Antitumor Effect Of DC Tumor Vaccine Activated By Combined Toll-like Receptor Ligations And The Related Mechanisms

Toll-like receptors (TLRs) are a group of transmembrane proteins mainly expressed on antigen-presenting cells (APC) such as dendritic cells (DC) and macrophages. They recognize pathogen-derived products and play a key role in the defense agaist invading pathogens by the host immune system, such as TLR4 is essential for the recognition of lipopolysaccharide (LPS) from gram-negative bacteria; TLR3 is involved in recognition and signal transduction induced by double-stranded (ds) RNA synthesized from viruses; TLR9 is involved in recognition of bacterial DNA containing unmethylated CpG motifs. TLRs control the activation of the adaptive immune response by APC. DC is the most potent APC with the unique ability to activate naive T cells and orchestrate innate and adaptive immune response. Maturation of DC is critical for priming T cells to initiate acquired immune response. TLRs expressed on DC can recognize various kinds of microbial or viral. Once binding with ligands, they induce DC activation, which is characterized by the production of inflammatory cytokines like IL-6, IL-12 as well as the upregulation ofcostimulatory molecules. Recent researches demonstrated that sitimulation of DC with combined TLR ligation could induce an even more quick and sustained DC activation. It is also demonstrated that DC sitimulated with combined TLR ligation favor the development of Th1 responses, whereas Th1 cellular immune response is crucial for anti-tumor and anti-microbial immunity. Moreover, more and more researchers proposed that TLRs have a close connection with regulatory T cells which can inhibit DC maturation and naive or effector T cells proliferation as well as IL-2 secretion of antigen-spencific effector T cells, and is another check-point to initiate acquired immune response. However, the presence of regulatory T cells at tumor site may one of the mechanisms tumor cells may take advantage of to protect themselves from immune attack. New evidence suggests that TLRs signaling may reverse the suppressive activity of regulatory T cells. All of above imply the possibility of antitumor effect by DC activated by combined Toll-like receptor ligations in vivo. So here we try to sdudy the characteristic of DC tumor vaccine activated by combined Toll-like receptor ligations in vitro as well as its antitumor activity in vivo.OBJECTIVETo observe the antitumor effect of DC tumor vaccine activatied by combined Toll-like receptor ligations on C57BL/6 murine model bearing EG7 (OVA-transfected EL4) thymoma and investigate the related immunological mechanisms, and also try to establish the basis for exploring the new tumor vaccination or immunotherapy.METHODS1. Stimulation of DC by single or combined TLR ligations in vitro: C57BL/6 mice at the age of 6 to 8 weeks were used in the expetiment .Afer removing all muscle tissues with gauze from the femurs and tibias, the bone marrow cells were flushed out using PBS with a syringe and red cells were lysed .After washing, the bone marrow cells were cultured with DC culure medium in 6 well plate and the majority of nonadherent cells were removed on day 2. On day 7, the DC cultures were coculturedwith LPS or Poly I:C (a synthetic mimic of dsRNA) or CpG-ODN (a synthetic mimic of bacterial DNA) alone or LPS in combination with Poly I:C or Poly I:C in combination with CpG-ODN for 24h after plused with OVA. Expressions of CD40, CD80, CD86, CD11c and Ia^b were then analyzed by flow cytometry. Secretion of IL-12p70, IL-6, IFN-β in culture supernatants and the capability of MLR were also analyzed.2. Establish the EG7 thymoma modle: 6-8 weeks female C57BL/6 mice were subcutaneously (s.c.) inoculated with EG7 cells at the outsite of the right leg (1× 10⁶cells per 50 μ 1 per mouse).3. Observe the antitumor effect of each kind of DC_ova on the tumor model using OVA as a mimical antigen: One week after tmor cells injection, 56 mice were randomly devided into 7 groups (each contained 8 mice) and subcutaneously injected with PBS (blank control), DC_ova, DC_ovaLPS, DC_ova PolyI:C, DC+(OVA)CpG-ODN, DC_ova (LPS+PolyI:C) ,DC_Ova (PolyI:C +CpG-ODN) at both sides of the groin skin (1 × 10⁶cells per 100 μ 1 per mouse and 50 μ 1 per side ), respectively. 7 days after the last injection, splenocytes were prepared from tumor-bearing mice for thr induction of cytokines and CTLs as well as the analysis of CD4⁺CD25⁺ regulatory T cells. The CTL and NK activity were further determined by a standard LDH release assay. The tumor size and survival period were observed.4. Statistical analyses: Statistical analyses of the date were performed by 2-way analysis of variance (ANOVA) for multivariate analyses using the SPSS software. Only P values less than 0.01 were considered statistically significant.RESULTS1. Activated DC by combined Toll-like receptor ligations resulted in higher degree of maturation in terms of surface molecules and fuctions. DC stimulating with combined Toll-like receptor ligations especially the combination of TLR3 and TLR9ligation resulted in a synergistic manner in terms of higher expression of costimulatory molecules like CD40, CD80, CD86 and cytokines secretion such as IL-12p70, IL-23, IFN-β , IL-6 as well as an increased MLR.2. More effective of protective antitumor immunity and prolonged survival period were elicited by treatment with DC_OVA activated by combined Toll-like receptor ligations in the mouse EG7 tumor modle. The tumor size of the group of DC_OVA (LPS +PolyI:C)or DC_OVA(PolyI:C +CpG-ODN)which was treated with DC_OVA activated by combined Toll-like receptor ligations was significantly smaller than either the single-TLR activated group or the PBS contol group(P<0.01), and the survival ratio was significantly improved.3. Augmented CTL and NK activity were induced by treatment with DC_OVA activated by combined Toll-like receptor ligations. NK cells or CTL which was derived from the mice which administrated by DC_OVA (LPS+PolyI:C) or DC_OVA (PolyI:C + CpG-ODN) has significantly increased killing activity compared with either the single-TLR activated group or the PBS contol group(P<0.01).4. TLR3 or 4 or 9 activated alone, or especially combined-activated DC_OVA could induce increased Th1 cytokines (such as IL-2 and IFN-γ) productions. The culture supernatants derived from the single-TLR activated groups secreted significantly increased Th1 cytokines compared with PBS contol group (P<0.01) and the production of these cytokines were further significantly increased by combined-activated DC_OVA treated groups (P<0.01).5. CD4⁺CD25⁺ regulatory T cells were reduced after treatment with TLR3 or 4 or 9 activated especially combined-activated DC_OVA in EG7 thymoma-bearing mice.CONCLUSIONS1. Activated DC by combined Toll-like receptor ligations resulted in highter degree of maturation in terms of surface molecules and fuctions as well as increased secretion of cytokines such as IL-12 and IL-23.2. More effective of protective antitumor immunity and prolonged survival period were elicited by treatment with DC tumor vaccine activated by combined Toll-like receptor ligations in the mouse EG7 tumor modle. The mechanisms maybe that combined-activated DC tumor vaccine induced augmented CTL and NK activity and increased secretion of Th1 cytokines as well as reduced CD4⁺CD25⁺regulatory T cells.3. DC tumor vaccine activated by combined Toll-like receptor ligations might provide a new insight into new DC-based vaccination strategies.