| Objective: The tumor-carrying C57BL/6 mice model was setted up by subcutaneously injecting B16 melanoma cells. Dendritic cells derived from C57BL/6 and CB6F1(BABL/C×C57BL/6 hybrid filial generation, female) rats were loaded with whole tumor antigen, then co-cultured with haploidentical T lymphocytes(CB6F1). 3 to 5 days later, the activated IFN-γ secreting T lymphocytes were enriched by magnetic beads cell sorting, then were subjected to the following cytotoxic assay in vitro or in vivo respectively. In vitro cytotoxic assay was detected with CCK-8 reagent.In vivo cytotoxic assay was tested with tumor-carrying C57BL/6 mice models by monitoring volumes of B16 melanomas, signs of graft-versus-host disease(GVHD) during the next two or three weeks, after treated with different reagents such as the semi-allogeneic effector T lymphocytes, cyclophosphamide and PBS as control, and the levels of IL-2 and IFN-γ in peripheral blood of testing mice models was also checked out with ELISA assay simultaneously. The survival period in different mice groups was observed within 80 days. This study explored the anti-tumor efficacy of the semi-allogeneic activated IFN-γ secreting T lymphocytes, and will provide more fundmental datas for constructing an innovative remedy also known as tumor microenvironment interfering induced adoptive cellular immunotherapy.Methods:1 The melanoma B16 cells were cultured in vitro for the use of setting up B16-carrying mice model.2 Preparation of tumor lysate-pulsed dendritic cells: Mouse bone marrow cells were collected from femora of C57BL/6 and CB6F1, then co-cultured with rm GM-CSF, rm IL-4, whereafter, tumor lysate as antigen and TNF-α were added to induce antigen-pulsed mature DC.3 Preparation of magnetic beads selected effector T cells: Isolating lymphocytes from CB6F1 spleen cells by density gradient centrifugation, and the purified T lymphocytes were prepared with nylon wool column adsorption method. 2 day’s later, the DCs sensitized by B16 antigen was added to the culturing T lymphocytes co-culturing for 3-4 days to obtain activated effector T cells. Then the IFN-γ positive haploidentical effector T cells were enriched by magnetic bead sorting.4 In vitro cytotoxicity assay to detect the killing rates of effector T cells stimulated by different source DCs with commissioned antigen: The C57BL/6 source DC(group A), CB6F1 source DC(group B) and the two mixed DC(group C) induced CTLs(as effector cells, E) and mouse melanoma B16 cells(used as target cells, T) were mixed at E;T ratios as 50:1, 20:1 and 10:1, and co-cultured for 24 hours, and added CCK-8 reagent(20 ul/ hole)for another 4h. Finally, the A values of each hole were determinated in the wavelength of 450 nm at the 1, 2 and 4h time points, then calculated to get the killing rates.5 In vivo cytotoxicity assay in different groups: The B16-carrying mice were divided into 3 groups with 40 mice in each group. PBS group(group A) : 0.2ml PBS was injected by vein; cyclophosphamide chemotherapy group(group B): CYC was previously intraperitoneally injected at 100mg/kg; MACS purified haploidentical CTL treatment group(group C): enriched IFN-γ secreting effector T cells from CB6F1 mouse were injected by vein at 1×106 per 0.2ml. Calculating the volume of tumor and the survival period in different groups.6 Mouse serum samples were collected on the 1, 4, 7, 10, 14 d after treatment. The levels of IL-2 and IFN-γ in serum samples were detected by ELISA kit, comparison of the difference between each group on its impact.7 Observing tumor tissue and pathological changes of liver, small intestine and spleen in mouse of different groups by HE staining.Result1 It’s about 7 days for successfully setting up B16-carrying mice model.2 The mature dendritic cells appear suspending, having dendritic prominencies on the 6th day.3 Cytotoxicity assay by CCK-8 method in vitro: At the ratios(E:T) of 50:1, 20:1, the killing rate of group A(CB6F1 DC) is better than that of group B(C57BL/6 DC) and C(mixed DC), their differences are statistically significant(P<0.05), but no statistic difference between group B and C(P>0.05); When the killing ratio comes to 10:1, also no significant statistic difference was found out among three groups(P>0.05).4 The tumor volumes in different B16-carrying mice groups: Tumor grew rapidly when the mice were injected only with PBS or cyclophosphamide chemotherapy. MACS haploidentical DC-CTLs can significantly inhibit tumor growth from day 7 to day 20.5 The survival periods of A, B and C groups: There is statistically significant difference between each group(P<0.05).6 The results of IL-2 and IFN-γ level in mice serum tested by ELISA: Levels of serum IL-2 and IFN-γ protein rised significantly after the treatment with MACS haploidentical DC-CTLs, and the difference between cell therapy group and control group is statistical significant(P<0.05).7 HE stain results: There are more necrosis areas observed in tumor tissues of cellular immunotherapy group and cyclophosphamide chemotherapy group than that of control group. Liver, spleen and small intestine have no obvious pathological changes in the cellular immunotherapy group.Conclusions:1 Group A(CB6F1 DC), group B(C57BL/6 DC) and group C(mixed DC) effector T cells all have anti-tumor activity in vitro. The killing rates of group A is better than others, no statistical difference was fonud out between group B and C have.2 The tumor grew slowly when mice were injected MACS haploidentical DC-CTLs, which significantly inhibited tumor growth from day 7 to day 20. Results: However, there was no significant difference between cyclophosphamide chemotherapy group and control group.3 Treatment with CB6F1 derived MACS haploidentical DC-CTLs or cyclophosphamide chemotherapy can prolong the survival period of the tumor-carrying C57BL/6 mouse. The survival period of the tumor-carrying C57BL/6 mouse received MACS haploidentical DC-CTLs treatment was longer than that of cyclophosphamide chemotherapy.4 The adoptive immunotherapy of MACS haploidentical DC-CTLs can significantly improve the levels of IL-2, IFN-γ in peripheral blood of the B16 carrying mice, the levels of IL-2 was higher than that of cyclophosphamide chemotherapy, but no statistically significant differences was found on the level of IFN-γ compaired with chemotherapy group.5 There was no obvious graft anti-host reaction in tumor-carrying mouse, which had been injected with MACS haploidentical gene DC-CTLs. |
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