Vivit peptide

Monocyte-derived DCs were obtained from a healthy human blood donor following a standard protocol [26 (link)]. Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats in lymphocyte separation medium (Corning) by density gradient centrifugation at 1500 rpm. Then, CD14+ monocytes were purified by using a MACS CD14 isolation kit (Miltenyi Biotech). Monocytes were then differentiated into DCs by incubating the cells during 5 days, at 37°C, in growth media containing RPMI 1640 (Invitrogen/Gibco), 8% FBS (Hyclone), 2 mM glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 500 U/mL hGM-CSF (PeproTech), and 1000 U/mL hIL-4 (PeproTech). Before infection, DCs were pretreated for 1 h at 37°C with RPMI medium containing the NFAT inhibitors cyclosporine A (CsA, Sigma, 1 μM) [27 (link)] or the VIVIT peptide (Tocris Biosciences, 100 μM) [28 (link)]. Then, the cells were infected with the NC virus grown in embryonated chicken eggs as described previously [29 (link)], diluted in DMEM, and added directly to pelleted cells at a MOI of 1. After incubation of 40 minutes at RT, fresh RPMI medium containing CsA (1 μM) or the VIVIT peptide (100 μM) was added back, and the cells were incubated at 37°C during 2 h. Mock-infected cells underwent the same experimental procedure.

Splenic B cells were negatively isolated with a MACS-based B Cell Isolation Kit (Miltenyi, 130090862) and purity above 90% was confirmed by flow cytometry staining for CD19 (eBio1D3, eBioscience, 1:100). B cells were stimulated in vitro with goat anti-mouse αIgM F(ab′)₂ fragments (Jackson ImmunoResearch) for the indicated times and with the respective concentrations. Cyclosporine A (CsA) (1 µM, Sigma Aldrich), FK506 (1 µM, Sigma Aldrich) and VIVIT peptide (10 µM, Tocris) were consistently added 1 h prior to αIgM stimulation.