Generation of dendritic cells from adherent cells of cord blood by culturewith granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha

Although dendritic cells (DC) can be cultured from cord blood (CB) CD34(+) progenitor cells, the generation of DC from CB monocytes has not been repor ted. In this paper, we explored the generation of DC from CB monocytes to e stablish the simplest way to obtain a substantial number of DC from CB. We isolated monocytes from CB mononuclear cells (CB-MNC) by the plastic adhere nce method. These adherent cells (monocyte-rich cells) were cultured in RPM I 1640 medium supplemented with 10 % fetal bovine serum (FBS) or in serum-f ree X-VIVO 15 medium (SFM) for 7 days, both of which contained 100 ng/ml gr anulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml interl eukin-4 (IL-4) with or without 10 ng/ml tumor necrosis factor-alpha, (TNF-a lpha) (added at day 5). In the presence of GM-CSF and IL-4, CB-adherent cel ls became nonadherent, acquired DC morphology, and showed increased express ion of CD1a, CD80, CD86, and HLA-DR; they lost membrane CD14 and some cells with the expression of CD83 and CMRF-44 were generated. With the addition of TNF-alpha to these cultures and culturing for further 2 days, the propor tion of CD83(+) cells was elevated in both the FBS and SFM culture systems, compared with the culture without TNF-alpha. In the culture with TNF-alpha , cells expressing CD1a, CD80, CD86, HLA-DR, and HLA-DQ were markedly incre ased. TNF-alpha-treated cells were demonstrated to be stronger stimulators for proliferation of both allogeneic CB lymphocytes and PB lymphocytes than were cells not treated with TNF-alpha. The yield of CD83+ DC at day 7 of c ultures was 4.9 +/- 1.1 x 10(5) or 3.0 +/- 0.5 x 105 per 1.2 x 10(7) CB-MNC plated initially when cultured in FBS or SFM, respectively. These results have shown that a substantial number of mature DC could be generated from C B-adherent cells even by serum-free culture. We then compared these CB-adherent cell-derived DC (CB-DC) with peripheral blood (PB)-adherent cell-derived DC (PB-DC) in cell-surface phenotype and f unction. We found day 7 CB-DC have lower expression of CD80, CD1a, CD83, an d CMRF-44 than day 7 PB-DC, but CB-DC have a similar capacity to stimulate the proliferation of both allo-CB lymphocytes and PB lymphocytes, compared with PB-DC. CB-DC cultured with GM-CSF and IL-4 have almost identical capac ity of phagocytosis to take up fluorescein isothiocyanate (FITC)-dextran an d Lucifer yellow (LY), compared with PR-DC, In summary, our findings suggest CB adherent cells, when cultured with GM-C SF, IL-4, and TNF-alpha, are a potent source of functional DC. Thus, CB-DC as well as PR-DC may become valuable tools for immunotherapy.