The Significance Of Three Distinct Subpopulations Of CD4+Foxp3+T Cells In The Peripheral Blood Of Patients With Non-small Cell Lung Cancer

Background and objectivesLung cancer is one of the leading causes of cancer-related deaths in the worldwide. Non-small cell lung cancer (NSCLC), which constitutes about80%of all cases of lung cancer, is a serious threat to human population. Despite the advances in treatment of lung cancer, the overall5-year survival rate is still not satisfactory. Because of its high incidence, low5-year survival rate and limited treatment efficacy, discovering new treatment is urgent. Studies have revealed that the occurrence of lung cancer is a multi-factorial and gene interaction process. With the significant development of the techniques in molecular biology, importance of the immune system in cancer development has been well established. It is understood that in patients with malignant tumors, immune system is in a state of tolerance or defects. Tumors continue to progress after escaping the host immune mechanisms. Regulatory T cell belongs to important subset of immunosuppressive cells that have a unique immune regulation function and play an important role in the process of cancer immune escape.So far a variety of surface markers of Treg has been reported, such as Foxp3CD127, CD39, CTLA-4, GITR, PD-1and TLR. Among which Foxp3is one of the most specific markers. However in recent years, studies have shown that human CD4+FoxP3+cells may not contain functionally homogenous population of cells; suggesting that some cells even if they express of Foxp3may not have have immunosuppressive function. Miyara et al. using CD4, CD25, CD45RA, and Foxp3as surface markers showed that human CD4+FoxP3+T cells comprised three distinct subpopulations with a precise phenotype and fate:CD45RA+FOXP3low(CD25++) resting Treg cells(rTreg cells), CD45RA-FOXP3hi(CD25+++) activated Treg cells (aTreg cells) and CD45RA-FOXP3low(CD25++) cytokine secreting non-Treg cells. Treg accumulation is observed in many kinds of cancer, and is related with the prognosis in some cancer models. But studies of rTreg, aTreg and non-Treg cells expression in non-small cell lung cancer patients is limited. So in the experiment, we attempted to investigate the expression of three CD4+Foxp3+T cell subsets in the peripheral blood of non-small cell lung cancer patients and their potential clinical significance. Among3subsets of CD4+Foxp3+T cell, knowing which group of Treg play a major role in immune suppression, in order to provide new targeted therapy for NSCLC.Methods6ml of peripheral blood from non-small cell lung cancer patients and healthy donors were collected and then peripheral blood mononuclear cells (PBMC) were obtained by centrifugation through Ficoll-Hypaque. The phenotype of CD4+Foxp3+Treg, rTreg, non-Treg and aTreg were determined by Flow cytomertry. Functional analysis was done by detection of the surface marker CD39and intracellular cytokine IFN-γ and TGF-β by Flow cytomertry. Plasma level of TGF-β1was measured by enzyme-linked immunosorbent assay (ELISA).Results1. Non-Treg expressed low CD39and rarely secreted high level IFN-γ and low level TGF-β. In contrast, rTreg and aTreg expressed high CD39and secreted low level of IFN-γ but higher level of TGF-β.2. The frequency of CD4+CD25+Foxp3+Treg and plasma level of TGF-β1was higher in patients with non-small cell lung cancer than healthy controls. However further analysis showed that only aTreg were highly expressed in the PBMC of NSCLC patients compared to healthy controls. The frequency of rTreg and non-Treg didn’t show any significant. 3. Further on histological analysis, no differences in expression of CD4+Foxp3+T cell were observed between adenocarcinoma and squamous cell carcinoma. But the level of CD4+CD25+Foxp3+Treg and aTreg is higher in patients with stage Ⅲ-Ⅳ than stage Ⅰ-Ⅱ patients.4. Non-small cell lung cancer patients receiving effective chemotherapy, non-Treg levels were significantly decreased in peripheral blood after initiation of chemotherapy. In addition rtreg and aTreg did not show significant change but had downward trend.Conclusion1. CD4+FoxP3+cells are not a functionally homogenous population of cells.2. The level of CD4+CD25+Foxp3+Treg and TGF-β1increased in the PBMC of NSCLC patients suggests NSCLC patients are in a immunosuppressive state.3. There is no association between CD4+Foxp3+T cell subgroup and pathological types. However high level of CD4+CD25+Foxp3+Treg and aTreg was associated with surgical stages.4. The level of peripheral blood Treg is reduced in the NSCLC patients who are responding to chemotherapy. Treg cells may be a potential indicator of effective evaluation of therapeutic effect and disease prognosis in patients with lung cancer. Also Treg may become a new target for effective therapy for lung cancer patients.