| PART Ⅰ DOCK8 REGULATES BCR SIGNALING AND ACTIVATION OF MEMORY B CELLS VIA WASP AND CD19Background: DOCK8 deficiency is an autosomal recessive-combined immunodeficiency characterized by chronic infections with diverse microbial pathogens,elevated serum IgE,severe allergic symptoms and autoimmunity.DOCK8 belongs to GEFs,and it involved in cytoskeleton regulation.The rucurrent sinopulmonary infections,bronchiectasis and life-threatening bacterical infection is possible due to defective production of long-term antibody responses and decreased memory B cells.Memory B cells play a key role in maintaining humoral immune memory,and the early activation is the first step of immune response of MBCs.The role of DOCK8 in B cell development and function has been revealed,but the role of DOCK8 on BCR signaling and function of memory B cells still remains elusive.Methods: In this study,we generated a Dock8 knockout mouse model and collected peripheral blood mononuclear cells from Dock8 patients to study the effect of Dock8 deficiency on the BCR signaling and activation of memory B cells with confocal microscopy and total internal reflection fluorescence microscopy.We detected the B cell subsets by flow cytometry,and the expression of WASP by WB.The relative mRNA levels of cd19,wasp,btk,cd21,and cd81 in splenic B cells examined by RT-PCR.The dual-luciferase repoeter system was used to detect the transcriptional regulation of DOCK8 on cd19.Results:(1)Dock8 is involved in BCR activation.The colocalization between BCR and Dock8 was increased after B cell activation.(2)The absence of Dock8 downregulates BCR signaling,the expression and activation of WASP.The activation of key,positive upstream BCR signaling molecules,pCD19 and phosphorylated Brutons tyrosine kinase(pBtk),is reduced.Interestingly,the total protein and activated levels of Wiskott–Aldrich syndrome protein(WASP)are decreased in Dock8-deficient mouse B cells.(3)The absence of Dock8 disrupts the differentiation of marginal zone and GC B cells.The CD19-mediated Btk signaling has been shown to control the generation of MZ B cells as well as the formation of germinal center(GC)B cells.Consistent with a previous report,We also found Dock8 is indispensable for the generation of peripheral MZ and GC B cells.(4)Our previous research has shown that WASP positively regulates cd19 transcription;furthermore,we found that Dock8 regulates cd19 transcription.What we found in Dock8 patients can be a phenotype copied from Dock8 mice.(5)The early activation of memory B cells from Dock8 patients is disrupted with reduced BCR clustering,B-cell spreading,and signalosome recruitment into the degree ofnaive B cells,as well as the transition from naive B cells to unswitched memory B cells.Conclusion: Our study provides a novel mechanism for Dock8 regulation of BCR signaling by regulating cd19 transcription,as well as the underlying mechanism of noncompetence of memory B cells in Dock8 patients.PART Ⅱ REGULATORY MECHANISM OF TREG FUNCTION IN DOCK2 DEFICIENCYBackground: DOCK2(Dedicator of cytokinesis 2)is a CDM family protein contains DHR1,DHR-2,SH3 domain,and C-terminal polybasic amino acid cluster.DOCK2 specificly expression in the hematopoietic system.DOCK2 patients suffer from early-onset of invasive infections.Functinally DOCK2 rugualtes cells motility and actin polarization via Rac activation.The role of DOCK2 on T cell,B cell and NK cell have been revealed,but the role of DOCK2 on the function of regulatory T cell remains elusive.Methods: Mouse airway resistance was detected by endotracheal intubation,the intestinal of wild-type mice and DOCK2 knockout mice were isolated and intestinal pathological changes were observed by HE staine,the Ig G deposition of the kidney was observed by immunofluorescence.Serum double-stranded DNA was detected by ELISA. We detected the frequency of CD4+ T cell and CD8+ T cell from spleen,peripheral lymphnode,mesenteric lymphnode and thymus between WT and DOCK2 KO mice by flow cytometry.We detected the frequency and MFI of CD25,Foxp3,KI67,Annex V,Neuropilin,CTLA4 and PD1.The expression of CD44 and CD62 L in spleen,peripheral lymphnode,and thymus were detected.The treg stability and proliferation were detected under the stimulation of CD3,CD28 and IL2,and we also analysed the Treg migration in vivo.The treg suppression assay was detected in vitro.We detected the production of IL4,IFN-γ,IL17 in CD4+ T cell and CD8+ T cell after the stimulation of golgistop,PMA and ionomycin.A bone marrow chimeric mouse model was used to verify whether the phenotypic changes after DOCK2 defects were intrinsic.Results:(1)The airway ventilation resistance of DOCK2 KO mice was increased,the intestinal villi were obviously destroyed,the renal Ig G deposition was obvious,while the serum double-stranded DNA was not statistically different from wild type.(2)We found that in DOCK2 knock out mice,the T cell homeostasis was disrupted.The cell number and frequency of CD4+T cell was decreased in DOCK2 knock out mice and it was intrinsic.The naive T cell(CD44low CD62high)in DOCK2 KO mice was decreased,while the actived T cell(CD44high CD62Llow)was increased.(3)DOCK2 deficiency instristiclly increased the percentage of CD25 and Foxp3 in thymic,spleen and pln.The expression o f KI67,Annex V and Neuropilin in Treg was comparable between WT and DOCK2 KO mice,while the expression of CTLA4 and PD1 in CD4+CD25+Foxp3+ cells was dramaticly increased in DOCK2 KO mice.(4)DOCK2 deficiency reduced the treg stability,proliferation and suppression in vitro and migration in vivo.(5)DOCK2 deficiency increases the production of inflammatory T cell lineages.Conclusion: DOCK2 knockout mice develope inflammatory disorder and autoimmune,DOCK2 knockout mice have excessive activation of T cells,and DOCK2 deficiency increases the production of inflammatory T cell lineages.DOCK2 can regulate the percentage,number and function of Treg cells,while the mechanism of how DOCK2 regulate treg is under further study.Overall,our study provides a new phenotype for DOCK2 can regulate Treg’s fuction. |
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