Method Details
White blood cells were incubated for 15 min in the dark with the monoclonal antibodies anti-CD14 FITC, anti-HLA-DR-PE, anti-CD45 PC5 (Beckman Coulter, Marseille, France) and Quantibrite HLA-DR/anti-monocyte PerCP-Cy5 (Becton Dickinson, Cockeysville Md). White blood cells were also incubated for 15 min with anti-CD3 FITC, anti-CD4 FITC and anti-CD19 FITC (fluorescein isothiocyanate, emission 525 nm, Beckman Coulter); with anti-CD4 PE, anti-CD8 PE, and anti-CD(16+56) PE (phycoerythrin, emission 575nm, Beckman Coulter); and with anti-CD45 PC5 (emission 667 nm, Beckman Coulter). Fluorospheres (Beckman Coulter) were used for the determination of absolute counts. Cells were analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co, Miami, Florida). Isotypic IgG controls stained also with anti-CD45 were used for each patient. Results were expressed as absolute molecules of HLA-DR on CD14/CD14+CD45+ cells, percentages, and MFI.
In separate experiments, whole blood was treated with 10 μg/mL brefeldin A (Invitrogen, Carlsbad, CA, USA) for 60 min at 37°C in 5% CO2 followed by incubation in the dark for 15 min at room temperature with anti-CD4 FITC, anti-CD14 FITC (Beckman Coulter) and anti-CD45 PC5 followed by red blood cells lysis. Stained cells were then washed with Dulbecco’s Phosphate Buffered Saline and permeabilized with the IntraPrep Parmeabilization Reagent kit (Beckman Coulter). Anti-IL-6 PE (Beckman Coulter) was added and analyzed after flow cytometry.
Peripheral blood mononuclear cells (PBMCs) were isolated after gradient centrifugation over Ficoll (Biochrom, Berlin, Germany) for 20 min at 1,400 g. After three washings in ice-cold PBS pH 7.2, PBMCs were counted in a Neubauer plate with trypan blue exclusion of dead cells. They were then diluted in RPMI 1640 enriched with 2mM of L-glutamine, 500 μg/mL of gentamicin, 100 U/mL of penicillin G, 10 mM of pyruvate, 10% fetal bovine serum (Biochrom) and suspended in wells of a 96-well plate. The final volume per well was 200 μL with a density of 2 × 106 cells/mL. PBMCs were exposed in duplicate for 24 h or 5 days at 37°C in 5% CO2 to different stimuli: 10 ng/mL of Escherichia coli O55:B5 lipopolysaccharide (LPS, Sigma, St. Louis, USA); 5 × 105 colony forming units of heat-killed Candida albicans; 25% of patient plasma; or 10 μg/mL of Tocilizumab (Roche, Athens, Greece). After incubation, cells were removed and analyzed for flow cytometry. Concentrations of TNFα, IL-1β, IL-6, IL-17A and IFNγ were measured in cell supernatants and/or serum in duplicate by an enzyme immunoassay (Invitrogen, Carlsbad, California, USA). The lowest detections limits were as follows: for TNF-α, 40 pg/mL; for IL-1β, 10 pg/mL; for IL-6, 10 pg/mL; for IL-17A, 10 pg/mL; and for IFN-γ, 12.5 pg/mL. Concentrations of ferritin were measured in serum by an enzyme-immunoassay (ORGENTEC Diagnostika GmbH, Mainz, Germany); the lower limit of detection was 75 ng/mL.
Six patients with immune dysregulation were treated with single intravenous infusion of 8 mg/kg of Tocilizumab (RoAcremra, Roche) based on the National Health Care off-label provision for COVID-19. Absolute lymphocyte blood counts were compared before and after Tocilizumab infusion.