The Potential Role And Mechanism Of Regulatory B Cells In The Immune Regulation In Gastric Cancer

Objective: Immune regulation palys an important role in the development and metastasis of gastric cancer. Recently, new researches have found a new subset of B lymphocytes, which plays a regulatory role —named regulatory B lymphocytes(regulatory B cells, Breg). A lot of researches about Breg have been made in autoimmune diseases, but the role of Breg in gastric cancer has not been clearly. To understand the characterization of regulatory B cells in gastric cancer, and to investigate the possible role and mechanism involved in the progression of human gastric cancer, we designed this study, providing further insight into the biology of Bregs in the context of tumor and other diseases.Methods:(1)In this study we characterized regulatory B cells in healthy controls( N=40) and primary gastric cancer patients( N=107). B cells, Bregs, serum IL-10 and CD4~+T subsets CD4~+ CD25- effetor T subsets, CD4~+ Fox P3~+ Tregs were analyzed by flow cytometry, immunohistochemistry, Real-Time PCR, ELISA, sorting, coculture, neutralization and so on. The percentage of CD19 ~+ B cells among CD45~+ lymphocytes was detected by flow cytometry, and the relationship between the pecentage and TNM stages was analyzed. The distribution of CD19 ~+ B lymphocytes in the tumor tissue from different TNM stages gastric cancer was detected by Immunohistochemical staining.(2)The percentage of CD19 ~+ B cells and CD19 ~+ IL-10 ~+ B lymphocytes in PBMC, normal tissues, adjacent tissues and tumor tissues from gastric cancer were detected by flow cytometry.(3)The mean fluorescence intensity(MFI) of different surface markers expressed in IL-10~+ B lymphocytes and IL-10- B lymphocytes was analyzed by multicolor flow cytometry.(4)The percentage of CD19~+ CD24 hi CD38hi Bregs in CD19~+ B lymphocytes in PBMC from gastric cancer patients was detected by flow cytometry.(5)The percentage of CD3~+ T lymphocytes, CD3~+ CD4~+ Th cells, CD3~+ CD8~+ Tc cells, CD3- CD19~+ B cells, CD3-(CD16~+56)~+ NK cells, CD3~+(CD16~+56)~+ NKT cells, functional cell subsets such as CD4~+ IFN-γ~+ Th1 cells, CD4~+ IL-4~+ Th2 cells and CD4~+ Fox P3~+Treg cells in gastric cancer patients was analyzed by flow cytometry.(6)Further analysis about the correlation among CD19~+ CD24 hi CD38hi Bregs and other cell subsets was peformanced.(7)The function of CD19~+ CD24 hi CD38hi Bregs to the proliferation of CD3~+ T cells, CD3~+ CD4~+ Th cells, CD3~+ CD8~+ Tc cells, to the cytokine secretion of Th cells, Tc cells, to the affection of Treg cells was analyzed by flow cytometry sorting, CFSE staining, cells co-cultured and other methods.(8)Functional blocking antibodies were used to determine the role of IL-10 and TGF-β in the process of Treg transformation. Statistical analysis was performed by Graph Pad Prism 5.0 software.Results:(1) A large number of B cells were detected both in peripheral blood from gastric cancer. But there was no statistic difference in the percentage of CD19~+ B cells between healthy controls and gastric cancer patients(14.03% ± 4.39% vs 15.06% ± 7.47%, P>0.05).(2) The relationship between the percentage of CD19~+ B cells and TNM stages was analyzed(stageⅠ18 cases, stageⅡ40 cases, stage Ⅲ 46 cases and stage Ⅳ 3 cases). The average percent of CD19~+ B cells in gastric cancer patients were decreased with TNM stages(Ⅰ>Ⅱ>Ⅲ) except TNM Ⅳ.