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Mature B cells remain in a quiescent state until activated. Rickert and colleagues identify a prominent role for the kinase Gsk3 in resting naive B cells and in activated germinal center B cells that restrains the production of Myc and reactive oxygen species and prevents metabolic collapse. B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.

Key Points Transformation of early B cells supplants the role of interleukin-7 receptor (IL-7R) and pre-B cell receptor (BCR) signalling. Continual BCR signalling is required for the propagation of many B cell malignancies. The transcription factor forkhead box protein O1 (FOXO1) and the B cell linker protein BLNK have distinct roles in early B cells versus peripheral B cells. The crucial BCR signalling pathways regulated by phosphoinsoitide 3-kinase (PI3K), spleen tyrosine kinase (SYK) and Bruton tyrosine kinase (BTK) are valid therapeutic targets for B cell chronic lymphocytic leukaemia (B-CLL) and B cell non-Hodgkin's lymphoma (B-NHL). Signalling via the BCR is modulated by additional receptors responding to microenvironmental cues. In this Review, the key signalling pathways that lie downstream of the pre-B cell receptor (BCR) and the BCR are discussed in terms of their contribution to B cell homeostasis and neoplasia, with a focus on the emerging distinctions between tonic and chronic active signalling. The B cell receptor (BCR) and its precursor (pre-BCR) control B cell homeostasis, differentiation and function. Moreover, aberrant pre-BCR and BCR signalling have a central role in B cell neoplasia; for example, enhanced positive signalling or disrupted negative signalling downstream of the pre-BCR promotes B cell acute lymphocytic leukaemia. The emerging distinctions between tonic and chronic active BCR signalling have contributed to the identification of oncogenic targets downstream of BCR signalling in mature B cell neoplasms. Indeed, the encouraging results of several ongoing clinical trials that target the activity of phosphoinositide 3-kinase δ-isoform (PI3Kδ), Bruton tyrosine kinase (BTK) or spleen tyrosine kinase (SYK) downstream of the BCR highlight the therapeutic potential of inhibiting BCR signalling.

Foxo transcription factors are linked to complex regulatory circuits governed by the availability of phosphatidylinositol-3,4,5-trisphosphate. Rickert and colleagues show that Foxo1 has nonredundant functions at many stages of B cell development. The transcription factors Foxo1, Foxo3 and Foxo4 modulate cell fate 'decisions' in diverse systems. Here we show that Foxo1-dependent gene expression was critical at many stages of B cell differentiation. Early deletion of Foxo1 caused a substantial block at the pro–B cell stage due to a failure to express interleukin 7 receptor-α. Foxo1 inactivation in late pro–B cells resulted in an arrest at the pre–B cell stage due to lower expression of the recombination-activating genes Rag1 and Rag2 . Deletion of Foxo1 in peripheral B cells led to fewer lymph node B cells due to lower expression of L-selectin and failed class-switch recombination due to impaired upregulation of the gene encoding activation-induced cytidine deaminase. Thus, Foxo1 regulates a transcriptional program that is essential for early B cell development and peripheral B cell function.

Successful B cell differentiation and prevention of cell transformation depends on balanced and fine-tuned activation of cellular signaling pathways. The phosphatidyl inositol-3 kinase (PI3K) signaling pathway has emerged as a major regulator of B lymphocyte homeostasis and function. Phosphoinositide-dependent protein kinase-1 (PDK1) is the pivotal node in the PI3K pathway, regulating the stability and activity of downstream AGC kinases (including Akt, RSK, S6K, SGK, and PKC). Although the importance of PI3K activity in B cell differentiation is well documented, the role of PDK1 and other downstream effectors is underexplored. Here we used inducible and stage-specific gene targeting approaches to elucidate the role of PDK1 in early and peripheral B cell differentiation. PDK1 ablation enhanced cell cycle entry and apoptosis of IL-7–dependent pro-B cells, blocking Ig synthesis and B cell maturation. PDK1 also was essential for the survival and activation of peripheral B cells via regulation of PKC and Akt-dependent downstream effectors, such as GSK3α/β and Foxo1. We found that PDK1 deletion strongly impaired B cell receptor (BCR) signaling, but IL-4 costimulation was sufficient to restore BCR-induced proliferation. IL-4 also normalized PKCβ activation and hexokinase II expression in BCR-stimulated cells, suggesting that this signaling pathway can act independent of PDK1 to support B cell growth. In summary, our results demonstrate that PDK1 is indispensable for B cell survival, proliferation, and growth regulation.

Phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN) phosphatase serve essential functions in the regulation of cell growth, differentiation and survival by modulating intracellular phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P 3 ) concentrations. Here we show that the conditional deletion of Pten in B cells led to the preferential generation of marginal zone (MZ) B cells and B1 cells. PTEN-deficient B cells were hyperproliferative in response to mitogenic stimuli, and exhibited a lower threshold for activation through the B cell antigen receptor. Inactivation of PTEN rescued germinal center, MZ B and B1 cell formation in CD19 −/− mice, arguing that recruitment and activation of PI3K are the dominant roles for CD19 in these B cell subpopulations. These findings establish the central role of PI-3,4,5-P 3 regulation in the differentiation of peripheral B cell subsets.

The PI3K pathway is integral for the germinal center (GC) response. However, the contribution of protein kinase B (AKT) as a PI3K effector in GC B cells remains unknown. Here, we show that mice lacking the AKT1 and AKT2 isoforms in B cells failed to form GCs, which undermined affinity maturation and antibody production in response to immunization. Upon B-cell receptor stimulation, AKT1/2–deficient B cells showed poor survival, reduced proliferation, and impaired mitochondrial and metabolic fitness, which collectively halted GC development. By comparison, Foxo1 T24A mutant, which cannot be inactivated by AKT1/2 phosphorylation and is sequestered in the nucleus, significantly enhanced antibody class switch recombination via induction of activation-induced cytidine deaminase (AID) expression. By contrast, repression of FOXO1 activity by AKT1/2 promoted IRF4-driven plasma cell differentiation. Last, we show that T-cell help via CD40, but not enforced expression of Bcl2, rescued the defective GC response in AKT1/2–deficient animals by restoring proliferative expansion and energy production. Overall, our study provides mechanistic insights into the key role of AKT and downstream pathways on B cell fate decisions during the GC response.

Detailed biochemical analysis of unmanipulated germinal center (GC) B cells has not been achieved. Previously, we designed and used a simple, economical and new magnetic bead separation scheme for the purification of 'untouched' mature GC and non-GC B cells from the spleens of immunized mice and reported the first biochemical assessment of the signaling cascades that contribute to cyclin D stability and GC B cell proliferation. Here we provide a detailed protocol for the method we used, which involves preparing single-cell suspension from the spleens of immunized mice, followed by labeling of nontarget cells with biotinylated antibodies specific for CD43, CD11c and IgD (for GC enrichment) or GL7 (for non-GC enrichment); these steps are followed by cell depletion using standard magnetic bead technology. This protocol can yield GC and non-GC B cells with purities exceeding 90%. The sorting process can be carried out in ∼1 h and provides a population of GC B cells of sufficient purity and quantity to allow ex vivo manipulation, including biochemical and genetic analysis as well as cell culture.

NF-κB-inducing kinase (NIK)-mediated IKKα phosphorylation activates the alternative NF-κB pathway, which is characterized by nuclear translocation of p52:RelB heterodimers. This alternative pathway is initiated by a select few receptors, including LT-βR, BAFF-R, and CD40. Although NIK, IKKα, and p52 are all critical regulators of LT-βR signaling in stromal cells during humoral immune responses, lymphocytes require NIK, but not p52, for optimal Ig production. This disparity suggests that NIK possesses critical cell-type-specific functions that do not depend on NF-κB. Here we use mice bearing targeted mutations of the IKKα activation loop Ser¹⁷⁶/¹⁸⁰ (IKKαAA) to address the B cell-intrinsic functions of NIK-IKKα signaling in vivo. We find that IKKαAA B cells mount normal primary antibody responses but do not enter germinal centers. This defect likely derives from ineffective early T-B cell collaboration and leads to impaired generation of humoral memory and relatively short-lived, low-affinity antibody production. Our findings contrast with those obtained by using p52⁻/⁻ B cells, which mount normal Ig responses, and alymphoplasia (NIK mutant) B cells, which produce very little primary Ig. Thus, the NIK-IKKα-p52 axis is not as linear and exclusive as previous studies suggest, and IKKα possesses critical NF-κB-independent functions in B cells.

The protein phosphatase LYP is known to regulate signaling in the immune system, but the regulatory mechanisms controlling LYP itself are less clear. Exploration of spatiotemporal dynamics and application of a newly identified chemical inhibitor now define a role for the kinase CSK in dialing down LYP activity. Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T-cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here we studied the spatiotemporal dynamics of the LYP–CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it downmodulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T-cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single-nucleotide polymorphism that confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a mutant LYP that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.