Antigen-loaded dendritic cells and CD40L triggers the killing effects of cytotoxic T lymphocytes on K562 cells in vitro

AIM:To investigate the anti-tumor effects of special cytotoxic T lymphocytes (CTLs) activated by dendritic cells (DCs) loaded with antigens and CD40L in vitro.METHODS:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation from normal human heparinized blood.The adherent cells were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF),interleukin-4 (IL-4),alpha tumor necrosis factor (TNF-α),DCs were co-cultured with frozen-thawed antigen of K562 cells and CD40L,then triggered T cells into specific CTLs.RESULTS:Most suspended cells exhibited distinctive morphological features of DCs which expressed CD40 96%,CD86 97%,CD80 77%,CD1a 69%,and gained the powerful capacity to stimulate proliferation of allogenic lymphocytes.Under the effector∶target ratio of 20∶1,CTLs derived from cultures with DCs and frozen-thawed antigen of K562 cells were showed 71.3% cytotoxicities against K562 cells.CTLs derived from cultures with DCs loaded with frozen-thawed antigen and CD40L were showed 86.9% cytotoxicities against K562 cells.Cytotoxicities by CTLs derived from cultures with unloaded DCs against K562 cells were 37.6% and cytotoxicities by monocytes were 21.1%.Cytotoxicities by CTLs derived from experiment groups were stronger than control groups (P<0.05).CONCLUSIONS:The tumor antigen-pulsed DCs induces efficient and specific anti-tumor immunity,CTLs derived from cultures containing DCs pulsed with CD40L show the strongest cytolytic activities on K562 cells.     

Effect of injecting immature dendritic cell intratumorally after microwave ablation under different temperature on the activity of cytotoxic T lymphocytes

AIM: To investigate whether the injection of immature dendritic cell (iDC) after ablation induce and upregulate tumor specific cytotoxic T lymphocytes (CTL).METHODS: The model of hepatoma was established and the tumors were ablated with microwave under (45±2) ℃, (50±2) ℃, (55±2) ℃, (60±2) ℃ and void ablation, after which the bone marrow derived iDC was injected intratumorally. The activity of CTL was detected, and the levels of IFN-γ and IL-4 secreted by activated spleic lymphocytes were measured.RESULTS: Compared with iDC injection intratumorally after the ablation of (45±2) ℃, (55±2) ℃, (60±2) ℃ and void ablation, the cytotoxic effect of CTL towards Hepa l-6 was heightened by injecting iDC intratumorally 72 hours after ablation of (50±2) ℃ (P<0.05), so was the secretion of IFN-γ (P<0.05). The level of IL-4 decreased after ablation of (50±2) ℃ subsequently with injecting iDC intratumorally (P<0.05). The cytotoxic effect of CTL towards Hepa l-6 was higher than that towards B16 lymphadenoma cell after iDC injection intratumorally and ablation of (50±2) ℃ and (55±2) ℃ (P<0.05).CONCLUSION: Thermal ablation of (50-55) ℃ and subsequent injection of iDC intratumorally may induce tumor specific CTL by the way of promoting the antigenicity of ablated tumor tissue and augmenting the presentation of TAA to effector cells.     

Effects of dendritic cell stimulation on cytotoxic activity of cord blood derived cytokine-induced killer cells,natural killer cells and CD45RO expression in CIK cells

AIM: To investigate the efficacy of dendritic cells (DCs) that augments the cytotoxic activity of cytokine-induced killer (CIK) cells, natural killer (NK) cells from a same donor and the CD45RO expression on CIK cells. METHODS: The expanded killer cells were divided into two groups: group A was pre-cocultured with DCs for 6 days, group B was the control that without any stimulation. Cytotoxicity of CIK and NK cells was measured at different effect-target ratio against K562 and HL-60. CD45RO and CD45RA expression on CIK cells in different groups were detected by flow cytometry. RESULTS: Cytotoxicity of CB derived killer cells was positive correlation with effect-target ratio. The cytotoxicity of group A against HL-60 was higher than that of group B significantly. At 20∶1 effector-target ratio, the lytic activity of group A CIK, NK cells against K562 was higher than that of group B significantly, but no significant difference between them at 10∶1 effector-target ratio. The CD45RO expression on CIK cells in groups A was significantly higher than that in groups B. CONCLUSION: CIK and NK cells cocultured with DCs can augment the killer's cytotoxicity against tumor cells and promote the CD45RO expression on CIK cells.     

