Odn 2216

PBMCs were thawed briefly in a 37°C water bath, washed in media (RPMI w/L-glutamine, 10% charcoal inactivated fetal bovine serum, 1% non-essential amino acids, and 1% penicillin/streptomyocin) and counted on a Beckman Coulter Cell Counter (Beckman Coulter, Brea, CA). 10^6 cells were plated per well and incubated at 37°C with 5% CO₂ for 2 hours prior to stimulation. Cells were treated with 2µM ODN2216 (Miltenyi Biotec, Bergisch Gladbach, Germany), 2µM ORN R2336 (Miltenyi Biotec, Bergisch Gladbach, Germany), 2µM ORN R2336 control (Miltenyi Biotec, Bergisch Gladbach, Germany) or media, and returned to incubator. After 2 hours, Golgi Plug (BD biosciences, San Jose, CA) was added to each well, and cells were subsequently incubated for another 4 hours until they were processed for flow cytometry staining as described above. The following antibodies were used in addition to those listed above and were all obtained from Miltenyi Biotec unless otherwise noted (Miltenyi Biotec, Bergisch Gladbach, Germany). PE-conjugated anti-IFNα, PE-conjugated anti-IL-6, PE-conjugated anti-human IgG1, APC-conjugated anti-TNFα, APC-conjugated anti-IL-10, APC-conjugated anti-human IgG1, APC-vio770-conjugated anti-HLA-DR, PerCP Cy5.5-conjugated anti-CD14 (ebioscience inc, Santa Clara, CA).

Cells were cultured in RPMI medium 1640 with GlutaMAX supplement (ThermoFisher Scientific) containing 10% (vol/vol) FBS and 100 U/mL penicillin/streptomycin. For cytokine production, PBMCs were stimulated with 2 μM class A CpG (ODN 2216; Miltenyi Biotec) or 2 μM ORN R-2336 (Miltenyi Biotec). For pDC/T-cell co-cultures, the cells were purified as described above (isolation of human peripheral blood cells). Purified pDCs (1 × 105) were cultured with autologous or allogeneic naive CD4⁺ T cells (5 × 105) for 5 days in the absence or presence of anti-CD3/CD28 beads (T-cell activation/expansion kit; Miltenyi Biotec) at a bead-to-cell ratio of 1:2. Cytokine production was measured by intracellular staining.

iPS-DCs were plated at 1 × 10⁴ cells per well in 200 μl of X-VIVO-15 medium without cytokines. TLR ligands were added directly to the medium, and supernatants were harvested after a 24-h incubation at 37°C. For the assays, TLR ligands were used at the following concentrations: Pam3CSK4, 300 ng/ml (InvivoGen); poly(I·C), 50 μg/ml (InvivoGen); lipopolysaccharide, 500 ng/ml (Sigma-Aldrich); imiquimod, 50 μg/ml (InvivoGen); and ODN 2216, 3 μg/ml (Miltenyi Biotech). For RIG-I stimulation, 1 μg of 3p-hpRNA was complexed with LyoVec (InvivoGen) for 15 min at room temperature and then added to iPS-DCs at 10 ng/ml.