pre-BCR Checkpoint Research Articles

Fc receptor-like 6 (FCRL6) delineates innate-like B cell progenitors that undergo pre-BCR dependent and independent pathways of natural antibody selection

Abstract Innate-like B-1a B cells produce ‘natural’ antibodies (Abs) to neutralize pathogens and clear neo self-antigens, but the early selection mechanisms that shape their polyreactive repertoires are poorly understood. Fc receptor-like (Fcrl) genes encode surface regulatory proteins preferentially expressed by B cells. Here, we identified a B cell progenitor subset defined by FCRL6 expression, harboring emerging Ighv repertoire, migration, and differentiation properties conducive for natural Ab generation. Receptor-specific monoclonal antibodies identified subpopulations of FCRL6+ pro B cells in E18 fetal liver (FL), neonatal tissues, and adult bone marrow (BM). While it was absent on Hardy Fr. A cells, FCRL6 expression became evident in Fr. B, peaked in Fr. C, and declined in Fr. C′. By the Dorshkind scheme, FCRL6 marked subsets of B-1P and pro B cells in the FL and BM, but was not detected on B-2P cells. Compared to FCRL6− pro B cells, the repressed mitotic, DNA damage repair, and signaling activity of FCRL6+ progenitors, yielded VH repertoires with biased distal Ighv segment accessibility, constrained diversity, and hydrophobic and charged CDR-H3 sequences. Similar features were apparent among B-1a and chronic lymphocytic leukemia (CLL) Ighv sequences. Beyond nascent autoreactivity, VH11 productivity, which predominates phosphatidylcholine-specific B-1a B cell receptors (BCRs), was higher for FCRL6+ cells as was pre-BCR formation, which was required for Myc induction and VH11, but not VH12, B-1a development. Thus, FCRL6 revealed unexpected heterogeneity in the developmental origins, regulation, and selection of natural Abs at the pre-BCR checkpoint with implications for autoimmunity and lymphoproliferative disorders.

Fc Receptor-Like 6 (FCRL6) Discloses Progenitor B Cell Heterogeneity That Correlates With Pre-BCR Dependent and Independent Pathways of Natural Antibody Selection.

B-1a cells produce “natural” antibodies (Abs) to neutralize pathogens and clear neo self-antigens, but the fundamental selection mechanisms that shape their polyreactive repertoires are poorly understood. Here, we identified a B cell progenitor subset defined by Fc receptor-like 6 (FCRL6) expression, harboring innate-like defense, migration, and differentiation properties conducive for natural Ab generation. Compared to FCRL6− pro B cells, the repressed mitotic, DNA damage repair, and signaling activity of FCRL6+ progenitors, yielded VH repertoires with biased distal Ighv segment accessibility, constrained diversity, and hydrophobic and charged CDR-H3 sequences. Beyond nascent autoreactivity, VH11 productivity, which predominates phosphatidylcholine-specific B-1a B cell receptors (BCRs), was higher for FCRL6+ cells as was pre-BCR formation, which was required for Myc induction and VH11, but not VH12, B-1a development. Thus, FCRL6 revealed unexpected heterogeneity in the developmental origins, regulation, and selection of natural Abs at the pre-BCR checkpoint with implications for autoimmunity and lymphoproliferative disorders.

C-Myb Coordinates Survival and the Expression of Genes That Are Critical for the Pre-BCR Checkpoint.

