| Objective: This study aimed to investigate the phenotype properties of CD19~+CD20~-B cells and analyse the inhibitory effects of lung cancer cell-induced B cells on T cell proliferation to provide supportive evidence for the immunosuppressive role for this B cell subset.Methods: Peripheral blood mononuclear cells(PBMCs) were collected from patients with lung cancer before any therapeutic intervention(n=26). The diagnosis of lung cancer was made on the basis of biopsy examination. Control samples were obtained from healthy donors(n=11). Flow cytometry was used to detect the ratio of CD19~+B cells as well as its subset CD19~+CD20~-B cells in both patient and control groups. The proportion of CD19~+CD20~-B cells in lung cancer patients was compared between different subgroups to assess their correlations with the clinicopathological features of patients. Multi-color flow cytometry was employed to analyze the phenotype properties of CD19~+CD20~- and CD19~+CD20~+B cells, respectively. The different expressions of Tim-1, PD-1 CTLA-4, CD62 L, CCR6, CD27, CD24 and CD38 were compared between the two subsets. Lung cancer tissues and corresponding para-cancer samples were collected to detect the density of infiltrating lymphocytes. Purified B cells were cultured in medium with B cell-activating factor belonging to the TNF family(BAFF), NCI-H1299 supernatant and NCI-520 supernatant, followed by detection of the changes of molecule markers on B cells. Meanwhile, B cells with different treatments were cocultured with CD3~+T cells to compare their inhibitory effects on CD4~+ and CD8~+T cells proliferation.Results: Multi-color flow cytometry analyse showed that the ratio of CD19~+ B cells to total lymphocytes in lung cancer patients was significantly reduced compared with that in healthy controls(p=0.0013). The level of CD19~+CD20~-B cell subset in CD19~+B cells were higher in patient group than control group(p=0.0379). Lung cancer patients were divided into non-metastatic and metastatic subgroups according to the presence or absence of lymphatic and/or diatant metastasis. We found that the proportion of CD19~+CD20~-B cells was significantly higher in metastatic group compared to that in non-metastatic group(p=0.0341). However, we failed to find any associations between CD19~+CD20~-B cell level and any other clinicopathological features of lung cancer patients, such as age, sex, tumor size and clinical stages.To get a further knowledge of CD19~+CD20~-B cells, we detected the different expression levels of several phenotype molecules between CD19~+CD20~- and CD19~+CD20~+B cells. Our results showed that the inhibitory molecules PD-1(p<0.0001), CTLA-4(p<0.0001) and Tim-1(p<0.0001) was significantly higher in CD19~+CD20~-B cells compared with that in CD19~+CD20~+B cells, suggesting that CD19~+CD20~-B cells might represent a new subset with immunosuppressive function. Meanwhile, CD19~+CD20~-B cells expressed significantly lower levels of CD62 L compared with CD19~+CD20~+B cells(p<0.0001), suggesting that CD19~+CD20~-B cells might have difficulty in accessing to the secondary lymphoid organs, where they encountered with antigens and developed into mature plasma cells. Additionally, the significantly lower expression of CCR6 in CD19~+CD20~-B cells relative to CD19~+CD20~+B cells(p<0.0001) further supported the hypothesis that CD19~+CD20~-B cells were not able to initial humoral response. Further, CD24 level was significantly lower in CD19~+CD20~-B cells than that in CD19~+CD20~+B cells(p<0.0001), while CD27(p<0.0001) and CD38(p<0.0001) were obviously higher in the former than that in the latter. These data indicated that CD19~+CD20~-B cells in lung cancer were different from regulatory B cells(Bregs) in the expression of some surface molecules. When compared with para-tumor tissues, tumor tissues were infiltrated with more CD19~+B cells(p=0.0070) and CD19~+CD20~-B cells(p=0.0120). The results from our functional assays showed that compared with the control group stimulated with BAFF, NCI-H1299 cancer supernatant can effectively induced a subset of B cells with the phenotype CD19~+CD20~-. While, CD19~+CD20~- B cells were not evoked by NCI-H520 cancer supernatant. Furthermore, we observed in vitro that after coculturing with CD3~+T cells, NCI-H1299–induced B cells have stronger inhibitory effect on the proliferation of both CD4~+ and CD8~+T cells than the other two groups.Conclusion: CD19~+CD20~-B cells from peripheral blood were higher in metastatic patients than that in non-metastatic patients. CD19~+CD20~-B cells express high levels of inhibitory molecules. They failed to migrate to secondary immune organs and to initial effective humoal response. B cells induced by NCI-H1299 supernatant in vitro have an inhibitory role on T cell proliferation. |
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