Salidroside regulates DC through TLR4 to increase the lethality of T cells to lung cancer 3LLcells / 中国肿瘤生物治疗杂志

@#Objective： ：To investigatetheeffectofsalidroside(SAL)onthephenotype of dendritic cells (DCs) and the antitumor ability of cytotoxic T lymphocytes (CTL). Methods： ：Lewis lung cancer cell line 3LL, wild type (WT) C57BL/6 mice and TLR4-/- C57BL/6 mice were chosen for this study. Mice bone marrow derived DC precursor cells were obtained to differentiate into immature DCs, which were harvested on the sixth day of culture. CD11c+ DCs were obtained by magnetic beads screening, and further divided into PBS group, SAL group and lipopolysaccharide (LPS) group.After being cultured for 48 h, the effects of SAL on surface molecules and phagocytosis of DCs as well as the efffect of TLR4 pathway on the killing effect of T cells were detected by Fow cytometry. Results： ： Compared with PBS group, expressions of DC surface molecules CD80, CD86 and MHC Ⅱ significantly increased (all P<0.05), phagocytosis significantly decreased (P<0.05), and TLR4 expression level significantly increased (P<0.01) in SAL group; Compared with WT group, after being treated with SAL or LPS, the expressions of DC surface molecules CD80, CD86 and MHC Ⅱ decreased significantly in TLR4-/- group (all P<0.05); ComparedwithPBSgroup,theactivatedCTLinSALgroupexhibited a significantly elevated killing effect against lung cancer 3LLcells (P<0.05). Conclusion：SAL can induce DC maturation by regulating TLR4, thus improving the killing ability of T cells.

Immunoregulation through IL-10 gene expression and the fate of cytotoxic T lymphocyte-mediated tumor immunotherapy.

Gene analysis of tumor associated antigens revealed that tumor antigens are all normal gene product. Inducing tumor reactive cytotoxic T lymphocytes (CT) in the patients is same as inducing autoimmunity in the patients. Immunosuppressive cytokine interleukin-10 (IL-10) plays an important role in maintaining homeostasis or tolerance. To break the tumor tolerance, blocking and IL-10 secretion or intervention in the pathways of IL-10 gene activation is indeed important.

Combined Therapy with Mixed Heat Shock Protein/Peptide Vaccine and Interleukin-12 Results in Enhanced Antitumor Effects / 中国康复理论与实践

@#Objective To investigate the antitumor immunity induced by tumor derived mixed heat shock protein/peptide(mHSPs),interleukin-12(IL-12)and Cyclophosphamide(Cy).MethodsPurified mixed HSP was prepared from tumor by S180 protein extraction and purification,SDS-PAGE,Western blot and animal experiment were applied for mixed HSPs analysis.ResultsThe proliferation of cytotoxic T lymphocyte(CTL)cocultured in the mHSPs+Cy+IL-12 group was significantly remarkable and the content of CD8+ CTLs was significantly more in comparison with the other groups(P<0.01).To the tumor bearing mice,mHSPs+Cy+IL-12 group showed partial therapeutic efficacy,the averaged survival period was over 60 d,and 90% of the mice in this group got long period tumor free survival(>90d),obvious difference(P<0.05)from the other groups.ConclusionTumor derived mixed HSPs can induce powerful antitumor immune efficacy and show favorable therapeutic efficacy.

Enhanced CEA-specific Immune Responses by Tat-LLO Fusion Protein

BACKGROUND: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. However, it is known that the induction of immune response to CEA is very difficult because CEA is a self-antigen expressed in fetal cells and weakly expressed in normal colorectal epithelial cells. To enhance anti-tumor immunity specific for CEA, recombinant CEA protein was modified using listeriolysin O (LLO) for endosomal lysis and transactivator of transcription (Tat) domain for transducing extracellular proteins into cytoplasm. METHODS: After immunization using dendritic cells pulsed with Tat-CEA, both Tat-CEA and LLO, and both Tat-CEA and Tat-LLO, antibody titer to CEA and LLO, cytotoxic T lymphocyte activity and the frequency of IFN-gamma producing T lymphocytes were measured. RESULTS: Immunization using DC pulsed with both Tat-CEA and Tat-LLO protein showed the increasement of production of CEA-specific antibody in serum, cytotoxic T lymphocyte activity, the frequency of IFN-gamma secreting T cells, compared with DC pulsed with both Tat-CEA and LLO. Furthermore the ratio of CD8+ T cell to CD4+ T cell among CEA-specific T cells was increased in group pulsed with both Tat-CEA and Tat-LLO. CONCLUSION: These results suggested that DC vaccine using Tat-LLO could be used for the development of effective immunotherapy for the treatment of tumor.

