| BackgroundPediatric malignant tumor has now become life-threatening diseases commonly seen in children. The therapeutic strategy for pediatric cancer has been derived from guidelines for adults, consisting of surgery, radiotherapy and chemotherapy. However, children have different pharmacokinetic and pharmacodynamic as compared to adults. Meanwhile, commonly-used radiotherapy and chemotherapy often result in both acute side effects and long-term sequelae, affecting growth and development of the patients and the quality of life in long-term survivors, accompanied by greater risks of metastasis and relapse. Therefore, there is a urgent need to develop therapeutic approaches that are highly efficient and safe for the treatment of pediatric malignancies.Dendritic cell (DC)-based immunotherapy has now been increasingly used as an important therapeutic strategy for cancer patients. Under normal circumstances, DC exists in an immature state, which has a relatively low capacity to activate T lymphocytes. Only mature DCs can stimulate innate T lymphocytes, rendering them activated and able to induce immune responses in the body. It has become an important and difficult issue as how to prepare DCs in large-scale in vitro and to enhance DC-induced anti-tumor immunopotency.The induction of immunity against self tumor-associated antigens is restricted by intrinsic inhibitory mechanisms, which usually lead to the reduction of anti-tumor potency, making many immunotherapy unable to achieve their desired efficacy. Recent studies found that suppressor of cytokine signaling 1 (SOCS1) is an intrinsic inhibitory molecule expressed in DCs that controls their ability to activate T cells and the magnitude of adaptive immunity. SOCS1 can inhibit Janus kinase (JAK) activity and the signal transduction of multiple cytokines such as IFN-γ. Therefore, SOCS1 is a key molecule that modulates antigen-presentation by DCs and the magnitude of adaptive immunity. Until now, there is no report concerning whether inhibiting SOCS1 in DCs can enchance the ability of DC vaccine to specificly kill pediatric malignant tumor.ObjectivesTherefore, we aimed to:1) To investigate the applicability of acquiring enough mature DCs from peripheral blood mononuclear cells (PBMC) and the strategies to stimulate DC maturation.2) To observe the effects of whole-tumor-antigen pulsed DCs on T cells activation and their ability to kill tumor cells.3) To explore the effects of silencing SOCS1 in DCs by siRNA on the ability of DCs to present antigen and the anti-tumor immunopotency and, therefore, to provide theoretical and experimental basis for the treatment of pediatric malignancy using DC-based vaccines.Methods1) Culture of tumor cells: Tumor cells were isolated from tumor tissues using tissue culture method. CNE-2Z and K562 cells were cultured using conventional cell cuture method.2) Preparation of whole-tumor antigen: Whole-tumor antigen was obtained by repetitive freezing and thawing of tumor cells followed by 60Coγirradiation (25 Gy).3) In vitro induction and proliferation of DC: By using PBMC isolation method. Cytokines GM-CSF and IL-4 were added into cell culture medium. Whole-tumor-antigen was added at the 3rd day of culture and TNF-αwas added at the 7th day to stimulate DC maturation.4) Morphologic characterization of DCs: Morphologic characteristics of DCs were examined on a contrast-phase microscope, a scanning electron microscope and a transmission electron microscope.5) DC surface antigen and level of maturation assay: Immunophenotyping of the DCs was performed using flow cytometry analysis (FCM).6) The ability of tumor-antigen-pulsed DCs to stimulate T cell proliferation was determined using methylthiazolyldiphenyl-tetrazolium (MTT) method.7) The ability of tumor-antigen-pulsed DCs to stimulate cytotoxic activity of T cells was determined using MTT method.8) The ability of whole-tumor-antigen-pulsed DCs to stimulate IFN-γsecretion from CTL was determined by ELISPOT assay.9) The expressions of SOCS1 mRNA and protein in DCs were determined by RT-PCR and Western blotting, respectively. Results1) Morphologic characterization of DCs using contrast-phase microscope and electron microscope showed typical dendritic-like protrusions on the surface of the cells.2) The positive rates for cell surface expression of marker molecules HLA-DR, CD1a, CD80, CD83 and CD86 on whole-tumor-antigen-pulsed DCs at the 5th day of culture (immature DC) were (60.86±5.42)%, (21.84±2.52)%, (5.49±6.32)%, (6.82±1.24)% and (1.24±0.90)%, while the rates for cell surface expression of marker molecules on whole-tumor-antigen-pulsed, TNF-α-stimulated DCs (mature DC) were (86.14±8.32)%, (78.28±11.42)%, (78.24±12.64)%, (67.25±14.24)% and (85.26±9.14)%, respectively, being significantly higher than those on immature DCs(P < 0.01~0.05)。