The c-Myb transcription factor is required for adult hematopoiesis, yet little is known about c-Myb function during lineage-specific differentiation due to the embryonic lethality of Myb-null mutations. We previously used tissue-specific inactivation of the murine Myb locus to demonstrate that c-Myb is required for differentiation to the pro-B cell stage, survival during the pro-B cell stage, and the pro-B to pre-B cell transition during B lymphopoiesis. However, few downstream mediators of c-Myb-regulated function have been identified. We demonstrate that c-Myb regulates the intrinsic survival of CD19+ pro-B cells in the absence of IL-7 by repressing expression of the proapoptotic proteins Bmf and Bim and that levels of Bmf and Bim mRNA are further repressed by IL-7 signaling in pro-B cells. c-Myb regulates two crucial components of the IL-7 signaling pathway: the IL-7Rα-chain and the negative regulator SOCS3 in CD19+ pro-B cells. Bypassing IL-7R signaling through constitutive activation of Stat5b largely rescues survival of c-Myb-deficient pro-B cells, whereas constitutively active Akt is much less effective. However, rescue of pro-B cell survival is not sufficient to rescue proliferation of pro-B cells or the pro-B to small pre-B cell transition, and we further demonstrate that c-Myb-deficient large pre-B cells are hypoproliferative. Analysis of genes crucial for the pre-BCR checkpoint demonstrates that, in addition to IL-7Rα, the genes encoding λ5, cyclin D3, and CXCR4 are downregulated in the absence of c-Myb, and λ5 is a direct c-Myb target. Thus, c-Myb coordinates survival with the expression of genes that are required during the pre-BCR checkpoint.

MLL1 Promotes IL-7 Responsiveness and Survival during B Cell Differentiation.

B lymphocyte differentiation is an exquisitely regulated homeostatic process resulting in continuous production of appropriately selected B cells. Relatively small changes in gene expression can result in deregulation of this process, leading to acute lymphocytic leukemia (ALL), immune deficiency, or autoimmunity. Translocation of MLL1 (KMT2A) often results in a pro-B cell ALL, but little is known about its role in normal B cell differentiation. Using a Rag1-cre mouse knock-in to selectively delete Mll1 in developing lymphocytes, we show that B cell, but not T cell, homeostasis depends on MLL1. Mll1-/- B progenitors fail to differentiate efficiently through the pro- to pre-B cell transition, resulting in a persistent reduction in B cell populations. Cells inefficiently transit the pre-BCR checkpoint, despite normal to higher levels of pre-BCR components, and rearranged IgH expression fails to rescue this differentiation block. Instead of IgH-rearrangement defects, we find that Mll1-/- pre-B cells exhibit attenuated RAS/MAPK signaling downstream of the pre-BCR, which results in reduced survival in physiologic levels of IL-7. Genome-wide expression data illustrate that MLL1 is connected to B cell differentiation and IL-7-dependent survival through a complex transcriptional network. Overall, our data demonstrate that wild-type MLL1 is a regulator of pre-BCR signaling and B cell differentiation and further suggest that targeting its function in pro-B cell ALL may be more broadly effective than previously anticipated.

Distorted antibody repertoire developed in the absence of pre-B cell receptor formation

The pre-B cell receptor (pre-BCR), consisting of the μ heavy chain (μHC) and the surrogate light chain (SLC, Vpre-B and λ5), plays important roles during B cell development. The formation of the pre-BCR, which enables the nascent immunoglobulin HC to associate with the SLC, is considered a prerequisite for B cell development. However, a significant number of peripheral mature (leaky) B cells exist in SLC-deficient mice. These leaky B cells develop in the absence of pre-BCR and do not undergo the pre-BCR checkpoint. The antibody repertoires of leaky B cells thus reflect the absence of pre-BCR function. To investigate how the absence of the pre-BCR is circumvented by these leaky-B cells and examine the effect of the pre-BCR checkpoint on the antibody system, we analyzed the antibody repertoires of λ5-deficient (λ5−/−) mice using next-generation sequencing. In λ5−/− mice, spleen B cells displayed different patterns of VDJ-usage, relative to those in wild-type (WT) mice. Moreover, leaky B cells were neither derived from unusual B2 cells, characterized by particular LC gene rearrangements in the absence of pre-BCR signaling, nor from B1 cells, originating from different B cell progenitors. Analysis of the CDR-H3 amino acid sequences of μ-chain repertoires revealed that certain bone marrow B cells with particular CDR-H3 profiles undergo clonal expansion in λ5−/− mice. Part of these CDR-H3s contain arginine(s) in the middle of the CDR-H3 loop in λ5−/− mice, whereas few arginine(s) exist in this middle loop in WT CDR-H3s in the absence of clonal expansion. This CDR-H3 feature in λ5−/− mice presumably reflects the role of the pre-BCR in autoantibody regulation, since arginine(s) are often found in the antigen-binding site of autoantibodies. Here, we present a unique viewpoint on the role of pre-BCR, by assessing the whole antibody repertoire formed in SLC-deficient mice.

