Odn2216

Leukopacks were obtained from the Etablissement Français du Sang. Peripheral blood mononuclear cells (PBMCs) were recovered using density gradient centrifugation. The pDCs were isolated using magnetic beads on AutoMacs (Miltenyi Biotech) as previously described (Ka et al., 2014 (link)). Briefly, pDCs were isolated by depletion of non-pDCs that were retained in the column, while unlabeled pDCs with high purity (90%) were collected in the flow-through. Plasmacytoid dendritic cells were then suspended in RPMI 1640, supplemented with 20 mM HEPES, 10% fetal calf serum, 2 mM L-glutamine, 100 U penicillin/ml, 50 μg/ml streptomycin (Life Technologies), and 10 ng/ml recombinant IL-3 (R&D Systems), as described previously (Dental et al., 2012 (link)) and were stimulated with heat-inactivated (100°C for 30 min) C. burnetii organisms (bacterium-to-cell ratio of 50:1), or CpG-A 10 μg/mL (ODN 2216; InvivoGen) for 8 or 24 h.

Whole venous blood was collected into tubes containing 1.8 mg/ml K₂EDTA (Becton Dickinson, Oxford, UK). PBMCs were isolated using Ficoll-plaque gradients (Cedarlane, Burlington, ON, Canada) as previously described [17 (link)]. Monocytes were isolated from PBMCs using CD14⁺ selection beads (Miltenyi Biotec, Bisley, UK) as per the manufacturer’s instructions before being cultured in RPMI1640 medium supplemented with 5% (v/v) fetal calf serum (PAN-Biotec, Aidenbach, Germany) and 1% (v/v) penicillin–streptomycin solution (PAA, Pasching, Austria). Cells were incubated with or without 100 ng/ml PAM₃CSK₄ (PAM3) or 10 ng/ml flagellin (Axxora, Exeter, UK), 1 ng/ml FSL-1, 10 ng/ml lipopolysaccharide (LPS), 2 μg/ml resiquimod (R-848), 2 μM ODN2006 or 2 μM ODN2216 (Invivogen, Toulouse, France) for 18 h at 37°C, 5% CO₂. The concentrations of the TLR ligands were previously determined by titrating each ligand to determine the threshold for maximal activation.

Purified NK cells were cultured in RPMI-1640 (Invitrogen, Burlington, ON, Canada) supplemented with 10% FBS at a concentration of 1 × 10⁶ cells/mL in 96-well round-bottomed plates for 20 h. NK cells were either unstimulated or stimulated by IL-15 (20 ng/mL; Miltenyi Biotec Inc., Auburn, CA, USA), IFN-α (1000 IU/mL; Merck, Kirkland, Quebec, Canada), or IL-2 (20 IU/mL; Novartis Pharmaceuticals Canada, Dorval, Quebec, Canada). For NK/pDC co-cultures, purified NK cells and pDCs were mixed at a ratio of 10:1 in the presence of CpG oligodeoxynucleotides (10 μg/mL; ODN 2216; InvivoGen, San Diego, CA, USA). For trans-well experiments, NK/pDC co-cultures were performed in 24-well plates using ThinCert inserts (Greiner Bio-One GmbH, Frickenhausen, Germany) with a 0.4-μm pore size. For type I IFN-blocking experiments in NK/pDC co-cultures, non-specific binding sites were blocked by adding 2 μg of mouse IgG2a, followed by the addition of mouse anti-human type I IFN-receptor chain 2 and anti-human IFN-α antibodies (20 μg/mL each; PBL Assay Science, Piscataway, NJ, USA).

Isolated spleen or BM pDCs (100k) were resuspended in 100 μL of complete 1640 medium and then stimulated in a 96-well U-bottom plate with 1-μM ODN2216 or 10-μg/mL poly U (Invivogen, San Diego, CA, USA) supplemented with Lipofectamine 2000 (ThermoFisher Scientific) (1.5 μL: 1000 μL) for 48 h. The supernatants were collected and stored at −80 °C for enzyme-linked immunosorbent assay (ELISA). Each group consisted of at least 3 mice, and each experiment was repeated at least 3 times.

The breast cancer cell lines, AU565 (MAM-A⁺/HLA-A2⁺) and MCF-7 (MAM-A⁻/HLA-A2⁺), and human monocyte-like cell line, THP-1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human CD8+T cells from HLA-A2⁺ healthy subjects were obtained from StemCell technologies (Cambridge, MA, USA). All cell cultures and incubations were performed as per provider’s recommendations and described by us before [19 (link),20 (link)]. Briefly, cells were cultured in RPMI-1640 medium at 37 °C in a 5% CO₂ incubator until they were 80% confluent. The presence of MamA and HLA-A2 expression in the breast cancer cell lines was confirmed by western blot analysis (data not shown). The ODNs—ODN2216, ODN2006, and ODN M362 (respectively controls represented by suffix ‘c’)—were at vaccigrade and obtained from InvivoGen (San Diego, CA, USA). For ODN stimulation, the breast cancer cells and the THP-1 cells were cultured in 24 well plates, 1 × 10⁵ per well pre-stimulated with PMA (phorbol myristate acetate, 10 ng/mL medium) for 16 h, washed once with RPMI-1640 media, and stimulated with ODNs (10 µM) for 5 h. These cells were later used for various experiments detailed below.

Purified splenic B cells and BM-derived cDCs and pDCs isolated from individual mice were stimulated or not with mouse IFN-α11 (1000 U/ml, Miltenyi Biotec), the TLR7 ligand R848 (1 μg/ml, InvivoGen), the TLR9 ligand ODN-2216 (CpG, 1 μg/ml, InvivoGen), anti-IgM (20 μg/ml, eBioscience), or combinations thereof. At the indicated time-points, cells were harvested, counted, and analyzed by flow cytometry, while supernatants were assayed for cytokines by ELISA. In vivo TLR9 stimulation was induced by injecting mice i.v. with 2 μg CpG-A (ODN-2216) mixed with the cationic lipid DOTAP. Six hours later, serum was collected and tested for the presence of IFN-α by ELISA.