Imaging the Interactions Between B Cells and Antigen-Presenting Cells

Abstract

In vivo, B cells are often activated by antigens that are displayed on the surface of antigen-presenting cells (APCs). Binding of membrane-associated antigens to the B cell receptor (BCR) causes rapid cytoskeleton-dependent changes in the spatial organization of the BCR and other B cell membrane proteins, leading to the formation of an immune synapse. This process has been modeled using antigens attached to artificial planar lipid bilayers or to plasma membrane sheets. As a more physiological system for studying B cell-APC interactions, we have expressed model antigens in easily transfected adherent cell lines such as Cos-7 cells. The model antigens that we have used are a transmembrane form of a single-chain anti-Igκ antibody and a transmembrane form of hen egg lysozyme that is fused to a fluorescent protein. This has allowed us to study multiple aspects of B cell immune synapse formation including cytoskeletal reorganization, BCR microcluster coalescence, BCR-mediated antigen gathering, and BCR signaling. Here, we provide protocols for expressing these model antigens on the surface of Cos-7 cells, transfecting B cells with siRNAs or with plasmids encoding fluorescent proteins, using fixed cell and live cell fluorescence microscopy to image B cell-APC interactions, and quantifying APC-induced changes in BCR spatial organization and signaling.

Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

“…APCs were generated as described previously (Freeman et al, 2011; Wang et al, 2017; Wang et al, 2018). Briefly, COS-7 cells (ATCC, #CRL-1651) or B16F1 cells (ATCC, #CRL-6323) were transiently transfected using Lipofectamine 3000 (Thermo Fisher, #L3000008) with plasmid DNA encoding either mHEL-GFP, mHEL-HaloTag, or a single-chain anti-Igκ antibody.…”

“…However, most subsequent studies have employed Ags that are attached to artificial planar lipid bilayers or to the cytoplasmic face of plasma membrane sheets in order to facilitate imaging of the contact site. We have developed systems for imaging APC-induced B cell immune synapse formation in real time (Wang et al, 2018) and now use this approach to investigate the role of Arp2/3 complex-dependent actin dynamics in B cell immune synapse formation and function. Here, we show that B cells interacting with live, intact APCs form BCR-Ag microclusters within dynamic actin-rich membrane protrusions that are dependent on the Arp2/3 complex.…”

Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and cell activation

“…mHEL-GFP is a transmembrane protein consisting of the complete HEL and GFP proteins in the extracellular domain, fused to the transmembrane region and cytosolic domain of the H-2K b protein ( Batista et al, 2001 ). mHEL-HaloTag consists of the complete HEL protein fused to the transmembrane region and cytosolic domain of H-2K b , with the the HaloTag protein fused to the C-terminus (see Figure 1 ) ( Wang et al, 2018 ). The single-chain anti-Ig, 𝜅 antibody is a transmembrane protein consisting of the single-chain Fv with the variable regions from the 187.1 rat anti-Ig, 𝜅 monocolonal antibody, fused to the hinge and membrane-proximal domains of rat IgG1 and the transmembrane and cytoplasmic domains of the H-2K d protein ( Ait-Azzouzene et al, 2005 ).…”

“…STED microscopy was performed as described previously ( Wang et al, 2018 ). Briefly, 5 x 10 4 A20 B cells in 0.1 ml imaging medium were allowed to spread on anti-IgG-coated coverslips before being fixed and stained with Alexa Fluor 532-conjugated-phalloidin (Thermo Fisher, #A22282).…”

“…However, most subsequent studies have employed antigens that are attached to artificial planar lipid bilayers or to the cytoplasmic face of plasma membrane sheets in order to facilitate imaging of the contact site. We have developed systems for imaging APC-induced B cell immune synapse formation in real time ( Wang et al, 2018 ) and now use this approach to investigate the role of Arp2/3 complex-dependent actin dynamics in B cell immune synapse formation and function. Here, we show that B cells interacting with live, intact APCs form BCR-Ag microclusters within dynamic actin-rich membrane protrusions that are dependent on the Arp2/3 complex.…”