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Objective: The metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free., Methods: The study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry., Results: OxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes., Conclusions: Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12 tfg KO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH 4 Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12 tfg KO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

Glucose uptake increases during B cell activation and antibody-secreting cell (ASC) differentiation, but conflicting findings prevent a clear metabolic profile at different stages of B cell activation. Deletion of the glucose transporter type 1 (GLUT1) gene in mature B cells (GLUT1-cKO) results in normal B cell development, but it reduces germinal center B cells and ASCs. GLUT1-cKO mice show decreased antigen-specific antibody titers after vaccination. In vitro, GLUT1-deficient B cells show impaired activation, whereas established plasmablasts abolish glycolysis, relying on mitochondrial activity and fatty acids. Transcriptomics and metabolomics reveal an altered anaplerotic balance in GLUT1-deficient ASCs. Despite attempts to compensate for glucose deprivation by increasing mitochondrial mass and gene expression associated with glycolysis, the tricarboxylic acid cycle, and hexosamine synthesis, GLUT1-deficient ASCs lack the metabolites for energy production and mitochondrial respiration, limiting protein synthesis. We identify GLUT1 as a critical metabolic player defining the germinal center response and humoral immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

B lymphocytes, as a central part of adaptive immune responses, have the ability to fight against an almost unlimited numbers of pathogens. Impairment of B cell development, activation and differentiation to antibody secreting plasma cells can lead to malignancy, allergy, autoimmunity and immunodeficiency. However, the impact of environmental factors, such as hyperosmolality or osmotic stress caused by varying salt concentrations in different lymphoid organs, on these processes is not well-understood. Here, we report that B cells respond to osmotic stress in a biphasic manner. Initially, increased osmolality boosted B cell activation and differentiation as shown by an untimely downregulation of Pax5 as well as upregulation of CD138. However, in the second phase, we observed an increase in cell death and impaired plasmablast differentiation. Osmotic stress resulted in impaired class switch to IgG1, inhibition of phosphorylation of p38 mitogen-activated kinase and a delayed NFAT5 response. Overall, these findings demonstrate the importance of microenvironmental hyperosmolality and osmotic stress caused by NaCl for B cell activation and differentiation.

The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body's own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca 2+ -mediated mitoflashes. Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca 2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.

B-cell development in the bone marrow comprises proliferative and resting phases in different niches. We asked whether B-cell metabolism relates to these changes. Compared to pro B and small pre B cells, large pre B cells revealed the highest glucose uptake and ROS but not mitochondrial mass, whereas small pre B cells exhibited the lowest mitochondrial membrane potential. Small pre B cells from Rag1 -/- ;33.C9 μ heavy chain knock-in mice revealed decreased glycolysis (ECAR) and mitochondrial spare capacity compared to pro B cells from Rag1 -/- mice. We were interested in the step regulating this metabolic switch from pro to pre B cells and uncovered that Swiprosin-2/EFhd1, a Ca 2+ -binding protein of the inner mitochondrial membrane involved in Ca 2+ -induced mitoflashes, is expressed in pro B cells, but downregulated by surface pre B-cell receptor expression. Knockdown and knockout of EFhd1 in 38B9 pro B cells decreased the oxidative phosphorylation/glycolysis (OCR/ECAR) ratio by increasing glycolysis, glycolytic capacity and reserve. Prolonged expression of EFhd1 in EFhd1 transgenic mice beyond the pro B cell stage increased expression of the mitochondrial co-activator PGC-1α in primary pre B cells, but reduced mitochondrial ATP production at the pro to pre B cell transition in IL-7 cultures. Transgenic EFhd1 expression caused a B-cell intrinsic developmental disadvantage for pro and pre B cells. Hence, coordinated expression of EFhd1 in pro B cells and by the pre BCR regulates metabolic changes and pro/pre B-cell development.

