Recruitment And Differentiation Of TH22 Cells In Malignant Ascites Due To Hepatic Carcinoma

Objective:To determine the distribution and phenotypic features of Th22 cells in both MA and blood from hepatic carcinoma patients, and elucidate the mechanism by which Th22 cells differentiate and recruit into the peritoneal space. Methods:Five hundred ml of ascites and 15ml Peripheral Blood samples were collected from 37 patients with newly diagnosed hepatic carcinoma with MA. None of these patients had received any anticancer therapy, corticosteroids, or other nonsteroid anti-inflammatory drugs. The cell pellets of MA were resuspended in HBSS, and mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation to Determine the distribution and phenotypic features of Th22 and Th17 cells in MA and Peripheral Blood. The expression markers on T cells from MA and blood were determined by flow cytometry after surface staining or intracellular staining with antihuman-specific Abs conjugated with either PE or FITC. Including anti-CD4, anti-CD45RA, anti-CD45RO, anti-CD62L, anti-CCR3, anti-CCR4, anti-CCR5, anti-CCR6, anti-CCR7, anti-IL-17, anti-IL-22 and anti-IFN-γmAbs. Purified naive CD4+T cells were cultured in 1ml complete medium with IL-2-containing medium providing a baseline for comparison, and stimulated with exogenous cytokines TNF-α(50 ng/ml)、IFN-γ (50ng/ml)、IL-1β (20ng/ml)、IL-6 (100ng/ml) for 7d then analysed by flow cytometry. Purified CD4+ T cells from blood were added into the top chamber resuspended in RPMI 1640 medium with 0.5% FBS, MA was placed in the bottom chamber, and the chambers were incubated 3h. Finally, the total cells migrated into the bottom chamber were harvested and intracellular stained for IL-22 and then analyzed by flow cytometry as above described. At the end of incubation, The chemotaxis index was calculated by dividing the numbers of cells migrated in response to test MA or recombinant human chemokines by the numbers of cells migrated in response to medium alone. To demonstrate that CCL20 or CCL22 was responsible for Th22 cell migration, blocking experiments were performed by mixing the MA with anti-CCL20, anti-CCL22, anti-CCL27. Results:Th22 cells in MA(3.22%±0.4%,n=37) were obviously higher than Peripheral Blood (0.79%±0.2%, n=37, P<0.01) from MA patients and healthy controls(0.63%±0.11%, n=10, P<0.01), Th17 cells in MA(3.30%±0.18%) were obviously higher than Peripheral Blood (0.79%±0.15%, n=37, P<0.01) from MA patients and healthy controls (0.67±0.12%, n=10, P<0.01)Th22 cells correlated positively with Th17 cells in MA(P<0.01). Th22 cells expressed low levels of CD45RA(10.51%±0.72%vs18.20%±0.78%,P<0.01), CD62L(7.05%±0.49%vs13.66%±0.68%, P<0.01), CCR3(5.77%±0.50%vs5.51%±0.53,P>0.05), CCR5(9.21%±0.82%vsl0.84%±0.71, P<0.01), high levels of CD45RO(83.34%±0.96%vs80.08%±1.14%, P<0.01), CCR4(72.48%±1.74%vs68.70%±1.61, P>0.05), CCR6(83.88%±1.68%vs85.61%±1.61,P>0.05), CCR10(88.13%±1.24%vs89.23%±1.31,P>0.05), CCR7 is (52.61%±2.67%vs42.33%±2.44%, P<0.05). Th22 cells were facilitated by interleukin-6 (IL-6), IL-lb, and/or tumor necrosis factor-α(IFN-α), and Inhibited by interferon-γ(IFN-γ) differentiate from CD4+T cells. Differentiation of Th22 cells showed a dose and time dependent rules by IL-6. MA was chemotactic for Th22 cells, that could be interdicted by anti-CCL20,-CCL22,and -CCL27 mAbs.Conclusion:Chemokines and cytokines may contribute to the increased Th22 cells in MA. Th22 cells probably participate in the pathogenic mechanism of MA and may play an important role in the immune regulation of the human peritoneal malignant environment.