树突状细胞摄取和提呈颗粒化HPV-E7抗原能诱导高水平体液和细胞免疫应答

目的 研究特定大小聚苯乙烯微粒作为抗原载体介导树突状细胞(DC)摄取和提呈抗原,诱导体液和细胞免疫应答的能力,并与DC-微粒-卵清蛋白(OVA)及DC-E7抗原表位的诱导能力进行比较。方法 用粒一巨噬细胞集落刺激因子(GM-CSF)联合白细胞介素(IL)-3从小鼠骨髓黏附细胞培养制备DC,流式细胞术分析表型,细胞经荧光素异硫氰酸酯(FITC)标记的微粒-OVA或微粒-E7饲育,或用OVA抗原表位(SIIFEKL多肽)或E7抗原表位(RAHYNIVTF多肽)负载,流式细胞术分析DC对微粒-OVA和微粒-E7的摄取能力,B3Z细胞活化试验检测DC提呈微粒-OVA和OVA抗原表位的能力。分别用DC-微粒-OVA、DC-微粒-E7、DC-OVA抗原表位、DC-E7抗原表位经双侧足掌免疫小鼠,36h后取髂部引流淋巴结,流式细胞仪分析微粒阳性细胞的表型,10d后用酶联免疫吸附实验(ELISA)法测定小鼠血清的抗体水平,酶联免疫斑点法(ELISPOT法)检测小鼠脾细胞干扰素γ(IFNγ)形成单位。结果 小鼠骨髓黏附细胞在GM-CSF和IL-3条件下培养5d后大部分细胞呈树突状形态和表型,经微粒-OVA或微粒-E7饲育后,能高效地摄取和提呈抗原,用DC-微粒-OVA或DC-微粒-E7免疫小鼠后引流淋巴结微粒阳性细胞,主要组织相容(抗原)复合物(MHC)分子和共刺激分子表达率分别为87．06％(MHC-I),63．57％(MHC-Ⅱ),73．40％(CD80),58．43％(CD86),37．62％(CD40)。第10天血清IgG和IgM水平显著升高,脾脏IFNγ形成单位与未免疫小鼠相比显著增多,DC-微粒-E7所诱导的细胞免疫应答与DC-微粒-OVA差异无显著意义。用负载E7抗原表位或OVA抗原表位的DC免疫小鼠后也能诱导细胞免疫应答,但应答强度与DC-微粒-OVA和DC-微粒-E7相比显著减弱。结论 DC-微粒-E7免疫小鼠后,细胞能迁移到引流淋巴结并诱导高水平的体液和细胞免疫应答,提示DC-微粒-抗原可能是一较理想的肿瘤疫苗形式。

Dendritic cells present particulate E7 protein of human papillomavirus and induce strong immunity

OBJECTIVE: To investigate the feasibility of using dendritic cell (DC)-beads-antigen (Ag) as novel cancer vaccine form with E7 as the target antigen. METHODS: C57BL/6 mouse was killed and the femora were taken out. Marrow cells were isolated and cultured with IL-3 and granulocyte-macrophage growth stimulating factor (GM-CSF) so as to prepare DCs. Flow cytometry was conducted to analyze the phenotype. FITC-labeled polystyrene beads-ovalbumin (OVA) or polystyrene beads-human papillomavirus (HPV) E7 protein were added into the culture fluid to be co-cultured with the DCs for 3 hours. Flow cytometry was conducted to analyze the up-taking rates of polystyrene beads-OVA and of polystyrene beads-HPV E7 protein by the DCs and B3Z T cell hybridoma cells were co-cultured and then beads-OVA or beads-SIIFEKL polypeptide was added into the culture fluid. The absorbance was read. Twelve mice were injected with DC-beads-OVA, DC-beads-E7, DC-OVA antigen epitope (SIIFEKL), or DC-E7 epitope (RAHYNIVTF) into the plantae and killed in 36 hours to take out the iliac lymph nodes. Flow cytometry was conducted to analyze the phenotypes of the bead-positive cells. Mice were killed 10 days after immunization and their heart blood was collected. Enzyme linked immunosorbent assay was used to detect the levels of antibodies. Immunized and non-immunized mice were killed and their spleens were taken out. ELISPOT was used to detect the number of cells secreting interferon (IFN)gamma. RESULTS: DCs were seen in the culture fluid of mice marrow cells 5 days after culture with IL-3 and GM-CSF. The bead-positive rates of bead-E7 and bead-OVA were 64% +/- 18% and 58% +/- 16% respectively (P > 0.05) in the IL-3DCs. The CD40 expression rate in the bead-positive cells in the graining iliac lymph nodes was significantly higher after feeding by beads-E7 in comparison with that before the feeding (P < 0.05), the NLDC145, and MHC-II expression rates were increased to a certain degree, however the F4/80 expression rate was decreased. The DCs fed with bead-OVA or with OVA antigen epitope SIIFEKL, especially the former, significantly activated the B3Z cells. The serum IgG level in the mice immunized by beads-E7 was significantly increases, the IgM level was increased slightly, however, the IgA level almost remained unchanged. The numbers of IFNgamma SFU in the splenic cells of the mice immunized by DC-bead-OVA and DC-bead-E7 were significantly higher than that in the unimmunized mice, especially those immunized by DC-bead-OVA. CONCLUSION: DCs fed with beads-E7 can migrate to the draining lymph nodes and induce high level humeral and cellular immunity. DC-beads-Ag seems better than DC-epitope according to the strength of induced immunity. DC-beads-Ag may be an ideal vaccine form and can be used to develop other cancer vaccine.