Experimental Study On The Inhibitory Effect Of Agonistic CD40 Monoclonal Antibody On Colon Cancer Cells Proliferation In Vitro

Objective: To investigate agonistic CD40 monoclonal antibody(CD40m Ab)to activate dendritic cells(DCs)loaded with tumor antigens in vitro and to research the inhibitory effect of the tumor-specific effector T lymphocytes on colon cancer cells(HCT116)proliferation.Methods : Tumor antigens were obtained by freeze-thaw method. Peripheral blood mononuclear cells(PBMCs)were isolated by density gradient centrifugation. DCs precursor cells and T lymphocytes were isolated from PBMCs by adherent culture. The immature DCs were induced by rh GM-CSF and rh IL-4 and loaded with tumor antigens. Then the DCs loaded with tumor antigens were activated by different methods. They were divided into three groups:blank control group, agonistic CD40 m Ab group and TNF-α positive control group. The concentration of cytokine IL-12(p70) in cell culture supernatant was determined with ELISA kit. The tumor-specific effector T lymphocytes were obtained by activated DCs stimulation. The proliferation activity of the T lymphocytes was evaluated by MTT. The tumor-specific effector T lymphocytes and colon cancer cells were co-cultured.The inhibition rate of colon cancer cells proliferation was assayed by MTT.Results :Soluble tumor antigen was successfully prepared and its concentration was 2.01mg/ml. DCs precursor cells and T lymphocytes were successfully isolated from PBMCs. The expression rates of surface molecules of the DCs activated by agonistic CD40 m Ab were:CD80( 92.5%±6.7%),CD83( 22.4%±5.3%),CD86(81.3%±7.5%)and HLA-DR(87.8%±3.7%).The concentration of cytokine IL-12(p70) was 716.80±53.43pg/ml in agonistic CD40 m Ab group. The proliferation activity of the T lymphocytes induced by the DCs was stronger in agonistic CD40 m Ab group than that of the blank control group(P<0.05). Compared with the blank control group(46.60%±1.10%), the inhibition rate of colon cancer cells proliferation was significantly increased in agonistic CD40 m Ab group(66.08%±0.41%)(P<0.05). Compared with the positive control TNF-α group(66.21% ± 1.73%),there was no significant difference(P<0.05).Conclusion:1、In vitro agonistic CD40 m Ab can better the activations of the DCs loaded with tumor antigens and promote their maturation. 2 、 The DCs activated by agonistic CD40 m Ab can significantly enhance the differentiation and proliferation activity of T lymphocytes and induce tumor-specific effector T lymphocytes. 3、The proliferation of colon cancer cells can be significantly inhibited by the tumor-specific effector T lymphocytes in vitro.