Antibody coated magnetic beads

Tonsil samples were digested as described previously^{56 (link)}. In brief, samples were cut into small fragments, digested with 0.1 mg mL⁻¹ Liberase TL (Roche) in the presence of 0.1 mg mL⁻¹ DNAse (Roche) for 40 min at room temperature before addition of 10 mM EDTA. Cells were filtered on a 40 μm cell strainer (BD Falcon) and washed. Light density cells were isolated by centrifugation on a Ficoll gradient (Lymphoprep, Greiner Bio-One). DCs were enriched by depletion of cells expressing CD3, CD15, CD19, CD56, and CD235a using antibody-coated magnetic beads (Miltenyi). Cell subsets were further isolated by cell sorting on a FACSAria instrument after staining for CD11c, HLA-DR, CD14, CD304, CD1c, and CD141 (BD Biosciences). Peripheral blood mononuclear cells (PBMC) were prepared by centrifugation on a Ficoll gradient. Blood CD14⁺ monocytes were isolated from healthy donors’ PBMC by positive selection using anti-CD14-coated magnetic beads according to manufacturer’s instructions (Miltenyi). DCs and macrophage population from ascites were isolated after centrifugation of total ascites cells on a Ficoll gradient, enrichment by depletion of cells expressing CD3, CD15, CD19, CD56, and CD235a using antibody-coated magnetic beads (Miltenyi), and cell sorting on a FACSAria instrument. Ascites DCs were gated as HLA-DR⁺CD11c⁺CD1c⁺CD16⁻ and ascites macrophages as HLA-DR⁺CD11c⁺CD1c⁻CD16⁺.

The activity of 28 was evaluated on LPS-stimulated
cytokine production in monocytes. Peripheral blood mononuclear cells
(PBMCs) were first isolated from blood using Lymphoprep-based separation,
a method which is based on the lower buoyant density of mononuclear
cells (monocytes and lymphocytes) compared with other blood cell types
such as erythrocytes and polymorphonuclear leukocytes (granulocytes).
From these PBMCs, CD14+ monocytes were selected using antibody-coated
magnetic beads (Miltenyi Biotec, DE). CD14+ monocytes were seeded
in 96-well plates and preincubated with a serial dilution of 28 for 1 h before LPS triggering (Sigma-Aldrich; 100 ng/mL
final concentration). TNFα and IL-10 were measured in the supernatant
after 4 h of LPS triggering using enzyme-linked immunosorbent assay
(ELISA)-based readouts.

Human MoDC (1 × 10⁶ cells/mL) were transduced with DAP12 adenoviruses or LPS for 48 h. Supernatants were collected and analyzed for the presence of IL-12p70, IL-10, TNF-α, IFN-γ, and IL-1β by ELISA (Ready-SET-Go kit) following the manufacturer’s instructions (eBioscience; San Diego, CA, USA). ELISA kit for IL-15 were purchased from BD Biosciences. For detection of CD8⁺ T cells secreting IFN-γ, EliSpot was performed using CD8⁺ T cells from spleens that were purified by positive selection using antibody-coated magnetic beads following the directions provided by the vendor (Miltenyi Biotec; Auburn, CA, USA). Responder (CD8 purified) cells were incubated at 3 × 10⁵, 1 × 10⁵ and 3 × 10⁴ cells per well together with 5 × 10⁴ stimulator cells (B16 cells). Cultures were incubated at 37 °C for 20 h and spots (IFN-γ producing cells) were developed as described by the EliSpot kit manufacturer (Mabtech, Inc.; Mariemont, OH, USA). Spot counting was done with an AID EliSpot Reader System (Autoimmun Diagnostika GmbH; Strassberg, Germany).