Orn r2336

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.

Cells were cultured in RPMI medium 1640 with GlutaMAX supplement (Thermo Fisher Scientific) containing 10% (vol/vol) FBS and 100 U/ml penicillin/streptomycin. For cytokine production, cells were stimulated with 2 μM class A CpG (ODN 2216; Miltenyi Biotec) or ORN R-2336 (Miltenyi Biotec). For pDC/T cell coculture, pre-enriched pDCs were treated with 10 ng/ml recombinant human TNF-α (R&D Systems) for 24 h and then washed twice to remove residual TNF-α before use in subsequent culture. Untreated or TNF-α–treated pDCs were cultured with allogeneic naive CD4⁺ T cells at 1:5 ratio for 5 d. For cytokine detection, cells were cultured in the last 4 h with GolgiPlug (BD Biosciences). Cell proliferation was measured using the CellTrace Violet Cell Proliferation kit (Thermo Fisher Scientific), according to the manufacturer’s instructions.