Interleukin 6 (il 6)

IL-6, IL-11, LIF, and OSM were purchased from Miltenyi Biotec. IL-27 was purchased from Bio-Techne, IL-6/IL-6R alpha was purchased from R&D Systems, and IFN-α (Intron A) was purchased from Merck. All cytokines were used at a final concentration of 100 ng/mL, with the exception of IFN-α, which was used at 10⁵ U/mL.

1 × 10⁴ purified CD34 + cord blood cells were cultivated with 10 μg/ml of anti-BCAM antibody or PBS as control for 3 days in 100 μl of serum-free expansion medium (SFEM) supplemented with 100 ng/ml Flt-3 ligand, 100 ng/ml stem cell factor (SCF), 20 ng/ml IL-3 (interleukin-3; Miltenyi) and 20 ng/ml IL-6 (interleukin-6). Cells were counted and 3 × 10³ cells were suspended in 300 μl of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 2% fetal bovine serum (Stem Cell Technologies, Vancouver, BC Canada). Cell suspensions were added to 3 ml methylcellulose medium (MethoCult H4434, Stem Cell Technologies) and 1 ml of cell suspension was plated in triplicates on 35-mm petri dishes and cultured in a humidified incubator with 5% CO₂ at 37°C for 14 days.
Cell aggregates containing >50 cells were scored as single colonies using an inverted microscope (Axiovert 135: Zeiss). Colonies were classified as burst-forming unit – erythroid (BFU-E), colony-forming unit – granulocyte/macrophage (CFU-GM) or colony-forming unit - granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) based on their morphology.

Naïve (CD25-) CD4+ T cells were isolated using the CD4+ T Cell Isolation Kit, mouse and the CD25 MicroBead Kit, mouse by Miltenyi Biotec. Instead of the recommended buffer in the kit, R10 medium (RPMI-1640 with 10% FCS (Gibco) and 1% Penicillin/Streptomycin (Sigma-Aldrich)) was used and the amount of reagents suggested per 10⁷ cells was instead used per 1.5 · 10⁷ cells.
For the polarization of the CD4+ CD25- T cells, a 24-well-plate was coated with 10 μg/ml anti-CD3 and 10 μg/ml anti-CD28 in PBS for 1 h at 37 °C and washed one time with PBS before the cells were seeded at a concentration of 10⁶ cells in R10 medium for Th0 and Th1 polarization. Lastly, 5 μg/ml anti-IL-4 (BioXcell) and 10 ng/ml IL-12 (R&D systems) diluted in PBS were added for Th1 polarization. For IL-6 treatment during polarization, 10 ng/ml or 100 ng/ml IL-6 (Miltenyi Biotec) were supplemented. The cells were in total cultured for five days at 37 °C and 5% CO₂, with a medium change on day four. On day five, the cells were stimulated by addition of eBioscience™ Cell Stimulation Cocktail (plus protein transport inhibitors) (500X) (Invitrogen) for 3.5 h before harvest of supernatant for ELISA and cells for flow cytometry. The shown data after in vitro polarization are representative of at least four (Figs. 2, 3) or two (Fig. 4) independent experiments.

Where indicated, CD45RA⁺RO⁻ naïve CD4⁺ T cells were cultured in X-Vivo 15 (Lonza, Cologne, Germany) media supplemented with 1% human serum and 1% penicillin-streptomycin (both Sigma-Aldrich, Saint Louis, USA). Cells were cultured for 4 days with the T cell Activation/Expansion Kit (Miltenyi Biotec) and recombinant human TGF-β (5 ng/μl; PAN™-Biotech GmbH, Aidenbach, Germany), IL-1β (12.5 ng/μl), IL-6 (25 ng/μl; both Miltenyi Biotec), and IL-23 (25 ng/μl; PeproTech, Rocky Hill, USA). Medium and cytokines were refreshed after 3 days.

PBMCs were rested for 2 hours at 37 °C (5% CO₂), left unstimulated or stimulated with indicated cytokine for 20 minutes then processed according to the manufacturer’s protocol (BD). The following were used: IL-6 (100 ng/ml, MiltenyiBiotec), IL-21 (100 ng/ml, MiltenyiBiotec).