Analysis of VpreB expression during B lineage differentiation in lambda5-deficient mice

The VpreB/lambda5 surrogate L chain complex is an essential component of the pre-B cell receptor, the expression of which serves as an important checkpoint in B cell development. Surrogate L chains also may serve as components of murine pro-B cell receptors whose function is unknown. We have produced two new mAbs, R3 and R5, that recognize a different VpreB epitope than the one recognized by the previously described VP245 anti-mouse VpreB Ab. These Abs were used to confirm the expression of surrogate L chains on wild-type pro-B and pre-B cell lines. Although undetectable on the cell surface, VpreB was found to be normally expressed within B lineage cells of lambda5-deficient mice. Nevertheless, VpreB expression was extinguished at the B cell stage of differentiation in these mice. The normal pattern of VpreB expression in lambda5-deficient mice excludes an essential role for pro-B and pre-B cell receptors in VpreB regulation.     

Cutting edge: signaling and cell surface expression of a mu H chain in the absence of lambda 5: a paradigm revisited

Pre-B cell receptor (pre-BCR) signals are essential for pro-B cells to mature efficiently into pre-B cells. The pre-BCR is an Ig-like transmembrane complex that is assembled from two mu H chains (mu HC) and two surrogate L chains consisting of the non-covalently associated polypeptides VpreB and lambda5. In lambda5(-/-) mice, pro-B cell maturation is impaired, but not completely blocked, implying that a mu HC induces differentiation signals in the absence of lambda5. Using a mouse model, in which transgenic mu HC expression can be controlled by tetracycline, we show that in the absence of lambda5, the transgenic mu HC promotes in vivo differentiation of pro-B cells, induces IL-7-dependent cell growth, and is expressed on the surface of pre-B cells. Our findings not only show that an incomplete pre-BCR can initiate signals, but also challenge the paradigm that an IgHC must associate with an IgLC or a SLC to gain transport and signaling competency.     

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.     

A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.

The murine gene lambda 5 is selectively expressed in pre-B lymphocytes. Of the three exons encoding lambda 5, exons II and III show strong homologies to immunoglobulin lambda light (L) chain gene segments, i.e. to J lambda intron and exon, and C lambda exon sequences respectively. We have now found, 4.6 kb upstream of lambda 5, another gene composed of two exons which is selectively expressed in pre-B cell lines as a 0.85 kb mRNA potentially coding for a protein of 142 amino acids including a 19 amino acid-long signal peptide. The 5' sequences of this gene show homologies to sequences encoding the variable regions of kappa and lambda L chains and of heavy (H) chains. The deduced amino acid sequence contains the consensus cysteine residues as well as other consensus amino acids at positions which characterize immunoglobulin (Ig) domains. We call the second gene VpreB. The 3' end of VpreB encoding the 26 carboxyl terminal amino acids shows no homology to any known nucleotide sequence. The putative protein encoded by VpreB is a potential candidate for association with the putative protein encoded by lambda 5, and thereby a candidate for association with H chains in pre-B cells. Southern blot analysis of DNA from liver (germ line) and 70Z/3 pre-B cell lines reveals two genes which hybridize to the VpreB gene. We call VpreB1 the gene which is found 5' of lambda 5. The other gene, called VpreB2, which has not yet been located within the genome, shows 97% nucleotide sequence homology to VpreB1 in an area of 1 kb which covers the coding region of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7R alpha, terminal deoxynucleotidyltransferase, and c mu expression

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Ig mu, lambda 5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cmu expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7R alpha (but not the common gamma-chain) chain. Similar defects were present in common gamma-chain and Jak3 KO mice, but not in lambda 5 or (excluding cytoplasmic Ig mu heavy chain (c mu)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7R alpha expression requires signaling through the high affinity IL-7R, but does not require cmu expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7R alpha, 2) initiating the production of cmu and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.     