microRNAs (miRNAs) are important posttranscriptional regulators during hematopoietic lineage commitment and lymphocyte development. Mature miRNAs are processed from primary miRNA transcripts in two steps by the microprocessor complex, consisting of Drosha and its partner DiGeorge Critical Region 8 (DGCR8), and the RNAse III enzyme, Dicer. Conditional ablations of Drosha and Dicer have established the importance of both RNAses in B- and T-cell development. Here, we show that a cre-mediated B-cell specific deletion of DGCR8 in mice results in a nearly complete maturation block at the transition from the pro-B to the pre-B cell stage, and a failure to upregulate Ig μ heavy chain expression in pro-B cells. Furthermore, we found that the death of freshly isolated DGCR8-deficient pro-B cells could be partially prevented by enforced Bcl2 expression. We conclude from these findings that the microprocessor component DGCR8 is essential for survival and differentiation of early B-cell progenitors., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ex vivo expansion of naive primary B cells is still a challenge, yet would open new possibilities for in vitro studies of the immune response or the production of monoclonal antibodies. In our hands, unstimulated murine B cells did not expand in significant numbers, while culture viability decreased rapidly within a few days. Activation mimicking in vivo stimulation through either T cell-independent or T-cell dependent signaling, led to several division cycles, albeit accompanied by irreversible differentiation. By co-culturing B cells under moderate hypothermia (30°C) on live feeder fibroblasts expressing recombinant CD40 ligand (CD154) and by repeatedly transferring cultured B cells to new feeder cell cultures, we could extend the growth of primary mouse B cells compared to cultures maintained at 37°C. B cells under these conditions showed an activated phenotype as shown by the presence of AID and IRF4, two factors required for IgH class switch recombination in antigen-activated B cells. In contrast to cells cultured at 37°C, B cells under hyperthermia did surprisingly not differentiate into Blimp-1 expressing plasmablasts. Thus, the repeated batch process under hyperthermic conditions represents a first step towards the development of a continuous cultivation system for the expansion of primary B cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:978-989, 2016., (© 2016 American Institute of Chemical Engineers.)

The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms involved in initiating downstream programs are incompletely understood. The pre-B-cell receptor (pre-BCR) is an important checkpoint of B-cell development and is essential for a pre-B cell to traverse into an immature B cell. Here, we show that activation of myocyte enhancer factor 2 (Mef2) transcription factors (TFs) by the pre-BCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the pre-B-cell stage in mice deficient for Mef2c and Mef2d TFs and that pre-BCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the Erk5 mitogen-activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development., (© 2016 by The American Society of Hematology.)

