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

Objective: Adoptive immunotherapy is one of the hotest subject in biotherapy of tumor. The treatment is not only the supplement to conventional therapy such as surgical operation,chemotherapy and radiotherapy ,but also apply hope to people who can not sustain the side effects of radiotherapy and chemotherapy any longer.Adoptive immunotherapy has good therapeutic effect to the tumor patients in immune reconstruction and eliminate remained pathological.Cytokine induced killer (CIK)cells is one of the new type immunocompetence cells, they have high efficiency in anti-tumor immunity. Cytokine induced killer cells have widespread using in adoptive immune therapy ,because they showed highly effcient cytolytic effector, faster proliferation speed rate then other immunocompetence cells in anti-tumor immunity.The key of studies to cytokine induced killer cells are how to enhance their cytolytic effcient and anti-tumor specific effcient,obtain more cytokine induced killer cells. Dendritic cells(DCs) are the most potent antigen presenting cells(APC) with the ability to acquire,process,present,antigens and the unique capability of initating primary immune responses against tumor-associated antigens. Dendritic cells(DCs) and cytokine induced killer(CIK) cells were separated and culture respectively,then dendritic cells(DCs) were loaded with specific tumor antigen. By using this methods ,we can co-culture tumor Antigen-sensitized Dendritic cells (DCs) with cytokine induced killer( CIK)cells. The tumor antigen-sensitized Dendritic cells (DCs) with cytokine induced killer( CIK)cells has higher cytolytic effcient and more specific anti-tumor specific effcient.We can obtain more quantity immunocompetence cells than cytokine induced killer( CIK)cells which did not loaded with specific tumor antigen. But in practice, at the tumor circumstances anti-tumor immunocompetence cells were be revulsanted into immune anergy state constantly . so the anti-tumor immunocompetence cells killing effect descend obviously. It has very importmant significance effect to increase the anti-tumor immunotherapy that delete immunodepression cells. CD4~+CD25~+ regulatory T cells is one of the new discovered cells,they are the main subsets of regulatory T cells that play an essential role in maintaining immunological self-tolerance. They have the function of down-regulates the tumor immunity mediated by T cells. CD4~+CD25~+ regulatory T cells also divided into two groups by produced channel.The two groups are natural regulatory T cells (nTreg) and induced regulatory T cells( iTreg). CD4~+CD25~+ regulatory T cells increases in the peripheral biood and tumor infiltration lymphoglandula in many tumor patients,such as breast cancer,lung cancer,gastric cancer. CD4~+CD25~+ regulatory T cells suppress immune responses mainly by cell contract-dependent interactions or secreting soluble cytikines.Depletion or attenuation of CD4~+CD25~+ regulatory T cells will evoke effective anti-tumor immunity,which may become a feasible immunotherapy for cancer.With this experimental, we use the specific antigen-sensitized DC-CIK cells as the anti-tumor immunotherapy cells.We observe the effect on B16 melanoma tumor animal experimental modle by DCs loaded with tumor antigen through morphological observation,cells proliferation and apoptosis,killing effect. By this way, we may analyse and discuss the relationship between CIK cells and regulatory T cells in anti-tumor immunity. We want to find more treament methods and theory foundation to clinical therapy in anti-tumor immunotherapy through the experimental.Methods:The B16 melanoma tumor cells were incubated in the C57 BL/6 mouse to build the animal models.Then using the total body irradiation by 60Co-ray to the animal models to eliminate it's immunol function. Then the animal models in a no-marrew lymphocyte deletion state.Peripheral biood mononuclear cells(PBMC) were isolated from C57 mouse. DCs were induced from adherent cells by some cytokines and CIK cells were generated from non- adherent cells. Mature DCs co-cultured with CIK cells,then loaded with specific tumor antigen, analyze specific antigen-sensitized DC-CIK immune epitope. Before and after culture ntigen-sensitized DC-CIK MACS (magnetic cell sorting) separating system was used to delete CD4~+CD25~+ regulatory T cells. So we have three gpoups anti-tumor immunocompetence cells. The first group: after by using MACS separating system to delete CD4~+CD25~+ regulatory T cells, then culture and acquire the specific antigen-sensitized DC-CIK cells. The second group: culture and acquire the specific antigen-sensitized DC-CIK cells ,then use MACS separating system to delete CD4~+CD25~+ regulatory T cells in this specific antigen-sensitized DC-CIK cells. The third group: the specific antigen-sensitized DC-CIK cells which did not delete CD4~+CD25~+ regulatory T cells.Then randomize divided the animal models into four groups. The first group: after by using MACS separating system to delete CD4~+CD25~+ regulatory T cells, then culture and acquire the specific antigen-sensitized DC-CIK cells