In old BALB/c mice, bone marrow pre-B cell and surrogate light chain reduction is associated with increased B cell reactivity to phosphorylcholine, but reduced T15 idiotype dominance

In young adult BALB/c mice, antibodies to phosphorylcholine (PC) bearing the T15 (TEPC 15) idiotype confer protection against pneumococcal infections. In old age, even though PC reactive B cells are often increased, the proportion of T15+ antibodies declines. We hypothesize that limited surrogate light chain (SLC) and compromise of the pre-B cell receptor checkpoint in old mice contribute to both reduced new B cell generation and changes in the anti-PC antibodies seen in old age. In old mice: 1) early pre-B cell loss is most pronounced at the preBCR checkpoint; however, the reduced pool of early pre-B cells continues to proliferate consistent with preBCR signaling; 2) increased PC reactivity is seen in bone marrow immature B cells; 3) deficient SLC promotes increased B cell PC reactivity and diminished T15 idiotype expression; and 4) as pre-B cell losses and reduced SLC become progressively more severe, increased T15 negative PC reactive B cells occur. These results associate a reduction in pre-B cells, imposed at the preBCR checkpoint, with increased reactivity to PC, but more limited expression of the protective T15 idiotype among PC reactive antibodies in old age.

The Expression Pattern of the Pre-B Cell Receptor Components Correlates with Cellular Stage and Clinical Outcome in Acute Lymphoblastic Leukemia.

Precursor-B cell receptor (pre-BCR) signaling represents a crucial checkpoint at the pre-B cell stage. Aberrant pre-BCR signaling is considered as a key factor for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) development. BCP-ALL are believed to be arrested at the pre-BCR checkpoint independent of pre-BCR expression. However, the cellular stage at which BCP-ALL are arrested and whether this relates to expression of the pre-BCR components (IGHM, IGLL1 and VPREB1) is still unclear. Here, we show differential protein expression and copy number variation (CNV) patterns of the pre-BCR components in pediatric BCP-ALL. Moreover, analyzing six BCP-ALL data sets (n = 733), we demonstrate that TCF3-PBX1 ALL express high levels of IGHM, IGLL1 and VPREB1, and are arrested at the pre-B stage. By contrast, ETV6-RUNX1 ALL express low levels of IGHM or VPREB1, and are arrested at the pro-B stage. Irrespective of subtype, ALL with high levels of IGHM, IGLL1 and VPREB1 are arrested at the pre-B stage and correlate with good prognosis in high-risk pediatric BCP-ALL (n = 207). Our findings suggest that BCP-ALL are arrested at different cellular stages, which relates to the expression pattern of the pre-BCR components that could serve as prognostic markers for high-risk pediatric BCP-ALL patients.

VpreB serves as an invariant surrogate antigen for selecting immunoglobulin antigen-binding sites.

