Research On The Mechanisms Of CD4～+CD25～+ Regulatory T Cell In Immune Escape Of Lung Cancer Cell

With the development of investigation on immunology, molecular biology and genetic engineering ,tumor immunotherapy will be developed into a new treatment model. Generous study indicate defection in the immune response may contribute to tumor growth .It has been found that there is a kind of unique T cell subgroup,CD4⁺CD25⁺ regulatory T cell. This kind of cell subgroup possess the characteristic of immuosupression and immunopotency, based on the above theory. this experiment will explore the mechanism of CD4⁺CD25⁺ regulatory T cell acting on lung cancer,By detecting the distribution of CD4⁺CD25⁺ regulatory T cell, we will demonstrate the mechanism of CD4⁺CD25⁺ regulatory T cell. The purpose is to provide a new way for lung cancer treatment.Part I Detection of CD₄⁺CD₂₅⁺ regulatory T cell of lung cancer patientsObjective : To detect the proportion of CD4⁺CD25⁺ regulatory T cell in the peripheral blood and resection tissue of lung cancer patients,and to explore the relative mechanism.Methods: Peripheral blood mononuclearcell(PBMCs) were separated by centrifugation over a ficoll density gradient of 46 lung cancer patients and 20 healthy controls. 21 case freshly resected lung cancer specimens were dealed with the method of enzymatic digestion,cancer cell and tumor-inflitrating lymphocytes were isolated and cultivarted .After stained with anti, all samples were performed on a cytomation MOFLO cell sorter by gating on lymphocytes. The level of TGF-β11 in the same blood sample was detected by ELISA.Results: The proportion of regulatory T cells in the peripheral blood of 46 lung cancer patients was(12.1±3.4) %,of 21 case resected lung cancer specimens, The proportion of regulatory T cells was(21.8±3.4)%, 22 cases of Squamous carcinoma patients, 19 cases of adenocarcinoma patients and 5 cases small cell carcinoma patients (12.1±0.9%,10.8±0.4%,11.2±0.4%) were obviously higher than that of healthy controls (4.6±0.6% ) , but there wasn't obvious discrepancy among different pathological type; The proportion of regulatory T cells in the peripheral blood of 20/15 lung cancer patients with â…¢/â…£ stages significantly higher than that of 11 lung cancer patients with â…¡ stages(11.4±2.1%,12.1±1.3% vs8.6±1.5%) ; Patients with different pathological type lung cancers and different stages all show an increase of the TGF-β11 level in the peripheral blood, compared with healthy controls; there wasn't obvious differentence among every lung cancer groups.Conclution: together these results suggest that the increase propotions of CD₄⁺CD₂₅⁺ regulatory T cells in the peripheral blood and local tissure of lung cancer patients , so to selectively inhibit the immune response,therefore could contribute to the progression of lung cancer.Part â…¡ The expression and significance of Foxp3 in human lung cancer tissueObjectives: To explore the role of FOXP3 in mechanism of human lung cancer tissue and its biological significance, we localized the expression of FOXP3 in lung cancer tissue of 21 patients and analysed the differentment of the expression of FOXP3 indifferent patho-type.Methods: Among 51 cases of lung resection tissue , 21 cases of lung cancer , 7 cases of bronchiectasis,5 cases of inflammatory pseudotumor,3 cases of tuberculoma and 15 normal health lung tissure, were studied for distribution of FOXP3 mRNA and protein by RT-PCR and Western blot. Compared with benign lesion tissure , we analysised expression level of FOXP3 mRNA and protein in different patho-type via imaging semi-quantity technique.Results: Positive stain of FOXP3 mRNA and protein were seen in 41cases human lung cancer tissue and 8 cases benign lesion tissure,but there was obvious difference of average degree of light suck value (P<0.05);those normal lung tissure showed negative stain; However, As for average degree of light suck value of FOXP3 mRNA and protein, there were not obvious differentment among different lung cancer patho-type.Conclusions: The positive expression of FOXP3 in lung cancer tissure suggest the infiltration of CD4⁺CD25⁺ regulatory t cell; Besides the normal physiological function, the expression change of FOXP3 in lung cancer can be thought to connected with local immuosupression of lung cancer.Part â…¢ The mechanism of TGF-β1 participate in the increased CD4⁺CD25⁺ regulatory T cell of lung cancerObjective : To explore on condition of directedly contacted with lung cancer cell,the role of TGF-β1 on the increased CD4⁺CD25⁺ regulatory T cell in lung cancer patients.Methods: The freshly resected lung cancer specimens were dealed with the method of enzymatic digestion. cancer cell and tumor-inflitrating lymphocytes (TIL)were isolated and cultivanted .Make lung cancer cell ,the culminated supernatant co-cultured with PBMCs,different group was stimulated by different level TGF-β1 factor(10, 5, 2ng/ml), detected the expression of FOXP3 mRNA in every group to illustrate the correlation between FOXP3 and TGF-β1.Results: The level of TGF-β1 in the supernatant origined from primary cultured lung cancer cell co-caltured with PBMCs was 1.4±0.7ng/ml, after stimulated with different level TGF-β1,the more expression of FOXP3 mRNA can be seen in every group with the increased level of TGF-β1. there was positive correlation between the two parameters. But after different level of TGF-β1 stimulated,there wasn't obvious change of the expression of FOXP3 mRNA in control groups.Conclusion: on condition of directed contacted with lung cancer cells,TGF-β1 can regulate the upexpression of FOXP3 mRNA,and it can play key role on the increased CD4⁺CD25⁺ regulatory T cell of lung cancer patients.Part â…£ Evaluate the effection and the mechanism of exogenous IL-2 biotherapy in malignancy pleural effusion of lung cancer patientsObjective: Detect the expression of TIL interleukin 2 receptorα (IL-2Rα) and the level of Soluble interleukin 2 receptor(SIL-2R) in malignancy pleural effusion of lung cancer patients to observe the change of the expression of TIL IL-2Ra in malignancy pleural effusion after exogenous IL-2 treatment ,in the same time ,we detected the disposition of CD8⁺ before and after situmated by exogenous IL-2 and to investigate the correlation between the level of SIL-2R and the expression of IL-2a of TIL,so to evaluate the effection of biotherapy and the mechanism of local immune recognition.Methods: The level of SIL-2Rα in malignant pleural effusion of lung cancer patients by ELISA. PBMCs were separated on a ficoll density gradient. After situmated by exogenous IL-2 to detected the expression of IL-2Ra and the disposition of CD8⁺ in malignant pleural effusion. All patients were treated with exogenous IL-2 by injection of local cavitas thoracis. After one week, these paraments were detected once more, we divided it into two parts on basis of median of SIL-2R to proceed statistical analysis by SPSS13.0. Results: There are positive correlation between the level of SIL-2R and the expression of IL-2Rα in malignant pleural effusion of lung cancer patients (r=0.872 ,p<0.05). It is become more obvious in high SIL-2R level group (r = 1.123, p<0.05). There were obvious difference about the level of SIL-2R in malignant pleural effusion of lung cancer patients .Before and after exogenous IL-2 treatment, the levels of SIL-2R in were (563.2±92.61 ng/l,390.12±101.12 ng/l,p<0.05).After exogenous IL-2 treatment, there wasn't difference of the expression of IL-2Rα (12.54±3.73%,13.37±2.65%, p>0.05) .Before treatment,the proportion of CD8⁺ in high and lower level groups were (29.3±7.9%, 28.7±6.3%,p>0.05). After treatment ,the proportion of CD8⁺ in high and lower level groups were (31.34±9.8%, 59.65±11.0%, p<0.001) .Conclusion: Exogenous IL-2 can stimulate the proliferation of CD8⁺.Difference SIL-2R level in malignant pleural effusion of lung cancer patients suggest there will be different effection after IL-2R treatment,perhaps by suppress the proliferation of CD8⁺ and down-regulated the sensitivity of IL-2Ra to exogenous IL-2R.