Studies On Lytic Activity Of Cytokine-induced Killer Cells Against MGC-803 Gastric Cancer Cell Lines After Co-culture With Antigen-pulsed Dendritic Cells

Objective: Gastric cancer is one of the most harmful malignant tumors to human health and life and the clinical therapeutic tools of gastric cancer is mainly radical excision assistant with chemo and radiation therapy both before and after operation. Most clinical cases are found to be middle or advanced stage, the curative effects of operation, chemo and radiation are not satisfactory, and thus the long term survival is relatively low. So improving the curing effects and quality of life for patients of gastric cancer and preventing the relapse and metastasis is the issue which clinicians have been exploring. The adoptive cellular immunotherapy (ACI) is the fourth oncotherapy mode after operation, chemo and radiation. Until now the adoptive therapy of NK, CTL, MΦ, TIL, LAK, CIK and DC cells has been well applied clinically, in which the antitumor immunotherapy based on CIK and DC has been warm spots for researchers both in home and abroad recently. The cytokine-induced killer cell(CIK) is a kind of cytotoxic T cell which can be induced by various cytokines such as IL-2, IFN-γand monoclonal antibody CD3. The tumor-lytic activity is non-MHC restricted for which the CIK cells can be termed as non-specific immuno-killer. Moreover, it can excrete kinds of cytokines(IL-4, IFN-γ,et al) and has a killing activity to tumor cells much stronger than LAK and CD3AK. Dendritic cell (DC) is regarded as a powerful antigen-presenting cells, which can exist in all parts of human body except in the brain and didymus. DC can capture low-density antigen effectively by receptors on its cell membrane, combine with MHC I and MHC II molecules on the antigen surfaces and stimulate the activation of na?ve CD8+ and CD4+ T cells. According to the researches in home an abroad, combination of DC pulsed with antigen and the effective killing cells of CIK to treat malignant tumors is a hopeful way of synergistic effect. My initial reseach use the MTT and FCM to detect the toxic activity to MGC-803 and apoptosis mechanisms of co-cultivation of DC pulsed with antigen cocultured with CIK, and do some foundation research for clinical use of CIK and DC in treatment of gastric cancer.Methods:1 Preparations of CIK and DC derived from PBMC: Extract PBMC, stimulate the non-adherent cell with interleukine-2, anti-CD3McAb, IL-1αand INF-γto obtain CIK; stimulate the adherent cells with granulocyte-macrophage CSF and IL-4 to obtain DC; test the phenotype of cells with FCM.2 Preparation and quantitation of MGC-803 soluble antigen: freeze thawing MGC-803 repeatedly with liquid nitrogen, filter and make into soluble antigen to stimulate DC; quantitative assay of antigen with Coomassie brilliant blue. 3 Comparison of killing function between three effective cell groups, with different E:T, in different days: The effector cells are divided into three groups: pure CIK group, non MGC-803 antigen pulsed DC with CIK group and MGC-803 antigen pulsed DC with CIK group. They are labeled as CIK, DC-CIK and Ag-DC-CIK. In Day 12 of co-culture, test the killing function of CIK, DC-CIK and Ag-DC-CIK groups with MTT, under the ratio of effective cells : target cells, 2.5:1, 5:1, 10:1 and 20:1; test the impact of different days.4 Test the level of excreting cytokines with enzyme linked immunosorbent assay(ELISA): extract the clear supernatant liquid of Ag-DC-CIK in Day 12 and 21, test the contents of IL-2, TNF-αand INF-γ.5 The proliferation and apoptosis of Ag-DC-CIK to MGC-803: co-cultivate the effector cells with the target cells (E:T=20:1)in Day 18 for 24h, collect cells, add Hoechst 33342 to the final concentration of 1ug/ml, incubate under the temperature of 37℃for 10min, centrifuge and draw off the staining solution; add PI, suspend, and incubate away from light; analyze the scatterplot or tonographic of blue fluorescence over the red; low blue/low red for the normal cells, high blue/low red for the apoptosis cell, low blue/high red for the necrosis cells.Results:1 CIK begins to multiply from Day3 and enter the multiplication period in Day5-6. DC-CIK and Ag-DC-CIK begin to proliferate fast from Day12, with the speed faster than CIK (p<0.05).2 The PBMC from healthy adults can derive into mature CIK and DC with the existing of IL-2, CD3McAb, IL-1α, INF-γ, GM-CSF and IL-4. (79.64±3.41)% of the cells present DC specific surface marker CD83,(95.17±1.22)% of HLA-DR, (94.66±2.42)% of CD86 and (86.59±1.09)% of CD40. DC-CIK in Day14 has a higher percentage of CD3+CD8+ and CD3+CD56+ cells of 59.39%-60.46% and 35.67%-36.90%. Ag-DC-CIK in Day14 has the highest percentage of the two cell populations of 63.63%-65.42% and 51.03%-53.15% and has significant difference (p<0.05) compared with CIK and DC-CIK groups.3 The killing activity of DC-CIK is generally stronger than CIK in the E:T range of 2.5:1-20:1. When DC is pulsed with MGC-803 antigen and co-cultured with CIK, the lytic activity of effector cells is strengthened and has significant difference (p<0.05) compared with CIK and DC-CIK.4 IFN-γhas a high concentration in the clear supernatant of co-culturing cells in Day21. TNF-αcan be detected in all periods with an obvious fluctuation, low in Day6 while high in Day21. On the contrary, the proinflammatory factor IL-2 has a high expression in Day6 and low or no expression in Day21.5 The 24h group shows a dominant percentage of early, middle and late period of apoptosis cells and normal cells in the FCM scatterplot. And 48h group is mainly composed of necrosis cells, with a obviously descending percentage of apoptosis cells than 24h group.Conclusions:1 DC pulsed with MGC-803 antigen can further improve the CIK proliferation rate.2 DC pulsed with MGC-803 antigen can elevate the percentage of CIK attacking cell populations: the CD3+CD8+ and CD3+CD56+ cells.3 Antigen-pulsed DC or non-antigen-pulsed DC can both strengthen the MGC-803 lytic activity of CIK.4 Ag-DC-CIK need cytokines to maintain culturing in vitro, and also excrete IFN-γ, TNF-αand IL-2 which influence the function of the cells.5 During the earlier period of the interaction of effectors and targets, MGC-803-DC-CIK kills MGC-803 mainly through inducing apoptosis of tumor cells and at the same time they themselves enter the process of apoptosis; in the late period of interactions, both of the targets and effectors tend to die of necrosis.