Roles And Potential Mechanisms Of Regulatory T/B Cells In Non-small Cell Lung Cancer

PART I Phenotype of circulating regulatory T/B cells in advanced NSCLC patientsObjective:To investigate the phenotype of circulating regulatory T/B cells in advanced NSCLC patients and their potential role in tumorgenesis.Methods:32 patients with advanced NSCLC and 20 healthy donors were enrolled in the study. Frequencies of Tregs and Bregs were measured by flow cytometry with antibodies against CD4, CD25, CD127, CD45RA, CD19, CD24, CD27 and IL-10.Results:Levels of CD4+T cells and CD19+B cells in PBMCs from patients with lung cancer significantly decreased as compared with healthy individuals (P<0.001, respectively). The frequency of peripheral naive Tregs (CD45RA+CD4+CD25+CD127") in CD4+T cells from lung cancer patients was significantly lower than in the healthy (P<0.05). The frequency of peripheral Bregs (CD19+CD24hiCD27+and CD19+IL-10+B cells) in CD19+B cells in lung cancer patients was significantly higher than in the healthy (P< 0.001 and 0.05, respectively).Conclusions:The frequency of peripheral naive Tregs in CD4+T cells from advanced NSCLC patients was lower than in the healthy, while the frequency of peripheral Bregs in CD19+B cells was significantly higher. The immune system plays a significant role in the control of tumor progression, although the regulatory mechanism of interaction between two systems remains unclear.Part II In vitro study of the interaction between tumor microenvironment and anti-tumor immunityObjective:To investigate the role of inflammation in shaping the phenotype of PBMC and to explore the interaction between tumor environment and anti-tumor immunity.Methods:PBMCs from healthy donors or NSCLC patients and lung cancer cell line were co-cultured in vitro. Frequencies of Tregs and Bregs were measured by flow cytometry with antibodies against CD4, CD25, CD127, CD45RA, CD19, CD24, CD27 and IL-10 before and after co-cultures.Results:In vitro co-culture of A549 cells with PBMCs polarized the lymphocyte phenotype in the same direction observed in the clinical trial. LPS-stimulated A549 cancer cells had profound effects in modulating regulatory cell number and function in an in vitro model.Conclusions:Inflammation-activated cancer cells could play the initiators and/or secondary sources of the development of cancer microenvironment and alterations of local immunity through the direct interaction and their products.Part Ⅲ Study of the potential mechanism by which lung cancer cells act on the antitumor immunityObjective:To investigate the crucial soluble factors in the tumor microenvironment and the role of LPS-related NF-κB signal pathway in the activation of lung cancer cells.Methods:The co-cultured A549 cells were harvested for qPCR for mRNA expression of RANTES and MIP-1α, while the co-cultured PBMCs were harvested for mRNA expression of TGF-β, IFN-γ, and IL-4. To investigate the role of LPS-related NF-κB signal pathway in the activation of lung cancer cells, A549 cells were pretreated with or without the NF-κB inhibitor PDTC at 10,50,100,300, or 500μM for 4hrs, followed by the stimulation of LPS at 500ng/ml. Treg frequencies were enumerated by flow cytometry.Results:The results showed a significantly increased mRNA expression of RANTES and MIP-1α in A549 in a concentration-dependent pattern after co-culture, which accompanied the up-regulation of Tregs. mRNA expression of TGF-β in PBMC significantly reduced, but IFN-γ and IL-4 in PBMC increased after co-culture. PDTC-pretreated A549 cells increased frequencies of CD4+T cells, while significantly decreased frequencies of Tregs and CD45RA+Tregs when A549 cells were pretreated with PDTC at 300μM.Conclusions:Tumor cells play a crucial role in antitumor immunity by providing soluble cytokines. LPS-related NF-κB signal pathway played a significant role in the activation of lung cancer cells.