B Cell Development

In B lymphocytes, immunoglobulin (Ig)M receptors drive development and construction of naive repertoire, whereas IgG receptors promote formation of the memory B cell compartment. This isotype switching process requires appropriate B cell activation and T cell help. In the absence of T cell help, activated B cells undergo Fas-mediated apoptosis, a peripheral mechanism contributing to the establishment of self-tolerance. Using Ig -deficient MT mouse model, where B cell development is blocked at pro-B stage, here we show an alternative developmental pathway used by isotype-switched B cell precursors. We find that isotype switching occurs normally in B cell precursors and is T independent. Ongoing isotype switching was found in both normal and MT B cell development as reflected by detection of IgG1 germline and postswitch transcripts as well as activation-induced cytidine deaminase expression, resulting in the generation of IgG-expressing cells. These isotype-switched B cells are negatively selected by Fas pathway, as blocking the Fas/FasL interaction rescues the development of isotypeswitched B cells in vivo and in vitro. Similar to memory B cells, isotype-switched B cells have a marginal zone phenotype. We suggest a novel developmental pathway used by isotypeswitched B cell precursors that effectively circumvents peripheral tolerance requirements. This developmental pathway, however, is strictly controlled by Fas/FasL interaction to prevent B cell autoimmunity.

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Agammaglobulinemia is a rare primary immunodefi ciency characterized by an early block of B-cell development in the bone marrow resulting in the absence of peripheral B cells and low/absent immunoglobulin serum levels. Mutations in the Bruton tyrosine kinase and in components of the pre-B-cell receptor (pre-BCR), such as μ heavy chain, surrogate light chain, and Ig α have been found in 85% to 90% of patients affected by this disease. Here we review the recent advances in the characterization of molecular defects underlying an early block in B-cell development, focusing on the novel fi nding of the fi rst two patients with agammaglobulinemia caused by mutations in Ig β , the transmembrane protein that associates with Ig α as part of the pre-BCR complex. Characterization of novel genetic defects involving components of the pre-BCR is crucial for a better understanding of the biology of early B-cell development and may have therapeutic and prognostic implications.

Seventy percent of peripheral immature conventional (B2) B cells fail to develop into mature B cells. The nature of this cell loss has not been characterized; the process that governs which immature B cells develop into long-lived peripheral B cells could be either stochastic or selective. Here, we demonstrate that this step is in fact selective, in that the fate of an immature B cell is highly dependent on its Ig receptor specificity. A significant skewing of the B cell receptor repertoire occurs by the time cells enter the mature B cell fraction, which indicates that there is selection of only a minority of immature B cells to become mature B cells. Because only a few heavy-light chain pairs are enhanced of the diverse available repertoire, we favor the idea that selection is positive for these few heavy-light chain pairs rather than negative against nearly all others. Because most immature B cells are lost at this transition, this putative positive selection event is likely to be a major force shaping the mature B cell receptor repertoire available for adaptive immune responses. † To whom reprint requests should be addressed at

Human interleukin-7 (IL-7) is a cytokine that appears to be critical for early T-and B-cell development and although IL-7 is currently under investigation as a therapeutic agent in a variety of hematolymphopoietic disorders, there have been few instances of the detection or investigation of this cytokine using a biological assay. This has been due, in the main, to the lack of a widely available, stable, easy to maintain and use, IL-7 responsive cell line. We have developed a pre-B-cell line, PB-1, from murine bone marrow, that is dependent on IL-7 for growth and has been maintained continually for up to 1 year without loss of responsiveness. The cells survive freezing and reviving, having been stored for periods of up to 4 years. The IL-7 bioassay is reproducible and sensitive, able to reliably detect 50 pg / ml IL-7. The assay is completely unresponsive to any other stimulatory cytokines tested and is not affected by a wide variety of inhibitory cytokines, with the exception of high levels of interferon alpha. The assay can be made completely specific for human IL-7 by including specific neutralizing antibodies for IL-7 and has been shown to be suitable for the estimation of IL-7 in both plasma and serum samples.

