The Expression Of Th17 Cells And Treg Cells In Primary Hepatic Carcinoma And The Potential Relationship With Development Of Carcinoma

Purpose To investigate the potential relationship between Th17 and Treg cells in the peripheral blood and their functions in the development of primary hepatic carcinoma (PHC) in human beings, to research the interplay mechanisms of tumor cells, Th17 and Treg, to detect the functions of related cytokines, costimulatory molecules and the fist T activation signal to Th17 and Treg in the tumor microenvironment. Methods and Materials Th17 and Treg cells in blood samples from 93 patients with PHC and 51 normal controls were evaluated with flow cytometry (FCM). The mRNA expression levels of IL-1R, IL-6, IL-17A, IL-17F, IL-21, IL-22, IL-23p19, TGF-β, RORc and Foxp3 in PBMC (Peripheral blood mononuclear cell) were detected by Quantitative real-time PCR. The potential interrelationship of Th17 and Treg cells in peripheral blood and the clinical characteristics of the 93 patients, including gender, age, the preoperative concentration of AFP, the diameter of the tumor, portal vein tumor thrombosis (PVTT), metastasis and TNM stages were analyzed. The distribution of Thl7 and Treg cells in tissues were stained with hematoxylin-eosin (HE) and immunohistochemisty with fluorescein labelled antibodies observed with Confocal Microscopy. CD4+T cells or CD4+CD25+ cells or CD4+CD25- cells were cocultured with tumor cells in contact-dependent or contact-independent. The costimulatory molecules CD80, CD86, ICOSL (CD275) in tumor cells were evaluated with FCM. The roles of IL-23p19, IL-6, TGF-β, CD80/CD86 and ICOSL (CD275) in regulating Th17 and Treg cells in the tumor microenvironment were evaluated with neutralizing antibody (antibodies). To eliminate the effect of cytokines and to evaluate the cell contact, tumor cells were irradiated with 150 gray y-ray. Statistical analysis was performed using the software SPSS 16.0. Results Th17 and Treg cells and the ratio of Treg:Th17 in 93 patients with primay hepatic carcinoma (PHC) were increased compared with nomal control group (Normal Control, NC) (4.621%±0.856% vs 3.148%±0.716%, P< 0.0001,8.618%±1.939% vs 4.967%±1.075%, P< 0.0001, 1.878%±0.402% vs 1.621%±0.315%, P<0.0001). Increased Th17 and Treg cells and the ratio of Treg:Th17 in 93 PHC patients were significantly higher than those in the normal controls (P<0.0001). The same tendency was also identified in the mRNA levels of IL-1R, IL-6, IL-17A, IL-17F, IL-21, IL-22, IL-23p19, TGF-β, RORc and Foxp3 in PBMC (P<0.05). The TNM stage was an independent factor for the Th17 and Treg cells increases in PHC patients (t=2.494, P=0.015; t=2.261, P=0.027). The percentage of Th17 and Treg cells in PHC patients had positive correlation with the TNM stage (P<0.05 for each group). The average percent of Th17 and Treg cells in PHC patients were increased with TNM stage (Ⅰ<Ⅱ<Ⅲ<Ⅳ, P=0.0008), where Th17 and Treg cells in patients with advanced stage of PHC (Ⅲ-Ⅳ) were significantly higher than those in early stage (Ⅰ-Ⅱ) (P<0.0001). Compared with peritumoral tissues, Th17 and Treg cells were enriched in tumor tissues of PHC. The percent of Th17 and Treg cells in CD4+T cells were increased after cocultured with tumor cells directively or indirectively, and the percentage of Th17 and Treg cells in CD4+T cells cocultured with tumor cells in cell-contact were much higher than those cocultured with tumor cells isolated by transwells. Neutralizing Ab(s) to IL-23p19, IL-6 and TGF-P singly or in combination, the percent of Th17 cells in CD4+T cells cultured with tumor cells in transwell were descended and the percentage of Treg cells in CD4+T cells cultured with tumor cells in transwell were also descended after blocking TGF-β.The percent of Th17 cells in CD4+T cells cultured with tumor cells in cell-cell contact were descended after blocking CD80, CD86, ICOSL(CD275) singly or in combination. However, the percent of Treg cells in CD4+T cells cultured with tumor cells in cell contact-dependent were increased after blocking CD80, CD86, ICOSL (CD275) singly or in combination. Irradiated tumor cells also promoted Th17 and Treg cells. Intriguingly, CD4+IL-17+ cells can be detected in CD4+CD25+Treg cells cultured with tumor cells and CD4+Foxp3+Treg can be detected in CD4+CD25-T cells cultured with tumor cells. Conclusion The increased percent of Th17 and Treg cells in peripheral blood of PHC are significantly correlated to TNM stages, which indicates that the Th17 and Treg cells might participate in promoting invasion and progression of PHC directly or indirectly. Synchronically increased Th17 and Treg cells in a positive linear correlation (r=0.5132, P< 0.001). The ratio of Treg:Th17 are increased in patients with PHC. Tumor cells in tumor microenviroment can promote Th17 and Treg cells both in direct and indirect manner. Antibodies neutralization certified that IL-23p19, IL-6 and TGF-βmay promoted the differentiation and proliferation of Th17 cells in tumor microenviroment, so as TGF-βto Treg cells, membrane costimulatory molecules CD80, CD86 and ICOSL (CD275) in tumor cells may regulate Th17 and Treg by enhangcing Th17 activation and decreasing expression of Treg cells. The T activation signal also palyed an important role in promoting Th17 cells and Treg cells in the tumor microenvironment. Th17 and Treg cells are promoted by IL-23p19, IL-6 and TGF-P in cell-indepented, where they are regulated by MHC and CD80/CD86 and ICOSL in cell-contact manner. And the phenotype of Th17 and Treg cells can be changed by tumor cells in the tumor microenvironment.