Relationship Between CTL Cells And Regulatory T Cells In Anti-tumor Immunity

Objective: Adoptive immunotherapy is one of the hottest subjects in biotherapy of tumor. The treatment is not only the supplement to conventional therapy such as surgical operation,chemotherapy and radiotherapy ,but also giving the found base for the clinical treatment to the tumor now. With effector cells being input back into the patient, they can kill the target cell at once with little side effects and toxicant effects during the treatment. Adoptive immunotherapy has good therapeutic effect on the tumor, because it can minimize the tumor and kill the cells and block their transfer and recidivism.Cytotoxic T lymphocyte (CTL)cells, as one of the new type immunocompetence cells, have high efficiency in anti-tumor immunity. CTL cells have widespread usage in tumor adoptive immune therapy, because they show highly effcient cytolytic effect, faster proliferation speed rate than other immunocompetence cells in anti-tumor immunity. The key of studies is to find how to enhance their cytolytic and anti-tumor specificity activity so that it will lead us to a bright future in tumor treatment.Dendritic cells(DC) are the most potent antigen presenting cells(APC) with the ability to acquire, process, present antigens and the unique capability of initiating primary immune responses against tumor-associated antigens. DCs can turn the T cells into the active cells with high ability to anti-tumor. The killer cells are well known as the CTLs. We can separate DCs, T cells from the spleen and culture respectively, then DCs are loaded with specific tumor antigen-B16 melanoma. We can co-culture tumor Antigen-sensitized DCs with T cells and then the DCs can turn the T cells into the CTLs. The CTLs induced by the tumor antigen-sensitized DCs have higher activity, cytolytic effect and more anti-tumor specificity. We can obtain the effector cells DC-CTLs with the tumor specific antigen to B16 melanoma. But in practice, the effector cells and tumor cells can be seen alive together peacefully in the tumor microenvironment. The anti-tumor immunocompetence cells are reduced into immune anergy and immunological tolerance constantly. So the effect of the CTLs to kill the tumor cells descends obviously. It is very important for us to delete the immunodepression cells and increase the anti-tumor effect. That is the new way to tumor immunotherapy.CD4~+CD25~+ regulatory T cells are a new member of the CD4~+ T cells. They are the more important subset of regulatory T cells(Treg) that play an essential role in maintaining immunological self-tolerance. They have the function of down-regulating the tumor immunity mediated by T cells. CD4~+CD25~+ Tregs also are divided into two groups by produced channel. The two groups are natural regulatory T cells (nTreg) and induced regulatory T cells( iTreg). CD4~+CD25~+ Tregs increase in the peripheral blood and tumor infiltration lymphoglandula in many tumor patients, such as breast cancer, lung cancer, gastric cancer and so on. CD4~+CD25~+ Tregs suppress immune responses mainly by cell contract-dependent interactions or secreting soluble cytokines. Therefore, to delete or attenuate the effect of CD4~+CD25~+ Tregs will evoke effective anti-tumor immunity, which may become a feasible immunotherapy for cancer.With this experiment, we use the specific antigen-sensitized DC-CTL cells as the anti-tumor immunotherapy cells to cure the B16 melanoma tumor animal model. We can observe not only the morphological difference, cell cycle,cell proliferation and cell apoptosis in vivo, but also the killing effect in vitro. Moreover, the deletion of the CD4~+CD25~+ Tregs by magnetic cell sorting (MACS) and observation the influence on the kill effection in the tumor treatment shows the relationship CD4~+CD25~+ Tregs and the adoptive immunotherapy in B16 melanoma. In this way, we may analyse and discuss the relationship between DC-CTL cells and regulatory T cells in anti-tumor immunity in order to find more treatment methods and theory foundation to clinical therapy in anti-tumor immunotherapy through the experiment.Methods: The B16 melanoma tumor cells are cultured in vitro and injected into the C57BL/6 mouse body to make the animal models. Then the animal models are irradiated by 60Co-ray on the total body in order to eliminate its immunol function. Then the animal models in a no-marrow lymphocyte deletion(NMLD) state. It is a special period for us to restitut the immune system with new immunocells.DCs and T cells are isolated from C57BL/6 mouse'spleen. By the analysis of the DC's immune epitope to show the mature stage, the DCs are cultured and loaded with the B16 melanoma tumor special antigen. DCs can induce the T cells into the DC-CTLs with tumor specificity. Before and after culture antigen-sensitized DC-CTLs were gotten, the method of MACS system was used to delete CD4~+CD25~+ regulatory T cells. So we have two groups anti-tumor immunocompetence cells: DC-CTL without CD4~+CD25~+ Tregs. They are after and before the culture of DC-CTL by using MACS system to delete CD4~+CD25~+ Tregs respectively. We also have the third group: the specific antigen-sensitized DC-CTL cells without deletion CD4~+CD25~+ regulatory T cells, the forth group the control group.80 cases of C57BL/6 mouse animal models have been randomly divided into four groups. The first group: the tumor specificity DC-CTL that gets before the culture to delete CD4~+CD25~+ Tregs; the second group :the tumor specificity DC-CTL that gets after the culture to delete CD4~+CD25~+ Tregs; the third group: the tumor specificity DC-CTL that gets without deletion CD4~+CD25~+ Tregs; the forth group: the control group that is to use normal sodium.To inspect DC-CTLs'killing effect to B16 melanoma in vitro by the method of mono-nuclear cell direc cytotoxicity assay(MTT) and the effect of the inhibitory to the B16 melanoma in vivo. Such as the change of tumor volume,tumor weight and the tumor inhibition rates, inhibitory effect of the specific antigen-sensitized DC-CTL cells on B16 melanoma cells are estimated. Morphological characteristics of immune cells are observed by electronic microscope. Moreover, the rate of tumor cells in G0/G1,S,G2/M stage, proliferation index(PI) and apoptosis index(AI)are respectively detected with florescene-actevated cell sorter(FACS). To study the effect of DC-CTL cells on B16 melanoma cells proliferation and apoptosis in the animal models, is to show the significant effect to kill the tumor cells.To check the CD4~+CD25~+ Tregs by FACS and the tumor necrosis factor-α(TNF-α), Interleukin-12(IL-12) by enzyme linked immunosorbent assay (ELISA) in patient with colorectal cancer at first. To find the change in the CD4~+CD25~+ Tregs, TNF-αand IL-12 and show the effect that the chemotherapy have done to the colorectal patients'immune system.Results: CTL cells with the tumor specificity of B16 melanoma induced by DCs are analyzed in the morphology. The specific antigen-sensitized DC-CTL cells are bigger than the normal lymphocytes and notch always appears on the cell nucleus observed under transmission electron microscope(TEM). Plenty of active-functioned organelles such as dilated endoplasmic reticulum,cytochondriomes were observed in the cytoplasm of the specific antigen-sensitized DC-CTL cells. Protrusions on the surface of the specific antigen-sensitized DC-CTL cells contacted closely with the tumor cells. Apoptosis and necrosis of the cells can be widely observed in tumor tissues.The kill effect in vitro by the method MTT, the tumor inhibition rate of the three experiment groups is significantly higher than the control group to B16 melanoma(P<0.05), and the inhibition rate increase according to the rate of the effect-target. The two groups that delete the CD4~+CD25~+ Tregs have higher tumor inhibition rate than the group that is without deletion. But there is no significant difference in tumor inhibition rate between the two groups that delete CD4~+CD25~+ Tregs before or after the culture (P >0.05).The kill effect in vivo to the B16 melanoma in the three experimental groups has an obvious inhibition to the tumor compared with the control group. The volume, weight of the tumor and the inhibition rate is higher in the groups with deletion the CD4~+CD25~+ Tregs than the groups without deletion CD4~+CD25~+ Treg(sP<0.05). But there is no significant difference between the groups that delete the CD4~+CD25~+ Tregs before or after the culture (P >0.05).By the method of FACS, compared with the three experimental groups and the control group, the rate of tumor cells in G0/G1 and AI are significantly higher; on the other hand, the rate of tumor cells in G2/M stage and PI is significantly lower. But there is no change in S stage. Comparing the two groups of deletion the CD4~+CD25~+ Tregs with the group without deletion, the difference in the cell cycle, AI and PI is significant in fact. But the difference between the two groups with deletion CD4~+CD25~+ Tregs befor and after the culture is not significant (P >0.05).The medicine of the chemotherapy can change the CD4~+CD25~+ Tregs, TNF-αand IL-12 in the colorectal cancer and have effect to the patients'immune system. But on the other hand CD4~+CD25~+ Tregs, TNF-αand IL-12 are the index to estimate the effect of the chemotherapy.Conclusion:1 The specific antigen-sensitized DC-CTL cells have high efficiency in inhibition on B16 melanoma. The specific antigen-sensitized DC-CTL cells may have an effect on the tumor cells cycle and induce them apoptosis. DC-CTL cells are a new generation of adoptive immunocell with high efficiency in killing tumors cells.2 The specific antigen-sensitized DC-CTL cells have high efficiency and more special in inhibition effect on B16 melanoma tumor cells after deleting CD4~+CD25~+Tregs. CD4~+CD25~+Tregs can exert inhibitory effect on The specific antigen-sensitized DC-CTL cells killing function. Deleting CD4~+CD25~+Tregs and recruitment of effector T cells will evoke effective anti-tumor immunity, which may have become a feasible immunotherapy for cancer.3 There is no significant difference between the two groups in different stage with the deleting CD4~+CD25~+ Tregs by observing the tumor inhibition rate, tumor cells apoptosis and proliferation, the killing effect. We must do more study to explain the possible mechanism and relationship among the main subsets of regulatory T cells.4 chemotherapy can change not only the CD4~+CD25~+Treg, TNF-αand IL-12 but also the immune system of patients. Combining with chinese medicine can reduce the side effects. It is a good way to cure the tumor.