flt3 Ligand in Cooperation With Transforming Growth Factor-β1 Potentiates In Vitro Development of Langerhans-Type Dendritic Cells and Allows Single-Cell Dendritic Cell Cluster Formation Under Serum-Free Conditions

Abstract

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-β1 (TGF-β1) 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-β1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, 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-β1. Upon omission of TGF-β1, 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-β1, 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-β1– 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-β1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-β1 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-β1 to show this costimulatory effect.