Effects And Mechanism Of WASp On The Regulation Of Immunological Synapse,cellselection And Viability In Germinal Center B-cell

PART ONE EFFECTS OF WASP-DEFICIENCY ON THE ARCHITECTUREANDFUNCTION OF IMMUNOLOGICAL SYNAPSE IN GERMINAL CENTER B-CELLBackground:B-cells undergo somatic hypermutation and affinity maturation in germinal centers.Somatic hypermutated germinal center B-cells(GCBs)compete to engage and capture antigen on follicular dendritic cells(FDC).Recent studies show that when encountering membrane-antigen,GCBs generate actin-rich pod-like structures with B-cell receptor(BCR)microclusters to facilitate affinity discrimination.While deficiencies in actin regulators,including Wiskott-Aldrich syndrome protein(WASp),cause B-cell affinity maturation defects,the mechanism by which actin regulates BCR signaling in GCBs is not fully understood.Objectives:To determine the unique architecture of immunological synapse in germinal center B cells which facilitates affinity discrimination,and to explore the mechanism by which WASp regulates the activation of germinal center B cells.Methods:Using SRBC-immunized WASp knockout(WKO)mice that express Lifeact-GFP and live-cell total internal reflection fluorescence(TIRF)imaging,this study examined the role of WASp-mediated branched actin polymerization in the GCB immunological synapse.Results:After rapid spreading on antigen-coated planar lipid bilayers,GCBs formed microclusters of phosphorylated BCRs and proximal signaling molecules at the center and the outer edge of the contact zone.The centralized micro-signaling clusters localized at actin-rich GCB membrane protrusions.WKO reduced the centralized micro-signaling clusters by decreasing the number and stability of f-actin foci supporting GCB membrane protrusions.The actin structures that support the spreading membrane also appeared less frequently and regularly in WKO than in WT GCBs,which led to reduction in both the level and rate of GCB spreading and antigen gathering.Conclusions:WASp showed essential role in the generation and maintenance of unique structures for GCB immunological synapses which facilitates the antigen gathering and BCR signaling upon activation.PART TWO EFFECTS OF WASP-DEFICIENCY ON THE B-CELL SELECTION AND VIABILITY IN GERMINAL CENTERBackground:Wiskott-Aldrich Syndrome protein(WASp)-deficiency causes severe immune disorders.WAS patients mount poor antibody responses to vaccination while accumulating autoantibodies.WASp-deficient B-cells form spontaneous germinal centers and produce class-switched autoantibodies in the presence of WT T-cells.We previously found that immunogen-specific Ig G levels and their affinity were significantly lower in WAS patients and WASp knockout(WKO)mice than in healthy and WT controls.Objectives:To examined the mechanism by which WASp regulates the B-cell selection in germinal centers and the maintenance of cell expansion.Methods:Using SRBC-immunized WASp knockout(WKO)mouse model,this study examined the ability of germinal center B cells to internalize antigen,to process and express antigen on surface MHC-II peptide as well as to cognate interact with T cells.Furthermore,we researched on the viability and its regulating factor of germinal center B cells.Results:Regarless of comparable level of germinal center B cell percentage and total Ig G production,we found reduced percentages of antigen-specific germinal center B-cells(GCB)in WKO mice.And the proximal signaling of the B-cell receptor(BCR)was only moderately altered by WKO,and the BCR internalized soluble antigen at a similar rate in WKO and WT GCB.Importantly,the ability of WKO GCB to present antigen and interact with cognate T-cells was defective,consistent with a decreased expression of ICAM-1.Surprisingly,WKO GCB had a higher surviving and a lower apoptosis rate than WT GCB,which was associated with lower production of ROS and mitochondrial superoxide in WKO GCB.Mitochondria in WKO GCB exhibited lower levels of Mito Tracker staining,larger volumes,and longer shapes than those in WT GCB.While f-actin levels were drastically reduced in WKO GCB.Mitochondria fission regulator DRP1 maturation and f-actin foci also colocalized less with mitochondria.Conclusions:Our data together demonstrate essential roles for WASp in both acquiring T-cell help for the activation of antigen-specific GCB and driving non-specific GCB to death through mitochondrial dysfunction.