Ro 25 1553

CD4⁺CD45RO⁺ T cells were cultured in 96 U-well plates (0.1 × 10⁶ cells/well) with RPMI-1640-GlutaMAX media (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Lonza, Basel, Switzerland) and 1% penicillin/streptomycin (Life Technologies). Cells were activated/expanded with anti-CD3/anti-CD28 coated beads (Life Technologies). CD4⁺CD45RO⁺ T cells were cultured in the absence (activation condition) or presence of 10 nM of VIP (Polypeptide group, Strasbourg, France), VPAC₁ agonist (K^{52 (link)}R^{1 (link),6 (link)}L^{2 (link),7 (link)}VIP^{53 (link),54 (link)} (1–7)/GRF (8–27)) or VPAC₂ agonist (RO 25–1553) (Bachem A.G., Bubendorf, Switzerland) at different days (1, 4, 7). Cells without any treatment or activation and recollected at day 0 were considered resting cells.

Levels of cAMP were determined by an ELISA kit (ADI-900-066, Enzo Life Sciences). PBMC obtained from 18 healthy subjects, 9 hyperthyroid GD patients and 5 euthyroid GD patients were cultured in RPMI-1640-GlutaMAX media (Life Technologies) supplemented with 10% FBS (Lonza,) plus 1% penicillin/streptomycin. After cells were treated with 10 nM VIP (Polypeptide Group), VPAC1 agonist [Lys¹⁵Arg¹⁶Leu^27-VIP (1-7)-GRF (8-27)], or VPAC2 agonist (RO 25-1553) (Bachem) for 60 min, the reaction was terminated by aspiration of the growth medium and addition of 0.1 N HCl. The concentrations of cAMP in the cell lysates were measured according to the manufacturer’s instructions. Protein concentration was determined using a QuantiPro BCA Assay Kit (Sigma- Aldrich).