Research Of The Potiential Of RHP-NAP As Adjuvant Of Anti-melanoma Dendritic Cells-based Vaccine

Purpose Melanoma is one of the most malignant tumors,takes up 80% of the mortality caused by skin cancers.With the Accumulation of the knowledge of the role immune system plays in tumor progression.Immunotherapy gained great focus among treatments of melanoma.Tumor vaccines can elicite immune response against these antigen and is considered to be a potential method to cure melanoma.Dendritic cells vaccine is a kind of tumor vaccine,in this vaccine,dendritic cells is generated in vitro,loaded with tumor antigens and stimulated with adjuvant to enhance the ability to stimulate immune response,then inject the dendritic cells vaccine to patients with cancer,the therapeutical effect is played by eliciting tumor specific immune response.During the generation of dendritic cells vaccine,besides loading tumor antigens to dendritic cells,stimulating the dendritic cells with adjuvants can promote the maturation of dendritic cells and enhance the anti-cancer effect of the vaccine.Thus screening the effective immunomodulator as the adjuvant of the dendritic cells vaccine is a key issue in the generating of dendritic cells vaccines.TLR agonists have the function to promote dendritic cells mature,is often used as adjuvants of dendritic cell vaccines.Recent years,a new TLR agonist,HP-NAP,gained great focus due to its function in the immune stimulating and anti-cancer effects.In this research,we expressed HP-NAP with E.coli.Expression system,and explored its function to promote dendritic cells maturation and its potential in acting as adjuvant of dendritic cells vaccines.This research aimed to find an adjuvant can be effectively used in generating dendritic cells vaccines.Methods1.We expressed HP-NAP with(p ET28a-HP-NAP)E.coli BL21(DE3),purified HP-NAP and eliminated the endotoxin by affinity chromatography.The purified HP-NAP was stored in-80 until used.2.Dendritic cells was generated from C57BL/6 bone marrow cells in the environment with GM-CSF and IL-4..3.The dendritic cells derived from wild type mice were treated with different concentration of HP-NAP and detected the phenotype of the cells by flow cytometry;and tested the cytokines expression in m RNA level.Then decide the best concentration used in follow-up work.4.The dendritic cells generated from wild type or TLR2-/-mice were treated with HP-NAP,then test the expression of costimulatory molecules CD80 on the cell surface.Determine whether HP-NAP stimulate dendritic cells through TLR2.5.Use B16F10-TCL as antigens and HP-NAP as adjuvant to stimulate dendritic cells.Test the expression of costimulatory molecules CD80,CD86 and chemokine receptor CCR7 on the cell surface by flow cytometry.Measure the phagocytic ability with FITC-Dextran assay.Measure the expression of cytokines IL-12,IL-23 and IL-1? with q RT-PCR.6.Purify T cells with nylon wool.Treat dendritic cells with HP-NAP and B16F10-TCL,cocultured with splenocytes or T cells,measure the proliferation of splenocytes and T cells with MTT assay;detect the expression of activation marker CD69 on T cells by flow cytometory;measure the concentration of IFN-?and IL-17 A by ELISA;determine the expression of IL-4,IL-10 in m RNA level through q RT-PCR.7.Subcutaneously inject C57BL/6 mice in the flank with 200?g B16F10-TCL twice in 5 days' interval.Purify T cells from spleen cells 5 days after the second injection.cocultured with dendritic cells treated with HP-NAP and B16F10-TCL.Measure the concentration of IFN-?with ELISA;determine the expression of granzyme B and proferine in m RNA level through q RT-PCR.Use the T cells as effector cells and cocultured with B16F10 at the ratio of 10:1,20:1 and 40:1,test the cytotoxicity of T cells with LDH assay.Results:1.The recombinant protein HP-NAP was successfully expressed and purified.2.The purity of the dendritic cells was more than 85% and could be used in the subsequent research.3.HP-NAP promote the maturation of dendritic cells.HP-NAP in the concentration of 1?M(17?g/ml)would be used in follow-up researches.4.HP-NAP up-regulate the expression of CD80 on the surface of dendritic cells derived from wild type mice but had no effect on the cells from TLR2 knocked out mice.5.Dendritic cells treated with B16F10-TCL and HP-NAP significantly up-regulate the expression of costimulatory molecules CD80,CD86 and chemokine receptor CCR7 but down-regulate phagocytic ability.Expression of cytokines IL-12,IL-23 and IL-1? were upregulated after treated with HP-NAP and B16F10-TCL.6.Dendritic cells treated with HP-NAP and B16F10-TCL significantly enhanced the ability to induce T cells activation and proliferation.HP-NAP and B16F10-TCL treated dendritic cells significantly upregulate T cells secrete IFN-?.7.Dendritic cells treated with HP-NAP and B16F10-TCL could stimulate antigen specific T cells express granzyme B and proforine and significantly enhance IFN-? secretion.Dendritic cells treated with HP-NAP and B16F10-TCL was significantly enhanced.Conclusions:1.HP-NAP induce the maturation of dendritic cells through TLR2.2.HP-NAP could act as adjuvant to promote the maturation of dendritic cells loaded with B16F10-TCL.3.HP-NAP enhanced the ability of dendritic cell loaded with B16F10-TCL to induce the activation and proliferation of T cells and induce Th1 immune response.4.HP-NAP significantly enhanced the ability of dendritic cells loaded with B16F10-TCL to induce antigen specific cytotoxic T lymphocytes.