Decomplemented

Undifferentiated U937 and U937-C5aR cells, which respectively do not express or stably express the C5aR receptor [51] (link), were a generous gift from Pr. J.A. van Strijp (Utrecht University, The Netherlands). Cells were cultured as 50 ml suspension at 37°C under a 5% CO₂ atmosphere in 250 ml flasks in RPMI-1640 medium supplemented with 10% (v/v) of decomplemented fetal calf serum (Life Technologies, Carlsbad, USA) and 0.1% (w/v) of both penicillin and streptomycin (InVitrogen, Paisley, UK).

Buffy coats from healthy donors were obtained from Zhongshan Hospital, Fudan University. This study has been approved by the Medical Ethics committee of Zhongshan Hospital, Fudan University. Peripheral blood mononuclear cells were separated by density-gradient centrifugation using Ficoll-Paque Plus (GE healthcare Bio-Science, Uppsala, Sweden) and were further processed for separation of T cells using CD3 MicroBeads (MiltenyiBiotec, Auburn, USA). T cells were cultured in RPMI medium (Eurobio, Les Ulis, France) supplemented with 20 IU/mL penicillin, 20 μg/mL streptomycin, and 10% decomplemented FBS (Life Technologies), and stimulated with Dynabeads® T-Expander beads coated with anti-CD3 and anti-CD28 Abs (Life Technologies) at a 1:1 cell/bead ratio in the absence of IL-2 (30 U/ml). Then IS stimulation experiments were conducted by treating T cells with different IS concentration for 96 h.