| Background Biotherapy of malignant diseases has become the fourth treatment modality besides surgery, chemo-and radiotherapy and the adoptive immune competent cells (AICs) infusion is one of the important mean for biotherapy. AICs are often derived from the cancer patient autologous immune cells which can do little in combating tumor cells due to the defected immune function of the cancer patient. A healthy donor with identical MHC would be ideal but it is very difficult to find such a donor. The allologous AICs has been demonstrated to be able to induce not tumor-specific CTLs but strong graft anti-host reaction (GAHR). On the other hand, it has been proposed that the semi-allogeneic AICs may use their allogeneic part to function as a powerful adjuvant to break the tumor immune tolerance or ignorance to some degree and use their autologous part to induce the MHC-restricted tumor-specific CTLs. So far, only semi-allogeneic CTL adoptive immunotherapy for the treatment of hematopoietic malignancies has been reported. Therefore, the semi-allogeneic CTL adoptive immunotherapy with less toxicity and more specific tumor targeting and adjuvant effects are to be studied.Objective This study was aimed at investigating the anti-tumor efficacy of the transfused tumor specific T cells with the surface semi-allogeneic antigens and to explore a new way on adoptive immunotherapy of cancer.Methods In order to produce the tumor-antigen specific semi-allogeneic lymphocytes, the lst-generation hybrid mice named F1 cross (H-2b/d) were bred by cross-mating C57BL/6 and Balb/c mice. The Fl tumor lysate-pulsed dendritic cells (TP-DC) and antigen specific CTLs (TP-DC-TCs) were prepared in vitro, and then the TP-DC-TCs were inactivated by MMC and transferred into the TC-1 bearing mice (C57BL/6). For evaluating therapeutic TP-DC-TCs infusion,1X1O5TC-1 tumor cells were injected s.c. into the right flank, and after the mice developed palpable tumor (approximately 5 mm diameter),3X107 antigen specific TP-DC-TCs were transferred into the recipients via the tail vein.In order to rapidly produce the tumor-antigen specific semi-allogeneic splenocytes (SPs), the Fl cross (H-2b/d) mice were then immunized with the mitomycin C (MMC) inactivated TC-1 tumor cells. These SPs were used as donor cells to transfuse into TC-1 bearing C57BL/6 mice (an artificial pulmonary metastases ainimal model was established by the i.v. injection of lxlO5 TC-1 cells) via intravenous injections three times at 7-d intervals in 3X107. On day 5 after the third treatment, the heparinized whole obital blood was collected and the plasma IL-2, IL-10, IFN-y, TGF-βwere examed by ELISA. To measure cell preparations of CD3+CD4+, CD3+CD8+, Treg, MDSC and NK mice were sacrificed on the 5th day after the third SPs administion, and their spleen cells were prepared and analysed by flow cytometry. The pathological characteristics of heart, spleen, liver and kidney were by HE staining. The phenotype of H-2 from recipients leucocyte was analysed by flow cytometry also.Results our results illuminated that the treatment of C57BL/6 tumor-bearing recipients receiving TP-DC-TCs from Fl mice resulted in a slower tumor growth and survived longer term. It was found in our study that both specific and nonspecific immune responses were triggered after SPs intra-vein transfusion (3x107/100ul, tail vein) with no obvious side effects. Intravenous transfusion of MMC inactivated semi-allogeneic SPs resulted in the increased IL-2 and IFN-y release, the decreased TGF-P excretion, the proliferation of specific CD4+and CD8+and the generation of nonspecific immune response (NK cells). In addition, the semi-allogeneic SP transfusion has shown to be able to delay TC-1 tumor growth and prolong survival in the established TC-1 tumor model. Our data indicate that the transfusion of tumor specific allogeneic SPs may induce anti-tumor therapeutic effects resulted from both the specific and nonspecific immune responses. There were less pathological characteristics of heart, spleen, liver and kidney were found by HE staining.Conclusion Adoptive transfer of semi-allogeneic tumor specific CTL provides a promising approach to cancer immunotherapy. Background Oncolytic virus (OV) therapy is based on the concept of using live viruses to selectively infect and replicate in cancer cells, with minimal destruction of non-neoplastic tissue. And the evidences have recorded their specificity, wild-oncolytic effecty, good safety, fast replication and facilitated process. oHSVl has enterⅠ-Ⅲclinical pilot. However, there a few studies demonstrated that oHSV2 is more oncolytic effective than oHSVl in the treatment of bladder cancer and peritoneal metastasis of ovarian cancer in mice models. Further more, HSV2 is an enveloped double-stranded DNA virus containing a large, well characterized, fully sequenced genome of about 152kb of DNA. While the large size makes genetic manipulations cumbersome, it also provides ample opportunities to remove genes that are not essential for replication and insert therapeutic transgenes within the viral backbone, just as a vector carrying DNA encoding GM-CSF, TNF-a, IL-12, TRPland prodrug-converting enzyme. Therefore, our lab has constructed a newly oHSV2hGM-CSF aiming at enhancing the safety and the activity of oncolytic. oHSV2hGMCSF may promote the anti-tumor immunity as adjuvant.Objective This study was to construct a new oHSV2hGM-CSF and evaluate the oncolytic activity in vitro and in vivo in parallel with oHSVlhGM-CSF.Methods oHSV2hGM-CSF is a replication-competent, attenuated HSV type 2 based on the HG52 virus (an HSV2 strain). It has been engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and inserted the gene encoding hGM-CSF. To measure the in vitro killing effect of the viruses,15 human tumor cell lines (HeLa, Eca-109 and PG etc.) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at 1 pfu/cell (Multiplicity of infection, MOI=1), or left uninfected. Cells were harvested 24 and 48 hours post infection, and observed at microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3x106 pfu for three times when tumor volume reached 7-8mm3. And the tumor volume was measured at 3 day interval and animal survival was monitored after treatment.Results Both oHSV2hGM-CSFand oHSVlhGM-CSF induce widespread cytopathic effects after 24h infection. oHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSVlhGM-CSF. In the in vitro killing experiments for the cell lines of HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSVlhGM-CSF under the same conditions. For the animal experiments, it is concluded that the oHSV2hGM-CSF significantly inhibited tumor growth. The long term therapitic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days post tumor cells inoculation whereas mice in the PBS group all were dead by day 22 (p<0.01). The anti-tumor mechanism of newly constructed oHSV2hGM-CSF against B16R cell carcinoma appeared to include the directly oncolytic activity and the induction of antitumor immunity in some degree.Conclusion These data demonstrate that the newly constructed oHSV2hGM"CSF have potent anti-tumor activity in vitro for many tumor cell lines and in vivo for the established B16R tumor models. |
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