In general, a long-lasting immune response to viruses is achieved when they are infectious and replication competent. In the mouse, the neutralizing antibody response to Friend murine leukemia virus is contributed by an allelic form of the enzyme Apobec3 (abbreviated A3). This is counterintuitive because A3 directly controls viremia before the onset of adaptive antiviral immune responses. It suggests that A3 also affects the antibody response directly. Here, we studied the relative size of cell populations of the adaptive immune system as a function of A3 activity. We created a transgenic mouse that expresses all seven human A3 enzymes and compared it to WT and mouse A3-deficient mice. A3 enzymes decreased the number of marginal zone B cells, but not the number of follicular B or T cells. When mouse A3 was knocked out, the retroelement hitchhiker-1 and sialyl transferases encoded by genes close to it were overexpressed three and two orders of magnitude, respectively. We suggest that A3 shifts the balance, from the fast antibody response mediated by marginal zone B cells with little affinity maturation, to a more sustained germinal center B-cell response, which drives affinity maturation and, thereby, a better neutralizing response., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Activated B cells are selected for in germinal centers by regulation of their apoptosis. The Ca2+ -binding cytoskeletal adaptor protein Swiprosin-1/EFhd2 (EFhd2) can promote apoptosis in activated B cells. We therefore hypothesized that EFhd2 might limit humoral immunity by repressing both the germinal center reaction and the expected enhancement of immune responses in the absence of EFhd2. Here, we established EFhd2(-/-) mice on a C57BL/6 background, which revealed normal B- and T-cell development, basal Ab levels, and T-cell independent type 1, and T-cell independent type 2 responses. However, T cell-dependent immunization with sheep red blood cells and infection with the helminth Nippostrongylus brasiliensis (N.b) increased production of antibodies of multiple isotypes, as well as germinal center formation in EFhd2(-/-) mice. In addition, serum IgE levels and numbers of IgE+ plasma cells were strongly increased in EFhd2(-/-) mice, both after primary as well as after secondary N.b infection. Finally, mixed bone marrow chimeras unraveled an EFhd2-dependent B cell-intrinsic contribution to increased IgE plasma cell numbers in N.b-infected mice. Hence, we established a role for EFhd2 as a negative regulator of germinal center-dependent humoral type 2 immunity, with implications for the generation of IgE., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Maturation as well as antigen-dependent activation of B cells is accompanied by alternating phases of proliferation and quiescence. We and others have previously shown that Krüppel-like factor 2 (KLF2), a regulator of T cell quiescence and migration, is upregulated in small resting precursor (pre)-B cells after assembly of the immature pre-B cell receptor (pre-BCR) and is downregulated upon antigen-induced proliferation of mature B cells. These findings suggest that KLF2, besides its function in maintaining follicular B cell identity, peripheral B cell homeostasis and homing of antigen-specific plasma cells to the bone marrow, also controls clonal expansion phases in the B cell lineage. Here, we demonstrate that enforced expression of KLF2 in primary pre-B cells results in a severe block of pre-BCR-induced proliferation, upregulation of the cell cycle inhibitors p21 and p27 and downregulation of c-myc. Furthermore, retroviral KLF2 transduction of primary B cells impairs LPS-induced activation, favors apoptosis and results in reduced abundance of factors, such as AID, IRF4 and BLIMP1, that control the antigen-dependent phase of B cell activation and plasma cell differentiation. Hence, we conclude that KLF2 is not only a key player in terminating pre-B cell clonal expansion but also a potent suppressor of B cell activation.

Small Ca(2+) binding adaptor proteins of the EF hand family play important roles in neuronal and immune cell Ca(2+) signaling. Swiprosin-1/EFhd2 (EFhd2) and Swiprosin-2/EFhd1 (EFhd1) are conserved and very homologous Ca(2+) binding adaptor proteins of the EF hand family, with possibly redundant functions. In particular, EFhd2 has been proposed to be involved in B cell signaling and neuropathological disorders. Little is known thus far about the expression of EFhd2 on the single cell level in tissue sections or blood cells. Here we describe the generation of four specific anti-EFhd2 monoclonal antibodies. These recognize murine and human EFhd2, but not murine EFhd1, and their binding site maps to a region in the N-terminal part of EFhd2, where EFhd2 and EFhd1 differ most. Moreover, to detect EFhd1 specifically, we also generated anti-EFhd1 polyclonal antibodies, making use of a singular peptide of the N-terminal part of the protein. Using anti-EFhd2 MAb, we reveal two EFhd2 pools in B cells, one at the membrane and one cytoplasmic pool. Staining of human peripheral blood mononuclear cells shows EFhd2 expression in B cells but a ∼5 fold higher expression in monocytes. Taken together, EFhd2 monoclonal antibodies will be valuable to assess the real subcellular localization and expression level of EFhd2 in healthy and diseased primary cells and tissues.

Epstein-Barr virus (EBV) replicates in superficial differentiated cells of oral hairy leukoplakia (OHL). Differentiation of squamous epithelial cells depends on B-lymphocyte-induced maturation protein 1 (Blimp1). Here we show that expression of the EBV immediate-early protein BZLF1 is restricted to Blimp1-positive epithelial cells in OHL. Luciferase assays revealed Blimp1-dependent induction of the BZLF1 promoter Zp in epithelial cell lines. Expression of ZEB1, a negative regulator of Zp, and of Xbp-1, which mediates the Blimp1 effect on Zp in B-cells, was not affected by enforced Blimp1 expression. Moreover, Xbp-1 protein expression was not detected in differentiated epithelial cells of OHL. Thus, Blimp1 induces BZLF1 expression in epithelial cells independently of ZEB1 and Xbp-1. In contrast to epithelial cells of OHL, BZLF1 expression was also observed in Blimp1-negative lymphoid cells in infectious mononucleosis tonsils, suggesting that EBV replication in B-cells may be induced independently of terminal differentiation.

