Staphylococcus aureus enterotoxin b seb

Purified genetically-detoxified Pertussis Toxin (PT; R9K, E129A; Limulus Amebocyte Lysate activity < 20 EU/mg) was purchased from LIST Biological laboratories Inc. (Campbell, CA, USA) and stored at 500 µg/mL at −20 °C. Purified Filamentous hemagglutinin (FHA) was kindly provided by Sanofi Pasteur (Marcy-l’Étoile, France) and stored at 700 µg/mL at 4 °C (Limulus Amebocyte Lysate activity < 66 EU/mg). A bacterial lysate of Bp strain B1917 [30 (link)] (Bp lysate/BPL) was kindly provided by Q Biologicals (Ghent, Belgium) and stored at 3.4 mg/mL at −80 °C. A peptide pool of 132 Bp immunogenic peptide sequences (referred to as peptide pool) from the five Bp vaccine antigens [15 (link)] was purchased from Pepscan (Lelystad, the Netherlands) and stored as a mix at 0.1 mM per peptide in PBS/10% DMSO at −20 °C. Staphylococcus aureus enterotoxin B (SEB) was purchased from Sigma (Saint Louis, MO, USA) and stored at 1 mg/mL at −20 °C. Purified anti-CD28 antibodies (stock concentration 1 mg/mL) and anti-CD49d antibodies (stock concentration 1 mg/mL) were purchased from eBiosciences (Landsmeer, The Netherlands). PT and Bp lysate were heat-inactivated for 10 min at 80 °C using a water bath to avoid any mitogenicity in the whole blood stimulations.

To study CD8⁺ T cell proliferation and functionality, 5000 DCs were stimulated as indicated and co‐cultured with 20 000 allogeneic naïve CD8⁺ T cells (CD8⁺, CD27⁺, CD45RO⁻, and CD45RA⁺) in the presence of 1 pg/mL Staphylococcus aureus enterotoxin B (SEB; Sigma–Aldrich). To determine proliferation, CD8⁺ T cells were incubated with 0.5 μM CFSE (Invitrogen) and washed extensively prior to co‐culture. At day 3 or 4, cells were incubated overnight with the modified thymidine analogue EdU (Click‐iT kit; Invitrogen) and further processed according to the manufacturer's instructions. The percentage of divided cells (EdU⁺ or CFSE⁻) was determined by flow cytometry (Canto II, BD Biosciences). To determine intracellular granzyme B expression, cells were harvested at day 4 or 5, washed with PBS, fixated with 4% formaldehyde (Sigma–Aldrich) for 15 min, washed again, permeabilized with 0.5% saponin (Calbiochem) in PBS containing 0.5% BSA (PAA) and 0.1% sodium azide (Merck), and stained with anti‐granzyme B‐PE (Sanquin Blood Supply) and analyzed by flow cytometry. For intracellular IFN‐γ or TNF staining, CD8⁺ T cells were restimulated at day 4 or 5 with 100 ng/mL PMA, 1 μg/mL ionomycin, and 10 μg/mL brefeldin A (all Sigma–Aldrich) for 6 h, washed, fixated, and permeabilized as described above, stained with anti‐IFN‐y‐ FITC and anti‐TNF‐APC (both BD Biosciences) and analyzed by flow cytometry.

Preparation of human inferior turbinate tissue was performed, essentially as described [28 ]. In short, human nasal tissue was cut in RPMI1640 tissue culture medium (Sigma-Aldrich, Belgium), complemented with 2mM L-Glutamine (Invitrogen, Belgium), 50 IU/ml penicillin, 50mg/ml streptomycin (Invitrogen) and 0.1% BSA (Sigma-Aldrich). Subsequently these pieces were passed through a mesh to achieve comparable sized fragments (±0.9mm³). After 1h equilibration, the obtained tissue fragments were washed with fresh culture medium, weighed and resuspended into 48-well plates (BD Falcon; VWR International, Belgium) as 0.04g/ml in 0.5 ml RPMI1640 tissue culture medium, prepared as above. Tissue suspensions were pre-incubated with either solvent, methylprednisolone (MP) (ranging from 10^-4M to 10^-11M) or compound A (ranging from 10^-4M to 10^-11M) for 1 hour at 37°C and 5% CO₂. Ensuing, tissue fragments were stimulated with 0.5 μg/ml (final concentration, fc) Staphylococcus aureus enterotoxin B (SEB, Sigma-Aldrich) for 24 hours. The SEB solvent PBS served as a negative control.