Effects of CpG ODN on dendritic cells in anti-hepatocarcinoma action

AIM: To study the effect of oligonucleotides containing unmethylated CpG motif (CpG ODN) on mouse bone marrow-derived dendritic cells (BMDCs) in anti-HcaF cytotoxicity. METHODS: BMDCs were stimulated by CpG ODN in combination with tumor antigen (TAg). The expression of CD80 on BMDC surface was analyzed by FCAS. Level of IL-12 (p70) in supernatants of BMDC culture was detected by ELISA. The proliferation of T cells was examined by MTT assay. Cytotoxicity of CTL induced by CpG ODN combining with TAg was detected by MTT assay. RESULTS: CpG ODN combining or not combining with TAg up-regulated the expression of CD80 on BMDC surface and stimulated BMDCs to produce a high level of IL-12. CpG ODN-activated BMDC promoted the proliferation of T cells. CTL induced by CpG ODN in combination with TAg appeared strong specific cytotoxicity on Hca-F cells. CONCLUSION: CpG ODN may effectively induce the functional maturation of mouse BMDC in vitro. CpG ODN in combination with TAg can enhance the anti-HcaF cytotoxicity of CTL.     

Anti-cancer effect of cytotoxic T lymphocytes activated by sensitized dendritic cells

AIM: To investigate the anti-cancer effect of cytotoxic T lymphocytes (CTL) activated by sensitized dendritic cells (DCs). METHODS: Immature DCs were induced in vitro from peripheral blood monocytic cells (PBMC) and sensitized by adding tumor cells antigen extract. DCs were identified by their morphology and surface markers. MTT assay was used to evaluate the killing activity of CTL activated by sensitized DCs. The effects of specific CTL cells on inhibiting transplanted tumor HT-29 growth and on preventing HT-29 tumor generation were evaluated by injecting CTL into nude mice. RESULTS: After cultured for seven days, a large number of activated DCs were obtained with typical morphology, extensive stimulatory proliferation capacity and high CD80 (63.5%), CD83 (67.6%) and CD3/HLA-DR (83.2%) expressions. The killing activity of CTL at 20∶1 ratio of effective cells to target cells was more than 75% to tumor cells, 35%-45% to homologous cell line and weaker to other germ cell line (P<0.01). Injection of CTLs activated by HT-29 cell antigen sensitized DCs inhibited HT-29 transplanted tumor growth and prevented HT-29 tumor occurring in nude mice (P<0.05). PCNA expression level in tumor cells in CTL therapy group was dramatically lower than that in control (P<0.05). CONCLUSION: CTL activated by sensitized DCs kill tumor cells specifically, inhibit transplanted tumor growth and prevent tumor transplantation in nude mice.     

Recent advances in the correlations between dendritic cells and regulatory T cells

Dendritic cells (DCs), representing a heterogenous population of professional antigen-presenting cells, are the initiators and modulators of the immune responses. Studies indicate that regulatory T cells contribute to immune nullipotency and immune suppression via cell-cell contact or cytokine secretion. These two kinds of cells may be valuable tools for modulating immunity in the setting of auto-immunity, cancer, chronic viral infections and graft rejection, etc. Here we discuss the current knowledge on the functions of regulatory T cells and denditic cells-based immunoregulation and the applications.     