MicroRNAs are small non-coding RNAs which are crucial for the fine regulation of gene expression. Recent results suggest that these molecules display multifaceted functions in B-cells and T-cells in rheumatic inflammatory diseases.

B-lymphocyte development is dictated by the protein products of functionally rearranged Ig heavy (H) and light (L) chain genes. Ig rearrangement begins in pro-B cells at the IgH locus. If pro-B cells generate a productive allele, they assemble a pre-B cell receptor complex, which signals their differentiation into pre-B cells and their clonal expansion. Pre-B cell receptor signals are also thought to contribute to allelic exclusion by preventing further IgH rearrangements. Here we show in two independent mouse models that the accumulation of a stabilized μH mRNA that does not encode μH chain protein specifically impairs pro-B cell differentiation and reduces the frequency of rearranged IgH genes in a dose-dependent manner. Because noncoding IgH mRNA is usually rapidly degraded by the nonsense-mediated mRNA decay machinery, we propose that the difference in mRNA stability allows pro-B cells to distinguish between productive and nonproductive Ig gene rearrangements and that μH mRNA may thus contribute to efficient H chain allelic exclusion.

Krüppel-like factor 2 (KLF2) controls T lymphocyte egress from lymphoid organs by regulating sphingosin-1 phosphate receptor 1 (S1Pr1). Here we show that this is not the case for B cells. Instead, KLF2 controls homeostasis of B cells in peripheral lymphatic organs and homing of plasma cells to the bone marrow, presumably by controlling the expression of β(7)-integrin. In mice with a B cell-specific deletion of KLF2, S1Pr1 expression on B cells was only slightly affected. Accordingly, all splenic B cell subsets including B1 cells were present, but their numbers were increased with a clear bias for marginal zone (MZ) B cells. In contrast, fewer peyers patches harboring fewer B cells were found, and fewer B1 cells in the peritoneal cavity as well as recirculating B cells in the bone marrow were detected. Upon thymus-dependent immunization, IgG titers were diminished, and antigen-specific plasma cells were absent in the bone marrow, although numbers of antigen-specific splenic plasmablasts were normal. KLF2 plays also a role in determining the identity of follicular B cells, as KLF2-deficient follicular B cells showed calcium responses similar to those of MZ B cells and failed to down-regulate MZ B cell signature genes, such as CD21 and CXCR7.

The proteasome inhibitor bortezomib is approved for the treatment of multiple myeloma and mantle cell lymphoma. We recently demonstrated that bortezomib eliminates autoreactive plasma cells in systemic lupus erythematosus mouse models, thereby representing a promising novel treatment for Ab-mediated diseases. In this study, we investigated the effects of bortezomib on the just developing and pre-existing T-dependent Ab response toward dinitrophenyl-keyhole limpet hemocyanin and the T-independent type 2 response toward (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP)-Ficoll in BALB/c mice. Bortezomib treatment strongly reduced T-dependent Ab titers mainly due to depletion of plasma cells. In contrast, the early T-independent type 2 response against i.v. administered NIP-Ficoll, which is predominantly dependent on marginal zone (MZ) B cells, resisted bortezomib. Upon bortezomib treatment, immunoproteasome subunits and the antiapoptotic unfolded protein response including NF-κB were induced in NIP-Ficoll-stimulated MZ B cells, but not in plasma cells and follicular B cells. In summary, bortezomib treatment decreases Ab titers arising from T-dependent immune responses predominantly by eliminating plasma cells. In contrast, the early T-independent type 2 response protecting the organism against blood-borne pathogens remains largely intact due to a remarkable resistance of MZ B cells against proteasome inhibition.

