Cytokine-Driven Differentiation Of Blasts From Patients With Acute Myelogenous Leukemia Into Dendritic-like Cells

Objective To investigate the induction method and function of dendritic cells (DCs) derived from acute myelogenous leukemia (AML) blasts in vitro. Methods Cytokine-supplemented suspension cultures of leukemia blasts in 25 AML patients were performed. Mononuclear cells (MNCs) were separated by density gradient centrifugation of whole bone marrow (BM) or peripheral blood (PB) over the isolation solution of lymphocyte. MNCs were cultured for 8 to 12 days using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), recombinant human interleukin-4 (rhIL-4) and recombinant human tumor necrosis factor-a (rhTNF-a). The growth process of cells was observed under invert light microscope. Cultured cells were collected when they showed significant DC morphology in the period of culture (days 8-9), and analyzed by flow cytometry with respect to DC-associated surface molecules (CDla, CD54, CD80, CD83, CD86 and HLA-DR). In2M3cases, these cells were determined to be of leukemic origin by fluorescence in situ hybridization (FISH) for chromosomal abnormalities t(15;17). The potent antigen-presenting capacity of generated DCs was measured by the allogeneic mixed lymphocyte reactions (Allo-MLR) by 3H-TdR incorporation assays. Results In this study, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DCs could be generated from 20/25 AML cases. After culture for 3 days, cells began to display an increase in size with dendritic morphology. The total number of cells in overwhelming majority cases decreased while that of dendritic-like cells increased during the period of culture. Phenotypic analyses of cells (18/20 cases) after culture were measured by flow cytometry. Among these 18 cases, phenotypic analyses of cells from 11 cases were performed before and after cultured. CDla, CD80, CD83 was increased from negative to 3. 2%, 18. 7%, 3. 8%, respectively; CD54 from 5. 2% to 53. 2%, CD86 from 2. 5% to 39. 7% and HLA-DR from 5. 4% to 10. 3%, respectively. The different expression rate of HLA-DR before and after was no significant (P>0. 05); Meanwhile, others were significant (P<0. 01).The percentage of each phenotype was individually correlated with the functional data in Allo-MLRs. FISH confirmed the leukemic origin ofgenerated cells. A marked increase of the T-cell stimulatory capacity could be generated in Allo-MLRs. Conclusion Leukemia-derived DCs can be generated from AML blasts using cytokine combination (GM-CSF, IL-4, and TNF-a ). Upregulating costimulatory and adhesion molecules increased the stimulatory capacity of these cells. These cells might useful for autologous immunotherapy in selected patients.Postgraduate Meng dongmei (clinical hematology) Directed by Zhao chunting...