Human dendritic cells transfected with MUC1 mRNA induce lethal effect of cytotoxic T-lymphocytes on non-small cell lung cancer in vitro

AIM：To investigate the specific anti-tumor effects of mature dendritic cells (DCs) transfected with amplified mucin 1 (MUC1) mRNA in vitro. METHODS：DCs separated and purified from the peripheral blood mononuclear cells were induced in vitro and then identified by flow cytometry. pcDNA3.1（+）-MUC1 plasmid was constructed and was able to transcribe MUC1 mRNA in vitro. The MUC1 mRNA was transfected into DCs by electroporation. MUC1-transfected DCs were used to induce T cells to be cytotoxic T-lymphocytes. Quantitative real-time PCR was performed to assess MUC1 mRNA expression in transfected DCs. The proliferation of T cells was examined by MTT assay. The proportion of CD8+ cells in the T cells was determined by flow cytometry and the specific cytotoxicity was measured by LDH assay. The secretion of IFN-γ was detected by ELISA. RESULTS：The marker gene expression in the DCs transfected with MUC1 mRNA was significantly increased compared with control group, peaking at 24 h. The transfection group showed the higher capacity to stimulate the proliferation of T cells compared with control group when the ratio of DCs to T cells was 1∶10. The proportion of CD8+ cells in transfection group was higher than that in control group. The lethal effect of special cytotoxic T-lymphocytes on target cells in transfection group was stronger than that in control group. The level of IFN-γ in the cell supernatant of transfection group was higher than that in control group. CONCLUSION：DCs plus MUC1 mRNA by electrical transfection induces specific anti-tumor effects, which provides an experiment evidence of using MUC1 as a target for immunotherapeutic strategy against non-small cell lung cancer.     

Effect of monoclonal antibody to CD47 molecule on dendritic cell differentiation and function

AIM: To explore the influence of CD47 molecules on the maturation and function of cultured dendritic cells (DCs). METHODS: Monocyte cell-derived DCs were propagated in granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) plus interleukin (IL)-4, in the presence or absence of anti CD47 monoclonal antibodies (anti-CD47 mAbs). Flow cytometry was used to detect the cell surface phenotype. The concentration of IL-12P70 in supernatant was measured by ELISA technique. The antigen-presenting functions of DCs were determined in one-way mixed leukocyte reaction by Brdu-ELISA. Electrophoretic mobility shift assays (EMSA) was used to examine NF-κB activity. RESULTS: The anti-CD47 mAbs markedly suppressed the expression of CD80,CD86,CD83,CD1a,HLA-DR on the surface of DCs (P＜0.05). The data of mixed leukocyte reaction and IL-12P70 production were consistent with the flow cytometry results (P＜0.01). Nuclear extracts from the anti-CD47 mAb-treated DCs revealed a decrease in NF-κB binding activity. CONCLUSION: The anti-CD47 mAb exerts a negative effect on the maturation and function of in vitro cultured DCs via inhibiting of NF-κB binding activity.     

Phagocytosis of viable apoptotic cells inhibits the activation of T lymphocytes

AIM: To investigate whether viable apoptotic cells and phagocytosis of them affect the activation of T lymphocytes. METHODS: Ultraviolet irradiation was used to induce apoptotic cells in vitro and the model of phagocytosis of these cells was established. Cytokine TGF_β1 was detected by ELISA. The rate of apoptotic cells and phagocytosis of them were assessed by flow cytometry. Furthermore, flow cytometry was also employed to examine the expression of activation signs, such as CD69, CD25, CD71, of T lymphocytes under the intervention of apoptotic cells and macrophage which ingested apoptotic cells, to reflect whether the apoptotic cells and the phagocytosis of these cells could influence the activation of lymphocytes stimulated by Con A. RESULTS: Ingestion of apoptotic cells increased TGF_β1 secretion. Only the macrophages that had ingested apoptotic cells could suppress the activation of lymphocytes. The expression of the markers of lymphocytes activation such as CD69, CD25, CD71 had been restrained. These inhibition effects were abolished by monoclonal anti-TGF_β1 antibody. CONCLUSION: The macrophages that have ingested apoptotic cells inhibit expression of CD69, CD25 and CD71 of T lymphocytes stimulated by ConA. This effect is dependent on the increase in TGF_β1 secretion in local site.     

