| Tumor microenviroment played an important role in cancer progression, the immune system plays a dual role in cancer:it can not only suppress tumor growth and invasion but also promote caner progression either by immunoselection or establishing an immune inhibition network within the tumor microenviroment that facilitate cancer development. Until now, much works have being focusing on the adaptive immune system, nonetheless, the role of innate system in the tumor microenviroment was not well defined yet. In this study, firstly, we analysis the roles of γδ T cells, which was recognized as subset of innate immune cells accumulating remarkably in the liver, in the microenviroment of hepatocellular carcinoma. Secondly, we evaluated the expression and prognosis value of interferon regulatory factor (IRF)-1and IRF-2, which were important mediator of IFN-y function, in hepatocellular carcinoma.PartⅠ The infiltration and phenotype profile of γδ T cells in different location of hepatocellular carcinoma patientγδ T cells, bearing γ-and δ-chain T-cell receptor (TCR) heterodimers and possessing innate properties, mediate tumor-inhibition effects in multiple malignancies. γδ T cells can recognize soluble protein and non-protein antigens that are up-regulated by transformed or otherwise dysregulated host cells, in a manner independent of major histocompatibility complex (MHC) and antigen presentation. Notably, the antigens recognized by y8T cells are not seen by αβ T cells. After activation, γδ T cells boost strong cytotoxic effector activity (using both cytolytic granule pathways and death receptor/death ligand) and produce various cytokines (frequently including tumor necrosis factor-a and IFN-y), thereby providing an essential additional pathway with immediate relevance for malignancy immunosurveillance. However, the immune status of γδ T cells in the tumor microenviroment was not defined yet.we compared the infiltration and immune phenotype of y8T cells from different location (peripheral circulation, peritumoral microenviroment and intratumoral microenviroment) of hepatocellular carcinoma (HCC) patients.Although the percentage of γδ T cells in peripheral T lymphocytes was low, a significant accumulation of γδ T cells in normal liver tissue was unraveled. Peritumor tissue which usually had HBV infection and cirrhosis had a decreased proportion of y8T cells compared with normal liver tissue. Furthermore, the proportion of γδ T cells in total T cells was significantly decreased in tumor tissue compared with peritumor tissue. It is worth mentioning that patients with advanced HCC stage had even lower infiltration of γδ T cells compared with those with early HCC stage. Then we examine the distribution of γδ T cells in a large HCC patient cohort by immunohistochemistry, in line with FACS analysis, the absolute counts of y8T cells in HCC tissue were also decreased compared with peritumor tissue.Most of the cell surface activation markers such as CD80, CD83, CD86and HLA-DR were expressed at similar levels in γδ T cells from both the tumor or peritumor tissue of HCC, no CD25expression was seen in both populations. We found the majority of γδ T cells were Vδ1+T cells in normal liver tissue and cirrhotic peritumor tissue, and a moderate skewing to Vδ2+T cells was seen in tumor tissue.Analyzing PBMCs, PILs and TILs of HCC patients revealed that the proportion of effector and later effector subpopulations was sharply attenuated among tumor-derived y8T cells. Moreover, the expression of NKG2D, which was essential for y8T cells activation, was downregulated in tumor infiltrating y8T cells.Taken these result together, the infiltration of y8T cells in HCC tissue was significantly decreased compared with paired peritumor tissue, and an cytotoxic function impairment may exist in tumor infiltrating y8T cells. Part Ⅱ The cytotoxic function of γδ T cells in different location of hepatocellular carcinoma patientBased on the results mentioned in part I, its reasonalbe to suppose that tumor infiltrating y8T cells may have funtional impairment, compared with peritumor infiltrating ones. In this part, we focusing on the functional differences of y8T cells from different location of hepatocellular carcinoma (HCC) patient.Firstly, we evaluated the expression differences of perforin, Granzyme B by FACS in y8T cells derived from both tumor and peritumor tissue of HCC. Then we evaluated the differences of degranulation and IFN-γ secretion ability in the two populations challenged either by no specific stimuli (PMA+Ionomycin) or HCC cell lines. Moreover, we purified the y8T cells from both tumor and peritunor tissue of HCC, and subjected to Mircroarray analysis.We found that tumor infiltrating γδ T cells had equivalent or even slightly higher expression of perforin and granzyme B compared with corresponding peritumor infiltrating ones. But the expression of CD107a by tumor infiltrating y8T cells was decreased. The in vitro analysis of cytotoxicity indicated that tumor infiltrating y8T cells could not kill HCC cell lines (HCCLM3; MHCC97H; MHCC97L; PLC; HuH7;7721) effectively as peritumor infiltrating ones did. IFN-y secretion ability of tumor infiltrating γδ T cells was also impaired challenged either by no specific stimuli (PMA+Ionomycin) or