Role Of Th9 Cells In The Pathogenesis Of Malignant Ascites Of Patients With Hepatic Carcinoma

Objective:Newly discovered IL-9-producing CD4+ helper T cells(Th9 cells)have been identified to be increased in some human cancers.However,the possible implication of Th9 cells in regulating antitumor responses in malignant ascites(MA)remain to be elucidated.Because lymphocyte migration is tightly regulated by chemokine-chemokine receptor interaction,we therefore sought to elucidate the effects of chemokinec-hemokine receptor interaction on recruitment of Th9 cells into MA from patients with hepatic carcinoma.The percentages of peritoneal Th9 cells in comparison with those in the corresponding blood and peripheral blood from healthy controls were also examined in our study.Design and Methods:We studied 30 hepatic carcinoma patients with MA,15 healthy controls.The expression of IL-9 and IL-17,as well as CCR4 and CCR6 were examined in MA and healthy controls by flow cytometry.The specimens IL-9 and IL-17 levels,as well as chemokines CCL20 and CCL22 in MA and sera were examined by Enzyme-linked immunosorbent assay.Naive CD4+ T cells were isolated from blood from hepatic carcinoma patients by MACS(Magnetic beads immune cell sorting)based on negative selection using the Naive CD4+ T cell isolation kit.Then the TranswellTM system was used to detect the chemotactic effect of chemokine CCL20 to Th9 cells.Results:1.The percentages of Th9 cells represented the higher values in MA,showing a signifcant increase in comparison with those in the corresponding blood and healthy controls.Similarly,significant increases in Thl7 cells was observed in MA compared with those in blood and healthy controls.A positive correlation was found between Th9 cells and levels of IL-9 in MA patients.Moreover,positive correlations were also found between serum levels of IL-9 and Th17 cells in MA patients.2.The levels of IL-9 were significantly increased in MA compared to those in the corresponding blood and healthy controls.The levels of IL-17 were significantly increased in MA patients compared to those in the corresponding blood and healthy controls.3.Th9 cells expressed high level of CCR6 in both MA and those in the corresponding blood,which are ligands for CCL20.However,Th9 cells expressed low levels of CCR4 in both MA and blood.Taken together with our observation that concentrations of chemokines CCL20 and CCL22 in MA were much higher than their compartments in serum.4.Indeed,an in vitro migration assay confirmed that MA could induce the migration of Th9 cells and that anti-CCL20 mAb significantly blocked the ability of MA to stimulate Th9 cell chemotaxis.5.There was a significant correlation between peritoneal Th9 cell numbers and patient survival(P=0.02).Patients with lower Th9 cells had significant longer overall survival than patients with higher Th9 cells.Individuals in the high Th9 cell group experienced a 2-fold higher death hazard as compared with those in the low Th9 cell group.6.In addition,the percentages of Th9 cells correlated positively with AFP,but not with CEA,CRP,LDH,AST and ALT.Conclusions:1.MA exerted potent chemoattractant activity for circulating Th9 cells,and anti-CCL20 mAb significantly suppressed Th9 cell chemotaxis.It could be concluded that the recruitment of Th9 cells into MA might be induced by a Chemokine-Dependent mechanism.The overrepresentation of Th9 cells into MA could be induced by CCL20/CCR6 axes.2.Moreover,the accumulation of Th9 cells in MA predicted poor patient survival.3.These findings suggest that Th9 cells may be implicated in the pathogenesis of MA,and Th9 cells may be reasonable cellular targets for therapeutic intervention.