Clinical Significance And Mechanism Of Up-Regulation Of CD4~+CD25~+FOXP3~+ Regulatory T Lymphocytes In Non-Small Cell Lung Cancer

Lung cancer is the most common malignant tumour and the leading cause of cancer death in the world. The major histologic subtypes of lung cancer are non-small cell lung cancer (non-small cell lung cancer, NSCLC,80%) and small cell lung cancer (small cell lung cancer, SCLC,20%). Despite medical advances, the overall 5-year survival rate is a dismal 15%. Even when diagnosed at an early stage, patients relapse at a rate as high as 50% after surgical resection. Conventional chemothery are only effective for 25% relapse ptients. These facts make clear the need for new therapeutic strategy that will allow better prognosis for this malignancy. CD4+CD25+regulatory T cells is a distinct population of CD4+T cells that constitutively express the interleukin-2 (IL-2) receptor (R)α-chain (CD25). These cells are both hyporesponsive and suppressive. It was defined as "professional" regulatory/suppressor T cells. Following T cell receptor (TCR) engagement, CD4+CD25+ T cells can suppress the activation and proliferation of other CD4+ and CD8+ T cells in an antigen-nonspecific manner. CD4+CD25+ T cells mediate the suppression of effectors T cell function both in vitro and in vivo via several mechanisms requiring either cell-cell contact or the production of immunosuppressive cytokines. Recent studies have suggested that the transcription factor FOXP3 is critical for the development and function of regulatory T cells in mice and humans. Several studies have suggested that the CD4+CD25+FOXP3+ regulatory T cells may impaire the antitumour immunity and subsequently contribute to malignancy progression. Based on the theory above, our study will focus on the quantity and clinical significance of CD4+CD25+FOXP3+ regulatory T cells in non-small cell lung cancr, and investigate the potential mechanisms about which CD4+CD25+FOXP3+ regulatory T cells increased in tumour microenviroment. The purpose is to help to find a new strategy for the immune treatment of non-small cell lung cancr.PART I:DETECTION AND SIGNIFICANCE OF CD4+CD25+FOXP3+REGULATORY T CELL IN NON-SMALL CELL LUNG CANCEROBJECTIVES:To detect the proportion, phenotypic profile, function of CD4+CD25+FOXP3+ regulatory T lymphocyte in non-small cell lung cancer and to determine whether the proportion is associated with clinicopathological parameters.METHODS:The percentages of CD4+CD25+FOXP+T lymphocytes in tumour tissue, non-tumour tissue and peripheral blood from patients with non-small cell lung cancer, and peripheral blood from healthy control were determined by four-color flow cytometry. The expressions of CD45R0, CTLA4, GITR, CD39, CCR4 were also examined. Meanwhile, the relationship between clinicopathological parameters and the percentages of CD4+CD25+FOXP+T lymphocytes was determined. CD4+CD25high and CD4+CD25-T cells from tumour tissue and peripheral blood were isolated, and were cultured to investigate the effects of CD4+CD25high cells on proliferation response of CD4+CD25-T cells in vitro.RESULTS:There were increased percentages of CD4+CD25+FOXP3+T cells in tumour tissue (13.05%±7.16%) compared with non-tumour tissue (3.86%±2.35%) from patients with non-small lung cancer, in peripheral blood from patients (3.43%±2.14%) compared with peripheral blood from healthy control (1.49%±0.80%), and that these cells have constitutive high-level expression of CD45RO,CTLA4,GITR,CD39,CCR4. CD4+CD25high T cells mediate potent inhibition of proliferation response of CD4+CD25- T cells in vitro. The proportion of CD4+CD25+FOXP3+ T cells is related to TNM stage, tumour size, and histologic type.CONCLUSIONS:The proportion of CD4+CD25+FOXP3+ T cells was increased in patients with non-small cell lung cancer, which consequently resulted in the impairment of anti-tumour immunity and advancement of tumor progression.PARTⅡ:RESERCH OF INDUCED-CONVERSION