Korean J Hematol.  2007 Sep;42(3):264-275. 10.5045/kjh.2007.42.3.264.

Generation and Qualification of Functionally Active Leukemia-derived DCs from Malignant Blasts in Acute Leukemia

• Baek S
• Jung CW
• Kim MJ
• Kim K
• Ahn JS
• Lee H

• KMID: 2305202
• DOI: http://doi.org/10.5045/kjh.2007.42.3.264

Abstract

BACKGROUND: Dendritic cells (DCs) are increasingly being utilized for anti-cancer immunotherapy. Acute myeloid leukemia (AML) blasts are able to generate leukemia-derived DC. Advances in culture techniques and AML-DC characterization justify possible clinical applications. We investigated the ability of AML, acute lymphoblastic leukemia (ALL) and biphenotypic acute leukemia (BAL) blasts to differentiate into DCs in vitro and the qualified function of the leukemia-derived DCs.
METHODS
Leukemia cells from 11 patients with AML, 3 patients with ALL and 2 patients with BAL were cultured with GM-CSF, IL-4 and with or without SCF. Cultured leukemia cells were evaluated by phenotype, mixed lymphocyte reaction (MLR), cytokine production and cytotoxic T cell (CTL) inducing activity.
RESULTS
DCs were generated with GM-CSF and IL-4 from the leukemic blasts in 72% of the AML patient cells. MHC class I/II, CD11c and ICAM-1 were highly expressed in the AML-derived DCs. MLR and enzyme linked immunospot (ELISPOT) assays demonstrated that AML-DCs were able to induce T cell proliferation and activation into IFN-gamma secreting effector cells. The ALL blasts from two out of three patients differentiated into DCs with MHC class I/II+, CD11c+ only in the presence of GM-CSF, SCF and IL-4 for 14 days.
CONCLUSION
These results suggest that functionallyactive DCs can be differentiated from AML blasts using GM-GSF and IL-4 and ALL, BAL blasts were differentiated into DCs only under stem cell-DC culture conditions.

Keyword

Acute leukemia; Acute myeloid leukaemia; Dendritic cells; Immunotherapy

MeSH Terms

Cell Proliferation
Culture Techniques
Dendritic Cells
Granulocyte-Macrophage Colony-Stimulating Factor
Humans
Immunotherapy
Intercellular Adhesion Molecule-1
Interleukin-4
Leukemia*
Leukemia, Biphenotypic, Acute
Leukemia, Myeloid, Acute
Lymphocyte Culture Test, Mixed
Phenotype
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Granulocyte-Macrophage Colony-Stimulating Factor
Intercellular Adhesion Molecule-1
Interleukin-4

Figure

• 

  Fig. 1 Phenotype of AML-derived DC. DCs were cultured from PBMC of AML patients with GM-CSF and IL-4 for 7days. (A) Most of the culture cells had dendritic morphology with fine fibrous cytoplasmic hairs (original magnification200). (B) Surface phenotypes of AML-derived dendritic cells. Cells were stained with fluorescent-labeled surface marker antibodies and analyzed with FACSvantage. Expression of MHC class I (HLA-ABC) was over 97%. Other antigen presentation related marker like MHC class II (HLA-DR), co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86), as well as CD11c were also expressed in high level. Data supported that the cultured cells were DCs.

• 

  Fig. 2 Comparison of phenotype between immature AML-derived DC and mature AML-derived DC. (A) Immature AML-derived DCs were expressed HLA-ABC+, HLA-DC+, CD80/86+, and CD11c++/CD54+++. (B) DCs were matured by maturation cocktail (IL-1β,TNF-α,IL-6,PGE2). Most of the cells were expressed CD80++/CD86+++ and CD83++.

• 

  Fig. 3 Cytokine profile of AML-derived DC. Most of the FAB type M1 patients were generated DCs secreting IL-10low, IL-12high. But DCs secreting IL-10high, IL-12low was also present. Columns represent means of duplicate samples: bars indicate SE.

• 

  Fig. 4 Function of AML-derived DC. (A) In vitro induction of lymphocyte proliferation by AML-derived DC. As a responder, lymphocytes (2×105cells/well) were co-cultured with mitomycin-C treated DCs (stimulator) from AML blast. AML-derived DCs cultured with GI (GM-CSF+IL-4) protocol make lymphocyte proliferate unlike cells cultured with GSI (GM-CSF+IL-4+SCF) protocol. Results are expressed as mean percentage of control of triplicate samples: bars indicate SE. (B) IFN-γ secreting T cell induction. T cell induced by DCs were differentiated into IFN-γ secreting T cell above 10 times than naive T cell.

• 

  Fig. 5 Surface phenotype of ALL-derived DC. (A) DCs were not shown when ALL blast cultured with GM-CSF and IL-4 for 7 days. (B) DCs were cultured from PBMC of ALL patients with GM-CSF, SCF and IL-4 for 14 days. Cells were stained with fluorescent-labeledsurface marker antibodies and analyzed with FACSvantage. Antigen presentation related marker like MHC class I/II, CD86, CD11c and ICAM-1(CD54) were expressed. Data supported that the cultured cells were DCs.

• 

  Fig. 6 Function of ALL-derived DC. (A) In vitro induction of lymphocyte proliferation by ALL-derived DC. As a responder, lymphocytes (2×105cells/well) were co-cultured with mitomycin-C treated DCs (stimulator) from ALL blast. ALL-derived DCs cultured with GSI (GM-CSF+IL-4+SCF) make lymphocyte proliferate. Results are expressed as mean percentage of control of triplicate samples: bars indicate SE. (B) Cytokine profile of ALL-derived DC. Patterns of cytokine secretion were different in patients. Columns represent means of duplicate samples: bars indicate SE.

• 

  Fig. 7 Surface phenotype of BAL-derived DC. DCs were culturedfrom PBMC of BAL patients with GM-CSF, SCF andIL-4 for 14 days or GM-CSF and IL-4 for 7 days. Cells were stained with fluorescent-labeled surface marker antibodies and analyzed with FACSvantage. Antigen presentation related marker like HLA-DR, CD11c were expressed by GI protocol and GSI protocol. Also, co-stimulatory molecules CD80 and CD83 were slightly manifested by both protocols.

• 

  Fig. 8 Function of ALL-derived DC. (A) In vitro induction of lymphocyte proliferation by BAL-derived DC. As a responder, lymphocytes (2×105cells/well) were co-cultured with mitomycin-C treated DCs (stimulator) from BAL blast. Lymphocyte were not proliferate by BAL-derived DCs. Results are expressed as mean percentage of control of triplicate samples: bars indicate SE. (B) Cytokine profile of BAL-derived DC. IL-10 was highly expressed by both of DCs cultured with GI or GSI protocols. Columns represent means of duplicate samples: bars indicate SE.

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