JCB_201607064 217..230

Production of high-affinity antibodies against pathogens is an effective mechanism of protection against a wide range of infections. Antibody responses are initiated when naive B cells bind foreign antigens on the surfaces of several types of cells, such as subcapsular sinus macrophages (Carrasco and Batista, 2007; Junt et al., 2007; Phan et al., 2007), dendritic cells (DCs; Qi et al., 2006; Gonzalez et al., 2010), and follicular dendritic cells (FDCs; Suzuki et al., 2009). These cells retain and display unprocessed antigen on their surfaces, and we refer to them here as antigen-presenting cells (APCs). The encounter of B cells with antigen on the APCs induces B cell receptor (BCR) signaling, BCR–antigen microcluster formation, contraction of microclusters into a mature immune synapse, and antigen internalization. The internalized antigens are processed, loaded onto major histocompatibility complex class II (MHC II) molecules, and presented to helper T cells (Batista et al., 2001; Fleire et al., 2006; Natkanski et al., 2013). After T cell engagement, B cells can enter the germinal center (GC), which is required for the development of affinity-matured plasma cells and memory B cells (Victora and Nussenzweig, 2012). The likelihood that a B cell will enter and expand within the GC depends on the affinity of the BCR for antigen and is limited by T cell help (Shih et al., 2002; Victora et al., 2010; Schwickert et al., 2011), suggesting that the quality of BCR–antigen binding regulates the efficiency of antigen internalization. The mechanisms that link antigen binding strength to antigen extraction and internalization remain, however, poorly understood. When presenting antigens to B cells, APCs use a variety of receptors including complement receptors, Fc receptors, and C-type lectins (Fang et al., 1998; Bergtold et al., 2005). However, it remains unclear how B cells extract antigen from these receptors. In two early studies, Batista and Neuberger showed that B cells can acquire antigen tethered to a surface and proposed that extraction occurs via mechanical forces (Batista and Neuberger, 2000; Batista et al., 2001). Direct evidence supporting this hypothesis was provided recently, in studies demonstrating that B cells physically pull on synaptic antigen through the BCR and deform flexible membrane substrates to promote antigen internalization (Natkanski et al., 2013; Nowosad et al., 2016). Mechanical forces provide an added benefit of allowing B cells to test the strength of synaptic antigen binding by applying tension to the BCR–antigen bond, resulting in affinitydependent extraction and internalization of BCR microclusters (Tolar and Spillane, 2014). An alternative mechanism of B cell antigen extraction based on enzymatic degradation of antigen in the synapse has also been proposed (Yuseff et al., 2011; Reversat et al., 2015). This mechanism is based on the observation that B cells polarize the microtubule-organizing center toward the synapse, leading to recruitment of lysosomal-associated membrane protein 1 (LAMP-1)–positive lysosomes to the plasma membrane. This Antibody production and affinity maturation are driven by B cell extraction and internalization of antigen from immune synapses. However, the extraction mechanism remains poorly understood. Here we develop DNA-based nanosensors to interrogate two previously proposed mechanisms, enzymatic liberation and mechanical force. Using antigens presented by either artificial substrates or live cells, we show that B cells primarily use force-dependent extraction and resort to enzymatic liberation only if mechanical forces fail to retrieve antigen. The use of mechanical forces renders antigen extraction sensitive to the physical properties of the presenting cells. We show that follicular dendritic cells are stiff cells that promote strong B cell pulling forces and stringent affinity discrimination. In contrast, dendritic cells are soft and promote acquisition of low-affinity antigens through low forces. Thus, the mechanical properties of B cell synapses regulate antigen extraction, suggesting that distinct properties of presenting cells support different stages of B cell responses. B cell antigen extraction is regulated by physical properties of antigen-presenting cells