(3) A large number of B lymphocytes in the accumulated in the tumor tissues were detected by flow cytometry and immunohistochemical staining.(4) Increased IL-10 in the serum from gastric cancer was detected by ELISA, which was significantly higher than that in the healthy control group(P<0.001). And increased IL-10 producing Bregs were detected both in peripheral blood and tumor local from gastric cancer. Compared with peritumoral tissues, IL-10~+ B cells were enriched in tumor tissues of gastric cancer(P<0.05).(5) The characteristics of IL-10~+ B cells were analyzed by multi-color flow cytometry. It was showed that the mean fluorescence intensity(MFI) of CD5, CD38, CD24 and CD25 in IL-10~+ B cells was significantly higher than that in IL-10- B cells(P<0.001), the MFI of CD1 d, Ig M and CD10 in IL-10~+ B cells was also higher than that in IL-10- B cells(P<0.01), the MFI of CD21 in IL-10~+ B cells was higher than that in IL-10- B cells(P<0.05), whereas there was no no significant difference between IL-10~+ B cells and IL-10- B cells in CD20(P>0.05). In addition, IL-10~+ B cells were CD5~+ CD1d~+ CD38~+ CD24~+ Ig M~+ CD25~+CD10~+ CD21~+ and CD19~+ CD24 hi CD38hi B subset cells possess robust immune regulatory capacity to produce IL-10.(6) Significantly higher percentage of CD19~+ CD24 hi CD38hi Bregs in gastric cancer than in healthy control were detected(P<0.001). Statistical analysis showed that the percentage of Bregs in the advanced gastric cancer(TNM stage III and TNM stage IV) was higher than that in early stage gastric cancer(TNM stage I and TNM stage II) although there was no statistical difference between the two groups(P>0.05).(7) The functional cell subsets such as CD4~+ IFN-γ~+ Th1 cells, CD4~+ IL-4~+ Th2 cells and CD4~+ Fox P3~+ Treg cells were analyzed by flow cytometry. Decreased CD4~+ IFN-γ~+ Th1 cells and increased CD4~+ Fox P3~+ Treg cells detected in gastric cancer(P<0.01,respectively).(8) The correlation between CD19~+ CD24 hi CD38hi Bregs and other cell subsets was further analyzed. There was a positive correlation between Bregs and CD3~+ T cells(R=0.3917, P<0.05) and a positive correlation between Bregs and CD3~+ CD8~+ Tc cells(R=0.5100, P<0.01). And a significant positive correlation between CD19~+ CD24 hi CD38hi Bregs and CD4~+ Fox P3~+ Tregs was revealed in gastric cancer(R=0.4252, P<0.01).(9) CD19~+ CD24 hi CD38hi Bregs do not suppressed the proliferation of CD3~+ T cells, CD3~+ CD4~+ Th cells, CD3~+ CD8~+ Tc cells(P>0.05). However, increased Breg cells suppressed the secretion of IFN-γ and TNF-α by CD4~+Th cells in gastric cancer(P<0.01, respectively).(10) We demonstrated that Bregs induced CD4~+ CD25- T cells to produce more Fox P3 than with other B cells subsets(P<0.01). Our results showed that significantly more TGF-β expression in CD19~+ CD24 hi CD38hi Bregs than other B cells subsets(CD19~+ CD24 int CD38int, CD19~+ CD24~+ CD38low) in gastric cancer(P<0.01 or P<0.05). Significantly reduced Fox P3 expression in CD4~+ CD25- T cells co-cultured with CD19~+ CD24 hi CD38hi Bregs after TGF-β neutralization(P<0.01) while there is no obvious change after IL-10 neutralization(NS, no significant difference, P>0.05).Conclusion: These findings indicated that increased CD19~+ CD24 hi CD38hi Bregs cells play significant immnunosuppressive roles in gastric cancer by inhibiting CD4~+ Th cell cytokine production meanwhile converting CD4~+ CD25- effector T cells to CD4~+ Fox P3~+Tregs dependented on TGF-β in gastric cancer.