Key Words B cell development, tolerance, receptor editing, RAG1 and RAG2 expression, antiself antibodies s Abstract Autoreactive antibodies are etiologic agents in a number of autoimmune diseases. Like all other antibodies these antibodies are produced in developing B cells by V(D)J recombination in the bone marrow. Three mechanisms regulate autoreactive B cells: deletion, receptor editing, and anergy. Here we review the prevalence of autoantibodies in the initial antibody repertoire, their regulation by receptor editing, and the role of the recombinase proteins (RAG1 and RAG2) in this process.

Human X-linked agammaglobulinemia (XLA) and murine X-linked immune defect (XID) are both immunodeficiencies mediated by mutations in Bruton's tyrosine kinase (Btk), yet the developmental stage(s) affected remain controversial. To further refine the placement of the XID defect(s), we used bromodeoxyuridine labeling to determine turnover, production and transition rates of developing B cell subsets in normal, xid and xid mice expressing a human Bcl-2 transgene (xid/bcl-2). We find the xid mutation manifest at two stages of B cell development. The first is early, reducing pre-B cell production by restricting pro-B to pre-B cell transit. Surprisingly, this impairment is offset by increased survival of cells progressing from the pre-to immature B cell pool, suggesting that Btk-independent homeostatic mechanisms act to maintain this compartment. The second point of action is late, substantially reducing mature B cell production. Together, these findings reconcile apparent discrepancies in the developmental stage affected by the murine versus human lesions and suggest previously unappreciated homeostatic processes that act at the pre-B to immature B cell transition. Finally, Btk likely functions differently at these two checkpoints, since ectopic Bcl-2 expression fails to directly complement the early xid lesion, yet reverses the defect impeding final B cell maturation.

We examined the generation and selection of the B cell antibody repertoire through crossing of mice bearing distinct Ig heavy (H) and light (L) chain rearranged variable region transgenes. Ig gene knockin and transgenic mice whose H and L chains pair to form a non-autoreactive, functional B cell antigen receptor (BCR) have significantly reduced pre-B cells in the bone marrow as their B cell progenitors rapidly differentiate into surface IgM 1 B cells. The presence of a pre-B cell compartment in these Ig transgenic mice, however, indicates the induction of receptor editing. Here, 18 distinct combinations of H and L chains were generated that we showed could pair in vitro to form BCRs of unknown specificities. Of these, nine induced receptor editing in vivo as evidenced by the presence of pre-B cells and endogenous L chain rearrangements in mice bearing these H and L chain transgenes. These data thus suggest that about half of the emerging antibody repertoire is negatively selected during B lymphopoiesis due to the likely encoding of autoreactive or non-functional BCRs. Fig. 4. Stromal cell cultures of bone marrow B cells derived from mice transgenic for certain H and 3-83j L chains. Figure shows the B220 versus CD43 profiles of the surface IgM-negative cells present in the stromal cell cultures. Numbers indicate the percentage of pro-B and pre-B cells as a fraction of total cultured cells recovered that are gated to exclude the ST2 stromal cells. Frequent generation of potentially autoreactive antibodies 349 by guest on December 17, 2015

Rabbits predominantly rearrange the most 3' VH gene (VHl); thus combinatorial diversity is very limited. In man and mouse, the most 3' D, gene, DQ52, is preferentially rearranged early in B-cell development. To test whether this preference for rearranging a DH gene segment based on 3' end proximity exists in rabbit, we cloned and sequenced the rabbit DQ52 gene. The 11 base pair coding region sequence is identical to a published mouse DQ52, and 81.8% similar to the human sequence. It is localized -805 bp upstream of the J,.,l gene. However, the 3' recombination signal sequence has an atypical nonamer. We prepared mRNA from 15-to 2%day fetal rabbits and amplified expressed VDJ sequences of p mRNA by RT-PCR. The PCR products with VDJ rearrangements were cloned and sequenced. As expected, 44 of 45 VDJ sequences reflected use of the 3' V,, 1 a2 gene. but the DQ52 gene was utilized very infrequently, if at all. We found only one VDJ sequence from 28-day fetal liver B-cells with 8 bp that matched the germline DQ52 sequence. Instead of expressing DQ52. another D, gene, Df was frequently expressed. We cloned the genomic Df gene and localized it about 32 kb upstream of the JH region. Thus, in contrast to man and mouse, rabbits preferentially express a Dti gene located in the middle of the D, region early in 3 cell ontogeny. This may correlate with more frequent initial rearrangement of V, to D, in rabbit B cells. Published by Elsevier Science Ltd.

