Large-scale isolation of immature dendritic cells with features of Langerhans cells by sorting CD34+ cord blood stem cells cultured in the presence of TGF-beta1 for cutaneous leukocyte antigen (CLA).
Epidermal Langerhans cells (LCs) are a subset of immature dendritic cells (DCs) and play a key role in the initiation and regulation of T cell responses. Upon antigenic stimulation, LCs differentiate into mature DCs undergoing profound morphologic and functional changes. Studies of the biological details of this conversion process have been hampered by difficulties in generating immature dendritic cells of a defined lineage. We propose a new method of purifying homogenous immature DCs in large numbers by sorting for CLA (Langerhans-like cells) from cord-blood-derived haematopoietic progenitor cells (HPCs). Established protocols describe the generation of LCs from CD34(+) HPCs by sorting for CD1a after 5 days of culture in the presence of GM-CSF and TNF-alpha. However, the numbers of LCs obtained by this method remain within the low range. Furthermore, CD1a is also expressed on interstitial DCs. LCs but not interstitial DCs express the cutaneous leukocyte antigen (CLA). The expression of CLA by cells stimulated with TNF-alpha and GM-CSF peaks on day 10. This expression can be raised further by stimulating the cells with TGF-beta1 and omitting TNF-alpha from day 6 onwards. CLA(+) cells were isolated on day 10 by AutoMACS. Their LC phenotype was established by the presence CD207. The immaturity of Langerhans-like cells was shown by the lack of CD83 and CD208 expression as well as their lower ability to activate allogeneic naive T cells as compared to maturing dendritic cells. However, CLA(+) cells cannot be termed Langerhans cells as they do not express Birbeck granules. Compared to sorting for CD1a (on day 6), sorting for CLA (on day 10) results in isolates of higher purity (80% vs. 50%) and a yield eight times higher (4.9x10(6) vs. 6.5x10(5) cells) when using identical numbers of input cells (5x10(5) cells). This novel method guarantees large numbers of pure and functionally active immature dendritic cells.
Bartz
H
H
Institute for Medical Microbiology, Philipps-University Marburg, Marburg, Germany.
Rothoeft
T
T
Anhenn
O
O
Bunse
D
D
Schauer
U
U
D018949:Antigens, CD1 / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D002021:Buffers; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002951:Citrates; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D005312:Fetal Blood / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D005947:Glucose; D006801:Humans; D016130:Immunophenotyping; D066298:In Vitro Techniques; D007231:Infant, Newborn; D007801:Langerhans Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008562:Membrane Glycoproteins / Q000378:metabolism; D008854:Microscopy, Electron; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D018949:Antigens, CD1; D018952:Antigens, CD34; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D002021:Buffers; C419393:CD1a antigen; C063419:CTAGE1 protein, human; D002951:Citrates; D008562:Membrane Glycoproteins; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C007077:citrate phosphate dextrose; D005947:Glucose