Immune function of dendritic cells in patients with hepatocellular carcinoma

AIM: To investigate the immune function of dendritic cells (DCs) in patients with hepatocellular carcinoma(HCC). METHODS: The DCs were cultured from human peripheral blood mononuclear cells (PBMC) by using GM-CSF and IL-4 for 7 days. Surface molecules CD86, HLA-DR of DCs were detected by flow cytometry. IL-12 production by DCs and IFN-γ production by T cells was measured with ELISA and ELISPOT, respectively. Allogenic mixed lymphocyte reaction was detected by MTT assay. RESULTS: CD86 expression in DCs in HCC patients were markedly lower than that in health control (91.7％ vs 83.5%, P<0.05). IL-12 production by DCs had no significant difference between the HCC patients and the health control ［(324.6±171.0)ng/L vs (436.5±142.7)ng/L, P>0.05］. However, after stimulated DCs with LPS, IL-12 production in HCC patients was significantly lower than that in health control ［(478.6±142.7)ng/L vs (630.0±151.9)ng/L, P<0.05］. IFN-γ production by T cells and T cell proliferation index (PI) in the HCC patients were all significantly lower than those in health control ［(IFN-γ: 133.4±51.2)103U/L vs (183.0±60.2)103U/L, P<0.05; PI: 2.3±0.7 vs 3.5±0.8, P<0.01］. CTL killing activity assay indicated that DC-induced CTL killing activity in HCC group was significantly lower than that in health group when the E/T ratio was 10∶1 and 20∶1, respectively. CONCLUSION: The immune function of DCs and T cells in the patients with HCC are significantly decreased. The CTL killing activity of HCC group is also markedly decreased.     

Anticancer effect of mouse marrow-derived dendritic cells transfected by pcDNA3-hCEA

AIM: To prepare the vaccine of DCs(pcDNA3-hCEA) and observ the immunity effect of the DCs(pcDNA3-hCEA) inoculating on CT26(hCEA⁺) loaded in BALB/c mice. METHODS: DCs were generated from bone marrow in the presence of rmGM-CSF and rmIL-4. A new recombinant plasmid, pcDNA3-hCEA, reformed with inserting a 2.4 kb human CEA cDNA into pcDNA3. DC vaccine was prepared by transfection with pcDNA3-hCEA using lipofectamine. CEA mRNA expressed in DCs(pcDNA3-hCEA) was confirmed by RT-PCR, CEA expression level was detected with RIA method, and CEA specific CTL was induced in vitro. After vaccination of DCs(pcDNA3-hCEA), the survival time of the BALB/c mice challenged with critical loading CT26(hCEA⁺) was observed. RESULTS: G₄₁₈ test showed that about14% DCs were transfected with pcDNA3-hCEA. And CEA mRNA and protein could be detected respectively by RT-PCR and RIA in the genetically modified DCs. Furthermore, the DCs coud be targeted by specific CTL, the survival time of the mice challenged with CT26(hCEA⁺) was prolonged1-4 weeks. CONCLUSION: These results demonstrate that specific antitumor immune responses could be induced efficiently by vaccination of DCs(pcDNA3-hCEA), which is transfected eukaryotic expression vector encoding tumor antigen gene.     

Function of dendritic cells in cord blood

AIM: To explore the function of dendritic cells in cord blood.METHODS: Dendritic cell precursor subsets pDC/pDC(CD11c+CD123- /CD11c-CD123+) in cord blood and adult peripheral blood were analyzed gated by CD34-Lin- HLA-DR+ cells and the levels of IL-12p40,IL-10,IFN-γ,IL-4 in the serums were tested by ELISA.RESULTS: The level of IL-12p40 in cord blood serum was higher than that in peripheral blood.pDC1/pDC2,IL-10,IFN-γ,IL-4 in cord blood were similar to that in peripheral blood.CONCLUSION: Function development of dendritic cells in cord blood may be consummate.     

Effects of CpG ODN on dendritic cells and its mechanisms

Oligodeoxynucleotide containing unmethylated cytosine phosphate-guanosine motif(CpG ODN) may induce high expression of CD80, CD86, CD83, HLA I and HLAⅡ molecules on dendritic cells(DC) and stimulate DC to produce high level of IL-6, IL-12, TNF-α and IFN-α. CpG ODN is demonstrated in vivo to be a very potent adjuvant for Th1 cells, regulating Th0 cells to develop toward Th1 cells. Its role for DC is characteristics of CpG ODN sequence specificity and species specificity. CpG ODN is, at present, considered as a pathogen associated molecular pattern which binds its specific receptor，Toll-like receptor 9，then functions through TLR/IL-1R signaling pathway. It may represent a new therapeutic drug for broad applications in infectious disease, autoimmune disease, allergy and cancer therapy.     

