To induce the production of inflammatory cytokines, sorted CD27+/−Tregs were cultured in a medium containing 10 IU/ml IL-2, 10 ng/ml IL-1β, 5
ng/ml IL-6, 25 ng/ml IL-21, 25 ng/ml IL-23, and 5 ng/ml TGF-β (R&D Systems,
Minneapolis, MN, USA) for 7 days. Cells cultured in medium containing only 10
IU/ml IL-2 were used as the non-induction control24 (link). Sorted Tregs were
cultured in the upper chamber of a transwell system (pore size:0.4 µm) with or
without anti-CD3/CD28 Dynabead-stimulated PBMCs in the lower chamber. Seven days
after stimulation, the Tregs were collected and further incubated with 50 ng/ml
phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, San Luis, MO USA), 1 µg/ml
ionomycin (Sigma-Aldrich, San Luis, MO USA), and 1 µl/ml GolgiStop protein
transport inhibitor (BD Biosciences, San Diego, CA, USA) for an additional 5 h
prior to cytometric analysis of a percentage of IL-17a-secreting cells.
ng/ml IL-6, 25 ng/ml IL-21, 25 ng/ml IL-23, and 5 ng/ml TGF-β (R&D Systems,
Minneapolis, MN, USA) for 7 days. Cells cultured in medium containing only 10
IU/ml IL-2 were used as the non-induction control24 (link). Sorted Tregs were
cultured in the upper chamber of a transwell system (pore size:0.4 µm) with or
without anti-CD3/CD28 Dynabead-stimulated PBMCs in the lower chamber. Seven days
after stimulation, the Tregs were collected and further incubated with 50 ng/ml
phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, San Luis, MO USA), 1 µg/ml
ionomycin (Sigma-Aldrich, San Luis, MO USA), and 1 µl/ml GolgiStop protein
transport inhibitor (BD Biosciences, San Diego, CA, USA) for an additional 5 h
prior to cytometric analysis of a percentage of IL-17a-secreting cells.
