flt3 ligand in cooperation with transforming growth factor-beta 1 potentiates in vitro development of Langerhans-type dendritic cells and allows single-cell dendritic cell cluster formation under serum-free conditions

Using a recently described serum-free culture system of purified human CD34(+) progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-beta 1 (TGF-beta 1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development, The addition of FL to serum-free cultures of CD34(+) cells supplemented with TGF-beta 1 granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and stem cell factor strongly increases both percentages (mean, 36% +/- 5% v 64% +/- 4%; P = .001) and total numbers (4.4- +/- 0.8-fold) of CD1a(+) dendritic cells. These in vitro-generated CD1a(+) cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-beta 1. Upon omission of TGF-beta 1, percentages of CD1a(+) DC decreased (to mean, 10% +/- 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a(-) cells that are lysozyme [LZ(+)]; myeloperoxidase [MPO+]; CD14(+)) increased approximately threefold (P < .05). Furthermore, in the absence of TGF-beta 1, FL consistently promotes generation of LZ(+), MPO+, and CD14(+) cells, but not of CD1a(+) cells. Serum-free single-cell cultures set up under identical TGF-beta 1- and FL-supplemented culture conditions showed that high percentages of CD34(+) cells (mean, 18% +/- 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-beta 1 supplementation, Lag(+) colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates, Total cloning efficiency in the absence and presence of TGF-beta 1 is virtually identical (mean, 41% +/- 6% v 41% +/- 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-beta 1 to show this costimulatory effect. (C) 1997 by The American Society of Hematology.