BCL6 protects germinal center (GC) B cells against DNA damage-induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7-dependent B cell precursors, we report that IL-7Ralpha-Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a mu heavy chain, however, activation of pre-B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Ralpha-Stat5 signaling is attenuated. At the transition from IL-7-dependent to -independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre-B cells from DNA damage-induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre-B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.

Background: Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line-dependent-manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T-cells., Methods: The performance of the tetracycline-regulated (Tet)-inducible promoters Tet-responsive element (TRE), mouse mammary tumor virus (MMTV)/TRE, TREtight and second generation TRE (SG/TRE) was compared in several B-cell lines and in Jurkat T-cells using transient transfections in combination with Tet-On. To monitor transgene expression in a Jurkat cell line containing a transsilencer and a reverse transactivator, expression cassettes encoding enhanced green fluorescent protein, CD123 or a constitutively active, cytotoxic caspase-3 were flanked with insulators and stably integrated. The performance of TREtight and SG/TRE was furthermore analysed in transiently transfected primary CD4(+) human T-cells., Results: The promoters exhibit greatly diverging characteristics. MMTV/TRE permits moderate, TRE and TREtight permits intermediate and SG/TRE permits very high expression levels. TRE and SG/TRE are leaky, whereas MMTV/TRE and TREtight provide stringent expression control. Tetracycline derivatives add flexibility to transgene expression by introducing intermediate expression levels., Conclusions: The different expression profiles of the promoters increase the flexibility to adjust transgene expression levels. The promoters provide an additional option to optimize system performance for many applications.

Precursor BCR (pre-BCR) signaling governs proliferation and differentiation of pre-B cells during B lymphocyte development. However, it is controversial as to which parts of the pre-BCR, which is composed of Igmu H chain, surrogate L chain (SLC), and Igalpha-Igbeta, are important for signal initiation. Here, we show in transgenic mice that the N-terminal non-Ig-like (unique) tail of the surrogate L chain component lambda5 is critical for enhancing pre-BCR-induced proliferation signals. Pre-BCRs with a mutated lambda5 unique tail are still transported to the cell surface, but they deliver only basal signals that trigger survival and differentiation of pre-B cells. Further, we demonstrate that the positively charged residues of the lambda5 unique tail, which are required for pre-BCR self-oligomerization, can also mediate binding to stroma cell-associated self-Ags, such as heparan sulfate. These findings establish the lambda5 unique tail as a pre-BCR-specific autoreactive signaling motif that could increase the size of the primary Ab repertoire by selectively expanding pre-B cells with functional Igmu H chains.

Pre-BCR signals are part of a checkpoint where early precursor (pre-) B cells with a pairing Ig muH chain (muHC) are clonally expanded before they differentiate into IgL-rearranging, resting pre-B cells. A pre-BCR consists of two muHCs, two surrogate L chains and the signal transducer Igalpha/Igbeta. The molecular circuits by which the pre-BCR controls proliferation and differentiation of pre-B cells are poorly characterized. Therefore, we identified the differential transcriptome by genome-wide expression profiling in progenitor (pro-) B cells from a Rag2-deficient mouse, in which the expression of a transgenic muHC and thus a pre-BCR as well as pre-BCR-mediated clonal expansion can be controlled by tetracycline (muHC-inducible mouse). This analysis revealed that pre-BCR signals upregulate components of the BCR signalosome, open the IgL chain (LC) locus and induce the krüppel-like transcription factor KLF2, a key regulator of quiescence and lymphocyte migration. Hence, pre-BCR signals establish the molecular network for BCR signaling even before the production of an IgLC and induce the expression of KLF2, a candidate for controlling clonal expansion and migration of functional pre-B cells.

