The Role Of Tumor Microenvironment In Follicular Regulatory T Cells And Its Mechanism

Objective: To study the change of the proportion of follicular regulatory T cells in the pathogenesis of lung cancer by using lung cancer xenograft mouse model, and analysis of the relationship between the disease and Tfr cells. In vitro, Lewis lung cancer cell culture supernatant was simulated to study the effect of tumor microenvironment on the differentiation of Tfr cells, and to explore the role of IL-6 in this process.Methods:(1) The mice model of transplanted tumor of lung cancer was given subcutaneous injection of Lewis cells. In the process of lung cancer, FCM was performed to detect the proportion of Treg cells Tfr cells and Tfh cells in mice abdominal drainage lymph nodes and tumor tissue. The change of the ratio of Treg/Tfr cells and the ratio of Tfh/Tfr cells were analyzed.(2) CD4+CD25+T cells were purified by MACS from the nomal murine spleens, and then co-culture with lung cancer cells, CD4+CD25+T cells were individual cultured as control. FCM was performed to detect the expression of CXCR5 and Bcl-6 on CD4+CD25+T cells. Analysis of the action between the two cells.(3) With serum-free RPMI 1640,murine Lewis lung cancer cells, CT26 colon cancer cells and MFC gastric cancer cells were cultured separately in the same conditions. Culture supernatants were collected and the expression level of IL-6 in different tumor cell culture supernatant was detected by ELISA.(4) CD4+CD25+T cells were purified by MACS from the nomal murine spleens.Each hole with 1 / 4 of total volume of Lewis lung cancer cell culture supernatant. The experimental group of Lewis lung carcinoma cells culture supernatant pretreatment with Anti-IL-6 neutralizing antibody treatment, in order to inhibit the biological activity of IL-6. Control group of Lewis lung cancer cell culture supernatant without any pretreatment. The expression of CXCR5 and Bcl-6 in CD4+CD25+T cells was detected by flow cytometry after 72 h culture.(5) CD4+CD25+T cells were purified by MACS from the nomal murine spleens, rm IL-6(10ng/ml) was added in the experimental group. The expression of CXCR5, Bcl-6 and Foxp3 in CD4+CD25+T cells was detected by flow cytometry after 72 h culture.(6) Lung cancer xenograft mouse model by tail vein injection anti IL-6 neutralizing antibody, with the same amount of PBS as control, and observation of tumor growth. FCM was performed to detect the proportion of Treg cells and Tfr cells in mice abdominal drainage lymph nodes and tumor tissue. The change of the ratio of Treg/Tfr cells was analyzed.Results:(1) With the development of cancer, The proportion of Tfr cells in the abdominal wall draining lymph nodes of the tumor bearing mice was increased, compared to wild mice, the difference was significantand(P<0.01).The proportion of Tfr cells in tumor tissue increased significantly in fourteenth days, and there was a significant difference in the seventh days(P<0.01), the proportion of cells decreased at twentyfirst days.The ratio of Treg/Tfr cells in the lymph nodes and tumor tissues of tumor bearing mice was significantly lower than that of wild-type mice(P<0. 01). The ratio of Tfh/Tfr cells in the lymph nodes of tumor bearing mice was lower than that of wild-type mice(P<0. 05). The results suggest that the tumor microenvironment may promote the differentiation of Tfr cells, and the imbalance of the proportion of Tfh cells and Tfr cells.(2) Compared with the control group, the expression levels of CXCR5 and Bcl-6 were significantly increased in Transwell co-culture group of CD4 + CD25 + T cells. The results indicated that, Lewis lung cancer cells can induce CD4+CD25+T express CXCR5 and Bcl-6 through the indirect action, in order to promote the differentiation of Tfr cells.(3) The expression level of IL-6 in the culture supernatant of Lewis lung cancer cells(276.3±29.9 pg/ml), CT26 cells and MFC cells were only(77.38±13.86 pg / ml) and(145.8±3.26 pg / ml). The result shows, Lewis lung cancer cells have the ability to secrete a large amount of IL-6, that is, a large number of IL-6 in the tumor microenvironment was simulated by Lewis lung cancer cell culture supernatant.(4) Compared with the control group,the expression level of CXCR5 on CD4+CD25+T cells decreased significantly in anti-IL-6 neutralizing antibody treatment group(P < 0.01), but the expression of bcl-6 had no obvious changes. The results suggested that: Lewis lung cancer cell culture supernatant simulation of tumor microenvironment can promote Tfr cell differentiation, which is related to the up regulation of CXCR5 expression and IL-6 content.(5) Compared with the control group, the expression level of CXCR5 in CD4+CD25+T cells was increased in the experimental group, while the expression of Bcl-6 and Foxp3 showed no significant changes. The results suggested that IL-6 could promote the expression of CXCR5 in CD4+CD25+T cells.(6) Compared with the PBS control group, the tumor growth rate of tumor bearing mice injected with anti IL-6 neutralizing antibody was slow,and the enlargement degree of lymph node and spleen decreased.The percentage of Tfr cells decreased significantly(P<0.05)and Treg/Tfr cell ratio was significantly increased(P<0. 05) in draining lymph node tumor tissue. The results suggested that the lack of IL-6 in the tumor microenvironment can inhibit the differentiation of Tfr cells.Conclusions:(1) With the development of cancer, the proportion of Tfr cells in tumor bearing mice was increased.(2) Tumor microenvironment promotes the differentiation of Treg cells into Tfr cells(3) IL-6 promotes the expression of CXCR5 on Treg cells...