Anti tnfr2

To control the purity of cell selection, the following antibodies (BD Pharmingen™, Franklin Lakes, NJ, USA) were used: anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD19 and anti-CD56. Antibodies used for TNF receptor expression (anti-TNFR1 and anti-TNFR2) were purchased from R&D Systems (R&D Systems). To bring out the T cell activation, the following antibodies (Beckman Coulter, Brea, CA, USA) were used: anti-CD3, anti-CD25, anti-HLA-DR. To bring out the early T cell activation, the marker CD69 was studied after TNFα stimulation by flow cytometry (BD Pharmingen™). To detect apoptosis in T cells after TNFα stimulation, a fluorescein isothiocyanate (FITC) annexin-V Apoptosis Detection Kit (Becton-Dickinson, Franklin Lakes, NJ, USA) was used. Appropriate isotype controls were used in all cases. Acquisition of samples was performed on a FACSCanto flow cytometer (Becton-Dickinson), and the data were analyzed with FlowJo software.

One million PBMCs, T or B cells/well were cultured into 24-well plates with 1 ml of RPMI 1640 medium supplemented with 10 % fetal calf serum (FCS), and 0.5 % penicillin and streptomycin. PBMCs or lymphocytes were cultured with or without 1 ng/ml of TNFα (R&D Systems, Minneapolis, MN, USA) and with or without adalimumab (1 μg/ml), etanercept (10 μg/ml), infliximab (100 μg/ml), certolizumab (1 μg/ml) or golimumab (100 μg/ml) for 24 or 48 hours in a 5 % CO₂ incubator at 37 °C. In neutralizing experiments, B and T cells were preincubated with anti-TNF receptor (TNFR)1 (9 μg/ml) or anti-TNFR2 (2.5 μg/ml) (R&D Systems) for 1 hour and TNFα was added for 48 hours. In activation experiments, T cells were incubated with IL-2 (60 U/ml) and anti-CD3 antibody (5 μg/ml) for 4 days. For experiments with Bryostatin-1, cells were incubated with TNFα for 48 hours. Cells were then activated with 50 nM Bryostatin-1 (Sigma-Aldrich, Saint Louis, MO, USA) for 5 hours. Cells were then harvested for flow cytometry analysis or RNA and protein extraction.

For flow cytometry, cells were detached using Alfazyme (PAA Laboratories), washed, and incubated in 50 μl of 3% (v/v) FCS in phosphate-buffered saline (PBS) with labeled antibody (phycoerythrin [PE]-conjugated) anti-TNFR1, anti-TNFR2, or the respective PE-conjugated isotype control (R&D Systems) for 45 min on ice in the dark. For the quantification of TACE/ADAM17 surface densities, cells were incubated with an antibody detecting the TACE ectodomain (MAB9301, R&D Systems) followed by the incubation with a PE-conjugated secondary antibody (Biolegend). After antibody staining, cells were washed three times and measured in the PE channel of a FACS Canto II (Becton Dickinson). For detection of TNFα binding, cells were incubated with TNFα-Biotin and Streptavidin-FITC or let unstained. Cells were washed three times and measured in the FACS Canto II FITC channel. Histograms were generated by the use of FlowJo (Tree Star).