PROJECT SUMMARY/ABSTRACT B cell signaling is responsible for the generation of humoral immunity. It is initiated when an antigen binds to a cell surface B cell receptor (BCR), triggering a signaling pathway that culminates in the secretion of neutralizing antibodies. A critical aspect underlying the B cell response to an antigen is the strength of the signal transmitted through the BCR, which is carefully regulated by various co-receptors in the B cell membrane. Aberrant BCR signaling is closely associated with B cell dysfunction, including autoimmunity, B cell malignancies, and immunodeficiency. Therefore, studying the regulatory mechanisms of BCR signaling by co- receptors is needed to understand B cell immunity, how B cell-derived diseases arise, and will enable selective targeting of malignant, autoreactive B cells to improve current therapeutics that cause severe immunosuppression through global depletion of both normal and pathological B cells. This project will focus on the primary stimulatory B cell co-receptor, CD19, and will elucidate and characterize the dynamic regulatory components it coordinates with during B cell activation. CD19 has an essential role in regulating the initiation of B cell signaling, and it is among the most important immunotherapy targets, meaning the results from this research program will inform both basic B cell biology and the design of novel B cell targeting therapeutics. A significant challenge in studying B cell signaling is identifying and characterizing dynamic regulatory complexes. To overcome this, we will leverage our expertise in structural biology, protein engineering, and cell biology to develop new cell-based and protein tools to study these dynamic complexes, allowing us to develop a mechanistic understanding of how CD19 coordinates with accessory co- receptor components to control the strength of the signal transmitted through the BCR. This will be achieved through three specific aims. First, we will identify and functionally characterize novel regulators of early B cell signaling through CD19 proximity labeling coupled to quantitative, multiplexed mass spectrometry (Aim 1). Then, we will determine the structure of a CD21-CD19 complex, which, combined with functional signaling assays, will elucidate how the strength of BCR signaling is regulated in response to antigen binding (Aim 2). Finally, we will engineer cell-state-specific, modulatory antibodies that stabilize the CD19-CD81 complex on resting B cells to attenuate the immune response to antigen (Aim 3). This proposed research will elucidate the biochemical and structural basis of regulation of CD19 signaling, identify new regulatory signaling proteins, and develop new ways of selectively targeting and modulating autoreactive B cell function.

PROJECT NARRATIVE B cell signaling is central to the adaptive immune response, serving as the basis for generation of humoral immunity to foreign agents. A multi-component co-receptor complex carefully regulates the strength of B cell signaling, and dysregulation of signaling through this complex can lead to autoimmunity, immunodeficiency, and B cell cancers. This research program will elucidate the molecular details of how the B cell co-receptor complex functions, is regulated by novel accessory components, and will engineer new tools to modulate B cell function, laying the foundation for new immunomodulatory therapeutics.