Abstract The generation of long-lasting humoral immunity is the purpose of almost all vaccines. Long-lasting humoral immunity is mediated through antibodies produced by long-lived plasma cells (LLPCs) and memory B cells (MBCs) that respond rapidly to pathogens. Despite the outstanding success of vaccinations overall, many clinical and experimental vaccines fail to induce long-lasting humoral immunity. One key example is the influenza vaccine, which requires administration every year. Upon encountering a foreign antigen, antigen-specific B cells proliferate and differentiate into germinal center (GC) B cells and subsequently generate LLPCs and MBCs. B cell receptor and CD40 receptor signaling result in the activation of NFkB in GC B cells. It is well established that transcription factors NFkB cRel and RelA are critical for physiological B cell responses and that their misregulation leads to B cell-mediated diseases. cRel and RelA are essential for generating GC B cells and plasma cells (PCs). The activation of cRel and RelA depends on the degradation of inhibitors of NFkB (IkBs). IkBa and IkBe have different binding preferences to cRel and RelA. IkBa binds to RelA-containing dimer (RelA:p50) with a higher affinity than cRel-containing dimer (cRel:p50), whereas IkBe binds to cRel:p50 with a higher affinity than RelA:p50. The network of IkBa, IkBe, cRel, and RelA is defined here as the NFkB signaling network. The role of the NFkB signaling network in generating LLPCs versus MBCs is unknown. This project will define the role of the NFkB signaling network in the generation of LLPCs versus MBCs. The objective of Aim 1 is to define how IkBa and IkBe regulate the generation of LLPCs and MBCs. The objective of Aim 2 is to identify how cell surface receptors regulate cRel and RelA expression in GC B cells, leading to the generation of LLPCs and MBCs. The objective of Aim 3 is to determine whether the vaccine-induced response of IkBa-deficient and IkBe-deficient mice can enhance humoral immunity against influenza infection. The proposed research is innovative because it connects the cell signaling and transcriptional network of GC B cells to the generation of LLPCs and MBCs and translates this mechanistic knowledge to generate long-lasting humoral immunity against influenza viruses. This work uses newly generated cRel and RelA fluorescence reporter mice and IkBe and IkBa knock-out mice. The combination of single-cell/bulk RNA-Seq, BCR-Seq, flow cytometry, and intravital imaging will reveal new insights into the generation of long-lived humoral immunity that can be applied to vaccination strategy for influenza and other infectious diseases. The proposed research is expected to define the mechanism that allows the NFkB signaling network to regulate different gene regulatory programs within phenotypically-identical GC B cells to generate LLPCs and MBCs, identifying a promising target for the development of longer-lasting vaccines for influenza and other highl...