| Dendritic cells(DCs) are the most potent antigen presenting cells that prime effective naive T-cell response. DCs play a pivotal role in the regulation of immue response. The ability of DCs to induce immunity or tolerance appears to be related to their state of functional maturation. Mature DCs induce active immune response, however, immature DCs induce immune tolerant, Therefore, DCs with a mature state play an important role in the induction of an antitumor immunity.Chronic myeloid leukemia (CML) is a malignant myeloproliferation disease rising from the clonal expansion of a stem cell expressing the bcr/abl oncogene. The use of allogeneic stem cell transplantation (alloSCT) and donor lymphocyte infusion (DLI) in the treatment of CML offers not only the possibility of cure, but also demonstrates the effectiveness of immunotherapy in CML; however, alloSCT is only available to a minority of CMLpatients. Despite the advances in understanding the molecular biology of CML and the development of new therapies, only a minority ofpatients are cured. Therefore, new treatments are also urgently needed in CML. Recently, the use of autologous DCs as the combination treatment of the first line therapy such as STI571 and(or) IFN-α in CML has been considered as an attractive approach of anticancer therapy in CML patients.In the present study, we investigated the effects of STI571, IFN-α on CML DC derived from bone marrow mononuclear cells(BMMNCs) aimed to discuss the probable new mechanisms of antitumor action of STI571, IFN-α in CML and further provide a theoretical and experimental basis for the treatment strategies of DC-based immunotherapy combination STI571, IFN-α for CML patients.There were two sections in this study.Section 1: Effects of STI571 on the development of dendritic cells derived from bone marrow mononuclear cells from patients with chronic myeloid leukemiaSTI571(Imatinib,Gleevce) is a potent, selective Bcr-Abl tyrosine kinase inhibitor and it has a substantial activity in CML in chronic phase(CP). Naoko et al's study showed that STI571 could promote co-stimulating molecules expression and enhance the ability of allogeneic antigen presentation of DC cultured from peripheral blood cells of patients with CML but without further study.In the present study, STI571 was introduced to the culture system of CML DCs to investigate the new probable mechanisms of the anti-CML action of STI571 and further provide a theoretical and experimental basis for the treatment strategy of DC-based immunotherapy combination STI571 for CML patients.Treatment bone marrow mononuclear cells(BMMNCs) of 11 patients with CMLin chronic phase with STI571+rhGM-CSF+rhIL-4(experimental group DCs) and rhGM-CSF+rhIL-4(control group DCs) for 7 days, and added TNF-α for 3 additional days. DCs morphology was monitored on Wright-stained cytospin slides under light microscope after cultured. Cells from both experimental and control groups showed similar morphological features. Fluorescence in situ hybridization (FISH) showed that DCs from both two groups expressed the bcr/abl fusion gene. Cultured cells were analyzed by flow cytometry for the expression of CD1a, CD80, CD86, HLA-DR and CD83. Cells from two groups shared the CD1a, a specific molecule of human DC. In comparison with control group, the experimental DCs showed an increased expression of CD80 73.16%±15.51 vs 60.11 %± 15.54(t=3.623, P=0.005); CD86 57.35%± 13.77 vs 48.08%± 11.75(t=4.278, P=0.002); CD83 76.96% ±9.94 vs 65.26% ± 13.77(t=4.187, P=0.002); HLA-DR 73.12% ±11.03 vs 55.26% ± 12.44(t=3.798, P=0.003); CD1a 61.28%±11.88 vs 57.53%±11.71(t=2.130, P=0.059). To determine the allostimulatory activity of DCs in vitro, one-way mixed leukocyte reaction(MLR) was performed with mitomycin C-treated DCs as stimulators and nylon wool-purified healthy donor T cells as responders. The T cell proliferation was determined using MTT assay after a 96-hour stimulation. In comparison with control group, DCs generated in the presence of STI571 stimulated a vigorous T cell proliferation in MLR