Experimental Study On Anti-tumor Effect Of Immunotherapy Based On Cyclophosphamide Regulating Treg

The focus of tumor immunotherapy is currently how to break through tumor immune tolerance and enhence the effective T cell response. Various types of tumor vaccine and adoptive cellular immunotherapy have gradually entered into the period of clinical application. And the phased Achievement have been got that was represented by DCs vaccine and the adoptive reinfusion of effective T cell cultured in vitro. However the total effective rate of individual use of these therapies was less than 30% in clinical practice, especially for these established solid tumors which are rich in vascular. Thus the application of immunotherapy were blocked in the stage of Phase I or II clinical trial. The reason is because the complicated immunosuppression network in tumor microenvironment limits the practical effect of immunotherapy, in which these inhibitory factors are the key to bother immunotherapy. With the development of the fundamental research on tumor immune, more attention has been increasingly paid to the Key-point, CD4+CD25+FOXP3+regulatory T cells (Treg) in the recent years.In tumor bearing host, the percentage of Treg is increasing significantly. More and more evidences have shown that Treg plays a negative role in tumor environment or in antitumor immune. Increased numbers of Treg have been one of important reasons of tumor immune tolerance, which leads to tumor immune escape and limits the immunotherapy effect. Therefore regulating Treg has been the crucial part of tumor immunotherapy. Now in Japan or European and American Countries, regulation of Treg have gradually entered into the period of clinical trial. But the related research in our country still rests in few preclinical experiments.Cyclophosphamide (CTX) has been widely used in clinic for cancer therapy, as a Conventional chemotherapeutic alkylating drugs. Besides its derect cytotoxic effect, CTX also plays a role of immunomodulation. Recently foreign researches in murine models have proved that low-dose CTX could delete Treg cells selectively in both dose-dependent and time-dependent manner. And current research Results showed consistently that the number of Treg declined to the lowest at 3rd to 4th day after CTX administration, while recoverd to the normal level after about 7th day. The application of CTX in those researches was single administration, meawhile there was much defferent about the optimal dosage of CTX deleting Treg cells and combination with immunotherapy. On the other hand, the duration of single CTX administration deleting Treg remains shorter, which is not enough to the consecutive or cyclical use of DCs vaccination and T cell adoptive reinfusion in clinical practice. If the duration of CTX deleting Treg remain more longer, more better result would be achieved. So in this study, C57BL/6 mice inoculated B16 cells was used as tomor model to select the optimal dosage of CTX. The effect of cyclical CTX administration referred to repeated administration at rhythmic interval on Treg was observed. Subsequently the antitomor effect of cyclical CTX administration combined with DCs vaccine or adoptive CTLs therapy was analyzed. And autoimmune Diseases of mice were also investigated after those therapies.Part 1 Influence of cyclical administration of low-dose cyclophosphamide on regulatory T cell in melanoma-bearing miceObjective:To observe the influence of cyclical low-dose cyclophosphamide administration on regulatory T cell of melanoma-bearing mice and explore its antitumor effect from the view of immunoregulation.Methods:Models of tumor-bearing mice were established by subcutaneous inoculation with melanoma cells (B16). mice were injected intraperitoneally with CTX at the dose of 25mg/kg to 200mg/kg, then the CD4+CD25+Foxp3+Treg in spleens were detected by flow cytometry to select the optimal dosage of CTX. The mice were treated with CTX at the optimal dose every 7 days, which were repeated 3 cycles. The changes of the CD4+CD25+Foxp3+Treg were detected by flow cytometry. The dynamic changes of tumor size were observed and the growth curves were drawn. The dendritic cells derived from the bone marrow of mice were cultured with spleen T lymphocytes, and the interferon-γ(IFN-γ) levels in culture supernatant were detected by ELISA.Results:1. the proportion of CD4+CD25+Foxp3+/CD4+was increased gradually in spleens of mice after tumor inoculation, which were significantly higher than those in mormal group at the 14th day.2. When the CTX was used at the dose of 100mg/kg, the proportion of CD4+CD25+Foxp3+/CD4+in tumor-bearing mice was decreased significantly (P<0.05).3. Single administration of CTX only inhibited Treg in short time, while cyclical administration of CTX could prolong the period in which Treg was kept at low level.4. Cyclical administration of CTX enhanced the IFN-γproduction of T lymphocytes in tumor-bearing mice higher than that in control group (P<0.05). And the IFN-γproduction in TL-DC group was higher than that in Unpulsed DC group.5. Neither single nor cyclical administration of CTX could delay the growth of tumor. Autoimmune vitiligo and other adverse side effect were not observed in mice treated with CTX.Conclusion:1. With the process of tomor growth, the proportion of Treg gradually increased in spleens of tumor-bearing mice.2. The optimal dosage of CTX regulating Treg cells may be 100mg/kg for the C57BL/6J mice.3. Cyclical administration of low-dose CTX may be of more clinical value, which could prolong the period in which Treg was kept at low level.4. Cyclical administration of low-dose CTX could regulate the level of Treg more effectively, and enhance the antigen-specific activation of T cells by DCs, which may enhence antitumor effect of DCs vaccine or ACI.Part 2 Anti-tumor effect of cyclical administration of low-dose cyclophosphamide combined with DCs vaccineObjective:To observe the actual Anti-tumor effects of cyclical administration of low-dose