Adaptation of the P1 phage-derived Cre /loxP site- specific recombination system to the gene targeting technique allows for the conditional deletion of genes in mice. To selectively modify genes in B lymphocytes, we have generated mice (designated CD19-Cre) which express cre under the transcriptional control of the B lineage-restricted CD19 gene. In a model system involving the cross of CD19-Cre mice with mice bearing a loxP -flanked substrate, we find a deletion efficiency of 75-80% in bone marrow-derived pre-B cells that increases to 90-95% in splenic B cells.

CD19 is the hallmark differentiation antigen of the B lineage. Its early expression has implicated a role for CD19 during the antigen-independent phases of B-cell development, whereas in mature B cells CD19 can act synergistically with surface immunoglobulin to induce activation. We have generated CD19-deficient mice and found that development of conventional B cells is unperturbed. However, mature CD19-/- B cells show a profound deficiency in responding to protein antigens that require T-cell help. This is accompanied by a lack of germinal centre formation and affinity maturation of serum antibodies. Thus CD19 is crucial for both initial B-cell activation by T-cell-dependent antigens and the maturation and/or selection of the activated cells into the memory compartment. An impairment in ligand-driven selection may also be responsible for the observation of a striking reduction in the B-1 (formerly Ly-1) B-cell subset, thought to develop under the control of self-antigens and bacterial antigens (reviewed in ref. 2).

The Foxo transcription factors (Foxo1, Foxo3, Foxo4) modulate cell fate decisions in diverse systems. Here we show that Foxo1-dependent gene expression was critical at multiple stages of B cell differentiation. Early deletion of Foxo1 caused a severe block at the pro-B cell stage, due to a failure to express interleukin 7 receptor α (IL-7Rα). Foxo1 inactivation in late pro-B cells resulted in an arrest at the pre-B cell stage due to a reduction in Rag1 and Rag2 expression. Deletion of Foxo1 in peripheral B cells led to fewer lymph node B cells due to reduced L-selectin expression, and failed class switch recombination due to impaired Aicda upregulation. Thus, Foxo1 regulates a transcriptional program that is essential for early B cell development and peripheral B cell function.

A classical cellular response to hypoxia is a cessation of growth. Hypoxia-induced growth arrest differs in different cell types but is likely an essential aspect of the response to wounding and injury. An important component of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Although this transcription factor is essential for adaptation to low oxygen levels, the mechanisms through which it influences cell cycle arrest, including the degree to which it cooperates with the tumor suppressor protein p53, remain poorly understood. To determine broadly relevant aspects of HIF-1 function in primary cell growth arrest, we examined two different primary differentiated cell types which contained a deletable allele of the oxygen-sensitive component of HIF-1, the HIF-1␣ gene product. The two cell types were murine embryonic fibroblasts and splenic B lymphocytes; to determine how the function of HIF-1␣ influenced p53, we also created double-knockout (HIF-1␣ null, p53 null) strains and cells. In both cell types, loss of HIF-1␣ abolished hypoxia-induced growth arrest and did this in a p53-independent fashion. Surprisingly, in all cases, cells lacking both p53 and HIF-1␣ genes have completely lost the ability to alter the cell cycle in response to hypoxia. In addition, we have found that the loss of HIF-1␣ causes an increased progression into S phase during hypoxia, rather than a growth arrest. We show that hypoxia causes a HIF-1␣-dependent increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27; we also find that hypophosphorylation of retinoblastoma protein in hypoxia is HIF-1␣ dependent. These data demonstrate that the transcription factor HIF-1 is a major regulator of cell cycle arrest in primary cells during hypoxia.Mammalian cells have evolved to utilize molecular oxygen for energy production. Cells can respond differently to wide ranges of oxygen through alterations in both their metabolic states and growth rates. In recent years, several lines of evidence have indicated that hypoxia can alter cell proliferation in two distinct ways: via programmed cell death and through growth arrest. In transformed cells, hypoxia can provoke apoptosis via the p53 pathway; ultimately, this can represent a potent mechanism for the selection of p53 mutants in tumor cell populations (30,34). Nontransformed hypoxic cells, on the other hand, can undergo cell cycle arrest at the G 1 /S interface without any alteration in their long-term viability (10).It has been proposed that hypoxically induced cell cycle arrest is caused by inactivation of enzymes responsible for nucleotide synthesis, ultimately inhibiting DNA replication (19, 39). However, inhibition of nucleotide synthesis occurs only under severe hypoxia (0.01% oxygen) or anoxia, but not under moderate hypoxia (0.1 to ϳ1% oxygen) (10).In the moderately hypoxic microenvironment, various biological reactions show significant changes relative to normoxia. Numerous studies on moderate hypoxia have indicated tha...

