| Background and Objective: Hepatocellular carcinoma is a principal contributor to cancer morbidity and mortality worldwide and poses a serious threat to human health and lives. The present method of treatment of hepatocellular carcinoma have mainly excised by the surgery primarily, radiotherapy, chemotherapy, biotherapy and auxiliary assist and recently the immunotherapy have attracted wide attention. T cell-mediated immune response played a critical role in the protection of the host against cancer invasion. Two signals were required in T cell activation and proliferation. T cell receptors firstly specifically engaged with the major histo-compatibility complexes(MHCs) on antigen presenting cells(APCs) to activate na?ve T cell and secondly bound to co-stimulatory ligands expressed on APCs to prevent anergy or inhibition of T-lymphocytes and immune suppression and escape.The co-stimulatory molecules mainly comprised B7, TNF and cytokine families.B7-H4 is a recently identified B7 family member, which is known to be a T cell co-stimulatory molecule down-regulated in the T cell-mediated immune response and related with tumor immune suppression. The study was designed to evaluate the significance of B7-H4 protein expressed in hepatocellular carcinoma, analyzed the associations between B7-H4 and tumor infiltration T-lymphocytes and cytokine secretion. Meanwhile, the expression levels of B7-H4, IFN-γ and PI3K/AKT/m TOR signaling molecules associated with autophagy in purified CD8 positive T-lymphocytes were detected by Western bloting. Further to analyzed the effect of B7-H4 expression on activation signaling pathways associated with autophagy inT-lymphocytes.Method:Part I, B7-H4 expression was detected by Immunofluorescence staining in 80 cases of hepatocellular carcinoma and adjacent hepatocellular carcinoma tissues and20 cases of liver tissue. The number of tumor-infiltrating CD4+T and CD8+T lymphocytes and their TGF-β1 and IFN-γ secretion in hepatocellular carcinoma was detected by Immunofluorescence staining. And the expression levels of B7-H4,TGF-β1 and IFN-γ in hepatocellular carcinoma and adjacent hepatocellular carcinoma tissues were detected by Western bloting.Part II, the purified CD8 positive T-lymphocytes were isolated by magnetic activated cell sorting from hepatocellular carcinoma and adjacent hepatocellular carcinoma tissues. And the expression levels of B7-H4, IFN-γ and PI3K/AKT/m TOR signaling molecules associated with autophagy in CD8 positive T-lymphocytes were detected by Western bloting.Result:(1) Co-stimulatory molecule B7-H4 weak positively expressed in adjacent hepatocellular carcinoma tissues but almost not in liver tissue, the positive rates in adjacent hepatocellular carcinoma tissues was 48.00%. The positive staining for B7-H4 was mainly localized in the cell membrane and cytoplasm and B7-H4 positively expressed in 81.00% of hepatocellular carcinoma, which was significantly higher than that of liver tissue and adjacent hepatocellular carcinoma tissues(**P=0.000, *** P=0.000). The expression of B7-H4 in hepatocellular carcinoma was significant positively correlated with lymph node metastasis, depth of infiltrations and TNM stages(P=0.010, P=0.023, P=0.007), particularly with lymph node metastasis and TNM stages(P=0.010, P=0.007), and it was not significantly correlated with patients’ gender, age, tumor size and degree of differentiation(P>0.05).(2) In 65 cases of B7-H4 positive hepatocellular carcinoma, the number of cases of light CD4 positive and CD8 positive T-lymphocytes infiltration were69.2%(45/65) and 80.0%(52/65), and their TGF-β1 and IFN-γ positive rates were67.7%(44/65) and 83.1%(54/65). The expression of B7-H4 in hepatocellularcarcinoma was significant negatively correlated with CD4 positive and CD8 positive T-lymphocytes infiltration and their TGF-β1 and IFN-γ secretion(P=0.034, P=0.005,P=0.014, P=0.002), particularly with infiltrating CD8 positive T-lymphocytes and their secretion IFN-γ(P=0.005, P=0.002).(3) The expression levels of B7-H4, TGF-β1 and IFN-γ in hepatocellular carcinoma and adjacent hepatocellular carcinoma tissues were detected by Western bloting. B7-H4 was markedly up-regulated and TGF-β1 and IFN-γ were markedly down-regulated in 80.0% of hepatocellular carcinoma. The expression of B7-H4 was significantly higher in hepatocellular carcinoma and negatively correlated with TGF-β1 and IFN-γ secretion.(4) The expression levels of B7-H4, IFN-γ and PI3K/AKT/m TOR signaling molecules associated with autophagy in CD8 positive T-lymphocytes were detected by Western bloting. B7-H4 was up-regulated and IFN-γ was down-regulated Similarity in hepatocellular carcinoma. Simultaneously, P-AKT, P- m TOR and LC3 II were markedly up-regulated, which confirmed that the expression levels of B7-H4 induced autophagy in CD8 positive T-lymphocytes indeed.Conclusion:(1) The expression of B7-H4 was significantly higher than that of liver tissue and adjacent hepatocellular carcinoma tissues, which significant positively correlated with lymph node metastasis, depth of infiltrations and TNM stages, but not significantly correlated with patients’ gender, age, tumor size and degree of differentiation. It confirmed that B7-H4 is expected to become a sensitive and specific tumor marker.(2) The expression of B7-H4 in hepatocellular carcinoma was significant negatively correlated with CD4 positive and CD8 positive T-lymphocytes infiltration and their TGF-β1 and IFN-γ secretion, which indicated that the expression of B7-H4 induced immune suppression through inhibited cell growth of T-lymphocytes and secretion of their cytokines.(3) B7-H4 was up-regulated and IFN-γ was down-regulated markedly in hepatocellular carcinoma. Simultaneously, PI3K/AKT/m TOR signaling molecules associated with autophagy in CD8 positive T-lymphocytes activated and marker ofautophagy, LC3 II up-regulated apparently, which further confirmed that the expression of B7-H4 induced autophagy in CD8 positive T-lymphocytes through activating PI3K/AKT/m TOR signaling. And it might inhibit cell growth of CD8 positive T-lymphocytes and secretion of IFN-γ and induce immune suppression. |
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