The chemokine receptor CXCR4 plays a central role in organspecific homing and tumor spreading and is induced by hypoxia. B lymphocytes are exposed to low oxygen tensions during their development, but the influence of hypoxia on their physiology is poorly understood. Here, we show that hypoxia is associated with up-regulation of CXCR4 expression in human normal and malignant B cells, through both transcriptional and posttranslational mechanisms. However, a dichotomic functional response to CXCR4 triggering was observed: both peripheral B cells and lymphomas arising from mature B cells displayed increased responses to CXCR4 triggering under hypoxia, whereas germinal center (GC) B cells as well as GC-derived lymphomas showed CXCR4 receptor desensitization. This phenomenon was associated with differential modulation of key signal-transducing molecules, including mitogen-activated protein kinase phosphatase-1 and regulator of G protein signaling molecule-1. The unresponsiveness of GC-derived lymphomatous B cells to CXCR4 triggering under hypoxia may have implications for the development and pathogenesis of GC-derived lymphoid tumors.

We describe the effect on the neonate of administration of rituximab to a woman with idiopathic thrombocytopenic purpura (ITP). Rituximab, an anti-CD20 antibody, was given weekly for 4 weeks to a woman with ITP in her third trimester of pregnancy. One month after the last rituximab administration a healthy girl was born. She had normal growth and development during the first six months. At birth, B-lymphocytes were not detectable. Rituximab levels in mother and neonate were 24000 and 6700 ng/mL, respectively. Only 7 cases of rituximab administration during pregnancy were described. No adverse events are described for fetus and neonate. We demonstrate that rituximab passes the placenta and inhibits neonatal B-lymphocyte development. However, after 6 months B-lymphocyte levels normalized and vaccination titres after 10 months were adequate. No infection-related complications occurred. Rituximab administration during pregnancy appears to be safe for the child but further studies are warranted.

We found that transgenic (tg) mice stably expressing a bacterial artificial chromosome (BAC)-derived human complement receptor type 2 (CR2/CD21) gene demonstrate B cell specific hCR2 protein expression, normal B cell development and no changes in B cell subpopulations. To determine whether this BAC-encoded human CR2 (hCR2) can replace mouse CR2/CR1 in Cr2 −/− mice and restore humoral immune responses to model foreign antigens (Ags), we generated hCR2 +/− Cr2 −/− tg mice and immunized them with sheep red blood cells (SRBC). We found that hCR2 +/− Cr2 −/− mice demonstrated anti-SRBC antibody (Ab) levels that were initially comparable to Cr2 −/− mice after a single injection of the Ag, but then showed marked increases in anti-SRBC IgM and IgG1 levels after a second immunization. Identical results were found with a second model Ag, NP-Ficoll. To further confirm that this improvement in Ag-specific Ab production over Cr2 −/− mice was indeed due to hCR2 expression, as well as to examine the effects of treating hCR2 +/− Cr2 −/− mice with an inhibitory anti-hCR2 monoclonal Ab (mAb) in vivo, we used mAb 171, an anti-hCR2 mAb that we have shown directly recognizes the C3d ligand binding site on hCR2. We first found that mAb 171 completely blocked hCR2-dependent co-activation of hCR2-tg B cells by anti-BCR/C3d complexes as measured in vitro by intracellular calcium influx. The i.p. injection of 1 mg of mAb 171 was then found to induce for at least three weeks only partial loss of hCR2 surface expression, without modifying B and T cell numbers or the apparent activation status of the cells. Treatment of hCR2 +/− Cr2 −/− mice with mAb 171 also substantially suppressed the development of anti-SRBC and anti-NP Abs following immunization with Ags. The development of this model system should allow the study of the effects of manipulating hCR2 function in vivo with potential therapeutic compounds.

