Modulation of T Cell Cytokines by CTLA4-Ig and ST6

Human PBMCs were seeded in triplicate in 6-well plates at 1×10⁵ cells per well in 2 mL of medium. The cells were cultured in RPMI 1640 medium containing 10% human AB serum (Gibco) supplemented with 1% penicillin/streptomycin at 37°C in a 5% CO₂ atmosphere for 24 h. Then, T cells and B cells were separated using a magnetic cell separation column. The B cells were inactivated using 10-Gy γ-ray irradiation at the Korea Institute of Radiological & Medical Sciences (KIRMS, Seoul, Korea) (Walcher et al., 2021 (link)). Inactivated B cells and isolated T cells were co-cultured for 24 h and then incubated with CTLA4-Ig (5 μg/mL) or ST6 (5, 10, 20 μg/mL) for 24 h. Then, the cells were centrifuged at 500×g at 4°C for 5 min. IL-2, TNF-α, and INF-γ cytokine levels were determined using the Quantikine^® ELISA kit (R&D Systems). The absorbance at 450 nm was measured using a FlexStation^® 3 Multi-Mode Microplate Reader (Molecular Devices).