Zaad cmk

The sensitizing effect was achieved with pre-treatment for 24 h with the bivalent Smac mimetic BV6 (kindly provided by Genentech). Cell death was induced with treatment with human recombinant TRAIL (R&D Systems, Wiesbaden, Germany). Cell death was inhibited with zVAD.fmk (Bachem, Heidelberg, Germany), anti-TRAIL antibody (No. 2E5; Enzo Life Sciences, Lörrach, Germany), zAAD-CMK (Calbiochem Merck, Darmstadt, Germany) and Etanercept (Enbrel, Merck, Darmstadt, Germany).

Spleen B cells were isolated (B cell isolation kit, Miltenyi) and cultured overnight in the presence of 25 μg/ml LPS (Escherichia coli 055:B5, Sigma-Aldrich, Diegem, Belgium) to maintain sufficient cell survival and support antigen presentation. Dead cells were removed by Ficoll centrifugation (Lympholyte-M, Cedarlane Labs, Atlanta, GA, USA) and remaining B cells were stained with Cyto-ID Red long-term cell tracer kit (Enzo Life Sciences, Lausen, Switzerland) following manufacturers’ instructions. B cells were then cocultured for 18 h with CD4⁺ T cells (ratio B:T, 1:5) in the presence of indicated peptide (2 μM, added to the culture media). Annexin V APC was used to detect cell death in B cells (Annexin V detection kit, BD Biosciences) according to manufacturers’ instructions. Gated B cells were then analyzed for Annexin V binding on flow cytometer. For inhibition of granzyme-B (GZB) activity, Z-AAD-CMK (Calbiochem/Merck, Overijse, Belgium) was added at 20 μg/ml during the entire coculture period. Inhibition of FasL was performed with functional grade anti-mouse CD178 antibody (clone MFL3, eBioscience) at 20 μg/ml during the coculture period.