B-cell receptor (BCR) signals are essential for B-cell differentiation, homeostasis and negative selection, which are regulated by the strength and quality of BCR signals. Recently, we identified a new adaptor protein, Swiprosin-1, in lipid rafts of B-cell lines that undergo apoptosis after BCR stimulation. During murine B-cell development, Swiprosin-1 exhibited highest expression in immature B cells of the bone marrow, but was also expressed in resting and activated splenic B cells and in non-lymphoid tissue, especially in the brain. Ectopic expression of Swiprosin-1 in the immature murine B-cell line WEHI231 enhanced spontaneous and BCR-induced apoptosis. In contrast, short hairpin RNA (shRNA)-mediated downregulation of Swiprosin-1 impaired specifically spontaneous and BCR-elicited apoptosis, but not BCR-induced G1 cell cycle arrest and upregulation of the cell cycle inhibitor p27(Kip1). In accordance, Swiprosin-1 abundance regulated net cell growth of WEHI231 cell populations through reciprocal regulation of Bcl-xL, but not Bim, thereby controlling spontaneous apoptosis. Swiprosin-1-enhanced apoptosis was blocked through nuclear factor kappaB-activating stimuli, namely B-cell-activating factor of the TNF family, anti-CD40 and lipopolysaccharide (LPS). This correlated with enhanced BCR-induced IkappaB-alpha phosphorylation and degradation in cells expressing a Swiprosin-1-specific shRNA. Finally, ectopic Swiprosin-1 expression enhanced BCR-induced cell death in primary, LPS-stimulated splenic B cells. Hence, Swiprosin-1 may regulate lifespan and BCR signaling thresholds in immature B cells.

Survival of mature B cells is thought to depend on the BCR signaling (BCR) because ablation of either H chain (HC) expression or BCR signaling causes B cells to rapidly disappear. Whether a complete BCR is required for survival of mature B cells is not known. To address this question, we generated a mouse in which we can repress the expression of a transgenic Ig L chain (IgL) by doxycycline (IgL-repressible mouse). Repression of IgL abrogated expression. Surprisingly, however, IgL-negative B cells survived longer than 14 wk, expressed signal-competent HC on the cell's surface, and active unfolded protein response factors. Like postgerminal center B cells, IgL-negative B cells were small lymphocytes, not dividing and expressed Bcl-6. Our results indicate that expression of unpaired HC, as it may occur as a consequence of Ag ligation, somatic hypermutation, or receptor editing, facilitates the survival of cells either by inducing receptor signaling or by inducing unfolded protein response and/or the expression of survival genes such as Bcl-6.

Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits alpha-1 and beta-2, vimentin, gamma-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.

Several studies have implicated a role for the cell surface glycoprotein CD44 in lymphocyte differentiation, adhesion to the matrix, homing, activation and apoptosis. However, the requirement of CD44 in B cell maturation remains elusive. To address this point, we analyzed the generation and activation of B lymphocytes in CD44-deficient mice. Unexpectedly, both maturation and autoreconstitution of early bone marrow B cell progenitors after sublethal irradiation as well as frequencies of splenic B cell subsets are indistinguishable in wild-type and CD44-deficient mice. Furthermore, splenic CD44-deficient B lymphocytes show a normal activation pattern after stimulation with LPS, anti-IgM/IL-4 or anti-CD40/IL-4. These results show for the first time that CD44 is clearly dispensable for development, reconstitution and activation of B lymphocytes.

The transmembrane receptor Notch1 plays a crucial role in differentiation and apoptosis of hematopoietic cells. To investigate the influence of Notch1 on apoptosis and cell growth of mature murine B cells, we transduced the murine B-lymphoma line NYC 31.1 with a constitutively active, intracellular form of human Notch1 (Notch1-ICT). NYC cells represent mature activated B cells that can be induced to undergo apoptosis by crosslinking of the B-cell receptor (BCR). In contrast to investigations in immature chicken B-cell lines, transduced Notch1-ICT did not affect cell cycle progression, cell growth or surface IgM levels in NYC cells and resulted only in a slight induction of apoptosis. However, BCR-crosslinking enhanced apoptosis, but did not influence cell cycle progression in Notch1-ICT-positive NYC cells. These data imply a distinct function of Notch1 in mature murine B-cells as compared to immature chicken B cells and provide further evidence for Notch1's involvement in B-cell differentiation and development.

