Phys. bonds and promoted internalization of only high-affinity, multivalent BCR microclusters. Thus, B cell contractility contributes to affinity discrimination by mechanically testing the strength of antigen binding. Efficient antibody responses require selective expansion of B cell clones that recognize foreign antigens with high-affinity. B cells are initially stimulated by the binding of their B cell receptors (BCRs) to antigens on the surfaces of antigen-presenting cells (APCs) (1-5). During these cellular contacts, termed immune synapses, B cells acquire the antigens from the APCs (1, 2, 6), which leads to B cell antigen processing and presentation to helper T cells. The extent of T cell help, and resulting B cell activation, depends on the BCR affinity for antigen (7-9). Therefore, efficient affinity discrimination during antigen acquisition is essential for B cell clonal selection. Although B cell synapse formation is sensitive to antigen affinity (10, 11), the TNP-470 mechanisms by which B cells extract antigens from APCs remain poorly TNP-470 understood (12, 13). To study this process, we developed an experimental model for studying HSPA1 immune synapses using immobilized plasma membrane sheets (PMS). PMS are made from plasma membranes of adherent cells (14) and are suspended approximately 10 nm above the coverslip (Fig S1). Decoration of the exposed surfaces of PMS with antigens, but not with control proteins, induced B cell spreading and antigen clustering that resembled B cell synapses with planar lipid bilayers (PLB), an alternative model substrate (10, 15). However, unlike synapses with PLB, B cells rapidly internalized the antigen from synapses made with PMS (Fig. 1A, B, Movie S1). The ability of B cells to internalize antigen was not a result of the composition TNP-470 of the PMS, as PLB prepared from plasma membranes (PM-PLB) did not support antigen internalization (Fig. 1B). In addition, the internalization did not correlate with lipid or antigen diffusion within these substrates (Fig. S2). Open in a separate window Fig. 1 B cells acquire antigens from flexible membranes. (A) Sideview reconstruction of B220-stained primary B cells forming synapses with DiI-stained and anti-Ig antigen-loaded (Ag) PMS or PLB. Arrowheads indicate the position of the substrate. Scalebars, 2 m. (B) Image quantification of primary B cell antigen internalization (meansSEM, n=23-60 cells). (C) AFM force retraction curves of streptavidin-coated AFM tip and biotinylated antigens. Antigens were anti-Ig for PLB, PM-PLB and PMS, and immune complexes of NIP antigen for DCs. Speed of retraction was 0.1m/s. (D) Rupture distances and forces, meanSD, n=31-109 retraction curves. (E) TNP-470 Colocalization of internalized antigen with DiI in primary B cells after internalization from the substrates. MeansSEM, n=12-21 cells. (F) B220-stained B1-8 primary B cell internalizing immune complexes of NIP antigen from a DC stained with DiI. Scalebar, 5 m. To investigate why B cells internalize antigens from PMS, but not PLB, we examined the flexibility of these substrates using atomic force microscopy (AFM) and compared them to live APCs. In these experiments, the AFM tip binds to the substrate and then retracts to measure forces between the tip and the substrate until the rupture of the bond (16, 17). On PLB and PM-PLB, forces during tip retraction increased rapidly to 30-40 pN and produced single-step ruptures of bonds a few nanometers from the surface (Fig. 1C, D), indicating high membrane stiffness. In contrast, on both PMS and dendritic cells (DCs), forces initially increased and then plateaued at approximately 20 pN, with bonds often rupturing hundreds of nanometers away from the surface (Fig. 1C, D). Thus, in contrast to PLB or PM-PLB, PMS were flexible, and similar in their viscoelastic properties to plasma membranes of APCs loaded with physiological antigen complexes. Labeling PMS with the hydrophobic dye, DiI, showed that B cells internalized antigen together with small pieces of the PMS membrane (Fig. 1A, E). We observed similar colocalization of antigen and lipid in B cells that acquired cognate immune complexes from DCs (Fig. 1E, F). In contrast, B cells forming synapses with PLB did not take up any DiI or antigen (Fig. 1A, E). These results resemble the acquisition of APC membranes by B cells in vivo (18), and, together with the.