Regulation And Mechanism Of GDF-15 On NKs Function Inhibition Which Induces Colon Carcinoma Immune Escape

The immunotherapy of tumor is a new strategy of tumor therapy besides surgery,radiotherapy and chemotherapy.In contrast to traditional therapies targeting cancer cells directly,the goal of immunotherapy is to treat the cancer by activating the patient's own immune system.However,the effect of immunotherapy of tumor is dissatisfactory,This phenomenon is due to immune escape in the tumor microenvironment.Tumor immune escape is the ability of monitor cancer cells to evade the immune system through its modification and metabolism changes.Rencently,research targeting immune cells in tumor microenvironment is a hotspots in the field of tumor immune escape.Natural killer cells are important members of the innate immune system,are large particles cells which different from T and B lymphocytes.At the same time NK is a kind of important immune cell involved in antiviral immunity and tumor immunity.According to the abundance of CD56 and CD16,human peripheral blood NK cells were divided into two groups: CD56^brightCD16^- and CD56^dimCD16⁺.Large number of immature subsets NK cell were found in tumors microenvironment,suggesting that TINK cells may represent the immature state and the function of TINK cells was inhibited.To identify new tumor derived factors which can promote tumor escape,we screened out a series of molecules by phage display technology.Among which,we pay more attention to GDF-15.Studies have shown that GDF-15 is closely related to the occurrence and development of many kinds of tumors,and can be used as the classification index of tumor staging.But,the function of GDF-15 in tumor microenvironment is undefined,and its interactionwith immune cells needs to be elucidated.In this study,we investigated the function of GDF-15 on NK cells and its influence of immune escape of colon cancer.Then we discussed the regulation mechanism of GDF-15 on NK cells.This study contains the following three aspects of experiments: 1.GDF-15 influences the phenotype and function of human peripheral blood NK cells;2.Effects of GDF-15 on the growth of colon cancer in mice by regulating the function of NK cells;3.mechanism study of GDF-15 on NK cells.The main methods include: 1.GDF-15 influences the phenotype and function of human peripheral blood NK cells.NK cells in the peripheral blood were separated by flow cytometry and Miltenyi immunomagnetic separation kit from healthy people;Real-TimePCR and flow cytometry were used to detect NK cell surface receptors NKG2 D,NKp30,CD16 a,CD56 expression;flow cytometry,ELISA method were used to detected expression of IFN-?,IL-10,IL-4,and IL-6;applied flow cytometry and CCK-8 method for detecting apoptosis and proliferation of NK cells after GDF-15 stimulation apoptosis and proliferation;double staining of CFSE/PI was used to detect NK cells cytotoxicity.2.SW480 cells bearing,NOD-SCID mice were used for small animal imaging experiments,the results confirmed that GDF-15 inhibited the cytotoxicity of NK cells and promoted the immune escape of colon cancer.3.Ranscription factor activity chip was performed to evaluated the molecular mechanisms how GDF-15 regulated NK cells,then verified the changes of transcription factors by Real-Time PCR and Western Blot.Through the above research,we finally got the following results: 1.in vitro,GDF-15 stimulation did not affect the survival of NK cells,but could inhibit NK cells' proliferation induced by IL-2 and IL-12;GDF-15 inhibited the expression of receptor of NKG2 D,NKp30,CD16 in NK cells,inhibited the secretion of IFN-?and promoted the expression of Th2 factor IL-10;GDF-15 could inhibit NK cell killing of target cells.2 in vitro,GDF-15 stimulation could inhibit NK cell killing of colon cancer cell line SW480,and in vivo,the cytotoxicityability of NK to colon cancer was inhibited after GDF-15 stimulation.3.In NK cells,GDF-15 stimulating down regulated of transcription factor E2A expression,and promoted the transcription of Id2.We first confirmed that GDF-15 can inhibit NK cell activation and cytotoxicity,and verified in NOD-SCID mice.GDF-15 influences the function of NK cells through promoting ID2 transcription and inhibiting E2A.