Developmental checkpoints eliminate B cells synthesizing defective immunoglobulin heavy (HC) and light (LC) chains. The first checkpoint tests for formation of a VpreB/λ5/µHC-containing preB-cell receptor (preBCR) and predicts whether µHCs will bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with µHC antigen-binding region CDR-H3, but whether it plays a role in immunoglobulin repertoire selection and function is unknown. On a position-by-position basis, we analyzed the amino acid content of CDR-H3s from H chains cloned from living and apoptotic preB cells and from IgG:Antigen structures. Using a panel of DH gene-targeted mice, we show that progressively reducing CDR-H3 tyrosine content increasingly impairs preBCR checkpoint passage. Counting from cysteine at Framework 3 position 96, we found that VpreB particularly selects for tyrosine at CDR-H3 position 101, and that Y101 also binds antigen in IgG:Antigen structures. VpreB thus acts as an early invariant antigen. It selects for particular CDR-H3 amino acids and shapes the specificity of the IgG humoral response. This helps explain why some neutralizing antibodies against pathogens are readily produced while others are rare.

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint.

A Targeted Functional Clone Tracking Assay for the Identification of Tumour Suppressor Genes in BCP- ALL Implicates the Transcription Factors FOXO3 and PRDM1

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is accompanied by genomic mutations and rearrangements that commonly affect cytokines, transcription factors or signalling molecules that drive B-cell development or contribute to the pre-B cell receptor (pre-BCR) checkpoint. Deletions of the long arm of chromosome 6 [del(6q)] occur in ~10% of BCP-ALL and are also frequent in mature B and T-cell malignancies. Loss of function of the 6q genes EPHA7 and PRDM1, have been implicated in the genesis of lymphoma and BACH2, as a mediator of pre-BCR negative selection, is functionally a candidate tumour suppressor gene. However loss of these or other 6q genes have not been demonstrated, for example through biallelic inactivation, to contribute to BCP-ALL.Analysis of our own and published SNP6.0 data from ALL patients defined 5 focal recurrent regions of deletion on 6q, 4 mapping to 6q15-6q21, coincident with previously published common regions of deletion in ALL. These 4 regions contain 22 candidate genes, including EPHA7 but not BACH2 or PRDM1, which nevertheless mapped close to focal deletions and were also classed as candidate tumour suppressors.To develop the clone tracking assay, we adapted the SIN-SIEW lentiviral construct that expresses EGFP under the control of a spleen focus forming virus (SFFV) promoter. Candidate gene consensus coding sequence (CCDS) or a control luciferase cDNA were cloned between the promoter and an internal ribosomal entry site immediately upstream of EGFP. Transduction of the control (pSLIEW) or candidate gene SIN-SIEW-CCDS constructs consistently expressed EGFP in 697, a BCP-ALL cell line with del(6)(q14.1-22.3). For clone tracking, SIN-SIEW-CCDS constructs were assigned to 4 pools that also included pSLIEW. Pools were transduced into 697 cells that were both cultured in vitro and transplanted by intra-femoral injection into NOD/LtSz-scid IL2Rƴ null (NSG) mice. DNA was isolated from transduced cells immediately before transplant and then at 3 to 5 day intervals from cultured cells or from cells recovered from mouse bone marrow, spleen or liver at end stage disease. The pSLIEW construct facilitated monitoring of disease progression by in vivo imaging and also served as a control to measure CCDS construct copy number changes against. To quantify changes in integrated SIN-SIEW-CCDS, we developed a multiplex targeted Illumina sequencing approach.In vitro, highly significant (p<0.01) reductions in copy number relative to pSLIEW over time, occurred for constructs expressing FOXO3, POU3F2, SIM1, PRDM13, C6orf168 and both α and β isoforms of PRDM1 (Fig 1a). With the exception of C6orf168, these genes also strongly suppressed leukemia development in vivo in all tissues analysed (Fig 1b). The known tumour suppressor genes, BACH2 and EPHA7, had no effect on cell growth in vitro. In vivo a moderate reduction for one of two EPHA7 CCDS was observed though curiously cells expressing BACH2 increased in relative copy number by approximately 3 fold. RNA sequencing data from 697 and published array data for normal pre-B cells and cases of BCP-ALL showed no, or extremely low, levels of expression for POU3F2, SIM1, PRDM13 and C6orf168 making it unlikely that they function as tumour suppressor genes in BCP-ALL. However significant expression of the transcription factors FOXO3 and PRDM1 were seen across data sets. Western blot confirmed expression of FOXO3 and PRDM1 in 697 and other BCP-ALL cell lines and demonstrated substantial increases in the corresponding proteins after transduction of 697 with FOXO3 and PRDM1 SIN-SIEW CCDS constructs. Over-expression of FOXO3 and both isoforms of PRDM1 decreased the proportion of cells in S and G2 phases of the cell cycle, but failed to induce apoptosis as measured by Annexin-5 staining. Comparison of total mRNA sequencing profiles of 697 cells, FACS sorted for ectopic expression of FOXO3, PRDM1 or control construct, showed distinctive patterns of up or down regulated mRNA. The roles of FOXO3 and PRDM1 in early B-cell development are currently undefined but notably our data suggests they influence expression of components of the pre-BCR and related signalling pathways and therefore may contribute to the pre-BCR checkpoint. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The pre-B-cell receptor checkpoint in acute lymphoblastic leukaemia.

