flt3 ligand in cooperation with transforming growth factor-beta1 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-beta1 (TGF-beta1) 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-beta1, 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-beta1. Upon omission of TGF-beta1, 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-beta1, 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-beta1- 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-beta1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-beta1 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-beta1 to show this costimulatory effect.
Strobl
H
H
Institute of Immunology-Vienna International Research Cooperation Center (VIRCC) at Sandoz Forschungsinstitut (SFI), University of Vienna, Austria.
Bello-Fernandez
C
C
Riedl
E
E
Pickl
W F
WF
Majdic
O
O
Lyman
S D
SD
Knapp
W
W
D018949:Antigens, CD1 / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D002449:Cell Aggregation; D016895:Culture Media, Serum-Free; D003713:Dendritic Cells / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006410:Hematopoiesis; D006801:Humans; D066298:In Vitro Techniques; D007801:Langerhans Cells / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D019089:Stem Cell Factor / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D018949:Antigens, CD1; D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D019089:Stem Cell Factor; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; C084881:flt3 ligand protein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor