Isolation and stimulation of PBMC and pDC

PBMC were collected from healthy donors and isolated and cultured at 2x10⁶/ml as described [68]. pDC were purified from isolated PBMC as previously described [69]. The purity of the recovered cells was greater than 95% as assessed by flow cytometry analysis with an anti-BDCA4 monoclonal Ab (Miltenyi biotech). Isolated pDC were plated at a density of 1x10⁶/ml and, for each experimental condition, 1x10⁵ pDC were used.

For pDC or monocyte cell depletion, PBMC were subjected to positive sorting using anti-BDCA-4 (CD304) or anti-CD14 conjugated magnetic microbeads (Miltenyi Biotech), respectively. The eluates, containing depleted populations, were collected.

Whole PBMC were stimulated with the 1:12.5 and 1:50 dilutions of Encepur and I-TBEV corresponding to 0.24 μg/ml and 0.06 μg/ml of antigen, respectively. Same dilutions were used for the excipient and sucrose solution. The TLR7/8 ligand Resiquimod (R848, 5 μM, Invivogen San Diego, CA), was used as positive control. Cells were stimulated for the indicated time, supernatants collected, and RNA extracted for further analyses. Where indicated, cells were also pre-treated for 30 minutes at 37°C with 1 μM TLR 7/8 antagonist ODN 2087 (Miltenyi Biotech) prior to I-TBEV stimulation. Where specified, I-TBEV was incubated with scAb (0.15, 0.3 or 0.6 μg/ml) against TBEV for 30 minutes at 37°C before PBMC treatment.