Sanne Hoeks

TH17 within first three months of life 55 4 For ex vivo cytokine production measurements, 1× 10 5 PBMCs were cultured in culture medium (RPMI 1640 supplemented with 1% l-glutamine and 100 mg/mL streptomycin and 100 U/mL penicillin; Gibco, Breda, The Netherlands) with 20 ng/mL phorbol myristate acetate and 1 μg/ mL ionomycin at 37°C. Then, either after 4 hours, supernatants were harvested for cytokine analysis, or after 30 minutes, monensin (Golgistop, BD Bioscience, San Jose, Calif) was added and cells were cultured for another 3.5 hours and stained for IL-17 production. For measurement of proliferation, differentiation, or cytokine production on immune activation, 1× 10 5 PBMCs were activated by plate-bound anti-CD3 (1 μg/mL, clone OKT3; eBioscience, San Diego, Calif) at 37°C. After 4 days, 3 H-labeled thymidine was added for another 16 hours for the measurement of proliferation. After 6 days of culture with plate-bound anti-CD3, supernatant was harvested for cytokine measurement and cells were used for flow cytometric analysis after restimulation with 20 ng/mL phorbol 12-myristate 13-acetate and 1 μg/mL ionomycin for 4 hours in the presence of monensin. Naive T cells and APCs were fluorescence activated cell sorting–sorted on an ARIA II (BD Bioscience) as CD4 + CD45RA + CD45RO − and CD3 − fractions, respectively. A total of 5× 10 4 naive T cells and 5× 10 4 APCs were activated by 1 μg/mL plate-bound anti-CD3 for 6 days and subsequently analyzed for FOXP3 expression by using flow cytometry. Flow cytometric analysis After thawing, an aliquot of cells was immediately stained with antibodies directed against CD3, CD4, CD8, CD14, and CD19 to quantify relative numbers of CD4 and CD8 T cells, monocytes, and B cells, respectively. Naive and memory cells were quantified by staining with CD45RA and CD45RO. For Treg-cell quantification, cells were permeabilized by using the FOXP3 staining kit from eBioscience and subsequently stained with an antibody against FOXP3 (clone PCH101, eBioscience). IL-17 (clone eBio64DEC17, eBioscience) was stained after restimulation with phorbol 12-myristate 13-acetate and ionomycin in the presence of monensin and permeabilization with the FOXP3 staining kit. After culture, cells were stained with a fixable viability dye (eBioscience) to discriminate between dead and viable cells. Cytokine analysis Supernatants of cell cultures were harvested and frozen at −80°C until analysis. Cytokine concentrations were measured by using Multiplex analysis (Luminex, Austin, Tex) as described elsewhere. 10 Statistics Significance of the differences observed was tested using Kruskal Wallis test and Dunn’s Multiple comparisons test using GraphPad Prism 5.03 for Windows. CB and infant samples were compared to samples derived from adult controls.