Plasmacytoid Dendritic Cell Activation

Purified pDCs were cultured in RPMI-1640 GlutaMax supplemented with 10% fetal calf serum (FCS) and 100U/ml of Penicillin-Streptomycin at 37°C in the presence of 5% CO2. pDCs were stimulated (in 96-well plates at 5x10⁴ cells/ml) by CpG (4 µg/mL) (Miltenyi Biotec), R848 (10 µg/mL) (In vivogen, Toulouse, France) or recombinant IL-3 (40 ng/mL) (R&D systems). After 24h, pDCs were stained for phenotypic analysis, or collected for the mixed lymphocyte reaction (MLR), or their supernatants were collected for cytokine measurement. For MLR experiment, activated pDCs were washed once with PBS and then co-cultured for 6 days in 96-well plates with allogenic T cells stained with 5μM of CFSE (Biolegend) (pDC: T cell ratio 1:5). Then, T cells were stained for phenotypic analysis or their supernatants were collected for cytokine measurement.