Enhanced Induction of CEA Specific Tumor Immunity by TatCEA Fusion Protein

PURPOSE: The human carcinoembryonic antigen (CEA) is expressed in several tumor types, including colorectal cancer, and is a tumor-associated antigen used as a target for antigen-specific immunotheraphy. CEA is a self-antigen associated with development, expressed in fetal cells and rarely expressed in normal colorectal epithelial cells. The induction of immune response to CEA is very difficult. In this study, we attempted to increase the tumor immunity specific to CEA by using dendritic cells pulsed with fusion proteins of CEA and Tat (transactivator of transcription), which transduces extracellular proteins into cytoplasm and causes antigens to be presented with MHC class I pathway. METHODS: The Tat gene was amplified in the PNL4-3 HIV plasmid and then inserted into PCEP4 plasmid vector. The CEA gene was cloned from cDNA from LoVo human colorectal cell line and then amplified through polymerase chain reaction method. After cloning of PCEP4 plasmid vector, the dendritic cell was sensitized and internalized with CEA and Tat-CEA protein. Then the Western blot analysis of the expression of CEA in the gene-modified dendritic cell and the immunofluorescent staining of the expression of CEA in CEA or Tat-CEA-pulsed dendritic cell were performed. A detection of IFN-gamma-releasing CD8 cell and a cytotoxicity of T-cell were was assesed using ELISPOT assay. The Immunoglobulin (Ig) G isotypes were analyzed with enzyme-linked immunosorbent assay. The statistical significance was assessed using Students t-test. RESULTS: CEA pulsed in dendritic cells was distributed over the cell surface and TatCEA was observed in the cytoplasm. The cellular immune responses by immunization with dendritic cells pulsed with TatCEA (322/10(4) lymphocytes) were significantly increased compared with those with CEA (244/10(4) lymphocytes) by IFN-gamma ELISPOT assay (P<0.05). The cytotoxic T lymphocyte (CTL) activity using mouse T-cell, EL-4 pulsed peptide (EAQNTTYL) as target cells was 23.3+/-2.75% (E:T=1:100) in the CEA group and 22.9+/-2.23% (E:T=1:100) in the TatCEA group. In ELISA analysis of the IgG isotype, the titer of IgG2a and IgG3, representing Th1 immune response, was lower than that of IgG1, representing Th2 immune response, in both the CEA group and the TatCEA group. CONCLUSIONS: These results suggest that TatCEA could be used for the development of a tumor vaccine and cellular immunotherapy using CTLs induced in vitro.

The biologic character and inducing to the HBV-specific cytotoxic T lymphocytes of DCs from the patients with chronic hepatitis B pulsed with HBsAg in vitro / 中国免疫学杂志

Objective:To study the functional status of the monocytes-derived dendritic cells (DCs) plused with HBsAg from patients with chronic hepatitis B and their capacity of inducing the HBV- special cytotoxic T lymphocytes (CTLs),and explore a new method of inducing the special anti-HBV cell-mediated immunity.Methods:Monocytes of patients were isolated from peripheral blood and incubated with GM-CSF+IL-4+TNF-? to induce DCs generation. Those DCs were pulsed with HBsAg to induce HBV- special DCs. The phenotype of DCs including CD1a,CD80,CD83,CD86,CD40 and HLA-DR was characterized by FCM and the stimulating reaction of allogenic T lymphocytes was detected by MTT assay. The concentration of cytokines such as IL-12 and IL-6 in supernate was tested by ELISA and the cytotoxicity of CTLs inducing by the DCs against HepG2 2.2.15 cells,HepG2 cells and K562 cells were detected by LDH assay.Results:The expression of CD1a, CD80, CD83,CD86, CD40, HLA-DR molecules on the DCs pulsed with HBsAg was higher than those of the control group (P

Induction of prolongation of allograft survival by immunization with immature DCS / 中国免疫学杂志

Objective:To prolong the allograft survival time in immunized mice with immature dendritic cell, and to analyze the mechanism of hypo allo immuno responsiveness induced by immature dendritic cells Methods:Based on mouse cardiac allograft model, the survival of cardiac recipients immunized with mature or immature DC were observed Meanwhile the CTL activity of splenocytes from immature DC immunized recipients was detected Results:The survival time of cardiac allograft was substantially prolonged and the mean survival time extended from 9 1?0 73 days to 25 4?4 27 days It became more effective if those immunized mice were treated in combination with adriamycin application, the mean survival time of allograft was extended to 30 5?3 98 days It was proved that the CTL activity in spleen cells from the mice immunized with immature DCs was much lower (specific release was 16 32%) than that from the immunized mice with mature DCs (specific release was 39 58%) Conclusion:Immature DCs could induce prolongation of allograft survival time It may be possible that low CTL activity in recipients immunized with immature DCs promised this prolongation