when DC/T ratio 1:1 and 1:10, while no significant different when DC/T ratio 1:100. DC:T=1:100, 0.507±0.015 vs 0.467±0.014(t=2.337, P=0.144); DC:T=1:10, 1.337 ±0.015 vs 0.835 ± 0.010(t=4.864, P=0.04); DC:T=1:1, 2.339±0.051 vs 1.256± 0.011(t=21.727, P=0.002). This observation indicated that DCs treated with STI571 were a more mature state and had a enhanced allostimulatory capacity.A number of tumor cells like as CML overproduce vascular endothelial growth factor(VEGF). VEGF directly promotes tumor growth, on the other hand, VEGF inhibits the activation of nuclear factor κB(NF-κB) in hematopoietic progenitor cells(HPCs) and then affects DC differentiation and activation, which contribute to tumor cells escape from immune attack. NF-κB family is one of the most important family which related to DC differentiation and activation including member P50, P52, RelA(p65), RelB and c-Rel. In a recent study, the phenomenon that defects of DC have a correlation with high serum VEGF level in patients with CML and upon STI571 treatment, plasmatic VEGF rapidly decreased, blood DC deficiency was recovery although not complete was observed. Therefore, VEGF concentrations in the culture supernatants were detected by sandwich enzyme-linked immunosorbent assay (ELISA). Interestingly, the concentration of VEGF was dramatically reduced in the culture supernatants of the experimental group compared with that of the control group, 855.90±620.47 pg/ml versus 1578.80±1353.26 pg/ml (Mean±S.D;t=2.517, P=0.033), indicating an inhibitory role of VEGF in CML DCs differentiation. We further estimated the activities of nuclear factor-κB (NF-κB) proteins involved in VEGF signaling pathway using TransAM? NF-κB Family kit.It was found that the NF-κB activity in experimental DCs was significantly increased: RelA 0.212±0.055 vs 0.167+0.081(t=2.548, P=0.044); RelB 0.187±0.067 vs 0.091±0.049(t=4.054, P=0.007); c-Re1 0.246±0.0195 vs 0.202±0.03(r=2.517, P=0.033); P50 0.388±0.150 vs 0.371 ±0.165(t=0.938, P=0.384); P52 0.217±0.067 vs 0.109±0.027 (t=5.475, P=0.002). The results suggested that STI571 may inhibit VEGF secretion from leukemic cells and then correct the activity of downstream key proteins NF-κB and ultimately promote the activation/maturation of DCs derived from BMMNCs of CMLpatients in vitro.Summary: (1) STI571 could promote co-stimulating molecules expression andenhance allostimulatory capacity of DCs cultured from bone marrow precursors of CMLpatients in vitro.(2) VEGF from CML may responsible for the defects of CML DC. (3) STI571 may inhibit VEGF secretion from CML and then correct the activity of downstream proteins NF-κB and ultimately promote the ctivation/maturation of DCs in vitro, which may contribute to therapeutic effect of STI571 in CMLpatents. (4) The therapeutic strategy of DC-based immunotherapy combination STI571 may be an attractive approach of anticancer therapy in CML patients.Section 2: IFN-α and GM-CSF induce the differentiation of bone marrow mononuclear cells into activated dendritic cells in chronic myeloid leukemiaAn historical advancement in the management of patients with CML has been achieved by the clinical use of interferon-α(IFN-α), which has represented the first line therapy in patients who are not candidates for allogeneic stem cell transplantation. In spite of many years of current use of IFN-α in patients with CML and other malignancies, the mechanisms of the antitumor action of these cytokines are still a matter of debate. In the present study, we evaluated the role of EFN-α in differentiation and activity of BMMNCs-derived dendritic cells (DCs) from CML patients as well as in modulation of the cell response to TNF-α. BMMNCs from 12 CMLpatients in chronic phase were cultured initially using cytokines as follows: recombined human granulocyte/macrophage colony-stimulating-factor (rhGM-CSF) plus rhIFN-α (IFN-α-DCs); rhGM-CSF plus recombined human interleukin-4 (rhIL-4) (IL-4-DCs) ; rhIFN-α alone; rhGM-CSF alone in 10% FCS RPMI1640 medium for 7 days and thenadded recombined human tumor necrosis