| ObjectiveThe prognosis of childhood acute myeloid leukemia (AML) is very poor because the minimal residual disease (MRD) always leads to relapse. In order to eliminate MRD, it is necessary to activate antigen specific cytotoxic T lymphocytes to start anti-leukemia cytotoxic response. However, AML cells may not present specific leukemic antigen to T lymphocytes because of low expression or absence of MHC molecules, co-stimulatory and adhesion molecules. Dendritic cells (DCs) are most potent antigen-presenting cells (APCs) that prime naive T lymphocytes and activate antigen specific cytotoxic T lymphocytes. The superior ability of DCs to present antigens to T cells has led to the development of DC-based strategies of enhancing the T-cell mediated cytotoxic killing against tumors. It is well known that DC-like cells can be obtained with conventional cytokine combinations in vitro. But the methods are expensive, time consuming, easily contamination, and sometimes AML cells can not be induced into DCs. Recent years, there were reports that normal monocytes(Mos) and CD34~+ hematopoietic progenitor cells (HPCs) could be induced rapidly into DCs by calcium ionophore (CI). In the studies presented here, we at first showed the morphology, immunophenotype and functions of DCs induced by CI-A23187 from HL-60 cells, and then investigated the role of protein kinase C (PKC) signal transduction in this process by using PKC inhibitor Bis-1. At last we explored the possibility of AML cells from patients with different FAB subtypes being induced into DCs by CI-A23187. The DCs derived from leukemia cells carry all auto-leukemic antigens and do not lead to autoimmune diseases. The appliment of DC vaccine to AML patients might thoroughly eliminate MRD and is very optimal in the near future.Methods1. HL-60 cells were cultured with CI-A23187 or plus rhIFN-γfor different times. The morphologic features of HL-60 cells were observed under inverted microscope and scanning electronic microscope, while the cell phenotypes of HL-60 cells treated with A23187 were determined by flow cytometry. The proliferation of allogeneic human T cells stimulated by DCs was tested by mixed lymphocyte reaction (Allo-MLR).2. HL-60 cells were pretreated with protein kinase C (PKC) inhibitor Bis-1 for 24 hours followed by cultured with A23187 for another 36 hours, and the morphology, immunophenotype and function of stimulating proliferation of allogeneic T cells were compared with the cells only treated with A23187.3. Bone marrow or peripheral blood mononuclear cells were isolated from 12 initially diagnosed AML patients and then cultured with A23187 for 3 to 4 days. DCs induced from leukemia cells were confirmed by morphology, immunophenotype and their capability of activating allogeneic T cells.Results1. After the HL-60 cells were treated with A23187 (180ng/ml) for 20 hours, the dendritic appearance of some cells was found and the expressions of CD83, a characteristic marker of mature DCs, and co-stimulating molecules CD80 and CD86 were up-regulated. When HL-60 cells were cultured with A23187 for 48 hours, the expression of CD83 began to decrease. A large number of cells with typical dendritic appearance were observed after cultured for 72 hours with A23187 and the expressions of CD80 and CD86 were up-regulated continuously. Allo-MLR revealed that DCs derived from HL-60 cells treated by A23187 were potent to stimulate the proliferation of allogeneic human T cells.2. The DCs were not derived from HL-60 cells cultured with rhIFN-γalone for 96h. When rhIFN-γcombined with A23187 were used to induced HL-60 cells, the DCs obtained expressed higher levels of CD83,CD80, CD86 molecules and were more potent to stimulate the proliferation of allogeneic T cells. However, there were no statistic differences in DC marker expression and capability of stimulating the proliferation of T cells when these DCs were compared with that induced only with A23187.3. The morphological changes, surface marker expressions and ability to stimulating the proliferation of allogeneic T cells in HL-60 derived DCs were inhibited by PKC inhibitor Bis-1.The percentage of CD83,CD80,CD86 expressing on HL-60 cells induced by A23187 decreased to (13.23±2.15) %,(9.70±1.69)%,(23.37±7.50) %, respectively (p<0.05) . These results indicated that PKC signal transduction pathway involved in the process of HL-60 cells induced into DCs by A23187.4. AML cells in 10/12 patients induced by A23187 for 3 to 4 days showed dendritic-like morphologic change, with the expression of CD83,CD80,CD86 increased from (2.06±0.95) %, (2.24±1.05) %, (3.61±1.43) % to (28.19±8.21)%,(45.32±9.90) %, (58.67±12.17) %, respectively (p<0.05). The DCs induced from AML cells also acquired potent capability of stimulating T cells proliferation. Based on immunophenotype analysis, we found that AML cells from patients with FAB subtypes of M4/M5 were induced more easily into DCs than leukemic cells with non-M4/M5 subtypes. There were no differences in expression levels of CD83,CD80,CD86 between peripheral blood and bone marrow derived leukemic DCs.Conclusion1. HL-60 cells can be rapidly induced into mature DCs with calcium ionophoreA23187. 2. The DCs were not derived from HL-60 cells cultured with rhIFN-γalone. However, when IFN-γcombined with CI-A23187 were used, DCs induced from HL-60 cells acquired were more mature and more potent to stimulate the proliferation of T cells.3. The induction of HL-60 cells into DCs by A23187 can be inhibited by PKC inhibitor Bis-1, suggesting signal transduction through PKC pathway might be involved in the process of AML cells differentiated into DCs by A23187.4. The majority of AML cells from patients with different FAB subtypes can be induced rapidly into DCs with A23187 cultured for 3 to4 days in vitro. Some cases of AML are not response to A23187 induction, indicating there is biological heterogeneity in AML patients which lead to different response to A23187. AML cells derived from patients with M4/M5 subtypes are more easily induced into DCs by A23187 than that of non-M4/M5 subtypes. Peripheral blood with high percentage of blast cells in AML patients can be used to replace bone marrow as an alternative source of leukemic DCs. |
PDF Full Text Request