Notch reporter mice were transplanted into Mib1 -null mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1-null microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre–B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible μHC transgene in Rag2−/− pro–B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-α. Furthermore, in wild-type mice and in mice lackin...

To study B-cell development from bone marrow (BM), we generated recombination-activating gene 1 (Rag1)–targeted mice lacking mature lymphocytes. B-cell development can be induced in such mice by B cell–specific restoration of a functional Rag1 transcription unit. Follicular and marginal zone B cells populated the spleen when Rag1 expression was permitted. Notably, the peritoneal cavity was dominated by bona fide B-1a cells, as judged by surface markers and functional properties. These BM-derived B-1a cells exhibited a polyclonal VDJ repertoire with substantial N nucleotide insertions. Nevertheless, physiologic frequencies of phosphatidylcholine-specific B cells were detected. Importantly, the BM of young and 5-month-old mice was indistinguishable with regard to the potential to generate B-1a cells.

Bruton's tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B ...

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection. ava i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s ev i e r. c o m / l o c a t e / yc l i m Clinical Immunology (2008) 129, 448-454

progenitor cells derived hematopoietic − embryonic stem cell and umbilical cord blood Differences in lymphocyte developmental potential between human http://bloodjournal.hematologylibrary.org/content/112/7/2730.full.html Updated information and services can be found at: (3132 articles) Hematopoiesis and Stem Cells Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.

Only mature B lymphocytes can enter the lymphoid follicles of spleen and lymph nodes and thus efficiently participate in the immune response. Mature, long-lived B lymphocytes derive from short-lived precursors generated in the bone marrow. We show that selection into the ...

Selective expansion of functional pre-B cells is accomplished by the assembly of a signaling-competent pre-B cell receptor (pre-BCR) consisting of immunoglobulin heavy chains (HC), surrogate light chains (SLC) and Ig␣/Ig␤. Here, we review recent data showing that HCs, in the absence of SLC, deliver autonomous differentiation signals. However, enhanced signaling necessary for pre-B cell expansion requires cross-linking of pre-BCRs via the non-immunoglobulin tail of SLC's subunit 5. We also discuss how SLC's ability to modulate the strength of pre-BCR signals is controlled by a HC's idiotype and its affinity to the chaperone BiP. In this model, BiP in concert with SLC functions as a pre-selector of the antibody repertoire.

The initial phases of B cell development depend on interactions between the cell surface molecules and secreted products of stromal cells with their receptor-ligand partners on lymphoid progenitors. Recent research in this area has greatly advanced our understanding of B cell development and differentiation. Antigen receptors on pre-B and B cells play key roles in the progression of this differentiation process, as revealed by targeted and inherited gene mutations that disrupt B cell development and by the transgenic repair of these mutations in mice.

All cells continually survey their environment and make decisions based on cues encountered. This requires specific receptors that detect such cues, then transduce signals that initiate the appropriate responses. B lymphocytes provide an archetypal model for such 'adaptive' cellular responses, where signals transmitted by the B cell Ag-receptor (BCR) influence not only cellular selection, maturation, and survival, but are imperative in generating the ultimate effector function of B cells, i.e. antibody production. While other extracellular stimuli and their cognate receptor signals can also influence B cell development, BCR-mediated signals and the way in which they are integrated and regulated are paramount in defining the cell's physiological fate.

