This study aimed at investigating the inhibitory effects and the anti-tumor mechanisms of co-adminis-tration of fusion proteins mGM-CSF/βhCG ( GC ) and hVEGF121/βhCG ( VC ) on RM-1 prostatic cancer and B16 F10 melanoma in the C57 BL/6 J mouse model. Two recombinant stains containing pET-28 a-mGM-CSF-X10-βhCGCTP37 and pET-28 a-VEGF-M2-X10-βhCG-CTP37 were induced by lactose to express fusion proteins. The fusion proteins were separated and purified to prepare the anti-tumor protein vaccines ( VC protein vaccine and GC protein vaccine) , which were then mixed to prepare a combined protein vaccine named VGC protein vac-cine. The prostatic cancer and melanoma tumor-bearing mice C57 BL/6 J were immunized with described vac-cines, then the growth of each tumor was measured;splenocyte proliferation of immunized mice was detected;and the cytotoxic effects of the vaccine on tumor cells were tested. After that, the in vivo concentrations of IFN-γ and anti-hVEGF antibodies were investigated by ELISA. The difference between each experimental group and normal saline group ( NS) was statistically significant in both tumor-bearing mouse models ( P <0. 05) respectively. Besides, VGC group exhibited significantly better anti-tumor effect compared with the GC and VC groups with the anti-tumor rate ( 41. 7 ± 0. 83)% and ( 46. 4 ± 1. 27)% for prostatic cancer and melanoma tumor, respectively. The co-administration of the two proteins, VC and GC, could inhibit the growth of RM-1 prostatic tumor and B16F10 melanoma effectively via anti-tumor immunity and anti-tumor angiogenesis.

Autophagosomes derived from tumor cells have been proved to induce potent T cell response both in mouse and human. In human in vitro study, dendritic cells(DC)loaded with cytomegalovirus(CMV)pp65 antigen-containing DRibble were capable to efficiently re-stimulate pp65-specific T-cell recall responses from freshly isolated or frozen humanperipheral blood mononuclear cell(PBMC). This study developed more robust assays using in vitro expanded antigen-specific T cells that contained a much higher percentage of antigen-specific T cells. DC cross-presentation efficiency of OX40 and CD80 modified pp65-DRibble was detected by intracellular IFN-γ staining. Compared with Ctrl/pp65 DRibble, the percentage of IFN-γ+ in total CD8+ T cells andCD4+ T cells was improvedwith OX40/pp65 DRibbleand CD80/pp65 DRibble stimulation. In addition, vaccine induced IL-12indendritic cells, whichpolarizes Th cells toward the IFN-γ high Th1 phenotype, evaluated by ELISA inco-culture supernatantwas dramatically higher in OX40/pp65 DRibble and CD80/pp65 DRibblegroups than in Ctrl/pp65 DRibble group. These results have implications for the immuneactivity of OX40 and CD80 modified DRibble and choice for prospective clinical use ofDRibble-based cancer immunotherapy.

To investigate the effects of Mycobacterium tuberculosis heat shock protein 65(HSP65)on Treg/Th17 immune balance in ApoE-knockout(ApoE-/-)mice, ApoE-/- mice with a high-cholesterol diet were immunized with M. tuberculosis HSP65. Sera were obtained for measurement of anti-HSP65 antibodies by ELISA; the effect of administration of different antigens was investigated, respectively, using flow cytometry analysis on the number of CD4+CD25+Foxp3+Tregs and CD4+IL-17+ Th17; the production of cytokines(IL-10, TGF-β1, IL-17 and IL-21)by these cells were determined by ELISA; total plasma cholesterol(TC), triglyceride(TG), high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)levels were detected by biochemical autoanalyzer. Atherosclerotic lesions were measured by lipid deposition stained with oil red O. The results demonstrated that the levels of anti-HSP65 IgG antibodies were increased significantly in Mycobacterium tuberculosis HSP65-treated ApoE-/- mice, revealed obvious decrease in Treg number, Treg related cytokines(IL-10, TGF-β1)levels and significant increase in Th17 number, Th17 related cytokines(IL-17 and IL-21)levels, the levels of TC, TG, HDL-C and LDL-C did not change between groups, while the atherosclerotic lesions significantly increased. Results indicate that M. tuberculosis HSP65 could interrupt the Th17/Treg immune balance in ApoE-/- mice, suggesting a potential role in the formation and progression of atherosclerosis.