Gm csf

Monocyte-derived DCs were generated as described previously (Heilingloh et al., 2014 (link)). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from different healthy donors using a Lymphoprep gradient (Nycomed Pharma AS, Oslo, Norway) and afterward monocytes were separated using plastic adherence. By addition of 800 U/ml granulocyte macrophage-colony stimulating factor (GM-CSF; CellGenix, Freiburg, Germany) and 250 U/ml IL-4 (Milteny, Bergisch-Gladbach, Germany) monocytes differentiated to immature DCs. Maturation was induced by adding 10 ng/ml TNF-α (Beromun, Boehringer Ingelheim, Germany), 1 mg/ml prostin E2 (PGE2; Pfizer, NY, USA); 200 U/ml IL-1β (CellGenix), 40 U/ml GM-CSF, 1000 U/ml IL-6 (CellGenix), and 250 U/ml IL-4 to the medium. The maturation status was controlled by flow cytometry, and mDCs were used for further experiments.

Monocytes were enriched from leukapheresis products as described before [25 (link)]. Monocytes were cultured in X-VIVO 15 medium (Lonza) supplemented with 2% human serum (HS; Sanquin), IL-4 (500 U/ml), GM-CSF (800 U/ml, both CellGenix) and KLH (10 μg/ml, Calbiochem). DCs were matured with a cocktail of 10 ng/ml TNF-α, 5 ng/ml IL-1β, 15 ng/ml IL-6 (all CellGenix) and prostaglandin E₂ (10 μg/ml, Pharmacia & Upjohn) [26 (link)]. Cells used for the DTH skin test were cultured without KLH. DCs were electroporated with mRNA encoding gp100 or tyrosinase, as previously described [27 (link)]. Patients could only participate if the predefined phenotypic minimal release criteria used in clinical trials were met [28 (link)]. DCs were administered both intradermally (maximum of 10 × 10⁶ cells) and intravenously (maximum of 20 × 10⁶ cells).

Blood from healthy subjects was obtained from the Blood collection center of Angers (agreement ANG 2003-2). Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density-gradient centrifugation. To generate Mϕ, monocytes were purified from PBMC by positive selection using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany); purity was >99% (data not shown). Purified CD14⁺ monocytes (1 × 10⁶ cells/ml) were differentiated into Mϕ by 5-day culture with 20 ng/ml M-CSF (Biotechne) and 2 ng/ml GM-CSF (CellGenix, Freiburg, Germany) in complete medium (CM), consisting of RPMI 1640 medium containing 2 mM l-glutamine, antibiotics (all from Lonza, Verviers, Belgium), and 10% (v/v) heat-inactivated fetal calf serum (Biowest, Nuaillé, France). After Ficoll-Paque centrifugation, neutrophils were isolated from erythrocytes by 1.5% (w/v) dextran (Amersham Biosciences) density-gradient sedimentation. Contaminating red blood cells were lysed by hypo-osmotic shock. Purity was routinely >98% (data not shown).

Peripheral blood monocytes were obtained using CD14^pos Microbeads kit (Miltenyi Biotech) or by elutriation and were cultured for 5 days with GM-CSF (800 UI/mL, Cellgenix, Breisgau, Germany) and IL-4 (250 UI/mL, R&D Systems). Immature DC were then washed and put again in culture for 2 days with or without agonist anti-CD200R antibodies (20 µg/mL, R&D Systems) or isotype control. Co-cultures were performed in parallel with stromal cells (40,000 cells/cm²) and/or immature DC (1 × 10⁶ cells/mL) that were cultured for 5 days with GM-CSF (800 UI/mL) and IL-4 (250 UI/mL). CD200R stimulation was then added for 2 supplemental days of culture. Culture supernatants were collected by centrifugation, whereas cells were used for RNA extraction. In co-culture conditions, DC were separated from stromal cells using CD45^posCD105^neg gating strategy and FACSARIA cell sorting.

For functional assays, DCs were isolated from buffy coats of healthy volunteers (Sanquin, Nijmegen, the Netherlands) after obtaining written informed consent per the Declaration of Helsinki and according to institutional guidelines. For RNA-seq measurements, cells were obtained from aphaeresis of 3 different donors. Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll density centrifugation (Lymphoprep; Axis-Shield PoC AS, Oslo, Norway). CD1c isolation kit of Miltenyi Biotec (Bergisch-Gladbach, Germany) was used to obtain CD1c⁺ mDCs, by following manufacturer's instructions. Subsequently, monocytes were depleted by either plastic adhesion, or by the use of CD14 microbeads (Miltenyi Biotec). Next, pDCs were purified by positive selection using anti–BDCA-4–conjugated magnetic microbeads (Miltenyi Biotec). DCs were cultured in X-VIVO-15 medium (Lonza, Basel, Switzerland) supplemented with 2% human serum (Sanquin). DCs were stimulated with: FSME (5%; Baxter AG, Vienna), pRNA (15μg/ml), CpG-P (5μg/ml; Miltenyi Biotech, Germany), GM-CSF (800 U/ml; (Cellgenix, Freiburg, Germany). pRNA complexes were prepared fresh 5-10 minutes before adding to the cell culture. pDCs were cultured with 10 ng/mL IL-3 (Cellgenix, Freiburg, Germany) as a survival factor in addition to the stimuli.

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood (Sanquin, Nijmegen, Netherlands) by density gradient centrifugation using lymphoprep (Axis-Shield). Adherent cells were obtained as previously described [32 (link)]. Immature DCs were generated by culturing the adherent cells in X-VIVO medium (Lonza) containing 2% human serum (Sanquin), with IL-4 (300 U/ml) and GM-CSF (450 U/ml; both CellGenix) for 6 days. On day 6, DCs were maturated with either a cytokine cocktail containing prostaglandin E₂ (PGE₂; 10 μg/ml; Pfizer), tumor necrosis factor (TNF)-α (10 ng/ml), IL-1β (5 ng/ml), and IL-6 (15 ng/ml; all CellGenix) or with toll-like receptor (TLR)3 and TLR7/8 ligands, poly[I:C] (20 μg/ml; Enzo), and R848 (4 μg/ml; InvivoGen), respectively, for 24 hours.