Sanne Hoeks

Chapter 3 42 METHODS CB samples were obtained from normal deliveries of full-term neonates (approved by the Medical Ethical Committee of the University Medical Center Utrecht). Following ethical guidelines of our hospital, no further information (eg, exact gestational age or sex) on the CB samples was provided, which limited the selection criteria for these samples. Adult samples were collected from healthy volunteers, aged between 21 and 35 years. Mononuclear cells from CB and APB were isolated by using Ficoll-Isopaque (Pharmacia, Uppsala, Sweden). Naive T cells and APCs were isolated by using magnetic cell sorting (CD4-T-cell enrichment and CD25 + CD45RO + T-cell depletion and CD3 + cell-depletion, respectively; BD Bioscience, San Jose, Calif, cell purity after magnetic cell sorting >95%) or by fluorescence-activated cell sorting by using ARIA II. A total of 50,000 T cells were cultured for 6 days with 100,000 autologous, nonirradiated (viable) APCs or 1 μg/mL anti-CD28 (eBioscience, San Diego, Calif), activated with 1 μg/mL plate-bound anti-CD3 (OKT3, eBioscience) and 30 units recombinant human IL-2/mL (Novartis, Arnhem, The Netherlands) in RPMI 1640 (Gibco, Breda, The Netherlands) supplemented with 10% human AB serum (Sanquin, Amsterdam, The Netherlands), 1% l-glutamine (Gibco), and 0.5% penicillin-streptomycin (Gibco), restimulated with phorbol 12-myristate 13-acetate (20 ng/mL) and ionomycin (1 μg/mL) in the presence of monensin (Golgistop, BD Bioscience) for 4 hours and subsequently analyzed for IL-17 (eBio64DEC17, eBioscience, phycoerythrin-conjugated) and FOXP3 (PHC101, eBioscience, eFluor450- conjugated) expression by using flow cytometric analysis. In other experiments, IL-21 (clone 3A3-N2) and IL-22 (clone 22URTI) production was also analyzed by flow cytometry. Recombinant IL-1β (Miltenyi Biotec, Bergisch Gladbach, Germany; 10 ng/mL), IL-6 (50 ng/mL), IL-23 (BD Bioscience, 10 ng/mL), GM-CSF (Immunotools, Friesoythe, Germany; 500 units/mL), and TGFβ (Peprotech, NJ, 5 ng/mL) were added to the cultures. Signaling of IL-1β and IL-6 was blocked with IL-1RA anakinra (Kineret) or IL-6 receptor blocker tocilizumab (RoActemra), both added as 1 μg/mL. Culture supernatants were analyzed for cytokine production by multiplex analysis, and cultured cells were analyzed for RORC2 and IL-17 mRNA expression. Detection limits for luminex were (lower limit-upper limit, pg/mL) as follows: IL-1β (0.6-4141), IL-6 (2.4-22643), IL-13 (0.43-10173), IL-17 (0.57-9940), IL-21 (12.2-87363), IL-22 (0.8-2294), IFNγ (6.24-20733), and GM-CSF (2.62- 40413).