A Role For GDF-15in The Generation Of Cd4~+Cd25~+Regulatory T Cells From Human Peripheral Blood

Objective: In recent years, studies have shown that GDF-15can upregulate the mRNAexpression of Foxp3which is the most specific marker of Tregs in peripheral bloodmononuclear cells. Therefore，the purpose of our studies is to investigate the roles of GDF-15in differentiation of na ve CD4~+T cells to human regulatory T cells and the function of theinduced Foxp3~+T cells and observe the correlation of CD4~+CD25~+Foxp3~+regulatory T cellNumbers of peripheral blood and serum GDF-15expression level in colorectal cancer. At last,we will preliminary explore the molecular mechanisms of GDF-15.Methods: To confirm these questions, this study contains3parts of experiments:1. Toidentify the differentiation and other functions of GDF-15-induced-Tregs. Human na veCD4~+T cells were negatively isolated from PBMC, and then stimulated with GDF-15. Afteractivation, mRNA was extracted and amplified for detecting relative genes about functionsusing real-time PCR. Moreover, the cells were collected to detect surface CD25, GITR,CD103and intracellular Foxp3expression levels by flow cytometery. Meanwhile we detected the inhibition effect of the induced Foxp3~+T cells by the BrdU incorporation and CFSE assaysand the secretion of suppressive cytokine IL-10and TGF-β by ELISA.2. To detect thepercentage of CD4~+CD25~+Foxp3~+regulatory T cells in CD4~+T cell by flow cytometry and thelevel of serum GDF-15by ELISA kit after clinical colorectal specimens collected. And thecorrelation was analyzed by the SPSS13.0.3. To elucidate the mechanisms of GDF-15indifferentiation of iTregs. We used laser confocal to detect the receptor on na ve CD4~+T cellmembrane which may binds to GDF-15and further detect the relevant gene transcription levelafter blocking TGF-βreceptor blocker SB431542.Results: In part one, in vitro studies indicated that GDF-15can induce Foxp3expression inhuman na ve CD4~+T cells and the Foxp3expression level is the highest in10ng/mL ofGDF-15after5days induced. Real-time PCR and flow cytometry showed thatTreg-associated cell surface markers such as Foxp3, CD25, GITR and CD103wereup-regulated. The BrdU incorporation and CFSE assay showed that iTregs can suppresseffctor CD4~+T cell proliferation. And Real-time PCR and ELISA showed the induced Tregscan promote the secretion of suppressive cytokines such as IL-10and TGF-β. In part two, theSPSS13.0analysis showed that peripheral CD4~+CD25~+Foxp3~+regulatory T cells and serumGDF-15expression level in colorectal cancer patients have a good correlation which is0.7909.In addition, in part three, confocal fluorescence assay showed that GDF-15may exist on themembrane of na ve CD4~+T cells. And real-time PCR indicated that SB431542can inhibitGDF-15-induced Foxp3expression in the differentiation of na ve CD4~+T cells.Conclusion: Peripheral CD4~+CD25~+Foxp3~+regulatory T cells and serum GDF-15expressionlevel in colorectal cancer patients have a good correlation. GDF-15can induce human na veCD4~+T cells to differentiate into Foxp3~+T cells which exhibit the natural regulatory T cellsphenotype and suppressive function in vitro. We found that GDF-15can bind to na ve CD4~+Tcell membrane which suggests that GDF-15receptor may exist on the surface of na ve CD4~+Tcells.