Staphylococcus enterotoxin b

4 DosR regulon-encoded latency antigens (Rv1733c, Rv1737c, Rv2029 and Rv2628) and 3 secretory Mtb antigens (Ag85A/B, TB10.4, ESAT6/CFP10) were synthesized as recombinant proteins at the Department of Infectious Diseases, Leiden University Medical Center. CMVpp65 and CandidaMP65 peptide pools were procured from JPT Peptide Technologies (Berlin, Germany). Phytohemagglutinin (PHA) was purchased from Remel, Thermo Fisher Scientific and Staphylococcus enterotoxin B (SEB) was purchased from Sigma-Aldrich. Purified protein derivative (PPD) was procured from Staten Serum Institute, Denmark. Fluorochrome conjugated monoclonal antibodies to cell surface and intracellular markers used in flow cytometry assays are listed: CD45RA-APC-H7 (HI100), CD27-BV785 (O323), CCR7-Alexa Fluor 647 (G043H7), γδTCR-PE-CF594 (B1), CD3-BV570 (UCHT1), CD4-BUV395 (SK3), CD8-BV711 (RPA-T8), IFNγ-V450 (B27), TNFα-FITC (MAb11), IL2-APC or IL2-Alexa Fluor 700 (MQ1-17H12), IL17A-BV605 (BL168), IL17F-BV650 (O33-782), IL10-BV786 (JES3-9D7), IL22-PE-Cy7 (22URTI), and MIP1β-PE (D21-1351). Antibodies were sourced from BD Biosciences, BioLegend and eBioscience.

A heparinised venous blood sample was collected at the beginning of anaesthesia. All samples were processed within two hours of blood collection. Whole blood was stimulated at 37 °C for 24 hours with phytohaemagglutinin (PHA, Merck chemicals LTD., Beeston, Nottingham, UK) at a concentration of 5 µg/ml, staphylococcus enterotoxin B (SEB, Sigma Aldrich GmbH, Schnelldorf, Germany) at a concentration of 10 µg/ml, Candida albicans (Greer Laboratories, Lenoir, USA) at a concentration of 4 µl/ml or left unstimulated (nil control) in the presence of CD 28 and CD 49d antibodies (Biolegend Inc., San Diego, CA 92121, USA). After stimulation supernatants were stored at −80 °C until further analysis. Short-term whole blood stimulation assays of fresh blood were used as these require small blood volumes, and show increased sensitivity and lower background as opposed to assays using peripheral blood mononuclear cells^{18 (link),19 (link)}. For Vitamin D measurement, whole blood was centrifuged at 1000 G for 5 minutes and plasma stored at −80 °C until later batched analysis.

Heparinized blood was diluted 1:4 in RPMI 1640 medium (BioWhittaker, Walkersville, MD) containing l-glutamine (BioWhittaker), 80 mg/mL gentamicin and 1 % HEPES (GIBCO BRL, Gaithersburg, MD). Diluted whole blood (0.5 mL) was cultured alone or in the presence of A. lumbricoides adult worm antigen prepared as described previously [16 (link)] at 10 μg/mL, D. pteronyssinus (glycerinated solution, standardized at 5,000 AU/mL; Greer Laboratories, Lenoir, NC, USA) at a dilution of 1/50, and the superantigen, Staphylococcus enterotoxin B (SEB; Sigma Aldrich, St Louis, MO, USA) at 1 μg/mL. The cells were cultured within 5 h of collection and were maintained in a humidified environment of 5 % CO₂ at 37 °C for 5 days for the detection of cyokines.

Luminex is a multiplex bead array that measures multiple cytokine and chemokine production in a single sample of culture supernatant. PBMCS were thawed and adjusted to 5 x 10⁶ cells/ml in tissue culture medium containing either mapped-positive peptide pools at 1.5 μg/ml per peptide, staphylococcus enterotoxin B (SEB; Sigma-Aldrich) at 1.0 μg/ml or R10 media as a negative control together with anti-CD28 and anti-CD49d mAbs both at 1 μg/ml in a final volume of 200 μl per well. The plates were incubated for 48 hours at 37°C, 5% CO₂, and 150 μl of supernatant was removed from each well and stored at -80°C until use [69 (link)]. A human pre-mixed multi-analyte kit (Magnetic Luminex screening assay, R & D Systems Ltd) was used to measure the following analytes; IFN-γ, TNF-α, TNF-β, IL-2, IL-3, IL-5. IL-6, IL-13, IL-17A, IL-17E, IL-17F, IL-27, SDF-1α (CXCL12), IP-10 (CXCL10), MIP-1α (CCL3), MIP-1β (CCL4), MIP-3α (CCL20), RANTES (CCL5), FasL (CD95L), GMCSF, Granzyme A, Granzyme B, MIG (CXCL9), Fas (CD95) and CD40L. The culture supernatants were diluted 1:1 and assayed in duplicate according to the kit instructions. The plate was read using Luminex 200 and XPONENT softwares. Levels seen in unstimulated wells were used as background controls.

Isolated mononuclear cells from the blood and skin were adjusted to a density of 1 × 10⁷ cells/mL in culture medium. Cells were stimulated with 1 μg/mL Staphylococcus Enterotoxin B (SEB) (Sigma-Aldrich), plate bound αCD3/αCD28 (Thermo Fischer; each 1 μg/mL) or PMA (1ng/mL) plus Ionomycin (1 μg/mL) (Thermo Fischer) for 7 h at 37°C, 5% CO₂, with 5 μg/mL Brefeldin A (Biolegend) added during the last 2 h. Cultured cells without added antigen served as negative controls.