Study Of Anti-tumor Immunity Induced By DC Vaccines Loaded By P53-modified Tumor Antigens

Object:To observe the anti-melanoma specific immunity induced by dendritic cells (DC)vaccine stimulated by p53-tumor antigens and the possibility to improve theanti-tumor immune response of DC vaccine by gene-modified tumor was discussed.Methods:1. Melanoma cells (B16) were transfected with wild-type plasmid p53.Expression of p53was detected before and after transfection. p53-modified tumorantigens were extracted by freeze-thaw method. Then the antigens were loaded ondendritic cells (DC) harvested from bone marrow of syngeneic Km mice into tumorvaccines.2. The ability to induce proliferation of T cells (MLR) activated by DC vaccinesand antigen-specific killing effect of cytotoxic T lymphocytes (CTL) in vitro wasdetected by MTT assay. Both experiments were divided into three groups: DC group(DC), DC loaded by B16antigens group (B16-DC) and DC loaded by p53-modifiedtumor antigens group (p53-DC). DCs were stimulator cells and spleen lymphocyteswere effector cells in experiment MLR. T cells were effector cells and B16weretarget cells in experiment CTL. Then cells culture and observation were carried oncommonly.3. B16cells were injected subcutaneously on the back of Km mice to build solidtumor models. The tumor-bearing mice were divided into four groups: B16group,DC group, B16-DC group and p53-DC group. DC vaccines were injected intoperitumoral on the1st,7th and14th day after inoculation. The length of the longestand shortest were measured every3days. Then tumor volume and the averagevolume of each group was calculated. Tumor growth curve was depicted. The micewere killed on the21th day. The tumor weight was weighed and the levels ofinterleukin-12(IL-12) and γ-interferon (IFN-γ) in blood were detected. Results:1. Tumor cells express p53gene and the product after transfection with p53plasmid. DC vaccines stimulated by p53-tumor antigens was consist with themorphological features of mature DC.2. Effect of DC vaccines in vitro MLR: The stimulation index of three groupswere1.49,2.49and2.93on the rate of1:40. The level of B16-DC and p53-DC werehigher than DC with a significant difference (P <0.05). p53-DC was also higher thanB16-DC (P <0.05). The levels of three groups were lower on the rate of1:80thanthat on the rate of1:40, but the difference between every two groups were same of thetwo rates (P <0.05). CTL: The killing rate of three groups were the highest when thetarget ratio was1:20. They were30.00%,37.89%and52.47%. The level of B16-DCand p53-DC were higher than DC with a significant difference (P <0.01). p53-DCwas also higher than B16-DC (P <0.01). The killing rate of three groups were lowerwhen the target ratio was1:40(24.24%,35.92%and46.63%), but the differencebetween every two groups were statistically significant (P <0.01).3. Anti-tumor effect of DC vaccines in vivo The tumor growth curves afterinoculation showed: the tumor of group B16formed early and grew rapidly, but thetumor of group DC formed later and grew slower. The tumor of group B16-DC andp53-DC formed latest and grew slowest. Tumor grew to the size of a grain of riceonly9days later and most of the tumor showed regression trend. The average tumorweight was0.453g of group B16and0.025g of group DC on the21th day. The tumorof group B16-DC and p53-DC cannot be dissected with a significant difference (P <0.01). Pathological observation showed that lymphocytic infiltrated in every groupand the numbers of group B16-DC and p53-DC were the most.4. Observation of the levels of interleukin-12(IL-12) and γ-interferon (IFN-γ)in blood: IL-12: the levels of experimental groups were higher than group B16with asignificant difference (P <0.01). The levels of group B16-DC and p53-DC werehigher than group DC with a significant difference (P <0.01). The levels of p53-DCwere higher than B16-DC with a significant difference (P <0.01) also. IFN-γ: thelevels of experimental groups except DC were higher than group B16with asignificant difference (P <0.01). The levels of group B16-DC and p53-DC were higher than group DC with a significant difference (P <0.01). The levels of groupp53-DC were higher than B16-DC with a significant difference (P <0.05) also. IL-12and IFN-γ levels of group p53-DC were the highest.Conclusion:1. p53gene modification could enhance the ability of inducing tumor antigen-specific immune response. The ability of stimulating lymphocyte proliferation ofp53-modified DC vaccines was stronger unmodified DC vaccines.2. p53-modified DC vaccines could significantly stimulate anti-tumor immuneresponse and improve the specific killing effect of T lymphocytes on tumor cells invitro.3. p53-modified DC vaccines could significantly inhibit the growth oftransplanted tumor and improve the levels of IL-12and IFN-γ in peripheral blood oftumor-bearing animals in vivo.