Abstract
B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The following additional data were added to the previous version: 1) An additional probe was used to demonstrate membrane permeabilization. We utilized the membrane-impermeant molecule Ponceau 4R, which quenches CFSE preloaded in B-cells upon entering the cytoplasm, to successfully detect B-cell permeabilization during interaction with surface-associated antigen (new Figure 2). Ponceau 4R potently absorbs emissions in the 490-560 nm range, but only when directly contacting the emitting fluorophore (in this case, CFSE in the B-cell cytoplasm). Thus, consistent with our prior results using the membrane impermeant dyes propidium iodide and lipophilic FM probes, these new results directly and independently demonstrate that the plasma membrane of B-cells is permeabilized when binding surface-associated antigen. 2) A malleable physiological substrate was used to trigger B-cell plasma membrane permeabilization (new Figure 3D-G). B-cells from MD4 mice become permeable to propidium iodide when binding to COS-7 cells expressing membrane-integral HEL. In contrast, significantly less permeabilization is observed when the same assays are performed with wild type B-cells that do not recognize mHEL, or with MD4 B-cells on mock-transfected COS-7 cells.