Effect Of Myeloid-derived Suppressor Cells On B Cell Function In Mice Bearing Breast Tumor

Objective:The effects of myeloid-derived suppressor cells(MDSCs)on various immune cells such as T cells and DC cells has been clearly reported,but the effects and mechanisms of MDSC on B cells have not been elucidated.Therefore,exploring the effect of MDSC on the function of B cells may help to more fully understand the negative regulation of MDSC on the multi-cell network of immune response in breast cancer.And it makes the researchers more deeply cognition on immune suppression of B cells.Moreover,it provides a new research approach for exploring the mechanisms of tumor,autoimmune tolerance and autoimmune diseases and the application of immunotherapy.METHODS:A BALB/c mouse 4T1 breast cancer model was established.The mouse spleen MDSC and normal mouse spleen B cells were sorted by magnetic beads,and the MDSC and B cells were co-incubated.Flow cytometry was used to test expression of PD-1,PD-L1,CTLA-4,CCR6,CD62L and MHCII on B cell;ELISA assay was used to detect the secretion of IgA,IgM and IgG on B cells in supernatant of the co-culture;BrdU detected B cell proliferation;Annexin V/PI detected B cell apoptosis.B cells in the co-culture system were again sorted by magnetic beads and were co-cultured T cells,BrdU was used to detect T cell proliferation,and Annexin V/PI was used to detect T cell apoptosis,and the secretion of T cells were detected by flow cytometry.Moreover,we have defined a new population of PD-1~-PD-L1~+B cells with immunoregulatory functions.To compare the immunosuppressive function of PD-1~-PD-L1~+B cells with traditionally defined regulatory B cells,we sorted different B cell subsets from the spleens of tumor-bearing mice and co-incubated with T cells.T cell proliferation was detected by BrdU kit;T cell apoptosis was detected by Annexin V/PI apoptosis kit;and cytokine secretions by T cells were detected by flow cytometry.To assess the significance of PD-1~-PD-L1~+B cells in the entire tumor microenvironment,we used flow cytometry to detect the proportion of PD-1~-PD-L1~+B cells in co-culture systems and spleen,bone marrow,peripheral blood,lymph nodes,and tumor tissues of tumor-bearing mice and the ratio of PD-1~-PD-L1~+B cells in peripheral blood of breast cancer patients.Results:Compared with B cell control group,in B+MDSC group the expression of PD-L1 on B cells was increased,and the expression of PD-1,CTLA-4,CCR6,CD62L and MHC?were decreased;The IgA,IgM and IgG secreted by B cells were significantly increased;the proliferation of B cells was increased and the apoptosis was decreased.Compared with the T cell control,the proliferation of T cells in the(B+MDSC 1:5)+T group was significantly lower,and there was no significant difference in T cell apoptosis.Compared with the inhibition of T cells by the traditional regulatory B cells,PD-1~-PD-L1~+B cells have much bigger inhibitory effect,including T cell proliferation,T cell apoptosis and the secretion of IFN-?.Compared with the B cell control group,the proportion of PD-1~-PD-L1~+B cells in the B+MDSC group was significantly increased,and it is same in the spleen,bone marrow,peripheral blood,lymph nodes and tumor tissues of the tumor-bearing mice,and in the peripheral blood of breast cancer patients.Conclusion:Tumor-derived MDSCs can regulate the normal immune function of B cells,such as the expression of B cell surface molecules,the antibodies secretion of B cell,B cell proliferation and apoptosis.Moreover,MDSC can induce normal B cells transform into PD-1~-PD-L1~+B cell with immunosuppressive effects,which can inhibit the proliferation of T cells and the secretion of cytokines and promote the apoptosis of T cells,thereby it can weaken the immune response of T cells.