via CD4 Microbeads (Miltenyi # 130-045-101). The regulatory T cells were isolated with the CD4⁺ CD25⁺ CD127^dim/− isolation kit (Miltenyi #130-094-775, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. For proliferation assays, CD4⁺ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, eBioscience #65-0850-84, San Diego, CA, USA) and cultured in 48 well plates at 10⁶ cells/mL, stimulated with 1 μg/mL anti-CD3 mAb (clone OKT3) plus 1 μg/mL anti-CD28 mAb (clone 28.2, eBioscience, San Diego, CA, USA) in the presence or absence of T98G in the ratio of 8:1, 10 μg/mL anti-GARP Ab (Origene AP17415PU-N, Rockland, MD, USA) and 1 μg/mL soluble GARP (recombinant human LRRC32/GARP protein #6055-LR-050, Minneapolis, MN, USA). For the activation of Treg, 1 x 10⁶ cells were stimulated with 1 μg/mL anti-CD3 mAb (clone OKT3) plus 1 μg/mL anti-CD28 mAb (clone 28.2, eBioscience, San Diego, CA, USA) with 10 U/mL IL-2 (Novartis #PZN 02238131, Basel, Switzerland) for 48 h.