PBMC-derived immature DCs. Immature DCs were obtained from PBMCs as described previously [13]. Briefly, PBMCs were freshly isolated with Ficoll-paque (Amersham-Pharmacia, Uppsala, Sweden) from the peripheral blood of healthy volunteers, and CD14+ monocytes were separated immediately by magnetic depletion using a monocyte isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) containing hapten-conjugated
TRAP-5 to CD3, CD7, CD19, CD45RA, CD56, and anti-IgE Abs, and a magnetic cell separator (MACS, Miltenyi Biotec) in accordance with the manufacturer’s instructions, routinely resulting in >90% purity of CD14+ cells. The cells were cultured in 24-well culture plates for 6–7 days in complete medium supplemented with 20 ng/ml IL-4 (Biosource Intl., Camarillo, CA, USA) and 50 ng/ml of hGM-CSF obtained from PeproTech EC, or from the conditioned culture medium of 293FT cells transfected with hGM-CSF gene or hGM-CSF-C4BPα gene, in order to obtain iDCs. After 4–6 days of incubation, fluorescent activated cell sorter (FACS) analysis was performed to analyze the phenotype of the cells.Immunoblotting. The cells were lysed in triple-detergent lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.1% SDS, 100 g/ml phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium deoxycholate). The obtained samples were run on a 4–12% NuPAGE Bis–Tris gel (Invitrogen) using MES (morpholine ethanesulfonic acid), SDS (sodium dodecyl sulfate) buffer (1000 mM MES, 1000 mM Tris, 70 mM SDS, 20 mM EDTA) under non-reducing conditions and transferred to a PVDF (polyvinylidene difluoride) membrane (ATTO, Tokyo, Japan). The membrane was incubated with rabbit anti-myc serum (Invitrogen) and immunoblotting was carried out using horseradish peroxidase conjugated-goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) and a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate kit for peroxidase (VECTOR Laboratory, Burlingame, CA).Electron microscopy. The conditioned culture medium containing hGM-CSF-C4BPα gene-derived multimeric hGM-CSF was dialyzed against 0.1 M NH4OAc/0.05 M NH4HCO3, pH 7.35, were adsorbed to thin carbon films and were negatively stained with 4.0% uranyl acetate. The photographs were taken at a primary magnification of 40,000 in a Hitachi H-7500 transmission electron microscope, operating at 80 kV.ResultshGM-CSF produced by 293FT cells could induce iDCsThe first question was whether 293FT cells could produce enough amount of hGM-CSF to prepare iDCs from PBMCs. Human embryonic 293FT cells were transfected with the hGM-CSF gene and the hGM-CSF concentration was analyzed by ELISA. As a result, the hGM-CSF concentration in the conditioned medium was always high enough for dendritic cell preparation; between 150 and 200 ng/ml after transient transfection, or with cloned 293FT
cells producing hGM-CSF.The second question was whether the conditioned medium of the hGM-CSF-transfected 293FT cells could induce iDCs together with IL-4, because dendritic cells are equipped with pattern recognition receptors such as toll-like receptors that enable DCs to respond to very scarce amounts of stimulants such as LPS or nucleotides. Flow cytometric analysis of DCs prepared with the conditioned culture medium of hGM-CSF-transfected 293FT and commercial IL-4 showed the typical phenotype of iDCs derived from PBMCs, suggesting that the conditioned medium of 293FT did not contain any stimulants that induced the maturation of DCs ( Fig. 3, right panels).Fig. 3. FACS analysis of the immature dendritic cells. (A) The hGM-CSF gene or the hGM-CSF-C4BPα fusion gene was transfected into 293FT cells and conditioned medium containing hGM-CSF or multimeric GM-CSF was used in combination with IL-4 to induce dendritic cells from peripheral blood monocytes. The resultant DCs either with hGM-CSF (right panels) or multimeric GM-CSF (left panels) showed an identical phenotype each other. The results from the hGM-CSF gene and the hGM-CSF-C4BPα fusion gene are shown as grey lines and negative control data are shown as black lines. (B) The immature DCs induced with multimeric hGM-CSF showed the same response to LPS and polyI:C as that of the iDCs induced with hGM-CSF. Multimeric hGM-CSF-induced iDCs were incubated with LPS (200 ng/ml) (solid line) or poly I:C (100 μg/ml) (filled line) for 48 h and FACS analysis was performed. The iDCs without LPS or poly I:C is shown as dotted line.Figure optionsDownload full-size imageDownload as PowerPoint slideProduction of multimeric hGM-CSFNext, it was attempted to produce multimeric hGM-CSF using the hGM-CSF-C4BPα gene to see whether it could induce iDCs from PBMCs as well as authentic monomeric hGM-CSF. A chimeric gene of hGM-CSF fused to C4BPα was transfected into 293FT cells and the conditioned culture medium was analyzed by ELISA for hGM-CSF. Immunoblotting was also performed to analyze the hGM-CSF-C4BPα chimeric gene product under non-reducing conditions, which was observed as a band with a molecular mass of 220 kDa as well as faint bands with molecular masses of 56 and 112 kDa ( Fig. 3). The predicted molecular weight of the monomeric hGM-CSF-C4BPα gene product is 28 kDa ( Table 1), indicating that the major product of the hGM-CSF-C4BPα gene was an octameric protein, but dimer and tetramer proteins were also produced.Table 1. Characteristics of hGM-CSF-C4Bpα monomer with myc and 6× histidine tag.Value?