3) With the increasement of DC: T cell ratio from 1:100 to 1:5, the stimulating index indicating the proliferation of T cells showed a tendency of increase. T cells stimulated with whole-tumor-antigen-pulsed mature DCs had higher stimulating indices than those stimulated with unpulsed DCs.4) T cells stimulated with whole-tumor-antigen-pulsed DCs had killing activity towards both autologous and allogeneic tumor cells. The killing rate of CTLs was proportional to effctor: target ratio.5) Number of IFN-γ-secreting T cells was greater in T cells stimulated with whole-tumor-antigen-pulsed DCs and non-stimulated T cells (P < 0.05).6) SOCS1 expression was increased in K562-DCs matured by TNF-α(1000 U/mL) added at the 7th day of culture. Mature K562-DCs transfected with SOCS1 siRNA (SOCS1 siRNA1 and siRNA2) showed decreased SOCS1 mRNA and protein expression.7) The positive rates for cell surface expression of marker molecules HLA-DR, CD1a, CD80, CD83 and CD86 on K562-DCs at the 5th day of culture (immature DC) were (54.65±3.28)%, (17.42±6.78)%, (6.27±5.29)%, (3.02±2.47)% and (3.28±2.79)%, while the rates for cell surface expression of marker molecules on whole-tumor-antigen-pulsed, TNF-α-stimulated DCs (mature DC) were (69.52±8.68)%, (58.97±4.25)%, (63.84±7.32)% , (72.69±6.23)% and (71.46±4.96)%, respectively, being significantly higher than those on immature DC(sP < 0.01~0.05)。The positive rates were (67.17±9.53)%, (59.46±5.17)%, (65.72±6.58)%, (71.47±5.68)% and (87.92±3.94)% after SOCS1 siRNA transfection into mature DCs for 24 h. The rate of CD83 was significantly higher than that of untransfected-DCs (P < 0.05).8) T cells stimulated with TNF-α-matured K562-DCs were more proliferative than those stimulated with immature DCs. With the increase in DC: T cell ratio, the stimulating indices enhanced. The stimulating capacity was most apparent when the effector: target ratio was 1:5 (both P < 0.01 when the effector: target ratio was 1:20 and 1:5). SOCS1 siRNA transfection into K562-DCs significantly increases their ability to stimulate T cell proliferation when compared to those transfected with scramble siRNA (P < 0.05).9) T cells stimulated with TNF-α-matured K562-DCs had higher killing capacity against K562 cells, with the killing rates being most apparent at effector: target ratios of 5~20:1 (P < 0.01, mature DC vs. immature DC). SOCS1 transfection significantly increased the killing rate of T cells against K562 cells (P < 0.05). The effects of SOCS1 transfection on the killing rate of K562 cells were proportional to effector: target ratios.10) Mature K562-DC-stimulation significantly increased the number of IFN-γ-secreting T cells (P < 0.01, mature DC vs immature DC). SOCS1 transfection further increased the number of IFN-γ-secreting T cells (P < 0.05).Conclusions1. PBMC-derived, cytokine-driven DCs pulsed with whole-tumor-antigen can generate both proliferative and cytolytic T-cells against tumor cells.2. SOCS1 siRNA transfection into DCs can effectively inhibit the expression of SOCS1 in DCs and bypass the limit of intrinsic inhibitory mechanisms and, therefore, renders mature DCs more effective in their stimulating capacity, which can be shown by increased T cell proliferation and killing capacity against K562 cells.In conclusion, our results indicate that using DCs pulsed with whole-tumor- antigen might be an effective method to treat pediatric malignancy and its application may have potential clinical significance. Meanwhile, silencing SOCS1 using SOCS1 siRNA can enhance CTL response against tumor cells and might represent a strategy to enhance adaptive immunity.
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