Recombinant canine il 4

Human and canine MoDCs were generated as previously described [9] . Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using an anti-human CD14 monoclonal antibody (clone TÜK4) conjugated to magnetic beads (MACS ® ; Miltenyi Biotec, Bergisch Gladbach, Germany), previously shown to cross-react with canine CD14 [11] . The CD14 + monocytes were seeded at 10 6 cells/ mL in RPMI-1640 (Life Technologies, Gaithersburg, MD, USA), containing 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine (Gibco Invitrogen Corp., Grand Island, NY, USA) and 100 U/mL polymixin B (Sigma-Aldrich, St. Louis, MO, USA).
Canine cell cultures were supplemented with 40 ng/mL recombinant human (rh) GM-CSF and 30 ng/mL recombinant canine (rc) IL-4 (R&D Systems Inc., Minneapolis, MN, USA), while human cells cultures were supplemented with 20 ng/mL rhGM-CSF and rhIL-4 (R&D Systems Inc.) every 2 days. Differentiated MoDCs were then primed with live B. canis (MOI=200), 1 µg/mL B. canis purified LPS or 1 µg/mL LPS from Escherichia coli strain 0128:B12 (Sigma-Aldrich, St. Louis, MO, USA) for 24 hours. Non-stimulated MoDCs were used as negative control. For each individual, experiments were performed separately.