The B-cell receptor (BCR) and its immature form, the precursor-BCR (pre-BCR), have a central role in the control of B-cell development, which is dependent on a sequence of cell-fate decisions at specific antigen-independent checkpoints. Pre-BCR expression provides the first checkpoint, which controls differentiation of pre-B to immature B-cells in normal haemopoiesis. Pre-BCR signalling regulates and co-ordinates diverse processes within the pre-B cell, including clonal selection, proliferation and subsequent maturation. In B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), B-cell development is arrested at this checkpoint. Moreover, malignant blasts avoid clonal extinction by hijacking pre-BCR signalling in favour of the development of BCP-ALL. Here, we discuss three mechanisms that occur in different subtypes of BCP-ALL: (i) blocking pre-BCR expression; (ii) activating pre-BCR-mediated pro-survival and pro-proliferative signalling, while inhibiting cell cycle arrest and maturation; and (iii) bypassing the pre-BCR checkpoint and activating pro-survival signalling through pre-BCR independent alternative mechanisms. A complete understanding of the BCP-ALL-specific signalling networks will highlight their application in BCP-ALL therapy.

C-Myb coordinates survival and expression of key signaling components required for the pre-BCR checkpoint (HEM1P.220)

Abstract The c-Myb transcription factor is abundantly expressed during B cell development and is down regulated as the cells mature. We targeted Myb in mice with loxP sites (Mybf/f). By crossing these mice to other strains that direct Cre expression to different stages of B cell development (Rag1-cre, Mb1-cre, CD19-cre) we are able to assess subsequent B-lineage maturation. In Mybf/f Mb1-cre mice, c-Myb is required for B cell differentiation beyond the pre-pro-B cell stage and survival of CD19+ pro-B cells. c-Myb controls survival of pro-B cells by suppression of the proapoptotic factors Bmf and Bim. We find that IL-7 signaling represses the expression of Bmf and Bim. c-Myb controls the expression of CD127 but forced expression of CD127 is not sufficient to repress Bmf and Bim or rescue survival of c-Myb deficient pro-B cells. Examination of negative mediators of IL-7 signaling revealed increased SOCS3 mRNA in c-Myb deficient pro-B cells and forced expression of SOCS3 suppressed proliferation and survival of CD19+ pro-B cells in the presence of IL-7. Surprisingly, Mybf/f CD19-cre Bcl2-Tg mice contain severely reduced large and small pre-B cell compartments. Examination of mRNA expression in these cells revealed that c-Myb controls expression of key regulators of the pre-BCR checkpoint, including CD127, lambda-5, cyclin D3 and Cxcr4. c-Myb coordinates survival in pro-B cells with the expression of key components of the signaling pathways that drive the pro-B to pre-B cell transition.

