Tim-3/galectin-9Signaling Pathway Mediates T-cell Dysfunction And Predicts Poor Prognosis In Patients With Hepatitis B Virus-associated Hepatocellular Carcinoma

Objectives To evaluate the expression of Tim-3and Galectin-9in hepatocellular carcinoma,investigate the relationship between Tim-3and clinical outcome of patients withHBV-associated hepatocellular carcinoma.Methods38paired fresh tissues were collected from HCC patients, including30HBV+cases and8HBV-cases in different experiments. Fresh HCC tissues and surroundingnontumor adjacent liver tissues (at least3cm distant from the tumor site) were used for theisolation of tumor and non-tumor-infltrating leukocytes. The expression of Tim-3andGalectin-9on immune cells were analyzed with flow cytometry analysis. The protein leveland mRNA level were detected with immunohistochemistry,western blotting and real Timequantitative PCR. For survival analysis, we followed99HBV-associated HCC patientsafter surgical resection from January2007to April2010.Results KCs in HCC expressed the highest levels of galectin-9in HCC, the expression ratewas40%, which was higher than in mDCs, pDCs, T cells and tumer cells. The KCsexpressed the higher levels of galectin-9in HBV+patients than in HBV-patients. Flowcytometry analysis showed that Tim-3was expressed on tumor-infltrating CD4+and CD8+T cells. In HBV+patients, the levels of Tim-3+CD4+T cells were higher than that of CD8+ T cells, and in HBV-patients the Tim-3had a rare expression. The number of Tim-3+cellsin HCC tissues was a negative prognostic factor of overall survival.Conclusion The Tim-3and Galectin-9were significantly overexpresion in human HBVassociated hepatocellular carcinoma, and the Tim-3expresion in HCC is associated withthe survival, which indicated a worse prognosis for patients, So Tim-3may be play aimportant role in regulating immune response in HCC microenvironment. Objectives To study the function of Tim-3+T cells and the corresponding mechanisms.Methods38paired fresh tissues were collected from HCC patients, including30HBV+cases and8HBV-cases in different experiments. Fresh HCC tissues and surroundingnontumor adjacent liver tissues (at least3cm distant from the tumor site) were used for theisolation of tumor and non-tumor-infltrating leukocytes. Phenotypes and cytokines ofTim-3+T cell were analyzed with flow cytometry analysis. Tim-3+CD4+T cells were sortedand applied for coculture with KCs and cell cycle analysis, IFN-γ production was examinedby ELISPAOT. CSFE was used to lable the Tim-3+CD4+T cells for T-cell expansion assay.RT-PCR was used to quantify the expression of key genes controlling cell cycle andcellular senescence.Results Tim-3+CD4+T cells have reduced proliferation and activation potential in HCC,and secreted less cytokines than Tim-3-CD4+T cells, such as IL-2and IFN-γ, also the Ki-67and HLA-DR which associated with cell proliferation were reduced in Tim-3+CD4+T cells.The impaired T cell effector function could be restored by blocking of Tim-3/Galectin-9pathway with specifc anti-Tim-3mAb. Cell cycle analysis revealed that the cell cycle wasarrested in G1/S phase, and G1/S phase-associated genes CDK2, CDK4, CCND1, CCNE1 was increased.Conclusion The study indicated that Tim-3/Galectin-9pathway may involved in animmunosuppressive niche in HCC. It impairs T cell function in antitumor affect. The affectmay be related to cell cycle arrest. These findings could provide us an immunotherapeutictarget in patients with HBV-associated HCC.