The assembly of a pre-B cell receptor (pre-BCR) composed of an Ig mu heavy chain (mu H-chain), the surrogate light (SL) chain, and the Ig alpha/beta dimer is critical for late pro-B cells to advance to the pre-B cell stage. By using a transgenic mouse model, in which mu H-chain synthesis is solely driven by a tetracycline-controlled transactivator, we show that de novo synthesis of mu H-chain in transgenic pro-B cells not only induces differentiation but also proliferation. This positive effect of mu H-chain synthesis on proliferation requires the presence of SL chain and costimulatory signals provided by stromal cells or IL-7. We conclude that pre-BCR signaling induces clonal expansion of early pre-B cells.

Our studies examined the expression and DNA binding activity of myocyte enhancer factor 2 (MEF2A-D) transcription factors in lymphopoietic tissues, cell lines, and primary lymphocytes. Our analyses demonstrate that mef2C expression is restricted to B cells within the lymphocyte lineage. Using in situ hybridization, mef2C is detected in foci in fetal liver and postnatal thymic medulla, and both mef2B and mef2C are expressed in areas of the postnatal spleen and lymph node that also express kappa light chain (Ckappa), a B cell-specific marker. Reverse transcriptase-PCR (RT-PCR) analyses demonstrate that all mef2 family members are expressed in B cell lines, and all except mef2C are expressed in T cell lines. Immunoblot analyses of cell lines and primary thymic and splenic lymphocytes show that MEF2C and MEF2D proteins are expressed in B cells and that MEF2D is expressed in T cells; however, MEF2A protein is not detected in lymphocytes. Electrophoretic mobility shift assays (EMSA) demonstrate that B cell lines have MEF2C-containing, MEF2-specific DNA binding complexes whereas T cells do not. Our data is the first to describe mef2C expression in the lymphocyte lineage, and this finding suggests possible roles for MEF2C activity in B cell development and function.

Leflunomide is an immunosuppressive drug capable of inhibiting T and B cell responses in vivo. A number of studies demonstrate that leflunomide functions both as a pyrimidine synthesis inhibitor and as a tyrosine kinase inhibitor. We previously reported that leflunomide inhibits LPS-stimulated B cell proliferation, cell cycle progression, and IgM secretion. This inhibition can be reversed by the addition of exogenous uridine, suggesting that leflunomide functions as a pyrimidine synthesis inhibitor in B cells. We report here that while the addition of uridine restored proliferation and IgM secretion to leflunomide-treated LPS-stimulated B cells, as determined by metabolic labeling and immunoprecipitation, it did not completely restore secretion of IgG Ab. We hypothesized that leflunomide inhibits LPS-induced IgG secretion by inhibiting tyrosine kinase activity required for isotype switch. We tested this hypothesis in a well-defined model of isotype switch, LPS plus IL-4 induction of IgG1. Leflunomide inhibited IgG1 secretion in this model in a dose-dependent manner. The signal transduction pathway utilized by IL-4 to induce IgG1 involves tyrosine phosphorylation of the IL-4 receptor, JAK1, JAK3, and STAT6 proteins induced by IL-4 binding to the IL-4R. Leflunomide diminished the tyrosine phosphorylation of JAK3 and STAT6 in the absence or presence of uridine. In gel mobility shift studies, STAT6 binding to the STAT6 DNA binding site in the IgG1 promoter decreased in the presence of leflunomide or leflunomide plus uridine. Taken together, these data suggest that leflunomide acts as a tyrosine kinase inhibitor to block IgG1 production.

NYC is a B lymphoma cell line derived from B/W mice. Upon fusion of NYC cells with a plasmacytoma, which itself produces no immunoglobulin, the resulting NYCH hybridoma cells are Mott cells; i.e., they contain large intracellular vesicles filled with immunoglobulin, the so-called Russell bodies. When NYCH.kappa, a variant of NYCH that had lost the ability to produce heavy chain, was transfected with a heavy-chain construct, this concentration of immunoglobulin in the intracellular vesicles occurred only when the transfected immunoglobulin heavy chain had the same variable region as NYC. Moreover, unlike conventional Mott cells, the hybrid cells secrete immunoglobulin at a normal rate.

