Depletion Of Regulatory T Cells Enhances The Efficacy Of SA-mGM-CSF Prostate Cancer Cells Vaccine

BackgroundProstate cancer(PC)is one of the most common malignant tumors in men in Europe and the United States.Radical surgery and local radiotherapy are effective for treatment of early-stage PC while endocrine therapy is used for late-stage treatment,but as time goes by,many patients develop into castration-resistant prostate cancer.Cancer immunotherapies,such as vaccines and cytokines,have been proven to be effective for cancer treatment.We have developed a novel therapeutic tumor cells vaccine,in which streptavidin-tagged mGM-CSF was immobilized on the cell surface of biotinylated prostate cancer cells.We found that the vaccine could enhance anti-tumor immune responses,but the number of Treg was increased in the vaccine-treated mice at the same time.Treg cells are a subset of T cells that have immunosuppressive functions and are involved in tumor immune escape.Therefore,inhibiting or eliminating Treg cells will effectively increase the anti-tumor immune response.Recent studies have found that ICOS+Treg is the major Treg subset that exerts immunosuppressive effects.Our previous study found that activation of ICOS signal in vitro can significantly promote the proliferation and immunosuppressive function of Treg cells.Based on the above findings,we put forward the hypothesis:depletion of regulatory T cells by anti-ICOS antibody enhances anti-tumor immunity of tumor cells vaccine.Norcantharidin(NCTD)has strong anti-tumor activity and could significantly inhibit proliferation and induce apoptosis of tumor cells.NCTD has a whitening effect which was distinguishes it from common chemotherapeutic drugs,that can reduce myelosuppression after chemotherapy.NCTD was reported to have an effect of immune regulation,but its effect on Tregs is not yet clear.Objectives1.Explorting whether anti-ICOS antibody has effect on Tergs and enhances anti-tumor immunity of tumor cells vaccine.2.Explorting whether NCTD has effect on the proliferation and apoptosis of Tregs,and the efficacy of NCTD combined with vaccine for mouse prostate cancer.MethodsICOS blocking antibody combined with SA-mGM-CSF tumor cells vaccine for prostate cancer:RM-1 cells were injected under the skin of the right hind leg of C57BL/6 male mice to establish subcutaneous tumor model and measured the volume of tumors.All mice were randomly assigned to four groups(PBS,Vaccine,Vaccine + IgG mAb and Vaccine + ICOS mAb group),the mice were injected subcutaneously with the RM-1 tumor cell vaccine on day 12 day after inoculation of tumor cells,the cure was repeated every four days with a total of four times.The mice were intratumor injected with 100 micrograms of anti-ICOS antibody on day 0(12 days after inoculation of tumor cells),the treatment was administered every three days with a total of four times.The frequency of dendritic cells,CD4+,CD8+ T cells and ICOS+FOXP3+Regulatory T cells were measured by flow cytometry about a week after the treatment.Immunohistochemical and immunofluorescence detected CD4+,CD8+ T cells and ICOS+Foxp3+ T cells in the tissue,respectively.The concentrations of IL-4,IL-10 and IFN-y were measured by ELISA.Effect of NCTD on proliferation and apoptosis of Treg cells:Tregs were isolated by magnetic bead separation.Cells were cultured in the presence of 1?g/mL soluble anti-mouse CD3 and anti-mouse CD28(1:1)antibody and then incubated with different concentrations of NCTD(0?80?M)for 24h or 20?M of NCTD for 0,24,48 and 72h.Cells were collected at every time point.The effect of NCTD on cells proliferation was determined by the CCK8 and the cells apoptosis was measured by flow cytometry.Total proteins and RNA were extracted after treatment with 20?M NCTD,and western blotting and Real Time PCR were used to assess the protein production and mRNA expression,respectively.Efficacy of NCTD combined with vaccine for prostate cancer:subcutaneous tumor model was established with the above method.The mice were randomly divided into four groups(PBS,NCTD,Vaccine and Vaccine + NCTD group),vaccine treatment with the same methods on day 10 after inoculation of tumor cells.The mice were intraperitoneally injected with NCTD beginning at day 10 after inoculation of tumor cells at a dose of 10 mg/kg/d for 14 days.The frequency of dendritic cells,CD4+,CD8+ T cells and Tregs were measured as mentioned above.Results1.ICOS antibody significantly enhanced the antitumor immunity of SA-mGM-CSF tumor cell vaccine:compared with PBS group,vaccine treatment could effectively inhibit tumor growth,and CD4+ and CD8+ T lymphocytes cells were increased after treatment,however,there are more ICOS+Treg cells infiltrated into the tumor tissues after vaccine therapy.Combination therapy could rapidly reduce the tumor volume,obviously decreased the proportion of ICOS+Treg cells,induced more T lymphocytes infiltration and greatly decreased the concentration of IL-10 and IL-4 compared with vaccine alone.2.Effect of NCTD on Tregs in vitro:NCTD could inhibit the proliferation and induce the apoptosis of Tregs in a time-and dose-dependent manner.Mechanism study found that NCTD down-regulated p-Akt and then increased the levels of FOXO1 and FasL protein.The mRNA levels of Fas,FasL,FOXO3a,FOXO4 and FOXO1 were significantly up-regulated in NCTD-treated Tregs.3.NCTD significantly enhanced the antitumor immunity of SA-mGM-CSF tumor cell vaccine:compared with PBS group,NCTD or vaccine treatment alone could modestly inhibit the tumor growth and increase the proportions of CD4+ and CD8+ T cells in blood and tissues.Tregs increased significantly after vaccine treatment,and NCTD could decrease the number of Tregs.The volume of tumor tissue was reduced faster in the combination group compared with PBS group.The number of T lymphocyte was increased significantly and the number of Treg was decreased significantly in the combination group than that in the vaccine group.Conclusions1.ICOS monoclonal antibody enhances the efficacy of SA-mGM-CSF surface-modified prostate cancer vaccine by depleting of Tregs.2.NCTD could inhibit proliferation and enhance apoptosis of Tregs in a dose-and time-dependent manner in vitro and enhance antitumor immunity of vaccine by reducing the number of vaccine-induced Tregs in vivo.