Clonal expansion and specific cytotoxicity of autologous or allogeneic T cells induced by M2a cells

AIM: To investigate clonal expansion and specific cytotoxicity of TCR Vβ subfamily T cells from acute myelogenous leukemia M_2a subtype (AML-M_2a)patients and normal individuals induced by AML-M_2a cells, respectively. METHODS: Complementarity determining region 3(CDR3) of TCR β with variable region genes was amplified in autologous or allogeneic T cells from mixed lymphocyte and tumor culture (MLTC) using RT-PCR. The positive products were further analyzed to identify the clonality of T cells by genescan. The specific cytotoxicity of T cells was analyzed by MTT. RESULTS: T cells from both M_2a patients and normal individuals after MLTC showed high response to M_2a cells with 4-17 TCR Vβ subfamily dominant utilization, one or two clonal expansion of T cells were identified in some predominant TCR Vβ subfamilies. Difference of distribution and clonal expansion of TCR Vβ subfamily T cells were related to source of T cells and the phase during MLTC. Compared with LAK cell, most of T cells from MLTC were CD3⁺CD8⁺T cells with higher and more specific cytoxicity to the induced cells, M_2a cells, but not HL60 or K562 cell line. CONCLUSION: Clonal expansion of TCR Vβ subfamily T cells stimulated selectively by M_2a cells may be a specific immune response of autologous and allogeneic T cells to M_2a cells associated antigen. The T cells induced by M_2a cells have the ability of specific cytoxicity to the AML-M_2a cells.     

The molecular mechanism of tolegenic dendritic cells induced by IL-10 in mouse

AIM:To investigate the relation of tolerogenic dendritic cells (DC) induced by interleukin-10 (IL-10) and the paired immunoglobin-like receptor (PIR) A and B (PIR-A and PIR-B) in mouse. METHODS:The mouse dendritic cell line, DC2.4 cells were cultured with the IL-10 to develope the IL-10-DC and were stimulated by lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small inference RNA (siRNA) molecule of PIR-B was chemically synthesized and was transfected into IL-10-DC by Lipofectamine 2000 (Si-DC). The expression of PIR A and PIR B on DC2.4 cells were measured by semi-quantitative RT-PCR and flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using ［3H］-thymidine incorporation test. The concentration of IFN-γ in supernatants of MLR from distinct groups was analyzed by ELISA. RESULTS:IL-10 up-regulated the PIR-B and down-regulated the PIR-A by semi-quantitative RT-PCR. On the contrary, LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. The expression of PIR, which is the common extra-membrane of PIR-A and PIR-B, was increased in both the IL-10-DC and the LPS-DC groups by FCM detection, but the higher expression was found in IL-10-DC group than that in LPS group. The IL-10 induced the higher PIR-B expression, inhibited allogenetic T cell proliferation and down-regulated the IFN-γ secretion. Special siRNA molecules of PIR-B in IL-10 group promoted the T cell proliferation and enhanced the IFN-γ secretion in MLR. CONCLUSION:IL-10 up-regulates the PIR-B expression and makes DC tolerance. Up-regulated PIR-B expression may be the molecular mechanism of tolerogenic dendritic cells induced by IL-10 in mouse.     

Effect of lung cancer cells transfected with interleukin 18 gene on the immunological activity of dendritic cells

AIM: To study the effect of interleukin 18 gene transfected lung cancer cells on the phenotype and immunological activity of dendritic cells (DC). METHODS: A secretive IL-18 expression vector containing IL-12 P40 signal sequence was constructed and transfected into NCI-H460 lung cancer cells. DC induced from human peripheral blood were divided into 4 groups (NT, PV, GT and PD). DC were stimulated by non-transfected NCI-H460 cells, pure vector transfected NCI-H460 cells and IL-18 transfected NCI-H460 cells respectively for group NT, PV, GT, and non-stimulated DC for group PD. CD54, CD80, CD83 and CD86 on DC in the 4 groups were detected with flow cytometry. T cell proliferation stimulated by DC in the 4 groups was assayed with MTT method. IL-12 release in cultured DC supernatant was measured by ELISA. RESULTS: Sequencing result of the secretive IL-18 was correct. The transfected cells expressed IL-18 fusion gene and 18 kD IL-18 protein. DC in GT group expressed more surface molecules than those in other 3 groups. T cell proliferation and IL-12 secretion in GT group were higher than those in other 3 groups. CONCLUSION: IL-18 gene transfected NCI-H460 cell increases surface molecule expressions on DC. It also enhances immunological activity and IL-12 secretion in DC.     