cyclophosphamide with DCs vaccine and explore its possible mechanism.Methods:Models of tumor-bearing mice were established by subcutaneous inoculation with melanoma cells (B16). The tumor lysate-pulsed dendritic cells (TL-DCs) were prepared for DCs vaccine by cultivation in vitro. Mice were were randomly divided into 4 groups:①cyclical CTX+DCs group,②single CTX+DCs group,③DCs group,④cyclical CTX group. mice were injected intraperitoneally with CTX at the dose of 100mg/kg, then received vaccination by subcutaneous inoculation with TL-DCs 4 days later. The growth curves of tumor and the survival curve of mice were drawn. The interferon-y (IFN-y) levels in serum were detected by ELISA. And CD8+T cells in lymph nodes were detected by immunohistochemical method.Results:1. The tumor growth was delayed more significantly in cyclical CTX+DCs group, compared with single CTX+DCs group, DCs group and cyclical CTX group (P<0.05).2. The cumulative survival rate and mean survival time in cyclical CTX+DCs group were significantly higher than other groups(P<0.05).3. The IFN-y levels in serum from cyclical CTX+DCs group increased significantly, compared with other groups(P<0.05).4. The number of CD8+T Cells and its average optical density were higher than other groups(P<0.05).5. Autoimmune vitiligo and other adverse side effect were not observed in mice treated with CTX.Conclusion:1. Cyclical administration of low-dose CTX could enhence the anti-tumor effect of DCs vaccine by regulating Treg, which provided a theoretical basis for the clinical application of DCs vaccine based on CTX regulating Treg.2. The combined treatment of CTX and DCs vaccine did not lead to the autoimmune disease such as vitiligo, that is safe.Part 3 Anti-tumor effect of cyclical administration of low-dose cyclophosphamide combined with CTLs adoptive transfusion.Objective:To observe the actual Anti-tumor effects of cyclical administration of low-dose cyclophosphamide with CTLs adoptive transfusion and explore its possible mechanism.Methods:Models of tumor-bearing mice were established by subcutaneous inoculation with melanoma cells (B16). The tumor antigen-specific CTLs were prepared by cultivation in vitro. And the killing effect of CTLs was detected by CCK-8. Mice were were randomly divided into 4 groups:①cyclical CTX+CTLs group,②single CTX+CTLs group,③CTLs group,④cyclical CTX group. The mice in combined treatment group were injected intraperitoneally with CTX at the dose of 100mg/kg, then received CTLs by veinous transfusion 4 days later. The growth curves of tumor and the survival curve of mice were drawn. The interferon-y (IFN-y) levels in serum were detected by ELISA.Results:1. The tumor antigen-specific CTLs induced by TL-DCs could kill the B16 cells specifically and strongly. Its killing rate for B16 cells (76.4±11.4)% was significantly higher than that for CT26 cells (15.3±6.5)% and BGC-823 cells (9.8±4.3)%, also higher than the killing rate of control CTLs (35.2±7.8)% (P<0.05).2. The tumor growth was delayed more significantly in cyclical CTX+CTLs group, compared with single CTX+ CTLs group, CTLs group and cyclical CTX group (P<0.05).3. The cumulative survival rate and mean survival time in cyclical CTX+CTLs group were significantly higher than other groups(P<0.05).4. The IFN-y levels in serum from cyclical CTX+ CTLs group increased significantly, compared with other groups(P<0.05).5. Autoimmune vitiligo and other adverse side effect were not observed in mice treated with CTX.Conclusion:Cyclical administration of low-dose CTX could enhence the anti-tumor effect of CTLs adoptive immunotherapy by regulating Treg, which provided a theoretical basis for the clinical application of CTLs adoptive immunotherapy based on CTX regulating Treg.2. The combined treatment of CTX and CTLs adoptive immunotherapy did not lead to the autoimmune disease such as vitiligo, that is safe.Part 4 Anti-tumor effect of cyclical administration of low-dose cyclophosphamide combined with DCs vaccine and CTLs adoptive transfusionObjective:To observe the actual Anti-tumor effects of cyclical administration of low-dose cyclophosphamide combined with DCs vaccine and CTLs adoptive transfusion, and explore its possible mechanism. Methods:Models of tumor-bearing mice were established by subcutaneous inoculation with melanoma cells (B16). The tumor lysate-pulsed dendritic cells (TL-DCs) and tumor antigen specific CTLs were prepared by cultivation in vitro. Mice were were randomly divided into 4 groups:①cyclical CTX+DCs+CTLs group,②cyclical CTX+CTLs group,③cyclical CTX+DCs group,④DCs+CTLs group. The mice in combined treatment group were injected intraperitoneally with CTX at the dose of 100mg/kg, then received CTLs by veinous transfusion and TL-DCs by subcutaneous inoculation 4 days later. The growth curves of tumor and the survival curve of mice were drawn. The IFN-y levels in serum were detected by ELISA.Results:1. The tumor growth was delayed most significantly in cyclical CTX+DCs+CTLs group, compared with other 3 groups (P<0.05). There were no significant difference among cyclical CTX+CTLs group, cyclical CTX+DCs and DCs+CTLs group (P>0.05).2. The cumulative survival rate and mean survival time in cyclical CTX+DCs+CTLs group were significantly higher than other groups(P<0.05). There were no significant difference among cyclical CTX+CTLs group, cyclical CTX+DCs and DCs+CTLs group (P>0.05).3. The interferon-γ(IFN-y) levels in serum from CTX+DCs+CTLs group increased significantly, compared with other groups(P<0.05).4. Autoimmune vitiligo and other adverse side effect were not observed in mice treated with CTX.Conclusion:1. Cyclical administration of low-dose CTX could enhence the anti-tumor effect of DCs vaccine and CTLs adoptive transfusion by regulating Treg, which provided a theoretical basis for the clinical application of immunotherapy based on CTX regulating Treg.2. The optimal anti-tumor effect may be achieved only when synthetic methods were adopted according to many links of tumor immune tolerance.