| Objective CD4+CD25+regulatory T cells (Treg) might contribute to tumor progression by suppressing antitumor immunity. To explore more views about the immune escape in ovarian cancer (OC), we aimed to investigate the role of Treg in antitumor immunity regulation in the peritoneal microenvironment of OC compared to the circulating Treg with different clinical characteristics. We further tended to disclose the potential mechanisms Treg mediated in OC ascites in order to supplement more ways about the immunotherapies towards immune escape. Besides, to gain a new view about the integrative treatments towards OC, we preliminarily detect the relationship of chemotherapy and Treg in OC patients.Methods①Flow cytometry was used to detect the proportion of CD4+CD25+Treg in CD4+T cells in ascites of OC patients compared to that in peripheral blood of OC patients with different clinicopathological variables, especially the differences in patients with primary disease, recurrent disease and after chemotherapy.②CD4+CD25+Treg were isolated from ascites and peripheral blood of OC patients by magnetic sorting (MACS) system and tested for regulatory function by coculture with carboxyfluorescein diacetate succinimidylester-labled autologous CD4+CD25-responder cells at different ratios.③To investigate the potential mechanisms Treg obtained from ascites mediate, Transwell insert and neutralizing anti-IL-10antibody or/and neutralizing anti-TGF-β1antibody were employed to explore whether Treg contact conducted suppression function by cell-to-cell contact or/and secreting suppressive cytokine.④Treg isolated from OC ascites were expanded in vitro and then were added in different concentrations to the culture system of human ovarian cancer line SKOV3/A2780. CCK8assay was used to test the influence of Treg on the efficacy of cisplatin treatment on SKOV3/A2780.Results①The Treg in ascites was detected to exhibit significantly increased frequency(27.17%±13.46%) than that(14.15%±4.80%) in peripheral blood of patients with OC (P<0.05). There is no significant difference of the Treg expression in patients age, histological type and tumor differentiation grade with P>0.05, but the-differences among tumor stages were significant (P<0.05). In addition, both in ascites and blood, the Treg of OC in RD or AC shows higher proportionthan that in PD with p<0.0001.②In vitro investigation identified CD4+CD25+suppressor Treg (S cells) obtained from OC ascites could effectively suppress the proliferation of autologous CD4+CD25-responder cells (R cells) at S/R ratios of1:1,1:2,1:5and1:10, and mediated strengthened suppressor ability than circulating Treg with P<0.05. Besides, the suppressor function of Treg from ascites showed significant differences in tumor stage at S/R ratios of1:1,1:2andl:5(P<0.05). And compared to Treg in ascites in PD, suppressor activity of Treg were enhanced in RD but subdued in AC patients with P<0.001.③Suppression of Treg in ascites was mediated by a unique subset of Treg, which produce IL-10and TGF-β1, and cell-to-cell contact between Treg and responder cells for inhibition could contribute to this suppression with P<0.05.④When cisplatin treated SKOV3and A2780for48h in the presence of different concentration of Treg (at SKOV3/Treg or A2780/Treg ratios of1:0.5,1:1,1:2,1:4), the survival rate of SKOV3and A2780both exhibit higher survival rate than that in24h and72h (p<0.01). And at the SKOV3/Treg or A2780/Treg ratios of1:0.5,1:1,1:2and1:4culture systems, after48h of cisplatin treatment, SKOV3and A2780both expressed significantly increased survival rate than the control group without Treg (p<0.05).Conclusion The frequency and suppressor function of Treg were significantly greater in ascites compared with peripheral blood, and showed significant relationship with tumor stage in OC patients, furthermore, Treg in OC ascites might mediate suppression by secreting IL-10and TGF-β1, and cell-to-cell contact could contribute to this suppression. And Treg in ascites and PBMC both showed higher expression in RD and AC patients than that in PD patients. However, compared to Treg in ascites in PD, suppressor activity of Treg were enhanced in RD patients but subdued in AC patients, indicating chemotherapy may favors expansion of Treg, and this expansion may promote the recurrence of cancer. Besides, Treg could decrease the efficacy of cisplatin treatment on ovarian cancer cells. |
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