Age-associated B cells (ABC) cause reduced surrogate light chain expression and loss of B cell precursors in old age (HEM2P.269)

Abstract Age-associated B cells (ABC, CD21/35- CD23-) accumulate in spleen and bone marrow of ~2 yrs. old mice and induce TNF-α-dependent apoptosis of pro-B cells. The remaining B cell precursors in aged mice exhibit reductions in surrogate light chain (SLC) and compromise of the preBCR checkpoint. Notably, the residual pro-B cells in aged bone marrow are resistant to ABC/TNF-α-mediated apoptosis. SLC is associated with cadherin 17 on the surface of pro-B cells. Like SLC-low aged pro-B cells, pro-B cells from λ5 SLC-deficient young adult mice were also resistant to TNF-α-induced apoptosis. This implies that expression of the SLC/cadherin 17 complex is required for normal susceptibility to apoptosis in pro-B cells. Resistance to apoptosis in aged pro-B cells and λ5-deficient young pro-B cells coincided with increased phosphorylation of the pro-apoptotic protein Bim, a modification that tags Bim for degradation. We hypothesize that, in old mice, expanded pro-inflammatory ABC within the bone marrow cause apoptosis of pro-B cells. However, this cell death occurs preferentially in pro-B cells which express relatively high levels of SLC. The remaining pro-B cells in aged mice are consequently skewed to lower SLC expression. We suggest that ABC, by inducing the loss of “high SLC” pro-B cells, deviate B lymphopoiesis into an ”SLC low pathway” with inefficient B cell production and altered B cell antibody repertoire.

Pre-B Cell Receptor Signaling Induces Immunoglobulin κ Locus Accessibility by Functional Redistribution of Enhancer-Mediated Chromatin Interactions

Author SummaryB lymphocyte development involves the generation of a functional antigen receptor, comprising two heavy chains and two light chains arranged in a characteristic “Y” shape. To do this, the receptor genes must first be assembled by ordered genomic recombination events, starting with the immunoglobulin heavy chain (IgH) gene segments. On successful rearrangement, the resulting IgH μ protein is presented on the cell surface as part of a preliminary version of the B cell receptor—the “pre-BCR.” Pre-BCR signaling then redirects recombination activity to the immunoglobulin κ light chain gene. The activity of two regulatory κ enhancer elements is known to be crucial for opening up the gene, but it remains largely unknown how the hundred or so Variable (V) segments in the κ locus gain access to the recombination system. Here, we studied a panel of pre-B cells from mice lacking specific signaling molecules, reflecting absent, partial, or complete pre-BCR signaling. We identify gene regulatory changes that are dependent on pre-BCR signaling and occur via long-range chromatin interactions between the κ enhancers and the V segments. Surprisingly the light chain gene initially contracts, but the interactions then become more functionally redistributed when pre-BCR signaling occurs. Interestingly, we find that the two enhancers play distinct roles in the process of coordinating chromatin interactions towards the V segments. Our study combines chromatin conformation techniques with data on transcription factor binding to gain unique insights into the functional role of chromatin dynamics.

The skewed heavy-chain repertoire in peritoneal B-1 cells is predetermined by the selection via pre-B cell receptor during B cell ontogeny in the fetal liver

As many as 5-15% of B-1 cells in the peritoneal cavity of adult mice produce antibodies reactive to phosphatidylcholine (PtC) and the vast majority of them express B cell receptors (BCRs) composed of V(H)11-muH chains utilizing the J(H)1 segment and Vkappa9-L chains. This extremely skewed repertoire of PtC-reactive B-1 cells is traditionally attributed to the expansion of particular clones in response to self or exogenous antigens. Here, we show that the strong bias toward the J(H)1 usage among V(H)11-muH chains is already established prior to the BCR assembly, namely at the transition from the large to the small pre-B cell stage during B cell ontogeny in the fetal liver. Among V(H)11-muH clones isolated from large pre-B cells where the J(H)1 skewing was not established yet, the J(H)1 users showed the highest ability to form pre-B cell receptor (pre-BCR) and to induce cellular proliferation and differentiation when expressed in fetal liver pro-B cells. Thus, the J(H)1 users were positively selected and amplified at the pre-BCR checkpoint. When co-expressed with Vkappa9-L chains to form BCR, the J(H)1 users almost exclusively conferred the PtC reactivity on BCR even though other J(H) users could also form BCR on the cell surface. Therefore, the pre-BCR-mediated positive selection of the J(H)1 users among V(H)11-muH chains appears to be beneficial to the efficient generation of 'innate-type' PtC-reactive B cells during the fetal B cell development, even before the self-renewal or the antigen-driven clonal expansion of B-1 cells takes place in the peritoneal cavity.