HCC cell lines (MHCC97H; HuH7). The Microarray analysis showed that the "NK cell mediated cytotoxicity","T cell receptor signaling" and "Primary immunodeficiency" pathway were impaired in tumor infiltrating y8T cells, which closely resembled the results mentioned above.These results confirmed that the cytotoxicity of γδ T cells was greatly impaired in HCC microenviroment. Part III Regulatory T cells mediated functional inhibition of y8T cells in an TGFβ-and IL-10-dependent mannerIn this part, we try to understand the reason why the infiltration and cytotoxic function of y8T cells were decreased in HCC microenviroment. Firstly, we unraveled that γδ T cells and regulatory T cells (Treg cells) had an negative association with each other, moreover, the infiltration and inhibitory function was enhanced in HCC microenviroment. This made us came up with a reasonable hypothesis that Treg cells may inhibit the function of γδ T cells in HCC.Firstly, we purified y8T cells from peritumoral liver tissue by immunomagnetic beads, and Treg cells from HCC tissue by flowcytometry. In vitro cytotoxicity inhibition assay and IFN-y secretion assay were conducted to assess whether Treg cells can inhibit the cytotoxicity function and IFN-y secretion of γδ T cells. Then we try to block the inhibitory effect by neutralization antibodies (namely anti-TGFP or anti-IL-10or anti-CTLA-4antibody). At last, we try to mimic the inhibitory function of Treg cells by recombinate cytokines (namely TGFp or IL-10).We found that the cytotoxicity and IFN-y secretion of y8T cells against HCC cell lines can be inhibited significantly by Treg cells, even at low effector/target ratio. The blocking experiments showed that both anti-TGFβ and anti-IL-10antibody, but not anti-CTLA-4antibody, can greatly block the inhibitory function of Treg cells, but no synergistic action between anti-TGFP and anti-IL-10antibody was seen. Moreover, TGFβ and IL-10can effectively mimic the suppressive effect.These results showed that Treg cells can inhibit the cytotoxicity ability of y8T cells in a TGFβ-and IL-10-dependent manner. Part IV The expression status of interferon regulatory factor (IRF)-1and IRF-2in hepatocellular carcinoma and their association with prognosis and invasion ability of HCCIFN-y put great tumor sculpting pressure on tumor cells, which may change their phenotype and immunogenecity to escape the pressure. The abnormality of signal transduction of IFNs mediated pathways had been observed. Interferon regulatory factor (IRF)-1and IRF-2are the important transduction factors mediated IFNs function in cells, these two factors also played an important role in tumor formation and progression.In this study, immunohistochemistry was used to analyze the nuclear expression of IRF-1/2in a cohort of332HCC patients. The expression of IRF-1and IRF-2in HCC cell lines with stepwise metastasis potential was determined by immunobloting. Down-regulation of IRF-1or IRF-2expression was mediated by shRNAs, a series of experiments were conducted to determine the changes of invasion ability and downstream molecular events.We found that high expression of IRF-1was associated with good outcome (p<0.001for OS/TTR), while high expression of IRF-2was relevant to increased recurrence probability (p=0.049) in HCC patients. The combination of the two IRFs showed better predictive power than either factor alone. Immunobloting analysis revealed that IRF-2/IRF-1ratio was positively correlated with the metastatic potential in human HCC cell lines. Down-regulation of IRF-2lead to sharply attenuated invasion ability, paralleled with a decreased expression of STAT3, p-STAT3Ser727and MMP9; while down-regulation of IRF-1cause a concurrent decrease in IRF-2, little or no change displayed in IRF-2/IRF-1ratio, invasion ability, and MMP9expression.This study unraveled that IRF-1and IRF-2expression were associated with prognosis of HCC patients with opposite predictive power. IRF-2/IRF-1ratio was associated with tumor invasion, probably through modulation of MMP9expression mediated by STAT3. Conclusions1The infiltration of γδ T cells in HCC tissue was significantly decreased compared with paired peritumor tissue, and tumor infiltrating y8T cells displayed immune inhibited phenotype.2The cytotoxicity and IFN-y secretion of y8T cells challenged by no-specific or specific stimuli were greatly impaired in HCC microenviroment.3Regulatory T cells can inhibit the effector function of γδ T cells in a TGFβ and IL-10dependent manner.4IRF-1and IRF-2expression were associated with prognosis of HCC patients with opposite predictive power. IRF-2/IRF-1ratio was associated with tumor invasion, probably through modulation of MMP9expression mediated by STAT3.Novelty1We firstly demonstrated that the infiltration and cytotoxicity function of γδ T cells were substantially decreased in hepatocellular carcinoma microenviroment.2We firstly showed that regulatory T cells mediated γδ T cells suppression in a TGFβ-and IL-10-dependent manner.3We firstly evaluated the prognosis value of IRF-1and IRF-2in hepatocellular carcinoma, and suggested that IRF-2/IRF-1ratio was associated with tumor invasion, probably through modulation of MMP9expression mediated by STAT3. |
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