Bacterial Metabolites In Tumor Immunotherapy

Tumors threaten people's health. Current treatment options include surgical resection, chemotherapy and radiotherapy. However, all these options have their limits. Thus, it is important to establish novel approaches for the treatment of tumor. Tumor immunotherapy is one of these new therapeutic ways.In section one , we evaluated the tumoricidal effect of staphylococcal enterotoxin C (SEC) which is one important superantigen(SAg). As potent activators of T lymphocytes, SEs (staphylococcal enterotoxins) have a potential to be used in tumor therapy. In this study, we evaluated SEC as an anti-tumor agent in vitro and in vivo. SEC activated PBMCs were able to induce cytotoxicity towards various human carcinoma cells in co-culture experiments in vitro at very low concentrations. Treatment of C57BL/6 mice systematicly with SEC resulted in a statistically significant decrease in tumor outgrowth. Treatment of rabbits also resulted in decrease in tumor outgrowth. Moreover, the therapy groups showed tumor necrosis and strong infiltration of lymphocytes in tumor area and prevented metastasis of tumor cell in contrast to control group. These preclinical results suggested that SEC might be a promising candidate as an anti-cancer agent for further clinical evaluation.Although SAg showed good curative effect clinically, the amount of tumor specific CTL was less in T cell pool activated by SAg. So if the tumor specific antigen is combined with SAg, more tumor specific CTL should be expected. In Section two, in order to evaluate if SE can enhance the NY-ESO-1 specific cellular immune response, a stable NY-ESO-1-B16 cell was established firstly by transfection. Full-length gene of NY-ESO-1 was generated by gene splicing and a mammalian expression vector NY-ESO-1-pcDNA3.1(+) was constructed. The recombinant plasmids were transfected into B16 cells by liposome, and the expression of NY-ESO-1 was proved by RT-PCR and Western blot. A B16-NY-ES0-1 cell line that expressed the NY-ESO-1 stably was consequently established after screening by G418.In order to obtain enough amount of NY-ESO-1 protein for further study, NY-ESO-1 gene was subcloned into PET-28a(+), a prokaryotic expression vector. After sequencing, the recombinant vector NY-ESO-1-PET-28a was transfected into BL-21 (DE3) and expressed protein by IPTG induction. The protein was purified by Ni²⁺ affinity chromatography. To see if SEA/SEB had the potential to enhance the specific cellular immunal response to NY-ESO-1, mice were devided into NY-ESO-1+SEA/SEB group, NY-ESO-1 group, SEA/SEB group and PBS group, respectively. After immunity, lymphocytes from murine spleen were obtained and pulsed with NY-ESO-1-B16 cell lysate. The result showed that the NY-ESO-1-specific lysis of CTLs from mice vaccinated with SEA/SEB+NY-ESO-1 was 44.33%, which is almost the same as that from mice vaccinated with NY-ESO-1, which is 44.34%. The result was in accordance with that of ELISPOT. The ELISPOT assay showed that the number of T cell that secreting IFN-γof NY-ESO-1+SEA/SEB group and NY-ESO-1 group was 134.81 and 157.44, respectively.The results in vivo also showed that SEA/SEB couldn't enhance the tumoricidal efficacy of NY-ESO-1. Mice of different groups were challenged with NY-ESO-1-B16 tumor cells after immunity. NY-ESO-1 group showed highest antitumor immune responses and had significant difference to PBS group. The reason why SEA/SEB could not enhance the specific cellular immune responses against NY-ESO-1 as expected in vivo and in vitro remained to be investigated.Tumor specific CTLs are one of the most crucial components among antitumor effectors. There is now substantial evidence that HSP65 of M. tuberculosis could produce cancer specific immunity when fused with tumor antigen peptide. In Section three, we evaluated the effect of heat shock protein 65 of Mycobacterium tuberculosis to enhance NY-ESO-1 specific cellular immune responses. In this study, the recombinant expression plasmid HSP65-NY-ESO-1-PET28a was constructed and confirmed by sequencing. The recombinant plasmids were transformed into BL-21 Escherichia coli. The target protein was induced by 1mM IPTG and was in the soluble fraction of bacterial extract and was checked by SDS-PAGE and Western blot.In vitro, ELISPOT was done as mentioned in section two. Mice were devided into HSP65-NY-ESO-1 group, NY-ESO-1 group and PBS group, respectively. The result of ELISPOT indicated that the number of spot-forming T-cell precursor specific for NY-ESO-1 in the splenocytes from mice vaccinated with HSP-65-NY-ESO-1 was 93.56, about 2-fold more than that from mice with NY-ESO-1 alone, with significant difference with PBS group. The result of LDH was in accordance with that of ELISPOT. NY-ESO-1-specific lysis of CTLs from mice vaccinated with HSP65-NY-ESO-1 was 39.97%, about 10 times greater than that from mice vaccinated with NY-ESO-1, also with significant difference with PBS group. The tumor inhibition rate of HSP65-NY-ESO-1 group is 2 times higher than that of NY-ESO-1 group, and is of significant difference to PBS group. The result in vitro and in vivo showed that HSP65 could enhance the NY-EO-1 specific cellular immune response.