“…22 The CFSE-labeled cells were cultured in Roswell Park Memorial Institute (RPMI; Nacalai Tesque, Kyoto, Japan) culture medium containing 10% fetal bovine serum (Sanko, Tokyo, Japan), 5 M 2-mercaptoethanol (Katayama, Osaka, Japan), 1% HEPES buffer (GIBCO, Grand Island, NY), and 100 IU/mL penicillin-100 g/mL streptomycin (GIBCO). The following stimuli were added at the start of the culture: 10 g/mL of goat anti-mouse IgM F(abЈ) 2 (Jackson ImmunoResearch Laboratories, West Grove, PA), 1 g/mL recombinant mouse CD40L, and 1 g/mL enhancer (Alexis Cor, Lausen, Switzerland), as well as 0.02 g/mL recombinant mouse IL-4 (R&D Systems, Minneapolis, MN). In the CsA inhibition assay, various doses of CsA (6.25-100 ng/mL; which was kindly provided by Novartis, Basel, Switzerland) were added at the start of the culture.…”