Intracellular Staining Procedure

Intracellular staining protocol was optimized by Beckman Coulter Immunotech (Marseille, France).

For monocytes, 100 μl of heparin anticoagulated whole blood was directly added to the stimulation tube (DurActive3^® tube containing dry coated lipopolysaccharide (LPS) and Brefeldin A, Beckman Coulter, Brea, US) or to an empty control tube. After 3h incubation at 37°C, cells were labeled with cell surface antibodies: FITC-labeled anti-CD16, ECD-labeled anti-HLA-DR, PB-labeled anti-CD14, and KrO-labeled anti-CD45 (all from Beckman Coulter, Brea, US). Thereafter, samples were washed with PBS and treated with the IntraPrep Permeabilization Reagent set (Beckman Coulter, Brea, US) according to the manufacturer’s instructions. Samples were then stained for 45min at room temperature in the dark with intracellular antibodies: PE-labeled anti-IL10 (BioLegend, San Diego, US) or Rat IgG2a PE-labeled isotype control antibody (BioLegend, San Diego, US) and AF700-labeled anti-TNFα (Beckman Coulter, Brea, US) or mouse IgG1 AF700-labeled isotype control antibody (BioLegend, San Diego, US). Isotype controls of anti-IL-10 and anti-TNF antibodies were used in order to evaluate non-specific binding both in stimulated and non-stimulated conditions and thus to set-up threshold of positivity when markers were expressed as percentages of positive cells.

For lymphocytes, 100 μl of heparin anticoagulated whole blood was directly added to the stimulation tube (DurActive1^® tube containing dry coated Phorbol 12-Myristate13 Acetate (PMA), Ionomycin and Brefeldin A, Beckman Coulter, Brea, US) or to an empty control tube. After 3h incubation at 37°C, cells were labeled with cell surface antibodies: PC7-labeled anti-CD19, KrO-labeled anti-CD45, PB-labeled anti-CD3 and APC-labeled anti-CD4 (all from Beckman Coulter, Brea, US). Thereafter, samples were washed with PBS and treated with the IntraPrep Permeabilization Reagent set (Beckman Coulter, Brea, US) according to the manufacturer’s instructions. Samples were then stained for 45min at room temperature in the dark with intracellular antibodies: PE-labeled anti-IL10 (BioLegend, San Diego, US) or Rat IgG2a PE-labeled isotype control antibody (BioLegend, San Diego, US).