AKT2 Promotes B Cell Activation And Function By Regulating CD19 Phosphorylation And Progranulin Regulates The Development And Function Of NKT2 Cells Through EZH2

PART ? AKT2 PROMOTES B CELL ACTIVATION AND FUNCTION BY REGULATING CD19 PHOSPHORYLATIONBackground and Objective:AKT2 is a serine/threonine protein kinase,which is highly abundant in embryonic brow fat,skeleton muscle as well as in immune cells.It is an important kinase involved in regulating cell metabolism,proliferation,survival and angiogenesis.At present,there are many studies on the role of AKT2 in the occurrence and development of diabetes,and some studies have reported that AKT2 is involved in regulating the functions of T cell,neutrophils and macrophages.However,no studies have been seen to reveal the role of AKT2 in B cells.Methods:In this study,we used AKT2 knockout mice model to study the role of AKT2 in BCR signaling and B cell differentiation.The effects of AKT2 on the development and differentiation of B cells were firstly determined by flow cytometry,and whether the effects on B cells are cell-intrinsic or not were determined by bone marrow chimeric models.An in vivo NP-KLH immune model was established to observe the effect of AKT2on the humoral immune response.The effect of AKT2 on BCR signalosome was detected by confocal microscopy and phosphorylation flowcytometry.The effect of AKT2 on the early activation of B cells and its mechanism were detected by total reflection fluorescence microscopy.Results:?1?There was no difference in bone marrow B cells between WT and AKT2 KO mice,but a decreased GC B cells in AKT2 KO mice compared with WT controls,and the impact of AKT2 deficiency on GC B cells is cell-intrinsic.?2?The levels of both NP-specific IgM and IgG₁ were decreased in AKT2 KO mice,which indicates the humoral immunity is impaired in AKT2deficiency mice.?3?After stimulated with sAg,The Prarson's correlation coefficient between BCR and pY in B cells were measured by confocal and the MFI of pY were analysed by flowcytometry,we found that the Pearson's correlation coefficient between BCR and pY was reduced in the AKT2 deficient B cells.Notably,the MFI of pY was much reduced in the AKT2-deficient B cells,which indicates that BCR signaling is decreased in AKT2 deficient mice.?4?Compared with WT controls,AKT2 KO mice showed impaired B cell spreading,BCR cluster formation and BCR signalosome during B cell early activation.?5?AKT2 deficiency leads to the defect of actin polymerization during BCR activation,and the phosphorylation of WASP was reduced in the B cells absent of AKT2 eventhough the mRNA expression of Was gene has no difference in WT and AKT2 KO mice.?6?The phosphorylation of CD19 is decreased during early B cell activation in AKT2-deficient mice,and there was no significant difference in the mRNA expression of Cd19,the expression of CD19 in MZ and FO B cells has no change between WT and AKT2 KO mice.Conclusion:AKT2 promotes the differentiation of GC B cells,and involves in the humoral responses.It also promotes the BCR signaling and actin remodeling to enhance the early activation of B cells via regulating CD19 phosphorylation,which is important for the function of B cells.PART ? PROGRANULIN REGULATES THE DEVELOPMENT AND FUNCTION OF NKT2 CELLS THROUGH EZH2Background and Objective: Asthma is one of the most common chronic inflammatory pulmonary diseases in children.The development of asthma involves many kinds of immune cells.Invariant natural killer T?i NKT?cells are a special subgroup of T cells that express surface markers of T cells and NK cells.More and more studies have shown that i NKT cells play an important role in asthma.Progranulin?PGRN?is an important secreted protein and expressed in many cell types including immune cells,which mainly antagonize TNF? signaling.The level of PGRN is lower in the serum of patients with bronchial asthma than in normal healthy control,and the lower the expression of PGRN,the more severe the infiltration of neutrophils in the airway.Studies have shown that macrophage derived PGRN could induce NKT cells to produce Th2 cytokines to promote airway inflammation,but the exact mechanism by which PGRN regulates NKT cells remains unclear.In this study,PGRN knockout mice were used to study the regulation and mechanism of PGRN on i NKT cells.Methods: The effects of PGRN on the development and differentiation of i NKT cells were firstly detected by flow cytometry.Whether these effects on i NKT cells are intrinsic was futher illustatrated in bone marrow chimeric models.Then in vivo and vitro stimulation was used to investigated the changes of i NKT secreted cytokines,and Ig E expression and airway resistance were further detected to understand the regulation of PGRN on i NKT cell function.Furthermore,the mechanism of PGRN regulating i NKT cells was investigated by RT-PCR,WB and confocal experiments.Results:?1?The frequency of i NTK cells was increased but the absolute number was decreased in PGRN KO mice compared with WT controls.The stage0 and stage1 i NKT cells were also decreased in PGRN KO mice,which indicates that PGRN deficiency impairs i NKT cell development.?2?The percentage and absolute number of NKT2 were decreased in PGRN KO mice,suggesting that PGRN deficiency results in impaired expansion of NKT2 cells.?3?PGRN KO mice showed impaired IL-4 production in vitro after stimulated with ?-Galcer,indicating that loss of PGRN leads to impaired IL-4 production in i NKT cells.?4?Ig E+ B cells and the titer of Ig E in serum were decreased in PGRN KO mice compared with WT controls,and PGRN could promote the production of Ig E.?5?After stimulated with different concentrations of acetylcholine,the lung resistance of PGRN knockout mice was significantly lower than that of the WT group,indicating that PGRN could promote airway hyperresponsiveness in mice.?6?Compared with WT group,the apoptosis of NKT2 cells in PGRN KO mice was significantly increased,suggesting that PGRN could inhibit the apoptosis of NKT2 cells.?7?The expression of PLZF and nuclear localization of PLZF in i NKT cells from PGRN KO mice were lower than WT controls,indicatin that the PGRN regulates the NKT2 differentiation by promoting the expression of PLZF.?8?Compared with WT group,the expression of Ezh2 was increased in i NKT cells from PGRN KO mice.After treatment of inhibitor of Ezh2,the i NKT cells,NKT2 cells and expression of PLZF were rescued to the degree of WT controls,which indicates PGRN couples with EZH2 to regulate the expression and stability of PLZF.Conclusion: PGRN regulates the development and function of NKT2 cells,which is regulated by inhibiting the expression of Ezh2 and upregulating the expression of PLZF.This is a novel mechanism of how PGRN links to the airway hyperactivity in mice,and might be a new target for the treatment of asthma.