Murine CD62L+CD4+ T cells were isolated using magnetic cell sorting from mouse splenocytes according to the manufacturer’s instruction (Miltenyi Biotec, Charlestown, MA, USA). In total, 3 × 105 cells were cultured in a 48-well plate coated with goat anti-hamster crosslinking antibody in RPMI 1640 medium complemented with penicillin/streptomycin, 10% fetal bovine serum, and ß-mercaptoethanol. To differentiate the cells into Tfh, the medium was supplemented with anti-CD3 (0.25 µg/ml, 145-2C11; BioLegend, San Diego, CA), anti-CD28 (0.5 µg/ml, 37.51; BioXcell, Lebanon, NH), interleukin-21 (50 ng/mL, R&D systems, Minneapolis, MN, USA) interleukin-6 (100 ng/mL, R&D systems), anti-TGFß (0.5 µg/mL, clone 1D11; R&D systems) anti-IFN-gamma (10 µg/ml; BioXcell, Lebanon, NH, USA), and anti-IL-4 (10 µg/ml; BioXcell). The cells were retrieved for analysis on day 3 of the culture.
For the coculture experiment using human Tfh and B cells, 50,000 freshly sorted autologous Tfh and B cells were cocultured at a 1:1 ratio in a 96-well plate with staphylococcal enterotoxin B (SEB, 1 ng/mL, Fisher Scientific) for 7 days at +37 °C. The CaMK4 inhibitor KN93 (Selleckchem, Houston, TX, USA) or its vehicle (dimethysulfoxide, DSMO) was used at the concentration of 10 µM.
For the coculture experiment using human Tfh and B cells, 50,000 freshly sorted autologous Tfh and B cells were cocultured at a 1:1 ratio in a 96-well plate with staphylococcal enterotoxin B (SEB, 1 ng/mL, Fisher Scientific) for 7 days at +37 °C. The CaMK4 inhibitor KN93 (Selleckchem, Houston, TX, USA) or its vehicle (dimethysulfoxide, DSMO) was used at the concentration of 10 µM.