Effects Of Myosin ⅡA On The Development And Function Of Regulatory T Cells And Study On The Mechanism Of Mst1/2 Involved In Early BCR Signal Transduction

PART Ⅰ EFFECTS OF MYOSIN IIA ON THE DEVELOPMENT AND FUNCTION OF REGULATORY TCELLSObjective: Myosin II is a member of the myosin superfamily that performs many mechanical tasks in cells.According to its heavy chain,it can be divided into myosin IIA,myosin IIB and myosin IIC.Among them,myosin IIA(coded by Myh9 gene)is mainly expressed in immune tissues and organs.Based on Treg cell-specific Myh9 gene knockout mouse models,this study explores the effect of myosin IIA on the development and function of regulatory T cells.Methods: Firstly,the control mice(YFP-Foxp3Cre/+)and Treg cellspecific Myh9 gene knockout mice(YFP-Foxp3Cre/+Myh9flox/flox)were acquired from hybridization between YFP-Foxp3Cre/+Myh9flox/+ female mice and YFP-Foxp3Cre/YMyh9flox/+ male mice.In control and Myh9 knockout mouse models,the survival status and immune status of mice were firstly observed.And their size change of the immune organs was observed.Meanwhile,other non-immune organs(lungs,kidneys,liver and skin)were isolated for H&E staining to observe their lymphatic infiltration.Secondly,flow cytometry was used to detect the proportion of CD4+ and CD8+ T cell subsets and their na?ve and effector-memory T cell in the thymus and peripheral lymphoid tissues(spleen,peripheral and mesenteric lymph nodes)of mice.Additionally,cytokine production was detected after in vitro stimulation.Further,flow cytometry was used to detect the proportions of CD25+ Treg cells and Foxp3+ Treg cells and their expression of suppressive molecules(ICOS,CTLA4 and PD1).Finally,splenic CD25+ Treg cells and CD4+ na?ve T cells were enriched with magnetic beads,and these two cell subsets were co-cultured in different ratios in vitro to detect the suppressive function of Treg cells on the proliferation of CD4+ na?ve T cells.Result:(1)Foxp3Cre/YMyh9flox/flox mice showed severe lethal autoimmune disease,shortened lifespan,and enlarged spleen and peripheral lymph nodes.H&E staining showed extensive lymphocyte infiltration in lung,kidney,liver and skin.(2)Foxp3Cre/YMyh9flox/flox mice showed increased inflammatory cytokine production,including IL2,IL4,IL17 and IFNγ of CD4+ T cells and IFNγ of CD8+ T cells in the thymus,spleen,peripheral and mesenteric lymph nodes.(3)The proportion of CD4+ T cells in Foxp3Cre/YMyh9flox/flox mice was significantly increased in the thymus,but decreased in peripheral immune organs(peripheral and mesenteric lymph nodes).Further,CD4+ and CD8+ na?ve T cells were significantly reduced,while CD4+ and CD8+ effector-memory T cells were significantly increased.(4)In Foxp3Cre/YMyh9flox/flox mice,the proportions of CD25+ Treg and Foxp3+ Treg in the thymus were significantly increased,while there was a decreased proportion of Foxp3+ Treg cells in the periphery.(5)It was found that suppressive molecule(ICOS,PD-1,CTLA4)expressions were increased in CD25+ Treg cells,and CTLA4 was also increased in Foxp3+Treg cells.(6)The suppressive function assay found that the splenic CD25+Treg cells of Foxp3Cre/YMyh9flox/flox mice had a stronger suppressive effect on the proliferation of na?ve CD4+ T cells than that in the control group.Conclusion: Treg-specific Myh9 deficient mice have lethal and severe autoimmunity,which is manifested by extensive lymphocyte infiltration in multiple organs,excessive activation of T cells and increased production of cytokines.Thymic Treg cells are significantly increased,while peripheral Treg cells are significantly reduced.In vitro experiments suggest that the function of splenic CD25+ Treg cells was enhanced.The current study revealed the importance of myosin IIA on Treg cell development and function.PART Ⅱ STUDY ON THE MECHANISM OF MST1/2 INVOLVED IN EARLY BCR SIGNAL TRANSDUCTIONObjective: Mst1 and Mst2(encoded by STK4 and STK3 genes)are two highly homologous mammalian STE20-like kinases in the Hippo signaling pathway.Loss of function mutations in Mst1 can cause immune deficiency and lymphocytopenia,as well as autoimmune manifestations.Previous studies have found that Mst1/2 were involved in regulating the central and peripheral development of B cells,and the early B cell receptor(BCR)signaling pathway is important for the development of B cells.However,the mechanism of Mst1/2 in regulation of early BCR signaling has not been elucidated.In this study,we explored the roles and molecular mechanism of Mst1/2 in early BCR signal transduction based on knockout mouse model.Methods: Firstly,knockout mouse models were generated as follows:Mst1KO mouse(Mst1-/-),Mst2 KO mouse(Mst2flox/floxCD19Cre/+)and Mst1/2DKO mouse(Mst1flox/floxMst2flox/floxCD19Cre/+).Based on these mouse models,splenic mononuclear cells were acquired by density gradient centrifugation,and splenic B cells of each group were enriched.Next,enriched splenic B cells were stimulated with soluble antigen(s Ag)in vitro,and the expression of key regulatory molecules and distal molecules in the BCR signaling pathway were detected by confocal microscopy,flow cytometry and western blotting.Meanwhile,an antigen-tether lipid bilayer was constructed to mimic membrane associated antigens(m Ag)activating B cells in vitro,total internal reflection fluorescence microscopy(TIRFm)was used to dynamically observe the activity of key molecules of BCR signal transduction in the contact zone of B cells at several time points.Result:(1)Using s Ag to stimulate mouse splenic B cells,compared with the control mice,the activation level of key regulatory molecules(p CD19,p BTK and p SHIP),and distal axis of AKT-S6 in the BCR signaling pathway were significantly decreased in Mst1 KO mice and significantly increased in Mst2 KO mice,while there were no significant changes or slightly lower in Mst1/2DKO mice.(2)Meanwhile,TIRFm was used to observe the splenic B cells activated by m Ag.The results showed that compared with the control mice,the formation of BCR clusters and expansion of B cells were attenuated in the Mst1 KO mice,but no significant change in the Mst2 KO mice,while B cell expansion and BCR cluster formation were also reduced in Mst1/2DKO mice,but to a lesser extent than that in Mst1 KO mice.(3)It was found that compared with B cells activated with m Ag in control mice,the key regulators of BCR signaling(p CD19,p BTK,p SHIP)were significantly decreased in Mst1 KO mice,and increased in Mst2 KO mice,and decreased in Mst1/2DKO mice but to a lesser extent than that in Mst1 KO mice.Conclusion: Mst1/2 have important regulatory roles in early BCR signal transduction.Among them,Mst1/2 are involved in early BCR signaling during B cell activation via CD19,BTK and SHIP.This study provides a new idea for analyzing the molecular mechanism of BCR signal transduction and Mst1 deficiency leading to humoral immune deficiency.