Purified PDCs isolated from freshly isolated PBMCs were stimulated with ODN-2006 or −2336 (both at 5 µM; InvivoGen, San Diego, CA; 10,000 cells/well) for 4 hours in the presence of 15 µg/ml of NTZ or IgG4 (Sigma) after which cells were lysed using RLT buffer and frozen at −80°C. Freshly isolated PBMCs were stimulated with ODN-2006 or -2336 (both at 5 µM; InvivoGen; 500,000 cells/well) for 18 hours in the presence of 15 µg/ml of NTZ or IgG4 (Sigma Aldrich, St Louis, MO). Cells were retrieved and stained for flow cytometric analysis.
For co-culture experiments, PDCs were isolated from freshly isolated PBMCs by negative selection using magnetic beads (Plasmacytoid Dendritic Cell Isolation Kit II, Miltenyi, Auburn, CA). More than 90% of separated cells were Lin−/CD123+/CD11c−/HLA-DR+. Cells were stimulated with 5 µM ODN-2006 for 18 hours (5,000 cells/well). CD4+ T cells were isolated from homologous blood kept at +4°C overnight by negative selection using magnetic beads (CD4+ T Cell Isolation Kit II, Miltenyi) and added to each well (20,000 cells/well). Co-cultures were incubated for five days in the presence of anti-CD3 (HIT3a, 1 µg/ml, BD Biosciences) and 20U IL-2. Live CD4+ T cells were isolated by FACS sorting and lysed using RLT buffer.
For co-culture experiments, PDCs were isolated from freshly isolated PBMCs by negative selection using magnetic beads (Plasmacytoid Dendritic Cell Isolation Kit II, Miltenyi, Auburn, CA). More than 90% of separated cells were Lin−/CD123+/CD11c−/HLA-DR+. Cells were stimulated with 5 µM ODN-2006 for 18 hours (5,000 cells/well). CD4+ T cells were isolated from homologous blood kept at +4°C overnight by negative selection using magnetic beads (CD4+ T Cell Isolation Kit II, Miltenyi) and added to each well (20,000 cells/well). Co-cultures were incubated for five days in the presence of anti-CD3 (HIT3a, 1 µg/ml, BD Biosciences) and 20U IL-2. Live CD4+ T cells were isolated by FACS sorting and lysed using RLT buffer.