The Study Of Immune Effect Of Recombinant E.coli LLO/OVA On Mice Dendritic Cells' Activation And Promoting Anti-Tumor T Cell Immunity

Malignant tumor is one of the main diseases which severely afflict humanity, but the results of various clinical therapies of tumors are not satisfaction. There is a phenomenon that the immune response is poor in the bodies of tumor patients, so tumor immune therapy was tried by researchers to induce anti-tumor responses in the bodies of tumor patients for inhibiting the growth and metastasis of tumors. In recent years, it is a hot point in the world that constructing various tumor vaccines which carry tumor antigens for tumor immune therapy. Some satisfied effects occurred in animal model experiments of various vaccines, and a few effects occurred in clinical trails also. In future the immune therapy may be used as the fourth measure of tumor therapy besides surgical resection, chemotherapy and radiotherapy.In the tumor immunotherapy experiments, the researchers use various vaccines including peptide vaccine, tumor cells vaccine, gene vaccines (DNA, RNA, recombinant virus and recombinant bacteria, et al) and dendritic cell vaccine. The vaccines may just carry tumor antigen, or carry cytokine/adjuvant together with tumor antigen to initiate stronger cytotoxic T cell responses. The vaccines are injected by subcutaneous, in muscle, in vein, in lymph node or in tumor nodule to activate the dendritic cells and prime the tumor antigen specific immune response.The core problem of tumor vaccine is how to improve the immune effect of vaccine to induce stronger tumor specific immunity in the bodies of tumor patients.Sole tumor antigen vaccination may not initiate strong enough anti-tumor immunity; however, the immune prime activation of the vaccine will be amplified by an effective adjuvant. Aimed to prime stronger immune responses against OVA positive tumors, our study was performed in construction of recombinant E. coli LLO/OVA that carries the gene segments of the nature adjuvant listeriolysin O (LLO) and the model tumor antigen ovalbumin (OVA) together, and elucidating the mechanism about the activation of mice dendritic cells and priming T cell immunity. In our experiment, LLO was expressed in E. coli LLO/OVA as its creating pore activity helping OVA antigen escape from lysosomal vesicle of dendritic cells, and the bacterial content can then be processed by the proteosome and the antigen motifs presented onto MHC class I to CD8+ T cells, initiating stronger cytotoxic T cell immunity. The distinct advantages of bacteria based DNA vaccines are that (1) The live attenuated vaccines induce the secretion of series cytokines and proinflammatory mediators that enhance early innate immunity and create a local environment conductive to antigen presentation, and (2) The bacterial cell components and unmethylated DNA as so-called pathogen-associated molecular patterns (PAMPs) provide an optimal"danger signal"through the activation of pattern recognition receptors on APCs. The innate immune response of antigen presenting cell (APC) will be triggered by PAMPs in the form of the production of pro-inflammatory cytokines and up-regulation of co-stimulatory molecules. These responses promote the maturation and migration of DCs to secondary lymph tissues, and in this way, PAMPs amplify the immune response against the antigen and act as adjuvant. Bacterial products are particular good at activation CTLs; therefore, they may act as natural adjuvant in cancer immunotherapy.In this study, E. coli OVA as a control, the C57BL/6 mice were vaccinated by E. coli LLO/OVA and E. coli OVA respectively, the inhibition effects of the B16-OVA melanoma were compared; in order to understand the role of CD8+ T cells in anti-tumor immune responses, these cells were deleted by specific antibody YTS169.4, and then the B16-OVA melanoma protection effects were compared by vaccination of E. coli LLO/OVA and E.coli OVA; In order to understand the role of recombinant E. coli LLO/OVA on dendritic cells of mice, the bone marrow dendritic cells (BMDCs) were infected with recombinant E.coli in vitro and the superarray were performed to check the genes transcription of NF-κB signal pathway, chemokines, chemokine receptors and cytokines; in addition RT-PCR and ELISA were done to confirmed some of the gene transcription and protein expression. Aimed to understand the immune prime effect of activated dendritic cells on T cell immunity, the vaccinated mice spleenic CD11c cells and autogenous CD4+ T, CD4+CD25- T and CD8+ T cells mixed reaction were studied by 3H examination, and the cytokines secretion in the supernatant were checked by ELISA respectively, and the OVA specific CD8+ T cells were calculated by flow cytometry also. The results and conclusion are:1. The plasmids which carrying LLO gene segment and OVA gene segment were successfully transferred into harmless, non replication strains of E. coli MC4100. The expressions of LLO and OVA were induced by IPTG (isopropyl-beta-D-thiogalactopyranoside), and were confirmed by coomassie brilliant blue staining in GEL and Weaternblot.2. After 3 times subcutaneous vaccination of recombinant E.coli LLO/OVA in C57BL/6 mice, the B16-OVA melanoma protection effects were observed in 75% mice, and the significant inhibition of tumor growth and metastasis were observed also. After deletion of CD8+ T cells, the effect of inhibition of tumor metastasis in mice were significantly decreased. This result confirmed that CD8+ T cells are the main effect cells in anti-tumor respnses in mice.3. After pulsed by recombinant E.coli LLO/OVA and E.coli OVA, the BMDC of C57BL/6 mice were activated through Toll like receptor 2(TLR2), TLR4, TLR7 and Card 4(NOD1), especial TLR4, and by NF-κB signal pathway, the series genes of chemokines, chemokine receptors and cytokines up-regulated; in addition, the expression of co-stimulatory molecules up-regulated also. The BMDC pulsed by E.coli LLO/OVA were activated more rapidly by NF-κB siganl pathway and produced higher level of CXCR4, IFN-γand G-CSF than that of BMDC pulsed by E.coli OVA.4. The spleenic CD11c cells of vacccination mice with E.coli LLO/OVA were significantly more effective at stimulating autogenous CD4+ T cells secreting IL-2, but shew same effect on stimulating autogenous CD4+ T and CD4+CD25- T cells proliferation than that of vaccination mice with E.coli OVA. In addition, the spleenic CD11c cells of vacccination mice with E.coli LLO/OVA were significantly more effective in stimulating autogenous CD8+ T cells proliferation and secreting IFN-γthan that of vaccination mice with E.coli OVA. In addition, The result of flow cytometry shew that there were more OVA specific CD8+ T cells were induced by vaccination with E.coli LLO/OVA in mice than that of vaccination with E.coli OVA.E.coli LLO/OVA induces stronger antitumor T cell immunity in C57BL/6, and showes significant effect on inhibition of tumor metastasis; it primes BMDCs mature and up-regulation genes of chemokine, chemokine receptors and cytokines through NF-κB signal pathway by activation TLR2,TLR4, TLR7 and Card4(NOD1), especial TLR4. Mature Spleenic CD11c cells stimulate autogenous CD4+T, CD4+CD25- T and CD8+ T cells proliferation and secretion of IL-2 and IFN-γ. The Flow cytometry results shew significantly more OVA specific CD8+ T cells were induced in vacccination mice with E.coli LLO/OVA.Taken together, recombinant E.coli LLO/OVA, which carries nature immune adjuvent lysteriolysin O and tumor antigen OVA together, induces a stronger T cell anti-tumor immunity than recombinant E.coli OVA. The vaccine vector (E.coli LLO) may be use as an effective vector of human tumor antigen for the research of human tumor immunothrapy in future.