The task of the adaptive immune system is to respond and clear pathogens. Tailoring responses based on pathogen type and retain memory of the pathogenic encounter for subsequent challenges are also requisite. For this, millions of individual B lymphocytes in the developing immune system express unique antigen receptors, creating a vast repertoire of potential antigen-recognition specificities. Regulation of lymphocyte selection and development is tightly controlled, as aberrant selection can result in autoreactive or diminished responses, thus pathogenesis. The NF[kappa]B transcription factor family is essential for lymphocyte proliferative responses during immune activation, but there is also evidence that it may play a role in lymphocyte development. The NF[kappa]B family comprises dimers of 5 related subunits; p50, p52, p65 (RelA), cRel, and RelB, which control gene expression by binding [kappa]B elements in the regulatory regions of target genes. Signals from antigen and innate immune receptors cause rapid, transient activation of NF[kappa]B through the NEMO/IKK-dependent, so called "canonical" signaling pathway, resulting in immediate degradation of I[kappa]Bs and nuclear translocation of pre-existing RelA and cRel containing dimers. A second "alternative" NF[kappa]B pathway has been described downstream of TNFR family members such as BAFF-R which results in persistent activation of NF[kappa]B via kinase NIKNF[kappa]B was initially discovered in B-cells. Since then the many components of the NF[kappa]B system have been identified and a mathematical model recapitulates the functioning of this dynamical system in fibroblasts, yet in B lymphocytes studies have generally focused on an individual NF[kappa]B component and systems level understanding remains lacking. This thesis describes our attempts to provide a predictive understanding of the functional role of canonical and non- canonical NF[kappa]B regulation in the context of B cell development and activation. Chapter one gives an overview of mechanism by which B cells are produced and maintained along with current understanding of NF[kappa]B roles within this progression. Chapter two reports on NF[kappa]B effectors required for B lymphocyte maturation and development. Chapter three describes how BAFF-R stimulation contributes to BCR-triggered proliferation via two distinct NF[kappa]B[ signaling mechanisms provided by the non-canonical pathway. Finally, chapter four summarizes our findings in the context of current and future studies in the field.

Understanding the functions of multi-cellular organs in terms of the molecular networks within each cell is an important step in the quest to predict phenotype from genotype. B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically. Based on tracking single-cell time-lapse trajectories of hundreds of B cells, single-cell transcriptome, and immunofluorescence analyses, we constructed an agent-based multi-modular computational model to simulate lymphocyte population dynamics in terms of the molecular networks that control NF-κB signaling, the cell cycle, and apoptosis. Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells. But as cRel deficiency causes growing B cells to die at similar rates to non-growing cells, our analysis reveals that the phenomenological decision model of wild-type cells is rooted in a biased race of cell fates. We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.

Targeted deletion of BAFF causes severe deficiency of splenic B cells. BAFF-R is commonly thought to signal to nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase dependent noncanonical NF-κB RelB. However, RelB-deficient mice have normal B-cell numbers. Recent studies showed that BAFF also signals to the canonical NF-κB pathway, and we found that both RelB and cRel are persistently activated, suggesting BAFF signaling coordinates both pathways to ensure robust B-cell development. Indeed, we report now that combined loss of these 2 NF-κB family members leads to impaired BAFF-mediated survival and development in vitro. Although single deletion of RelB and cRel was dispensable for normal B-cell development, double knockout mice displayed an early B-cell developmental blockade and decreased mature B cells. Despite disorganized splenic architecture in Relb(-/-)cRel(-/-) mice, generation of mixed-mouse chimeras established the developmental phenotype to be B-cell intrinsic. Together, our results indicate that BAFF signals coordinate both RelB and cRel activities to ensure survival during peripheral B-cell maturation.

The transcription factor NF-κB is a regulator of inflammatory and adaptive immune responses, yet only IκBα was shown to limit NF-κB activation and inflammatory responses. We investigated another negative feedback regulator, IκBε, in the regulation of B cell proliferation and survival. Loss of IκBε resulted in increased B cell proliferation and survival in response to both antigenic and innate stimulation. NF-κB activity was elevated during late-phase activation, but the dimer composition was stimulus specific. In response to IgM, cRel dimers were elevated in IκBε-deficient cells, yet in response to LPS, RelA dimers also were elevated. The corresponding dimer-specific sequences were found in the promoters of hyperactivated genes. Using a mathematical model of the NF-κB-signaling system in B cells, we demonstrated that kinetic considerations of IκB kinase-signaling input and IκBε's interactions with RelA- and cRel-specific dimers could account for this stimulus specificity. cRel is known to be the key regulator of B cell expansion. We found that the RelA-specific phenotype in LPS-stimulated cells was physiologically relevant: unbiased transcriptome profiling revealed that the inflammatory cytokine IL-6 was hyperactivated in IκBε(-/-) B cells. When IL-6R was blocked, LPS-responsive IκBε(-/-) B cell proliferation was reduced to near wild-type levels. Our results provide novel evidence for a critical role for immune-response functions of IκBε in B cells; it regulates proliferative capacity via at least two mechanisms involving cRel- and RelA-containing NF-κB dimers. This study illustrates the importance of kinetic considerations in understanding the functional specificity of negative-feedback regulators.