MACHENISM AND UP-REGULATION OF CD4+CD25+FOXP3+ REGULATORY T LYMPHOCYTE IN PATIENTS WITH NON-SMALL CELL LUNG CANCEROBJECTIVES:To investigate whether the induced-conversion mechanism is concerned with the up-regulation of CD4+CD25+FOXP3+ T cells in tumour microinviroment.METHODS:Lymphocytes isolated from peripheral blood, tumor tissue and non-tumor tissue of lung cancer patiens(n=17) were analyzed for expression of TGFβon CD4+CD25+T lymphocytes and of TGF βRⅡon CD4+CD25-T lymphocytes by flow cytometry. Lymphocytes from health adults(n=11) were served as control.The expression of TGFβin tumor and nontumor tissue was determined by immunohistochem-istry. CD4+CD25-T cells isolated from tumour tissue and peripheral blood were cocultured with hrTGFβor monoclonal TGFβantibody to investigate whether hrTGFβcan convert CD4+CD25-T cells into CD4+CD25+FOXP3+T cells in vitro.RESULTS:The expression of membrane TGF 0 on CD4+CD25+T lymphocytes from tumour tissue was significantly higher than peripheral blood. The expression of membrane TGFβRⅡon CD4+CD25-T lymphocytes from tumour tissue and peripheral blood was also significantly higher than peripheral blood from healthy donors. TGFβ, mainly produced by stromal cells, was prominent in tumour tissues. hrTGFβcan actually convert CD4+CD25-T cells into CD4+CD25+FOXP3+T cells at higher concertration in vitro.CONCLUSIONS:Up-regulation of CD4+CD25+FOXP3+T cells in tumour microenviroment may be partially due to TGFβinduced-conversion. Both TGFβ,produced by stromal cells, and higher expression of TGFβRⅡon CD4+CD25-T lymphocytes contribute to this process.PARTⅢ:RESERCH OF CHMOTATIC MACHENISM AND UP-REGULATION OF CD4+CD25+FOXP3+ REGULATORY T LYMPHOCYTE IN PATIENTS WITH NON-SMALL CELL LUNG CANCEROBJECTIVES:To investigate whether the chemotatic mechanism is concerned with the up-regulation of CD4+CD25+FOXP3+ T cells in tumour microinviroment. METHODS:Lymphocytes isolated from peripheral blood, tumor tissue of lung cancer patiens were analyzed by flow cytometry for expression of CCR4 on CD4+CD25highT lymphocytes. Lymphocytes from health adults were served as control. The proportion of CCL22+ CD14+cells and CD4+CD25+FOXP3+T cells in tumor tissue were also analyzed by flow cytometry. CD4+CD25highT cells isolated from peripheral blood, tumour supernatants and malignant pleural effusion were used for in vitro chemotatic experiments.RESULTS:The expression of CCR4 on CD4+CD25+T lymphocytes from tumour tissue was significantly higher than peripheral blood. The proportion of CCL22+CD14+cells was positively related to that of CD4+CD25+FOXP3+T cells in tumor tissue. CD4+CD25high T cells were attracted by tumour supernatants and malignant pleural effusion in vitro, and monoclonal antibody to CCL22 can block this chemotactic activity.CONCLUSIONS:CD4+CD25+FOXP3+T cells in peripheral blood can be selectively attracted into tumour microenviroment by CCL22-CCR4 axis.PART IV:RESERCH OF FOXP3+CELLS, CD8+CELLS AND THE PROGNOSIS OF PATIENTS WITH NON-SMALL CELL LUNG CANCEROBJECTIVES:To investigate whether the numbers of FOXP3+cells CD8+ cells in tumour tissue impact the prognosis of patients with non-small cell lung cancer.METHODS:Immunohistochemistry was used to evaluate the epithelial and stromal FOXP3+, CD8+ cells. Combined with clinicopathological parameters and follow up data, the clinical significances of FOXP3+ cells and CD8+ cells were determined.RESULTS:The numbers of FOXP3+ cells and CD8+ cells from tumour tissue were significantly higher than non-tumour tissue. Stromal FOXP3+ cells related to differentiation and TNM stage, but had no effect on survival. Stromal CD8+ cells related to nodal status and TNM stage. Increasing numbers of stromal CD8+ cells significantly correlated to an improved survival. The ratio of CD8+ cells to FOXP3+ cells had no effect on prognosis.CONCLUSIONS:Stromal CD8+ cells, not FOXP3+ cells were positive prognostic indicators for resected NSCLC patients.