Dissecting the contributions of epitope accessibility in B cell activation

Antibody-mediated immunity is highly specific and is activated by B cell exposure to pathogens or antigens. Antigen bound to the B cell receptor (BCR) triggers signaling cascades that can lead to B cell activation, antigen phagocytosis, and the eventual secretion of antibodies. Although our understanding of the biochemical and biophysical events involved in B cell activation have advanced considerably in recent years, the specific features of antigens that contribute to BCR engagement, B cell activation, and ultimately the production of antibodies are not well-defined. Among the factors that may contribute, the accessibility of particular epitopes on the antigenic surface is thought to play an important role. In this line of thinking, accessible epitopes are more easily bound by the BCR, directing the adaptive immune response to these sites. However, because different epitopes also differ in their sequence and structure, decoupling the contributions of accessibility from other contributing factors has been challenging. To address this challenge, we have developed an experimental system to specifically dissect the contributions of epitope accessibility in BCR engagement and B cell activation. Using engineered Ramos B cells that express BCRs specific to different epitopes on the fusion protein of respiratory syncytial virus (RSV) that can be presented in either membrane-proximal or membrane-distal conformations, we are able to compare the efficiency of BCR engagement and B cell activation to a single epitope while varying its accessibility. We track these processes using fluorescence microscopy combined with site-specific labeling of viral antigens and the BCR. These experiments will help to identify physical constraints imposed on BCR binding and B cell activation which may contribute to the immunodominance of particular epitopes on RSV and other viral pathogens.