| Primary liver cancer(PLC)is one of the most common cancer in the world.HCC,which account for about 90% of PLC,is the main type of PLC and the fifth most common malignant tumor and its mortality rate ranks third among malignant tumors in the word.The microenvironment composed of liver cancer cells,hepatic stellate cells,epithelial cells,lymphocytes,cytokines and other interstitial components plays an important role in the occurrence and development of HCC and immunotherapy for liver cancer microenvironment has become a treatment strategy.T lymphocytes are an important part of the tumor microenvironment and programmed death 1(PD-1)and cytotoxic T lymphocyte-associated antigen-4(CTLA-4)are the main immunosuppressive molecules on the surface of T lymphocytes.PD-1 is mainly expressed on activated CD8+ T lymphocytes and negatively regulates its function.CTLA-4 is also an important costimulatory molecule that down-regulates T cell function.It belongs to the immunoglobulin superfamily and is expressed on the surface of activated CD4+ and CD8+ T lymphocytes and B lymphocytes.Transforming growth factor β1(TGF-β1)is one of the cytokines that play an important regulatory role in the occurrence and development of liver cancer.It can inhibit the immune function of T lymphocytes and promote the occurrence and development of tumors.However,whether TGF-β1 can up-regulate the expression of PD-1 and CTLA-4 on T cells to attenuate the killing of liver cancer cells by T cells is still unknown.Therefore,this study aims to explore the effect of TGF-β1 on the expression of PD-1 and CTLA-4 on T lymphocytes,and whether TGF-β1 attenuates the killing of T cells on liver cancer cells.In this study,normal human T lymphocyte line H9 cells were used for in vitro experiments.The purpose of this study was to explore the effect of TGF-β1 on the expression of PD-1 and CTLA-4 on T lymphocytes,and whether TGF-β1 attenuated the cytotoxicity of T cells towards liver cancer cells.At the same time,explore the possible mechanism of TGF-β1 up-regulating the expression of PD-1 and CTLA-4 on T lymphocytes.This study will provide some experimental basis for liver cancer immunotherapy based on the tumor microenvironment.Objectives:To investigate TGF-β1 induces HCC immune escape by up-regulating the expression of T lymphocytes PD-1 and CTLA-4.To reveal the mechanism of TGF-β1up-regulating the expression of T lymphocytes PD-1 and CTLA-4.Methods:1.ELISA method was used to detect the concentration of TGF-β1 in the serum of patients with liver cancer.2.Flow cytometry was used to detect the expression of PD-1 and CTLA-4 on CD8+T cells in the peripheral blood of liver cancer patients and liver cancer tumor-bearing mice.3.Flow cytometry was used to detect the expression of PD-1 and CTLA-4 and T cell apoptosis on T lymphocytes.4.Western blot was used to detect the expression of PD-1,CTLA-4,NFATc1,p-NFATc1 in T lymphocytes.5.qPCR was used to detect the m RNA levels of PD-1 and CTLA-4 in T lymphocytes.6.CCK-8 assay,LDH release detection assay,clone formation assay were used to detect the cytotoxicity of T lymphocytes for SMMC-7721 and Bel-7404 HCC cells.7.Chromatin immunoprecipitation(ChIP)assay was used to detect the binding of NFATc1 to PD-1 and CTLA-4 promoter regions in T lymphocytes.8.Subcutaneous transplanted tumor mouse was established to study the effect of TGF-β1 on the expression of CD8+ T cells PD-1 and CTLA-4 in the peripheral blood of mice,and the effect of TGF-β on the growth of mouse liver cancer tumors.Results:1.The concentration of TGF-β1 is positively correlated with the expression of PD-1and CTLA-4 on CD8+T cells in the peripheral blood of patients with liver cancer.2.TGF-β1 up-regulates the expression of PD-1 and CTLA-4 on T lymphocytes,and attenuates the cytotoxicity of T lymphocytes for HCC cells.3.The apoptosis of T cells stimulated by TGF-β1 is increased after co-cultured with HCC cells.4.TGF-β1 up-regulates the expression of PD-1 and CTLA-4 on CD8+T cells in peripheral blood of subcutaneous transplanted tumor mouse and promotes the growth of H22 tumors in vivo.5.TGF-β1 promotes NFATc1 to enter the nucleus,and NFATc1 directly targets the promoter regions of PD-1 and CTLA-4 genes.6.Blocking the TGF-β receptor or CaN/NFATc1 pathway can both down-regulate the expression of PD-1 and CTLA-4 on H9 cells induced by TGF-β1.Conclusions:1.TGF-β1 induces HCC immune escape by up-regulating the expression of PD-1and CTLA-4 on T lymphocytes2.The mechanism by which TGF-β1 up-regulates the expression of PD-1 and CTLA-4 in T lymphocytes may be related to the Ca N/NFATc1 pathway. |
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