Theses and Dissertations from UMD
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Item ROLES OF PLASMA MEMBRANE WOUNDING AND REPAIR IN B CELL ANTIGEN CAPTURE AND PRESENTATION(2022) van Haaren, Jurriaan Jan Hein; Song, Wenxia W; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell-mediated humoral immune responses play a crucial role in neutralizing pathogens and unwanted foreign substances. B cells become activated upon antigen binding of their B cell receptor (BCR), and then internalize and process antigen for presentation on their MHCII for T cell recognition. acquiring T cell signaling through antigen presentation is essential for B cell differentiation into high-affinity antibody-producing cells and memory B cells. In vivo, Antigen encountered by B cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. When B cells engage surface-associated antigen, BCR signaling induces reorganization of the cytoskeleton, causing spreading and contraction of the B cell on the antigen-presenting surface. This allows the B cell to engage more antigen and gather the antigen into a central cluster for internalization. Internalization of surface-associated antigen has been shown to require myosin-generated forces and the exocytosis of lysosomal enzymes. However, the mechanism that initiates lysosomal exocytosis remains unknown.This research explored a possible mechanism for the triggering of lysosomal exocytosis of B cells during interaction with surface-associated antigen. We showed that BCR interaction with antigen tethered to beads, to planar lipid-bilayers (PLBs) or expressed on the surface of live cells causes permeabilization of the B cell plasma membrane (PM), an event that required strong BCR-antigen affinity, BCR signaling, and activation of non-muscle myosin IIA (NMIIA). Moreover, we showed that B cell PM permeabilization triggers a repair response that includes the exocytosis of lysosomes at the site of antigen interaction. Importantly, we showed that B cells undergoing PM permeabilization and subsequent repair internalize more antigen; and better activate T cells compared to unpermeabilized B cells. Thus, our research reveals a novel mechanism for B cells to capture surface-associated antigen: antigen affinity-dependent binding of the BCR indices localized B cell PM permeabilization and lysosome exocytosis as a repair response, which facilitates antigen internalization and presentation through the extracellular release of lysosomal hydrolases. In addition, we explored the molecular mechanism required for B cell PM permeabilization in response to surface-associated antigen. We showed that B cells that undergo PM permeabilization in response to PLB-associated antigen spread over the PLB at a faster rate and to a larger area in comparison to cells that remain intact. Furthermore, we showed that B cells that undergo PM permeabilization recruit more NMIIA at a faster rate, and display a higher level of NMIIA organization at the immune synapse. We additionally discovered a 2o B cell spreading and NMIIA recruitment event, approximately 25-30 minutes after antigen engagement, that facilitates B cell PM permeabilization. Thus, B cell PM permeabilization requires the engagement of a large amount of antigen through B cell spreading on the presenting surface, as well as strong NMIIA recruitment and organization at the immune synapse. This research suggests that B cell PM permeabilization in response to surface-associated antigen plays an important role in distinguishing B cells with various levels of BCR activation, providing novel insights into the mechanisms responsible for affinity differentiation during B cell activation.Item The crosstalk between B-cell receptor mediated signaling and the actin cytoskeleton(2008-08-15) Sharma, Shruti; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Oligomerization of the B-cell receptor (BCR) by antigen leads to both signal transduction and antigen internalization for processing and presentation. Previous studies indicate that these processes intersect at the actin cytoskeleton to coordinate the two cellular processes for the optimal activation of B-cells. The exact mechanism by which signals are transduced via the actin cytoskeleton into the efficient internalization and transport of BCR-antigen complexes is not well delineated. In this thesis, I demonstrate that Bruton's tyrosine kinase (Btk), a Tec kinase in the early signaling pathway of the BCR, is able to transduce signals from the BCR to actin regulatory proteins such as WASP and N-WASP. Upon BCR activation, Btk modulates actin dynamics by increasing the levels of phosphorylated, active WASP and N-WASP in B-cells. Btk regulates the activity of WASP and N-WASP by increasing the levels of PtdIns-4,5-P2 and phosphorylated Vav, both of which are required for WASP and N-WASP activation. Inhibition of Btk activity by a point mutation or a specific inhibitor prevents BCR-induced increases in PtdIns-4,5-P2 as well as in phosphorylated WASP, N-WASP and Vav. Furthermore, Btk deficiency or inhibition leads to a severe reduction in BCR-mediated antigen internalization, processing, and presentation to cognate T-cells. Further studies on the role of WASP show no significant effect of WASP deficiency on BCR internalization, while WASP deficiency affects B-cell development, decreasing the numbers of T1/T2 immature B-cells and marginal zone B-cells. Intriguingly, the protein expression levels of N-WASP and WAVE-2, homologues of WASP, increase in WASP-/- B-cells, implicating a compensatory role for WASP homologues in the absence of WASP. Over-expression of N-WASP's proline-rich domain inhibits BCR-mediated antigen uptake and intracellular transport. All of these data indicate that Btk, which is activated upon BCR binding to antigen, regulates actin dynamics and consequently antigen uptake and transport, by activating WASP and N-WASP via Vav and phosphatidylinositides. This presents a novel mechanism by which BCR-mediated signaling regulates BCR-mediated antigen processing and presentation.