Role of mtCLIC/CLIC4 in injured C6 glioma cells induced by hydrogen peroxide

AIM: To explore the changes and the possible function of mtCLIC/CLIC4 (mitochondrial chloride intracellular channel 4) proteins in malignant C6 glioma cells treated with hydrogen peroxide (H₂O₂). METHODS: The viability of C6 cells was measured by MTT assay, LDH release rate was detected by ultraviolet spectrophotometry, CLIC4 mRNA level was determined by RT-PCR and CLIC4 protein level was measured by Western blotting. RESULTS: Compared with the control group, the cell viability was constant, the LDH release rate increased obviously, and the CLIC4 protein level also increased significantly in 500 μmol/L H2O2 treated group (P<0.05, respectively). However, the cell viability decreased, LDH release rate increased significantly (P<0.01, respectively), and the CLIC4 protein level increased obviously in 1 000 μmol/L H₂O₂ treated group (P<0.05). CONCLUSION: The CLIC4 protein may be involved in the process of C6 injuries induced by H₂O₂.     

Effect of adriamycin combined with sensitized dendritic cells on cervical tumor-bearing mice

AIM：To explore the immunotherapeutic effect of adriamycin (ADM) combined with frozen-thawed antigen-sensitized dendritic cells (DCs) on cervical tumor-bearing mice. METHODS：The U14 cervical cancer model of Kunming mice was established by subcutaneous implantion of U14 cells in axillary fossa. DCs vaccine was prepared by U14 cervical cancer cell frozen-thawed antigen-sensitized mouse bone marrow-derived DCs. Mature phenotype of sensitized DCs was identified by flow cytometry. Tumor-bearing mice were randomly divided into 4 groups and treated for 3 cycles with PBS (control), DCs vaccine, ADM and ADM combined with DCs vaccine, respectively. The tumor volume was evaluated. The tumor weight and the levels of interleukin-2 (IL-2), IL-12 and interferon γ (IFN-γ) in the serum were determined by ELISA on the 21st day. RESULTS：Cancer cell frozen-thawed antigen-sensitized DCs had higher expression levels of CD11C, CD80 and CD86. The volume and weight of the tumor in ADM combined with DCs vaccine group were less than those in ADM group, DCs vaccine group and control group. The tumor inhibitory rate in combination group was higher than that in the other 3 groups. Compared with the other 3 groups, the serum levels of IL-2, IL-12 and IFN-γ in combination group significantly increased. CONCLUSION：ADM combined with tumor antigen-sensitized DCs vaccine can strengthen the animal antitumor immune response and effectively inhibit the growth of tumor in cervical tumor-bearing mice.     

Role of TRAF6 in maturation of human PBMC-derived dendritic cells

AIM: To investigate the immunological effect of tumor necrosis factor receptor-associated factor 6 (TRAF6) on the maturation of dendritic cells in vitro.METHODS: The human peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), induced by recombined human granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, were stimulated to mature with TNF-α, LPS or cocktail of cytokines (TNF-α, IL-6, IL-1β and PGE₂).The cell surface markers, T-cell stimulatory proliferation capacity and IL-12 p40 production of the DCs were determined by the methods of flow cytometry, ELISA and mixed lymphocyte reation (MLR), respectively.The mRNA expression of TRAF6 was detected by real-time PCR.RESULTS: The expression of TRAF6 was observed in all groups, which in cocktail group was the highest in the DCs with optimum state of maturation.Furthermore, TRAF6 enhanced the production of IL-12 and the ability of T-cell stimulation of mature DCs.CONCLUSION: TRAF6 might play an important role in inducing the maturation of human PBMC-derived DCs.