factor-α(TNF-α) for another 3 days. IFN-α-DCs displayed features in morphology which were similar to those of IL-4-DCs with delicate membrane projections observed on Wright-stained cytospin slides under light microscope. No dendritic cells were observed when BMMNCs treated with rhGM-CSF alone or rhIFN-α alone. Fluorescence in situ hybridization (FISH) showed that DCs from both IFN-α-DCs and IL-4-DCs expressed the comparable bcr/abl fusion gene. Compared with the control groups of IL-4-DCs, IFN-α-DCs showed an increased expression of CD80, CD86, CD83 and HLA-DR analyzed by flow cytometry before and after treated with TNF-α. Before treated with TNF-α: CD80 56.91%±6.05 vs 40.21%±15.54(t=3.952, P=0.017); CD86 41.77%± 9.20 vs 25.89%± 7.87(t=7.200, P=0.002); CD83 53.87% ±10.04 vs 36.08%± 5.74(t=2.901, P=0.044); HLA-DR 56.32% ± 10.21 vs 36.08% ± 7.98(t=4.933, P=0.008); CDla 51.82%±10.55 vs 43.39%±6.38(r=2.053, P=0.109). After treated with rhTNF-α: CD80 74.02%±6.05 vs 60.54%±14.80(t=3.213, P=0.008); CD86 59.54%±13.39 vs 47.14%±11.28(t=4.382, P=0.001); CD83 76.08%±13.94 vs 64.46% ± 11.76(t=3.273, P=0.007); HLA-DR 71.65% ± 16.05 vs 55.25% ± 10.52(t=4.074, P=0.002); CDla 60.82%±11.13 vs 60.02%±11.82(t=0.452, P=0.660). In comparison with control group, IFN-α-DCs stimulated a vigorous allogeneitic T cell proliferation in MLR when DC/T ratio 1:1 before treated with TNF-α and when DC/T ratio 1:1, 1:10 after treated with rhTNF-α. Before treated with TNF-α: DC:T= 1:100, 0.243±0.011 vs 0.247+0.015(t=0.238, P=0.834); DC:T=1:10, 0.586±0.087 vs 0.446 ± 0.014(t=2.460, P=0.133); DC:T = 1:1, 0.998 ± 0.023 vs 0.726 ± 0.066(t=9.412, P=0.011); After treated with rhTNF-α: DC:T= 1:100,0.419±0.034 vs 0.392 ± 0.013(t=2.704, P=0.054); DC:T = 1:10, 0.865 ± 0.099 vs 0.691± 0.044(t=6.003, P=0.004); DC:T=1:1, 1.279 ±0.031 vs 0.983 ± 0.085(t=9.971, P=0.001). This observation indicated that IFN-α-DCs were a more mature state andhad the ability of stimulating stronger allogeneitic T cells proliferation compared with IL-4-DCs. In addition, it was found that the percentages of viable cells counted by Trypan Blue Dye Exclusion Method recovered after DC differentiation/maturation under the different culture conditions were comparable before treated with TNF-α: the average (Mean ± S.D) were (1.26 ± 0.18) × 106cell/well vs (1.20 ± 0.16) × 106cell/well(n=10, t=1.548, P=0.156) for IL-4-DCs and IFN-α-DCs, respectively. However, when the two different DC types were further treated with TNF-α, a reduction in the number of recovered viable cells was observed in IFN-α-DCs as compared to IL-4-DCs:(1.01 ±0.13)× 106cell/well vs (0.83±0.08)× 106cell/well(n=8, t=7.747, P=0.000).Then the percentage of apoptotic cells of DCs labeled with annexin V fluorescein isothiocyanate(FITC) and propidium iodide (PI) were further detected by flow cytometry. It showed that apoptotic cells were comparable before treated with TNF-α: the average (Mean±S.D) were 9.64%±3.48 vs 8.65%±2.35 (n=5, t=1.580, P=0.189) for IL-4-DCs and IFN-α-DCs, respectively. However, after treated with TNF-α apoptotic cells were increased remarkably in IFN-α-DCs: 38.26%±7.01 vs 18.29% ± 5.39(n=5, t=14.958, P=0.000).These results suggest that induction of apoptosis occurring in these cultures at a final stage of maturation may represent an homeostatic mechanism regulating survival/death of activated DCs.Summary: (1) IFN-α and GM-CSF induce the dfferentiation of bone marrowmononuclear cells into activated dendritic cells in CMLpatients in vitro. (2) IFN-α-DCs were a more mature state and had the ability of stimulating stronger allogeneitic T cells proliferation compared with IL-4-DCs. (3) The induction of apoptosis occurring in IFN-α-DCs at a final stage of maturation may represent an homeostatic mechanism regulating survival/death of activated DCs. (4) The phenomenon of generation of activated DCs in vitro might contribute to therapeuticeffect of IFN-α on CML. (5) The IFN-α-DCs may be an attractive approach of anticancer therapy in CML patients.
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