Expression And Significance Of Tim-3on INKT Cells In Patients With Hepatitis B Virus-associated Hepatocellular Carcinoma

Objective: T cell immunoglobulin mucin-3(Tim-3), a member of thenovel Tim family, has been established as a negative regulatory moleculeand plays a critical role in immune tolerance. Exposure to the Tim-3ligand,galectin-9, triggers cell death in Tim-3+T cells. Evading tumor immuneresponses is an important aspect of the pathogenesis of hepatocellularcarcinoma. Invariant CD1d-restricted natural killer T (iNKT) cells play anallegedly pivotal role in such responces via transactivation of immuneeffector cells. Tim-3is upregulated in exhausted CD8+T cells in bothchronic infection and tumor. It has been reported that Tim-3/galectin-9signaling pathway mediates T-cell dysfunction in patients with hepatitis Bvirus (HBV)-associated hepatocellar carcinoma (HCC) and regulates thehomeostasis of hepatic NKT cells in a murine of nonalcoholic fatty liverdisease. Here, we investigated the expression of Tim-3on iNKT cells inpatients with HBV-associated HCC to reveal the effect of Tim-3in HCCgenesis.Methods: We enrolled15patients with HBV-associated HCC ascases. As controls, another7patients with hepatic hemangioma withnon-HBV infection were included in this study. All patients werepathologically confirmed. Paired tumor and nontumor tissue samples fromthe HCC patients and relatively normal liver tissue samples from thehepatic hemangioma patients were processed to isolate fresh tissueinfiltrating lymphocytes. Fresh blood samples from both HCC and hepatichemangioma patients were processed with the lysing solution to obtainperipheral blood leukocytes (PBLs) after staining. Tumor-infiltratinglymphocytes (TILs) and nontumor-infiltrating lymphocytes (NILs) from HCC tissue samples along with intrahepatic lymphocytes (IHLs) fromnormal liver tissue samples were obtained after Percoll density gradientcentrifugation. Immune cells were stained extracellularly withfluorochrome-conjugate-specific antibodies against human antibodies. Weperformed a comprehensive ex vivo analysis of iNKT cell populations(defined as the clonotypic mAb6B11) and the representation of the CD4+,(CD4-CD8-)DN and CD8+iNKT subsets in PBLs, NILs and TILs ofpatients in cases together with PBLs and IHLs of patients in controls.Meanwhile, we sought to characterize the expression of Tim-3on bothcirculating and intrahepatic human iNKT cells, including their subsets incases and controls.Results:1Levels of Tim-3expression on T cells and subsets increased inHBV-associated HCC TILs. As compared with the levels of Tim-3expression on CD3+T, CD4+T and CD8+T cells among HCC NILs andcontrol IHLs, the counterparts among HCC TILs were the significantlyhighest (Tim-3+CD3+T cells：2.729±1.436%vs.0.729±0.373%and0.600±0.265; Tim-3+CD4+T cells：3.343±1.976vs.1.500±0.896%and1.086±0.367%; Tim-3+CD8+T cells：3.300±1.854vs.1.300±0.635%and0.657±0.244, respectively, P<0.05).2Circulating and intrahepatic iNKT cell populations and thedistribution of iNKT subsets in HBV-associated HCC cases and thecontrols. The frequency of CD3+iNKT cells among HCC TILs wassignificantly lower than that among HCC NILs and control IHLs(0.123±0.089%vs.0.227±0.140%and0.462±0.145%, both P<0.05). Thefrequencies of CD8+iNKT and (CD4-CD8-) DN iNKT cells among TILssignificantly decreased when compared with those among HCC NILs(14.8±9.4%vs.25.2±10.8%;24.4±14.0%vs.41.3±12.6%; respectively,both P<0.05). The frequency of CD4+iNKT cells among HCC TILs wassignificantly higher than that among HCC NILs (37.3±12.9%vs.24.3±7.2%, P<0.05). As compared with the frequency of CD3+iNKT cells among control PBLs, the counterpart among HCC PBLs significantlyreduced (0.152±0.362%vs.0.373±0.200%, P<0.05). The distributions ofthe PBL iNKT subsets beween HCC and hepatic hemangioma patientswere not statistically significant.3Levels of Tim-3expression on intrahepatic iNKT cells and subsetsin HBV-associated HCC TILs, NILs and control IHLs. The level of Tim-3expression on CD3+iNKT cells among HCC TILs was significantly higherthan that among control IHLs and HCC NILs (51.2±8.3%vs.18.4±3.4%and24.1±4.5%, both P<0.05). Meanwhile, the level of Tim-3expressionon CD4+iNKT cells among HCC TILs was significantly higher than thatamong control IHLs and HCC NILs (62.0±6.9%vs.35.4±4.2%and36.8±6.5%, both P<0.05).Conclusions:1Levels of Tim-3expression significantly accumulated on CD3+Tcells, CD4+and CD8+T cells among HCC TILs as compared with thoseamong HCC NILs and normal IHLs.2The frequencies of iNKT cells among HBV-associated HCC PBLs,NILs and TILs decreased as compared with those among controls. It wasworth noting that the distributions of iNKT subsets among HCC NILs andTILs were heterogeneous. The frequency of CD4+iNKT among HCC TILswas significantly higher than that among HCC NILs. By contrast, thefrequencies of CD8+iNKT and (CD4-CD8-) DN iNKT cells among HCCTILs significantly reduced as compared with those among HCC NILs.3As compared with the level of Tim-3expression on CD3+iNKT cellsamong control IHLs and HBV-associated HCC NILs, the counterpartaccumulated among HCC TILs, and the majority of Tim-3+iNKT cellswere the subset of Tim-3+CD4+iNKT cells. Our finding reveals that Tim-3 may play a pivotal role that involves in the HBV-associated HCC immuneresponses by inhibiting the iNKT cells.