Generation of antigen-loaded dendritic cells in a serum-free medium using different cytokine combinations.
Dendritic cells (DCs) are antigen-presenting cells that play a critical role in the induction of cytotoxic T-lymphocytes. An optimal method for the generation of DC for clinical use remains to be established. The aim of our study was to find an optimal cytokine combination for DC generation from peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) in serum-free conditions. Serial immunophenotyping enabled us to observe changes in DC content during the culture as well as the development of maturation and activation markers. As a source for DC culture, we used PBSC from patients with multiple myeloma after stem cell mobilization using cyclophosphamide and G-CSF, or PBMC from healthy donors without mobilization. The cells were cultured in a serum-free medium with different cytokine combinations including GM-CSF, TNF-alpha, Flt-3, CD40L, IFN-gamma, IL-1alpha, IL-6, PGE1, and IL-4. The cell cultures were evaluated by immunophenotyping. For PBMC, interleukin-12 assay was performed. For PBSC, the yield of DC as determined by CD83+ cell count ranged from 0. 6 x 10(5) to 30.1 x 10(4) (mean: 9.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated nucleated cells from apheresis. This yield corresponded to (0.3-19.1) x 10(5) (mean: 4.3 x 10(5)) per 1 x 10(6) of CD34+ cells in the apheresis products. For PBMC, the yield was (0.4-24.8) x 10(4) (mean: 2.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated mononuclear cells from venous blood. The cultured cells expressed the mature immunophenotype. No significant differences in cell yield or immunophenotype were detected when comparing different cytokine combinations.
Büchler
Tomas
T
Department of Clinical Hematology, Masaryk University Hospital, Brno, Czech Republic. tbuchler@fnbrno.cz
Hajek
Roman
R
Bourkova
Lida
L
Kovarova
Lucie
L
Musilova
Romana
R
Bulikova
Alena
A
Doubek
Michal
M
Svobodnik
Adam
A
Mareschova
Iveta
I
Vanova
Pavlina
P
Tuzova
Eva
E
Vidlakova
Petra
P
Vorlicek
Jiri
J
Penka
Miroslav
M
D000941:Antigens / Q000008:administration & dosage*; D019496:Cancer Vaccines / Q000008:administration & dosage; D002454:Cell Differentiation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000008:administration & dosage*; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000008:administration & dosage; D006801:Humans; D016130:Immunophenotyping; D007167:Immunotherapy; D018664:Interleukin-12 / Q000096:biosynthesis; D015847:Interleukin-4 / Q000008:administration & dosage; D011994:Recombinant Proteins / Q000008:administration & dosage; D014409:Tumor Necrosis Factor-alpha / Q000008:administration & dosage
D000941:Antigens; D019496:Cancer Vaccines; D016895:Culture Media, Serum-Free; D016207:Cytokines; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor