| Objectives:Along with the development of the cellular immunology and molecular biology, cell-mediated immunotherapy have been developed in the clinical experiment, and have got significant curative effect. Cytokine Induced Killer cells (CIK), as a new type of immune competent cells, have already been used in the treatment of many kinds of malignant tumors. Dendritic Cells (DC), as the most efficient antigen presenting cells, can handle and present antigen to mediate humane immune reaction. As many tumor-suffering hosts can not induce antigenic specificity T cell immunologic response because of the lost of DC. Inducing functional DC to present antigen effectively and initiate immun reaction are of important meanings. Several studies shown that FLT3-L, as a cytokine, can successfully induce the maturation of DC (including plasmacytoid lymphocyte DC) .GM-CSF has been well known as a standard cytokine to induce DC. Therefor, the present study was designed to compare the DC-inducing effect of FLT3-L and GM-CSF and to investigate the optimal method for coculturing DC-CIK and analysis the IFN-γsecreting function of CIK.Methods: The first step of the experiment: Cells of DC and CIK were collected from peripheral blood mononuclear cells (PBMNC). The cells were cultured with FLT3-L(the experiment group) or GM-CSF(the control group). Flow cytometry was used to analysis immunophenotype of DC. The second step of the experiment: Cells of DC and CIK were collected from two resources, the first patient was a hemophagocytic syndrome (HPS) patient who infected EBV, the second one was a AML patient who was in complete remission condition. EBV antigen peptide was added to the culture of DC and the co-culture of DC-CIK of the HPS patient. On the other hand, FLT3-L or GM-CSFwas added to the culture of DC and the co-cultured DC-CIK of the AML patient. After cultured for the designed time point, flow cytometry, immunofluorescence quantitative analysis were used to analysis immunophenotype of DC and CIK, and IFN-γwas measured to evaluate the function of CIK.Results:1 Comparision of the effect of FLT3-L and GM-CSF in inducing DC.The number of cultured cells in both experiment group and control group decresed in the process of culturing, and there was no significant difference in the cell number found between these two group. The total number of cells in each group was 2.00±0.10×107 before culture. The total number of cells in FLT3-L group was 1.10±0.04×107 on the third day, 0.80±0.04×107 on the seventh day and 0.60±0.02×107on the tenth day; The total number of cells in GM-CSF group was 1.20±0.03×107 on the third day, 0.80±0.02×107 on the seventh day and 0.53± 0.01×107 on the tenth day.The analysis of immunophenotype by flow cytometry: the percentage of different kines of surface antigen on the cells before culture: CD14+ cell 11.41%,CD83+ cell 0.28%,CD80+ cell 0.01%,CD86+ cell 1.32%,CD4+CD123+ cell 1.23%;the percentage of different kines of CD on the cells cultured with FLT3-L: CD14+ cell 49.22%,CD83+ cell 6.96%,CD80+ cell 33.48%,CD86+ cell 52.05%,CD4+CD123+ cell 4.25%;the percentage of different kines of CD on the cells cultured with GM-CSF:CD14+ cell 72.21%,CD83+ cell 2.90%,CD80+ cell 41.23%,CD86+ cell 68.31%,CD4+CD123+ cell 1.16%; The number of matured DC increased after culture,the phenotype of DC in the two group have significant deviations. As the cellular percentage of matured DC was higher in GM-CSF group.The phenotype of CD4+CD123+ cell was higher in FLT3-L group,as the phenotype of plasmacytoid lymphocyte DC.2 The optimal cultural method for DC-CIK induction in patient with hemophagocytic syndrome who infected EBVCell growth conditions and cell number: the number of DC had no significant change after 3 days' culture. Barb shape of tuber can be seen on the cells under the inverted microscope at the second day of culture and they showed half adherence.CIK proliferated after MabCD3 and MabCD28 added in the fourth day. The speed of cell proliferation have no significant differences between the group of DC-CIK and CIK. After 14 days' culture, the cell population were 6.3 and 5.8 times of that before culture. The percentage of alive cells in each culture was more than 95%.The analysis of immunophenotype by flow cytometry: the percentage of different kines of surface antigen on the cells before culture:HLA-DR+CD86+ 12.05%,CD83+2.32%,CD80+ 0.00%; the percentage of different kines of surface antigen on DC after cultured with GM-CSF : HLA-DR+CD86+ cell 91.17%,CD83+ cell 7.28%,CD80+ cell 36.20%。CIK before culture: CD3+ cell 74.88%,CD8+ cell 30.02%,CD3+CD8+ cell 28.08%,CD3+CD56+ cell 1.27%; the percentage of different kines of surface antigen on DC-CIK without EBV Ag stimulation: CD3+ cell 97.59%,CD8+ cell 55.49%,CD3+CD8+ cell 54.95%,CD3+CD56+ cell 8.50%;the percentage of different kines of surface antigen on DC-CIK with EBV Ag stimulation:CD3+ cell 95.62%,CD8+ cell 53.55%,CD3+CD8+ cell 52.89%,CD3+CD56+ cell 8.44%。The analysis of genescan before and after culture:there were no significant peak before culture but there was a mono-peak at TCRβ5.2 out of the whole 24 TCRβafter culture. Gaussian distribution can be seen in both DC-CIK without EBV Ag and DC-CIK with EBV Ag after culture。The analysis of IFN-γsecretion:the ratios of effective cells to target cells were 5:1,10:1,20:1, separately. Using IFN-γELISA kit to measure the concentration of IFN-γ. The concentrations of IFN-γsecreted by CIK before culture, by DC-CIK without EBV Ag stimulation , by DC-CIKstimulated with EBV Ag were1:1.4:2.7(at 5:1 ratio);1:1.2:1.7(at 10:1 ratio);1:1.1:1.7 (at 20:1 ratio), separately. It idicated that the concentration of IFN-γsecreted by DC-CIK stimulated with EBV Ag was 1.4~1.9 times higher than that secreted by DC-CIK without EBV Ag stimulation.3 The optimal cultural method for DC-CIK induction in a AML patient in complete remission conditionCell growth conditions and cell number: the number of DC had no significant change after 3 days' culture. Barb shape of tuber can be seen on the cells under the inverted microscope at the second day of culture and they showed half adherence.CIK proliferated after MabCD3 and MabCD28 added in the fourth day. The speed of cell proliferation have no significant differences between the group of DC-CIK and CIK. After 14 days' culture, the cell population were 6.6 and 6.2 times of that before culture. The percentage of alive cells in each culture was more than 95%.The analysis of immunophenotype by flow cytometry: the percentage of different kines of surface antigen on DC:HLA-DR+CD86+ cell 6.90%,CD83+ cell 0.27%,CD80+ cell 0.13%,CD14+ cell 73.85%;the percentage of different kines of surface antigen on DC after cultured with FLT3-L : HLA-DR+CD86+ cell 76.79%,CD83+ cell 3.17%,CD80+ cell 11.87%,CD14+ cell 55.33%;the percentage of different kines of surface antigen on DC after cultured with GM-CSF :HLA-DR+CD86+ cell 43.87%,CD83+ cell 1.40%,CD80+ cell 6.74%,CD14+ cell 19.80%。The number of matured DC were increased in both group. Comparing the effect of FLT3-L and GM-CSF on inducing matured DC, the rusult showed that degree of maturity,FLT3-L was effective than GM-CSF。The percentage of different kines of surface antigen on CIK before culture: CD3+ cell 78.57%,CD8+ cell 61.542%,CD4+ cell 22.85%,CD3+CD8+ cell 55.20%,CD3+CD56+ cell 5.03%;the percentage of different kines of surface antigen on DC-CIK cultured with FLT3-L: CD3+ cell 91.43%,CD8+ cell 81.83%,CD4+ cell 12.59%,CD3+CD8+ cell 80.29%,CD3+CD56+ cell 47.31%;the percentage of different kines of surface antigen on DC-CIK cultured with GM-CSF:CD3+ cell 91.97%,CD8+ cell 78.6%,CD4+ cell 12.56%,CD3+CD8+ cell 76.90%,CD3+CD56+ cell 57.72%. The the number of CD3+,CD8+,CD3+CD8+,and CD3+CD56+cells increased after culture , the average amplification percentage were 12.09%, 18.67%, 23.40% and 47.48%;the number of CD4+ cells decreased after culture,the average decrease was 10.27%。This result showed that FLT3-L had no significant amplification effect than GM-CSF,but the inumber of CD3+CD56+ cell induced by FLT3-L was 10.41% larger than that induced by GM-CSF.The analysis of genescan before and after culture:There were no significant peak before culture. Gaussian distribution can be seen in both group after culture and there was no significant differences between the two group.The analysis of IFN-γsecretion: IFN-γELISA kit was Used to measure the concentration of IFN-γ. The concentrations of IFN-γsecreted by CIK before culture, by DC-CIK treated with FLT3-L,DC-CIK treated with GM-CSF were1:4:3, separately.It indicated that the concentration of IFN-γsecreted by DC-CIK treated with FLT3-L was 33.3% higher than that secreted by DC-CIK treated with GM-CSF.Conclusion:1 we induced DC and CIK successfully by a 14-day's co-culture system and optimized the culture system.2 It is feasible for the patient with hemophagocytic syndrome (HPS) who infected EBV to carry out DC-CIK adoptive immunotherapy, and it can be an adjunctive therapy for HPS patients. The addition of EBV antigen in the process of DC's culturing can enhance the production of single clone T lymphocyte .3 It is feasible for patient with AML who is in paracmasia to carry out DC-CIK adoptive immunotherapy , and it can be an adjunctive therapy in the clinical practicce to decrease microresidual disease.4 Comparing the DC inducing effect of these two cytokines, FLT3-L showed few superiority in inducing DC, but FLT3-L was more effective than GM-CSF in stimulating plasmacytoid lymphocyte DC.
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