Germline inactivation of c-myc in mice causes embryonic lethality. Therefore, we developed a LoxP/Cre-based conditional mutation approach to test the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes. Cre expression resulted in reduced proliferation of wild-type MEFs, but c-Myc-deficient MEFs showed a further reduction. In contrast to fibroblasts, Cre expression had no apparent affect on wild-type B cell proliferation. Deletion of both c-Myc genes in B cells led to severely impaired proliferation in response to anti-CD40 plus IL-4. However, treated cells did upregulate several early activation markers but not CD95 or CD95 ligand. We discuss these findings with respect to potential c-Myc functions in proliferation and apoptosis and also discuss potential limitations in the Cre-mediated gene inactivation approach.

B cells predominate in a quiescent state until antigen is encountered, which results in rapid growth, proliferation and differentiation. These distinct cell states are likely 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 naïve recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, GSK3 was selectively required for CD40-mediated regulation of B cell size, mitochondria biogenesis, glycolysis and reactive oxygen species (ROS) production. GSK3 was required to prevent metabolic collapse and ROS-induced apoptosis when glucose became limiting, functioning in part by repressing c-Myc-dependent growth. Importantly, 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.

IkappaB Kinase (IKK)alpha is required for activation of an alternative NF-kappaB signaling pathway based on processing of the NF-kappaB2/p100 precursor protein, which associates with RelB in the cytoplasm. This pathway, which activates RelB:p52 dimers, is required for induction of several chemokine genes needed for organization of secondary lymphoid organs. We investigated the basis for the IKKalpha dependence of the induction of these genes in response to engagement of the lymphotoxin beta receptor (LTbetaR). Using chromatin immunoprecipitation, we found that the promoters of organogenic chemokine genes are recognized by RelB:p52 dimers and not by RelA:p50 dimers, the ubiquitous target for the classical NF-kappaB signaling pathway. We identified in the IKKalpha-dependent promoters a novel type of NF-kappaB-binding site that is preferentially recognized by RelB:p52 dimers. This site links induction of organogenic chemokines and other important regulatory molecules to activation of the alternative pathway.

Summary Members of the tumor necrosis factor receptor superfamily (TNFRSF) participate prominently in B-cell maturation and function. In particular, B-cell activating factor belonging to the TNFR family receptor (BAFF-R), B-cell maturation antigen (BCMA), and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) play critical roles in promoting B-cell survival at distinct stages of development by engaging a proliferation-inducing ligand (APRIL) and/or BAFF. CD40 is also essential for directing the humoral response to T-cell-dependent antigens. Signaling by the TNFRSF is mediated primarily, albeit not exclusively, via the TNFR-associated factor (TRAF) proteins and activation of the canonical and/or noncanonical nuclear factor-κB (NF-κB) pathways. Dysregulated signaling by TNFRSF members can promote B-cell survival and proliferation, causing autoimmunity and neoplasia. In this review, we present a current understanding of the functions of and distinctions between APRIL/BAFF signaling by their respective receptors expressed on particular B-cell subsets. These findings are compared and contrasted with CD40 signaling, which employs similar signaling conduits to achieve distinct cellular outcomes in the context of the germinal center response. We also underscore how new findings and conceptual insights in TNFRSF signaling are facilitating the understanding of B-cell malignancies and autoimmune diseases.