were injectedinto the animal models. The second group: culture and acquire the specific antigen-sensitized DC-CIK cells ,then use MACS separating system to delete CD4~+CD25~+ regulatory T cells in this specific antigen-sensitized DC-CIK cells were injectedinto the animal models.The third group: the specific antigen-sensitized DC-CIK cells which did not delete CD4~+CD25~+ regulatory T cells were injectedinto the animal models.The fourth group:control group.According to the change of tumor volume,tumor weight and the tumor inhibition rates, inhibitory effect of the specific antigen-sensitized DC-CIK cells on B16 melanoma cells were estimated. Morphological characteristics of immune cells were observed by electronic microscope.With flow cytometry equipment,the rate of tumor cells in G0/G1,S,G2/M stage,proliferation index and the apoptosis index were respectively detected. To study the effect of cytokine induced killer cells on B16 melanoma cells proliferation and apoptosis in the animal models.Determine the killing effect on tumors cells by MTT method.Results:Co-culture tumor antigen-sensitized Dendritic cells (DCs) with cytokine induced killer (CIK) cells,analyze the immune epitope. The specific antigen-sensitized DC-CIK cells were bigger than the normal lymphocytes and notch always appears on the cellar nucleus observed under TEM. Plenty of active-functioned organelles such as dilated endoplasmic reticulum were observed in the cytoplasm of the specific antigen-sensitized DC-CIK cells. Protrusions on the surface of the specific antigen-sensitized DC-CIK cells contacted closely with the tumor cells. Apoptosis and necrosis of the cells could be widely observed in tumor tissues.The tumor volume of the first group is 0.0377±0.0128cm3 ;the second group is 0.0359±0.0131cm3;the third group is 0.1278±0.0362 cm3;control group is 0.4052±0.0429cm3. The tumor volume of the three experiment groups were significant smaller than the control group(P<0.05). The first and second groups were significant smaller than the third group(P<0.05).There was no significant difference between the first groups and the second groups in tumor volume (P >0.05).Tumor inhibition rate of the third group(59.19%) was lower than that of either the first group(91.70% ,P<0.05) or the second group(92.64% ,P<0.05) . There was no significant difference between the first groups and the second groups in tumor inhibition rate (P >0.05). The tumor inhibition rate of the three experiment groups were significant higher than the control group(P<0.05).The tumor weight of control group is 3.361±1.07g, the first group is 0.958±0.32g ;the second group is 0.939±0.41g;the third group is 1.651±0.57g. The tumor weight of the three experiment groups were significant lower than the control group(P<0.05). The first and second groups were significant lower than the third group(P<0.05). There was no significant difference between the first groups and the second groups in tumor weight (P >0.05). By using flow cytometry equipment,compared with the three experiment groups and the control group, the rate of tumor cells in G0/G1 and apoptosis Index were significantly higher,on the other hand , the rate of tumor cells in G2/M stage and proliferation Index were significantly lower,but there was not greatly change in S stage. The first and second groups compared with the third group have the same circumstances. There was no significant difference between the first groups and the second groups in the rate of tumor cells in G0/G1,apoptosis index,G2/M stage and proliferation index(P >0.05).The specific antigen-sensitized DC-CIK cells showed strong killing effect on B16 melanoma tumor cells. The killing effect on B16 melanoma tumor cells of the three experiment groups were significant lower than the control group(P<0.05). The first and second groups were significant higher than the third group(P<0.05). There was no significant difference between the first groups and the second groups in killing effect on B16 melanoma tumor cells (P >0.05).Conclusion:1. The specific antigen-sensitized DC-CIK cells have high efficiency in inhibition on B16 melanoma. The specific antigen-sensitized DC-CIK cells may have an effect on the tumor cells cycle and induce them apoptosis.CIK cells is a new generation of adoptive immunocell has high efficiency in killing tumors cells.2. The specific antigen-sensitized DC-CIK cells have high efficiency and more specifical in inhibition effect on B16 melanoma tumor cells after delete CD4~+CD25~+ regulatory T cells. CD4~+CD25~+ regulatory T cells could exert inhibitory effect on The specific antigen-sensitized DC-CIK cells killing function. Deletion CD4~+CD25~+ regulatory T cells and recruitment of effector T cells will evoke effective anti-tumor immunity,which may have become a feasible immunotherapy for cancer.3. There was no significant difference between the deletion CD4~+CD25~+ regulatory T cells in different stage through tumor inhibition rate,tumor cells apoptosis and proliferation,the killing effect. We must do more study to explain the possible mechansim and relationship between the main subsets of CD4+CD25+ regulatory T cells.