Project Summary Cellular and molecular mechanisms that control self-specificity and the magnitude of humoral (antibody, Ab) responses are still poorly understood. To evoke high-affinity Ab response, B cells have to acquire foreign antigen and receive help from cognate T helper (Th) cells. This leads to generation of short-lived plasma cells and Germinal Center (GC) B cells. Within GCs, B cells mutate their B cell receptors (BCRs), receive help from follicular helper T cells (Tfh), undergo affinity maturation, and differentiate into memory B cells and long-lived plasma cells that enable long-term humoral immunity. However, random mutation of BCRs in GC B cells can also lead to generation of self-reactive GC B cells and thus enable development of plasma cells that produce self-reactive Abs and promote autoimmunity. Multiple studies indicate an important role of follicular regulatory T cells (Tfr) in the control of self-specificity and magnitude of Germinal Center responses. However, whether Tfrs that recognize self antigen-acquiring and presenting GC B cells in a cognate fashion can directly act on these GC B cells to promote their apoptosis or modify B cell interactions with Tfh cells in vivo is unknown. The objective of this application is to determine whether Tfr cells exert significant negative regulation of cognate self-reactive GC B cells in vivo and address the mechanism of this control by intravital two-photon imaging of GC B cell, Tfr cells, and Tfh cell interactions in murine lymph nodes (LNs). Our central hypothesis is that direct interactions between cognate Tfr cells and GC B cells promotes elimination of self antigen-presenting GC B cell clones. This hypothesis is based on the previously published work suggesting that Tfr deficiency results into accumulation of bystander GC B cells and generation of anti-nuclear antibodies and autoimmunity. We will test our central hypothesis by combining standard immunological approaches with novel intravital imaging of cognate and non-cognate interactions between GC B cells, Tfr cells, and Tfh cells in the LNs of living mice. The expected out-come of the proposed work is to better understand the mechanisms that control self- specificity of humoral responses in vivo. Such results are expected to have an important positive impact because they could lead to development of novel strategies for control and treatment of self-reactive Ab- mediated autoimmunity and for better manipulation of humoral responses against pathogens.