T Cell Differentiation and Cytokine Profiling

Naïve T cells were activated with plate-bound anti-CD3 (10 µg/mL) plus soluble anti-CD28 (2 µg/mL, BD Bioscience, San Jose, CA, USA) for 2 days. For T cell differentiation, CD4⁺ T cells were differentiated into Th17 or iTreg cells as previously reported [27 (link),29 (link)]. Briefly, CD4⁺ T cells were stimulated by anti-CD3/CD28 for 4 days with anti-IFN-γ and anti-IL-4 (both 10 µg/mL) in the presence of TGF-β1 (5 ng/mL, for Treg), or TGF-β1 plus IL-6 (10 ng/mL, for Th17) (all obtained from R&D Systems, Minneapolis, MN, USA). Cells were restimulated with phorbol 12-myristate 13-acetate (PMA, 25 ng/mL) plus ionomycin (500 ng/mL; Sigma, St. Louis, MO, USA) for 5 h with GolgiStop (BD Bioscience, San Jose, CA, USA) in the last 2 h for intracellular cytokine staining; or without GolgiStop for cytokine assays in the culture supernatants by enzyme-linked immunosorbent assay (ELISA). Where indicated, fasudil (Selleck Chemicals, Houston, TX, USA) was added to the cultures.