Optimizing preparation of normal dendritic cells and bcr-abl+ mature dendritic cells derived from immunomagnetically purified CD14+ cells.
The goal of this work was to optimize dendritic cell (DC) preparations obtained from patients suffering from chronic myeloid leukemia (CML) and compare them with DC prepared from normal CD14+ mononuclear cells (MNC). We studied normal DC and bcr-abl+ leukemic DC (CML-DC) yields, expression of membrane molecules, differentiation status, and ability to stimulate T cells. We isolated DC precursors from PBMC by CD14-specific immunoadsorption and cultured them for 7 days in GM-CSF and IL-4, followed by a 3-day incubation to fully differentiate the cells. We evaluated cultures of CML-DC using RPMI 1640 medium supplemented with FBS and X-VIVO 15 medium containing human AB serum. In contrast to cells matured in RPMI 1640, virtually all cells incubated in X-VIVO 15 expressed CD83, a marker of mature DC. CML-DC and normal DC were indistinguishable in expression of CD83, resulting in the highest percentage reported so far. The yields of normal DC and CML-DC from CD14+ cells were indistinguishable. The percentage of bcr-abl+ cells in PBMC varied among patients between 65% and 97% and the final CML-DC preparations were >98% bcr-abl+ the highest purity of bcr-abl+ cells to date. Normal DC and CML-DC were equally effective in stimulating proliferation of allogeneic and autologous T cells. These techniques provide highly enriched, mature, functional CML-DC.
Dietz
A B
AB
Stem Cell Laboratory, Mayo Clinic Cancer Center, Rochester, MN 55905, USA.
Bulur
P A
PA
Erickson
M R
MR
Wettstein
P J
PJ
Litzow
M R
MR
Wyatt
W A
WA
Dewald
G W
GW
Tefferi
A
A
Pankratz
V S
VS
Vuk-Pavlović
S
S
D002455:Cell Division; D018920:Coculture Techniques; D003470:Culture Media; D016895:Culture Media, Serum-Free; D016207:Cytokines; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects; D016044:Fusion Proteins, bcr-abl / Q000097:blood*; D006801:Humans; D018189:Immunomagnetic Separation; D017404:In Situ Hybridization, Fluorescence; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood; D007963:Leukocytes, Mononuclear; D018950:Lipopolysaccharide Receptors / Q000097:blood*; D013048:Specimen Handling; D013601:T-Lymphocytes / Q000166:cytology
D003470:Culture Media; D016895:Culture Media, Serum-Free; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D016044:Fusion Proteins, bcr-abl