Etn enbrel

RPMI-1640 Dutch modified (Gibco, Massachusetts, United States) culture medium, containing sodium bicarbonate and 20 mM HEPES, supplemented with penicillin/streptomycin (100 U/ml), sodium pyruvate (1 mM), glutamine/glutamax, and 10% human pooled serum (HPS, Radboudumc), was used in all experiments. After cell isolation, 2.5 × 10⁴ cells/well were cultured in 96-well U-bottom plates and stimulated with Dynabeads^® Human T-Activator CD3/CD28 (αCD3/CD28 beads, 1:5 of bead:cell ratio) (Gibco, Massachusetts, United States) in the presence of recombinant human (rh) IL-2 (rhIL-2, 100 U/ml) (Proleukin Prometheus Laboratories, California, United States). In some conditions, cultures were supplemented with rhTNFα (50 ng/ml, R&D, Minnesota, United States), or TNFα inhibitors etanercept (5 μg/ml; ETN—Enbrel, Pfizer, New York, United States), or TNFR2 agonist (2.5 μg/ml, Clone MR2-1, Hycult Biotech, Uden, the Netherlands). To examine the effect of a pharmaceutical inhibitor, tofacitinib (0.112 μM, Pfizer, New York, United States), PKC inhibitor Sotrastaurin (1 μM), Lck inhibitor A420983 (1 μM), or p38α/β kinase inhibitor UR13870 (10 μM) was pre-incubated with the FACS-sorted cells for 30 min before the addition of any stimulus. In some cases, cells were stimulated with PMA (12.5 ng/ml) and ionomycin (500 ng/ml) for 20 h.

FACS-sorted CD4⁺CD25^high Treg were cultured for 7 days with IL-2 (200 U/mL) containing medium alone as non-stimulated control, or together with different combinations of CD28 superagonist (CD28-SA, 1 µg/mL, Clone ANC28.1/5D10, Cat# 177-820, preservative free; Ancell, Bayport, MN, USA), rapamycin (Rap, 1 µM, Sigma-Aldrich, St. Louis, MO, USA), and TNFR2 agonist mAb (2.5 µg/mL, Clone MR2-1, Hycult, Netherlands). Exogenous recombinant human (rh) TNFα (50 ng/mL, R&D, Minneapolis, MN, USA) was used to replace TNFR2 agonist where indicated. Etanercept (10 µg/mL, ETN-Enbrel^®, Pfizer) was added to cell culture for the depletion of TNFα. Cells were harvested at day 7 of culture for phenotypic analysis, and culture supernatants were collected and stored for the subsequent cytokine analysis.