Study On Function And Mechanism Of Regulatory Lymphocyte In Graves’Disease

Part I Study on Effect and Mechanism of Regulatory B Lymphocyte in Graves’Disease(1) Phenotypic Characterization of Regulatory B Lymphocyte and EnumerationObjectives:The objective of this study was to investigate phenotypic characterization of IL-10-competent B cells and compare the quantity of regulatory B cells subset between Graves’Disease and healthy individuals.Methods:Peripheral blood was obtained from36healthy human controls,15new-onset and19recovered GD patients in endocrine outpatient and ward since February2011to December2011. Isolated leukocytes or purified cells were resuspended with CpG, phorbol12-myristate13-acetate (PMA), ionomycin, and brefeldin A (BFA) for5h. The proportion of CD19+IL-10+regulatory B cells in the peripheral blood were detected by flow cytometric analysis. CD19+B cells were isolated from PBMCs.The expression of IL-10mRNA was analyzed using quantitative real-time polymerase chain reaction (PCR) methods. The phenotypic characterization was determined by analyzing cell surface markers (IgM, IgD, CDld, CD5, CD10, CD20, CD24, CD27, CD38, CD40, CD138and B220) after in vitro stimulation with CpG and PIB for5h. The proportion of regulatory B cells subset in the peripheral blood were detected by flow cytometric analysis.Results:In this study, we demonstrated that B10cells in human peripheral blood belonged to a CD24hiCD27+B cell subpopulation. The proportion of B10cells along with the CD19+CD24hiCD27+B cell subset was significantly lower in new-onset patients compared with healthy individuals. In recovered patients, these proportions were restored to levels similar to those seen in healthy individuals.Conclusions: A deficiency in the numbers of CD19+IL-10+regulatory B cells and CD19+CD24hiCD27+B cells is related to the pathogenesis of Graves’disease.(2) Study on Function and Mechanism of Regulatory Lymphocyte in Graves’Disease Objectives:The objective of this study was to compare the suppressive effect of regulatory B cells subset between Graves’Disease and healthy individuals and explore the detailed approach how Bregs from the peripheral blood of GD patients play immunosuppressive role in vitroMethods:Peripheral blood was obtained from6healthy human controls,6new-onset and6recovered GD patients in endocrine outpatient and ward since July2011to December2011.CD19+CD24hiCD27+B cells from the blood of Graves’Disease and healthy individuals were cultured with isolated CD4+T cells in the presence of CpG, CD40L and a plate-bound CD3monoclonal antibody, with or without an IL-10monoclonal antibody. Cells were then harvested and CD4+IFN-γ+,CD4+TNF-a+cells or CD4+CD25hiCD127loFoxp3+were detected by flow cytometry. For T cell proliferation analysis, BrdU was added for the last5hours and detected according to the manufacturer’s instructions for the BrdU ELISA colorimetric assay, we will aim at identify if Bregs from the peripheral blood of GD patients play immunosuppressive role through an IL-10-dependent pathway.Results:CD19+CD24hiCD27+B cells from new-onset and recovered GD patients produced less IL-10compared with healthy individuals. The suppressive effect of CD19+CD24hiCD27+B cells on CD4+T cell proliferation was significantly impaired in both new-onset and recovered GD patients compared with that in healthy individuals. Additionally, in new-onset and recovered GD patients, the ability of CD19+CD24hiCD27+B cells to inhibit the production of IFN-γ and TNF-α in CD4+T cells was also impaired. CD19+CD24hiCD27+B cells from healthy individuals inhibited proliferation and TNF-a production of CD4+T cells via an IL-10-independent pathway. They also inhibited IFN-γ production by CD4+T cells, through an IL-10-dependent pathway.Conclusions: A breakdown of regulatory B cell immunosuppressive functions on proliferation and pro-inflammatory cytokine production in CD4+T cells may be responsible for GD pathogenesis. Incomplete recuperation from these immunosuppressive functions in recovered GD patient may lead to a GD relapse. Part Ⅱ Study on Function and Mechanism of CD4+CD25+regulatory T cells in Graves’DiseaseObjectives:To analysis peripheral blood CD4+CD25+regulatory T cells (Tregs)’ role in the pathogenesis of Graves’ disease (GD), explore their function and changes of peripheral lymphocyte subsets in different periods of GD patients.Methods:New-onset and recovered GD patients together with healthy human controls were included in this study. The proportion of lymphocyte subsets in the peripheral blood were detected by flow cytometric analysis. By using immunomagnetic beads, Tregs were isolated from peripheral blood of these subjects. MTT assay was performed to analyze Tregs’ immunosuppression function.Results:Compared to healthy controls, the proportion of CD3-CD19+B lymphocyte and CD3+CD4+T lymphocyte was significantly higher in new-onset GD patients (P<0.05), while the proportion of CD3-CD16+CD56+NK lymphocyte was significantly lower (P<0.05).The proportion of different lymphocyte subsets was normal in recovered GD. Compared to healthy controls, the proportion of CD4+CD25+Foxp3+regulatory T cells was normal in new-onset and recovered GD, but their immunosuppression function was decreased. Furthermore Tregs’ immunosuppression function was decreased in new-onset GD compared to recovered GD (P<0.05).Conclusions:Breakdown of Tregs immunosuppression function lead to the wild proliferation of CD4+T lymphocyte, and may take an important role in the pathogenesis of GD. Tregs’ immunosuppression function in recovery GD was difficult to fully restore normal which bring about the easy relapse of GD.