Understanding of the signal transduction pathways that lead to B cell development is of extreme interest to learn how alterations in these pathways might initiate malignant transformation. Long-term cultured early pre-BI cells can differentiate into IgM þ B cells after transplant into NOD/SCID mice, offering the possibility to explore checkpoints in B cell development. Using DNA microarray and Western blot analysis of IgM þ B cells vs parental early pre-BI cells, we demonstrated that zetaassociated protein 70 (ZAP-70) is upregulated in our B cell differentiation model. Unlike parental ZAP-70 À early pre-BI cells, ZAP-70 þ IgM þ B cells exhibited a transformed phenotype, as indicated by BCL-6 expression, a high Ki-67 proliferation index, resistance to IL-7 deprivation-induced apoptosis, and an increased repopulation rate in NOD/SCID mice. These data show the characterization and generation of a novel murine leukemia model with many similarities to human ZAP-70 þ B cell chronic lymphocytic leukemia.

Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4 + T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.

Background-Despite best current therapy, up to 20% of pediatric patients with acute lymphoblastic leukemia (ALL) have a relapse. Recent genomewide analyses have identified a high frequency of DNA copy-number abnormalities in ALL, but the prognostic implications of these abnormalities have not been defined.

Summary The germinal center (GC) is an anatomic compartment found in peripheral lymphoid organs, wherein B cells undergo clonal expansion, somatic mutation, switch recombination, and reac- tivate immunoglobulin gene V(D)J recombination. As a result of somatic mutation, some GC B cells develop higher affinity antibodies, whereas others suffer mutations that decrease affinity, and still others may become self-reactive. It has been proposed that secondary V(D)J rearrange- ments in GCs might rescue B cells whose receptors are damaged by somatic mutations. Here we present evidence that mature human tonsil B cells coexpress conventional light chains and recombination associated genes, and that they extinguish recombination activating gene and terminal deoxynucleotidyl transferase expression when their receptors are cross-linked. Thus, the response of the recombinase to receptor engagement in peripheral B cells is the opposite of the response in developing B cells to the same stimulus. The...

The matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the extracellular matrix, thus involved in cellular migration. The extent and role of MMPs secretion in primary non-transformed B cells, and specifically during early stages of development in the bone marrow (BM), has been barely unveiled. Herein, we investigated the secretion of MMP-9 during B lymphopoiesis and its modulation in response to different mitogens and cytokines. To do so, we used our BM culture system and well-studied mutated mouse models to isolate the different B cell populations. Our results show that MMP-9 is spontaneously secreted throughout B lymphopoiesis, and that the level of secreted MMP-9 is developmentally regulated. Using reverse transcription-PCR, we found that IFNbetaR is expressed throughout B cell development, while tumor necrosis factor (TNF)-alphaR-p55 and IFNgammaR expressions are initiated only at the pre-B stage. We found that TNFalpha stimulates MMP-9 secretion in transition...

V(D)J recombination at Igh and Igk loci takes place sequentially during successive stages in B cell development. Using 3-dimensional DNA fluorescence in situ hybridization (FISH), we identified a lineage-and stage-specific interchromosomal association between these two loci which marks the transition between Igh and Igk recombination. Co-localization occured between pericentromerically located alleles in pre-B cells, and was mediated by the 3' Igk enhancer. Deletion of this regulatory element prevented association of the Igh and Igk loci, inhibited pericentromeric recruitment and locus decontraction of an Igh allele, and resulted in increased distal V H gene rearrangement. Our data indicate a role for the Igk locus and its 3' enhancer in directing the Igh locus to a repressive nuclear subcompartment, and in rendering the Igh locus unable to contract.

In mouse mutants incapable of expressing chains, V J joints are detected in the CD43 ϩ B cell progenitors. In agreement with these earlier results, we show by a molecular single cell analysis that 4-7% of CD43 ϩ B cell progenitors in wild-type mice rearrange immunoglobulin (Ig) genes before the assembly of a productive V H D H J H joint. Thus, chain expression is not a prerequisite to Ig light chain gene rearrangements in normal development. Overall, ‫ف‬ 15% of the total CD43 ϩ B cell progenitor population carry Ig gene rearrangements in wild-type mice. Together with the results obtained in the mouse mutants, these data fit a model in which CD43 ϩ progenitors rearrange IgH and Ig loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors V J joining rapidly initiates chain expression, irrespective of the presence of a chain.