The Distribution, Phenotypic Characteristics And Function Of CD20~+B Cells In Gastric Cancer

Gastric cancer(GC) is one of the common malignancies, and its morbidity and mortality are the top three. The tumor immunity companies with GC development and the clinical outcome of GC patients, while tumor-infiltrating immune cells and their function are the key factors to affect the progress of GC.It has been demonstrated that several immune suppressive cells including Tregs, myeloid derived suppressor cells and tumor-associated macrophages, etc have promoted the progress of GC, but other immune cells such as CD8+ T cells and NK cells have entailed antitumor effects. In recent years, studies have found that CD20+ B cells could be detected in tumor tissues from a variety of malignancies including breast, ovarian, head and neck, non-small cell lung, and colorectal carcinoma as well as melanoma and tumor-infiltrating CD20+ B cells showed strongly correlation with the prognosis of patients. However, the nature and role of CD20+ B cells in GC is unknown. Therefore, the distribution, phenotypic characteristics and function of CD20+ B cells in GC were studied to investigate whether it can be used as a novel marker of diagnosis for GC prognosis to investigate whether it can be used as a novel marker of diagnosis for GC prognosis.Objectives1. To definite the distribution and phenotypic charaterisitics of CD20+ B cells in gastric cancer.2. To investigate the function of CD20+ B cells in gastric cancer.Methods1. The distribution and phenotypic charaterisitics of CD20+ B cells in gastric cancer.The peripheral blood and different tissues(intratumor tissues, peritumor tissues and nontumor tissues) of 30 GC patients were collected from General Surgery of Southwest hospital of Third Millitary Medical University in Chongqing. The proportion of CD20+ B cells was detected by flow cytometry. The surface molecules(CD80, CD86, CD40, IgM, Ig G, HLA-ABC, HLA-DR, IgD, CD39, CD73, CD69 and TRAIL) and chemokine receptors expression of CD20+ B cells were detected by flow cytometry. Moreover, the quantity of CD20+ B cells in tumor tissues and nontumor tissues were also examed by immuno-histochemical staining.2. The preliminary study on the function of CD20+ B cells in gastric cancer.CD20+ B cells were sorted from tumor tissues and nontumor tissues of GC patients, and then co-cultured with purified CD3+ T cells from autologous peripheral blood at 1:1 ratio for 5 days. The proliferation of T cells and the expression of IFN-γ were detected by flow cytometry. In addition, the expression of IL-10 in CD20+ B cells was detected by immunohistochemical staining in tumor tissues.3. Statistical AnalysisStatistical analysis was performed using the GraphPad Prism 5.0 Softwar e. The differences between two groups were analyzed by the Student T test and multiple groups were analyzed by a one-way analysis of variance(ANOVA). P<0.05 was considered statistically significant.Results1.The distribution and phenotypic charaterisitics of CD20+ B cells in gastric cancer.1.1 The percentages of CD20+ B cells in CD45+ cells of peripheral blood, nontumor tissues, peritumor tissues and intratumor tissues were 2.27%±0.35%, 9.23%±1.46%, 8.86%±1.55% and 17.92%±2.24% respectively. Statistical analysis showed that the percentages of CD20+ B cells in intratumor tissues were significantly higher than those in peripheral blood, nontumor tissues and peritumor tissues of GC patients(P<0.001, P=0.002 and P=0.002 respectively), and the percentages of CD20+ B cells in nontumor tissues and peritumor tissues were also higher than those in peripheral blood(P<0.001). However, there was no significance between peritumor and nontumor tissues(P=0.863).1.2 The quantity of CD20+ B cells in tumor tissues was significantly higher than that in nontumor tissues by immunohistochemical staining(P<0.01).1.3 The phenotypic characteristics of CD20+ B cells in GC patients1.3.1 The expression of immunoglobulin on CD20+ B cellsIgD, IgM but not IgG were expressed on CD20+ B cells of peripheral blood, nontumor tissues and tumor tissues obtained from GC patients, however, the expression of IgD and Ig M on CD20+ B cells of nontumor tissues and tumor tissues were lower than those in peripheral blood, suggesting that CD20+ B cells of nontumor tissues and tumor tissues may be at mature status.1.3.2 The expression of CD5 and co-stimulatory moleclues on CD20+ B cellsCD5 molecule was not expressed on CD20+ B cells of nontumor and tumor tissues.High expression of CD40, HLA-ABC and HLA-DR, but low expression of CD80 and CD86 on CD20+ B cells were detected in peripheral blood, nontumor and tumor tissues. However, the expression of CD40 and HLA-ABC on CD20+ B cells of tumor tissues were lower than those in nontumor tissues and peripheral blood, suggesting that CD20+ B cells of tumor tissues have weakly capability to activate CD8+ T cells.1.3.3 The expression of function-associated molecules on CD20+ B cellsThe expression of TRAIL and PD-L2 were not detected on CD20+ B cells in tumor tissues, nontumor tissues and peripheral blood. Compared with peripheral blood, higher expression of CD69 and PD-L1 but lower expression of CD39 and CD73 on CD20+ B cells was detected in tumor and nontumor tissues. The expression of CD73 on CD20+ B cells of tumor tissues was lower than that in nontumor tissues.1.3.4 There was no significance of Ki-67 expression of CD20+ B cells between tumor and nontumor tissues. The expression of chemokine receptor CXCR4 on B cells in peripheral blood and tumor tissues was high, but no expression of CXCR1, CXCR2, or CXCR3. The high expression of CXCR5 on B cells in blood was detected, but no expression on tumor-infiltrating CD20+ B cells.2. The preliminary study on the function of CD20+ B cells in gastric cancer.2.1 Compared with nontumor-infiltrating CD20+ B cells, tumor-infiltrating CD20+ B cells could inhibit the proliferation and IFN-γ production of CFSE-labeled CD3+ T cells.2.2 The expression of IL-10 was detected in CD20+ B cells of tumor tissues by doubleimmunohistochemical staining.Conclusion1. The proportion and quantity of CD20+ B cells were increased in tumor tissues and showed a tendency of immunosuppressive phenotype. In addition, the highly expression of chemokine receptor CXCR4 was detected on CD20+ B cells of tumor tissues and peripheral blood, suggesting that increased tumor-infiltrating CD20+ B cells may be chemotaxised from peripheral blood.2. CD20+ B cells of tumor tissues could inhibit the proliferation and IFN-γ production of CD3+ T cells. CD20 and IL-10 were co-localized in tumor tissues by immunohistochemical staining, suggesting that CD20+ B cells could suppress the antitumor function of CD3+ T cells, and it may depend on the expression of IL-10.