EVIDENCE FOR THE EXISTENCE OF AN ALTERNATIVE B CELL DEVELOPMENT PATHWAY (B3)

Abstract During B-cell development rearrangement of immunoglobulin heavy and light chain genes occurs sequentially, separated by a pre-BCR checkpoint where the quality of the heavy chains is tested by pairing with a surrogate light chain (SLC). Here we demonstrate the existence of a second B-cell development pathway, that is characterized by a high expression of Sca1 and LFA1, is less dependent on SLC expression and does not have a late pre-B stage. This pathway becomes predominant with age and in conditions of lymphopenia, and is biased towards marginal zone B cells. However, cell fate switches occur between marginal zone and follicular B cells, likely to maintain marginal zone homeostasis. FO B cells derived directly from MZ B cells are distinguished by a higher expression of Sca1. Thus, an alternative, and perhaps ancestral, B-cell development pathway exists that is biased toward to the generation of MZ B cells.

Pre-B Cell Receptor Assesses the Quality of IgH Chains and Tunes the Pre-B Cell Repertoire by Delivering Differential Signals

It is well understood how a variety of Ig H and L chains, components of BCR, are generated in the DNA level during B cell development. However, it has remained largely unknown whether and how each component is monitored for its quality and selected before the assembly into the BCR. Here we show that muH chains produced by pre-B cells display a wide spectrum of ability to form the pre-BCR, which is composed of muH and surrogate light (SL) chains and is crucial for B cell development. The level of surface pre-BCR expression varies among pre-B cells, depending on the ability of their muH chains to pair with SL chains. The higher the level of pre-BCR expression by pre-B cells, the stronger their pre-BCR signaling, and the better they proliferate and differentiate. Thus, the extent of survival, proliferation, and differentiation of individual pre-B cells is primarily determined by the SL-pairing ability of their muH chains. Furthermore, IgH chains with higher potential to assemble with IgL chains appear to be positively selected and amplified through the assessment of their ability to pair with SL chains at the pre-BCR checkpoint before the assembly into the BCR. These results indicate that the pre-BCR assesses the quality of muH chains and tunes the pre-B cell repertoire by driving the preferential expansion and differentiation of cells with the higher quality of muH chains.

Selection of Ig mu heavy chains by complementarity-determining region 3 length and amino acid composition.

Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring V(H)81X and J(H)4 gene segments from J(H)(+/-) and lambda5(-/-)mice. V(H)81X-J(H)4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental in shaping the diverse B cell repertoire.

Negative Selection at the Pre-BCR Checkpoint Elicited by Human μ Heavy Chains with Unusual CDR3 Regions

Approximately 9% of in-frame μ heavy chain transcripts found in normal human pro-B cells encode proteins that can be expressed on the cell surface in the absence of surrogate or conventional light chains. These unusual μ heavy chains demonstrate preferential use of certain VH genes (VH3-23), frequent expression of DH regions in underrepresented reading frames, and an increased number of positively charged amino acids within the CDR3 region. Transcripts for these proteins are not found in pre-B cells or in mature B cells. When expressed in Jurkat T cells with the Igα/Igβ signal transduction module, these aberrant μ heavy chains induce cell activation and apoptosis. These results suggest that some μ heavy chains elicit negative selection at the pro-B cell to pre-B cell transition.