| PART ONEStudy on the phenotypic characterization and function of B10 CellsObjective:To investigate phenotypic characterization of IL-10-competent B cells,and identify the regulatory function of B10 cells in healthy donors.Methods:PBMCs were purified,the phenotypic characterization was determined by analyzing cell surface markers after in vitro stimulation.The mean fluorescence intensity(MFI)of different surface markers expressed in IL-10 and IL-10-B lymphocytes was analyzed by multicolor flow cytometry.The proportion of IL-10~+cells in B10 cells were detected by flow cytometric analysis.B10 cells and CD4~+CD25-T cells were sorted by FACS.Cells co-culture was used to explore the function of B10 cells.Functional blocking antibodies were added to the co-culture system to explore the mechanism of B10 cells.Results:(1)The MFI of CD24 and CD38 in IL-10~+B cells was higher than that in IL-10-B cells(P<0.01).The expression of IL-10 was significantly higher in B10 cells than other B cell subsets(P<0.001).(2)B10 cells significantly suppressed the frequencies of CD4~+IFN-γ~+(P<0.01),TNF-α~+(P<0.01)and IL-17~+(P<0.05)T cells and promoted the frequencies of CD4~+IL-4~+(P<0.01)and Foxp3~+(P<0.05)T cells.(3)Blockade of IL-10 inhibited B10 cells from suppressing the frequency of IFN-γ(P<0.01),TNF-α(P<0.001)and IL-17(P<0.05)and promoting the frequency of IL-4(P<0.05)and Foxp3(P<0.05).Blockade of CD80 and CD86 only inhibited B10 cells from suppressing the frequency of IFN-γ(P<0.05)and TNF-α(P<0.05).Conclusion:In this study,we demonstrated that B10 cells belonged to CD19~+CD24hiCD38hi B cell subpopulation.B10 cells inhibit proinflammatory cytokines and promote regulatory cytokines production by CD4~+T cells.B10 cell regulation is dependent on IL-10.PART TWOStudy on the levels of B10 cells in T1DObjective:To compare the levels of B10 cells between TID patients and healthy donors.Methods:Participants of this study include 62 T1D patients and 74 healthy donors.PBMCs were purified,the percentage of different T cell subsets and B10 cells was detected by flow cytometry.Analysis of the correlations among B10 cells and other cell subsets were performed.Results:(1)Increased CD4~+,CD4~+IFN-γ~+,CD4~+TNF-α~+,CD4~+IL-17~+T cells(P<0.05,respectively)and decreased CD4~+IL-4~+T cells(P<0.001)were detected in T1D patients.(2)Significantly lower percentage of B10 cells in T1D patients than in healthy donors were detected(P<0.05).(3)There was a positive correlation between B10 cells and CD4~+CD25~+Foxp3~+(P<0.01)T cells.And there was a negative correlation between B10 cells and the following T cell subsets:CD4~+,CD4~+IFN-y~+and CD4~+TNF-a~+T cells(P<0.05,respectively).(4)There was a negative correlation between B10 cells and HbAlc(P<0.05).Conclusion:The proportions of B10 cells and inhibitory T cell subsets were significantly lower in T1D patients.The positive correlation between B10 cells and inhibitory T cell subsets and the negative correlation between B10 cells and HbAlc demonstrated that the decrease of B10 cells might participate in the development of T1D.PART THREEIdentification of the B10 cells regulating function in T1D and its mechanismsObjective:To identify the function of B10 cells in T1D,and explore whether B10 cells were dysfunction during the development and progressive of T1D.Methods:PBMCs were purified,B10 cells depletion was used to explore the function of B10 cells in T1D.The proportion of IL-10~+cells in B10 cells were detected by flow cytometric analysis.Results:(1)In contrast to healthy donor PBMCs,depletion of B10 cells from the PBMCs of T1D patients did not lead to any significant increase in the percentages of CD4~+IFN-γ~+,TNF-α~+and IL-17~+T cells(P>0.05,respectively).(3)The proportion of IL-10~+cells along with B10 cells was significantly lower in T1D patients(P<0.001).Conclusion:B10 cells in T1D lack these capacities to inhibit proinflammatory cytokines and promote regulatory cytokines production by CD4~+T cells.The dysfunction of B10 cells in T1D may due to the apparently decrease of IL-10. |
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