Il 10

CB mononuclear cells were prepared by density centrifugation using Ficoll-Paque^TM premium (GE Healthcare) and HSC were isolated using the CD34 MicroBead kit48 (link) and frozen for future use. The purity of CD34+ cells ranged from 90 to 98%. Thawed CD34⁺ cells were plated on irradiated EL08.1D2 cells and cultured as described by Grzywacz et al.30 (link) except for the addition of 50 ng/mL IL-15 for the last two weeks31 (link). Where indicated, different concentrations of human recombinant TGF-β (R&D Systems) and IL-10 (Prospec) were added.

Recombinant human cytokines used for DC treatments were as follows: LT-α (TNF-β), GM-CSF, M-CSF, IL-1β, IL-6, IL-4, TNF-α, IL-13, IFN-γ, IL-10 (ProSpec-Tany Technogene, Rehovot, Israel) and TGF-β1 (R&D Systems, Minneapolis, Minnesota, USA) were used in culture within a final concentration range of 5–80 ng/ml. Dexamethasone was used at a final concentration in culture at 30 ng/ml (Sigma-Aldrich, St. Louis, MO). All cell culture experiments utilized RPMI 1640 tissue culture medium, heat-inactivated (56°C/20 min) fetal calf serum (FCS), penicillin/streptomycin and L-glutamine (SAFC Biosciences, Lenexa, KS).

Recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage (M)-CSF, interleukin (IL)-1β, IL-6, IL-4, IL-10, IL-13, interferon (IFN)-γ, lymphotoxin (LT)-α, tumor necrosis factor (TNF), M-CSF and GM-CSF were purchased from ProSpec-Tany Technogene (Rehovot, Israel), transforming growth factor (TGF)-β1 from R&D Systems (Minneapolis, MN), and dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO). A mouse monoclonal antibody (clone 3C9) that recognizes the IgV domain of human CRIg was kindly provided by Dr. Menno van Lookeren Campagne (Genentech, San Francisco, CA). RPMI 1640 tissue culture medium, foetal calf serum (FCS) and L-glutamine were purchased from SAFC Biosciences (Lenexa, KS).