Therefore, the ability of vaccines to elicit effective antitumor immunity was impaired. CY has immunomodulatory effects, and low-dose CY (20 mg/kg) was found to selectively deplete CD4+CD25+ T cells (Treg) and impede the tolerance [42]. CY can preconditioning enhance the CD8+ Kinase Inhibitor Library in vitro T-cell response to peptide vaccination, thus

leading to enhanced antitumor effects against pre-existing tumors [43]. Cy markedly enhanced the magnitude of secondary but not primary CTL response induced by vaccines and synergized with vaccine in therapy but not in prophylaxis tumor models [44]. With our enhanced vaccine, IFN-γ secretion was significantly increased. In addition, CD8+ and NK cells were triggered

to release IFN-γ and mediate cytotoxic activity. The increased IFN-γ secretion may also be due to the combined effects of HSP60 in mHSP/P and IL-12. Hsp60-inducing IFN-γ depends strictly on the ability of the macrophages to produce IL-12 [45]. Activation and expansion of tumor-specific T cells by HSP/Ps were identified [46]. Our study showed that mHSP/Ps purified PD-1 antibody from S180 sarcoma cells activated tumor antigen-specific T cells in vitro, and the induction of tumor-specific CTLs with enhanced vaccine was stronger than that with mHSP/Ps alone, possibly because of the combined effect of HSP60 and IL-12. HSP60 induces a strong non-specific immune reaction, but when it meets IL-12, it can activate cytotoxic T cells. HSP60 can mediate the activation of cytotoxic T cells, which depends on production of IL-12

[47]. Our data showed that inflammatory cells infiltrated tumors with mHSP/P vaccination and that a preexisting antitumor immune response was elicited, which was required for an effective IL-12 response for tumor rejection. Conclusions To enhance the current immunotherapeutic efficacy, novel strategies designed in the laboratory and proven in preclinical 3-mercaptopyruvate sulfurtransferase animal tumor models are now entering the clinic trials [48, 49]. These novel strategies involved breaking tolerance to tumor self-antigens by inhibiting regulatory T cells, boosting T-cell co-stimulation and using combinations of recombinant cytokines and other defined molecules with “”immuno-enhancing”" activities. Our immunization protocol of a combination immunotherapeutic regimen of vaccination with mHSP/Ps followed by low-dose CY plus IL-12 resulted in enhanced immunologic antitumor activity that was better than that of either treatment alone. Acknowledgements and Funding This study was supported by the National High Technique Research and Development Program of China funded by the Chinese government (863 No. 2007AA021806). We are thankful of Dr.