During the switch in expression of an immunoglobulin class, the gene segment encoding the constant region of the heavy chain is replaced in a way that leads to a deletion. Three different models of how this deletion is generated have been proposed: recombination between homologs, unequal sister chromatid exchange, and looping out and deletion. While none of the predicted recombination products of the first two models have been found, the products of the looping out--inversions and circular DNA--have been isolated. Thus looping out and deletion appears to be the appropriate model to explain the genetic events leading to the immunoglobulin heavy chain class switch.

During differentiation of B lymphocytes, the change in the amount of immunoglobulin heavy chain produced is reflected by a change in the steady state level of heavy chain mRNA. At the pre-B cell stage, the earliest stage at which immunoglobulin chain is produced, and later at the small resting B cell stage, there is a low steady state level of heavy chain mRNA. After the small B cell has differentiated to become a plasma cell, the steady state level of heavy chain mRNA is much higher. We confirm that the transcription rate at the immunoglobulin mu heavy chain gene does not change during differentiation from the pre-B cell to the plasma cell stage. In contrast, we show here that differences in the stability of mu mRNA are sufficient to account for the differences in the steady state level at the various differentiation stages.

In the mouse pre-B-cell line 18-81, cells can switch production in vitro from immunoglobulin mu chain to gamma 2b chain. The gene encoding the gamma 2b chain is created by a rearrangement of the mu gene. This rearrangement always takes place within a homolog. In cells with a gamma 2b gene, most of the time the gene segment encoding the constant region of the mu chain is deleted, but often the rearrangement leads to cells that produce no immunoglobulin, and all DNA sequences are retained. The latter result is due to an inversion. Inversions exclude the unequal sister chromatid exchange model of the heavy-chain class switch. Looping out is an intermediate step in the process of generating an inversion. Our findings demonstrate that the switch rearrangement occurs by looping out and deletion.

It is established that somatic mutation is an important source of antibody diversity in vivo. It is also established that Igh-V gene segments are hypermutable in vitro. This is not a completely satisfactory situation. While there is no reason to believe that Igh-V genes are not hypermutable in vivo as well, direct experimental evidence is lacking. Perhaps experiments with transgenic mice will soon fill this gap. It is not so clear how much higher than normal the rate of hypermutation is. As far as we are aware, there are no direct measurements of mutation rates per base pair per cell generation in mammals, certainly not for lymphocyte cell lines. For a variety of reasons, it is difficult to measure very low mutation rates. The general consensus is that the normal rate should be somewhere between 10(-10) and 10(-12) mutations per base pair per cell generation. Therefore, an experiment designed to directly determine a rate using the compartmentalization test would involve hundreds of cultures, each containing at least 10(9) cells. It is not a trivial problem to find one or a few mutants among so many cells. It is simple to study mutation to resistance to a drug, for example, ouabain or azaguanine, but, as we discussed, there are technical and conceptual pitfalls. The vast excess of dead cells influences the growth of a few mutant cells, particularly in lymphocyte cell lines. Even if this problem could be solved, the mutation rate so obtained would be "per gene(s)" and not "per base pair". The problems associated with cytotoxic agents can be avoided by immunofluorescence methods in conjunction with selective cloning or cell sorting. Using these techniques, we have carried out extensive experiments to determine whether the immunoglobulin mutator system acts, at least partially, on genetic elements other than those in or near the heavy chain variable region gene segment. For an opal termination codon in a heavy chain constant region gene segment, the rate of reversion was less than 10(-7) per base pair per cell generation. This upper limit was fixed by the high rate of small deletions at the heavy chain locus. For an allotype mutation at B2m, the gene encoding beta 2 microglobulin, the rate of mutation was less than 10(-8). This upper limit could be lowered by at least two orders of magnitude by using a high-speed cell sorter.

In a pre-B-cell line that rearranges its heavy chain gene segments in vitro, we found that the rate of productive rearrangement on one allele was not influenced by the presence of heavy chain protein encoded by the other allele. This shows that allelic exclusion of heavy chain genes is not regulated at the genetic level.