Br3 fc

The isolated PBMCs were washed with pre-cold PBS and cultured with supplemented RPMI 1640 medium in 24-well plates. Cells were stimulated with rhBAFF (20 ng/ml, R&D Systems, Minneapolis, MN, USA) or a combination of rhBAFF and BR3-Fc (100μg/ml, R&D Systems, Minneapolis, MN, USA) at 37 °C with 5% CO₂ for 48 h. Cells were harvested for assessment of apoptosis of CD4⁺ and CD8⁺ cells using flow cytometry. Isolated CD4⁺ or CD8⁺ cells were cultured with supplemented RPMI 1640 medium and phytohemagglutinin (Sigma-Aldrich and Merck KGaA, Darmstadt, Germany) in 24-well plates. Cells were stimulated with rhBAFF or a combination of rhBAFF and BR3-Fc at 37 °C with 5% CO₂ for 72 h. For CD4⁺ cells culture, the supernatant and cells were seperately obtained for the detection of IL-4 and IFN-γ expression. For CD8⁺ cells culture, the cells were harvested for the detection of mRNA expression of perforin and granzyme B.

The binding abilities and affinities of BAFF-Trap to BAFF were measured by a specific and sensitive ELISA (R&D Systems, Minnesota, USA), which detects unbound human BAFF at 25 pM (R&D Systems, Minnesota, USA) mixed with BAFF-Trap (ranging in concentration from 0.04 nm to 655.36 nm) incubated overnight at room temperature. The same serial amounts of TACI-Fc and Br3-Fc (R&D Systems, Minnesota, USA) were added as controls.