VpreB1/VpreB2/lambda 5 triple-deficient mice show impaired B cell development but functional allelic exclusion of the IgH locus

At the precursor B cell stage during bone marrow B cell development, Ig muH chain associates with surrogate L (SL) chain, which is encoded by the three genes VpreB1, VpreB2, and lambda 5, to form the pre-B cell receptor (pre-BCR). Surface expression of the pre-BCR is believed to signal both proliferation and allelic exclusion of the IgH locus. Mice which lack either VpreB1/VpreB2 or lambda 5 show a lack of precursor B cell expansion but normal IgH allelic exclusion. This would suggest that one of either lambda 5 or VpreB can make a pre-BCR-like complex which is still able to signal allelic exclusion but not proliferation. To investigate this, we established mice lacking all components of the SL chain. These mice showed severely impaired B cell development which was similar to that previously found in mice lacking either lambda 5 or VpreB1/VpreB2. Surprisingly, the IgH locus was still allelically excluded and thus the SL chain appears not to be involved in allelic exclusion.     

The unique region of surrogate light chain component lambda5 is a heavy chain-specific regulator of precursor B cell receptor signaling

Signals transduced by precursor-BCRs (pre-BCRs) composed of Ig mu heavy chains (HCs) and the surrogate L chain components lambda5 and VpreB are critical for B cell development. A conserved unique region (UR) of lambda5 was shown to activate pre-BCR complexes in transformed cells and to engage putative ligands, but its contribution to pre-B cell development is not known. It is also not clear why the lambda-like sequences in lambda5 are used to select HCs that will associate mainly with kappa L chains. In this study, we show that, in transformed and primary mouse B cell progenitors, receptors containing full-length HCs and lacking the lambda5UR were expressed at higher surface levels, but exhibited reduced activity compared with normal pre-BCRs in supporting developmental changes that accompany the progenitor to pre-B cell transition in primary cell culture systems and in the bone marrow in vivo. In contrast, deletion of the lambda5UR did not change net signaling output by the Dmu-pre-BCR, a developmentally defective receptor that exhibited impaired activity in the primary cell culture system. Moreover, the lambda-like sequences in lambda5 were more accommodating than kappa in supporting surface expression and signaling by the different HCs. These results show that the lambda5UR is important, although not essential, for surrogate L chain-dependent receptor signaling in primary cells, and furthermore may help allow discrimination of signaling competency between normal and Dmu-pre-BCRs. That the lambda-like portion of lambda5 in the absence of the UR was nondiscriminatory suggests that the lambda5UR focuses pre-BCR-dependent selection on the HC V region.     

Structure and pre-B lymphocyte restricted expression of the VpreB in humans and conservation of its structure in other mammalian species.

DNA from several mammals, including humans, was found to contain one or more restriction enzyme digested DNA fragments which hybridized to the mouse VpreB gene under stringencies demonstrating at least 70% nucleotide sequence homologies, indicating that the VpreB locus may be widespread and highly conserved among mammals. A human VpreB genomic clone was isolated and sequenced. Two exons and the intervening intron are spaced almost identically as in the mouse VpreB1 gene, and show 76% sequence homology to the mouse gene. As in the mouse VpreB1 gene, the 5' end of the human VpreB gene contains characteristic features of Ig domains, while the 3' end is Ig non-related. This 3' Ig non-related structure of the VpreB gene(s) may, therefore, have existed before the speciation of humans and mice over 65 million years ago. Sequences encoding the entire putative second framework region and a stretch in the third framework region are identical in human and mouse VpreB. the human VpreB gene appears to be selectively expressed in human pre-B cell lines as an 0.85 kb poly(A)+ RNA. Its expression promises to be a useful marker for the detection of normal and malignant human pre-B lymphocytes.     

A critical role of lambda 5 protein in B cell development.

The lambda 5 gene is a homolog of immunoglobulin J lambda-C lambda genes, expressed specifically in immature B-lineage cells. Lambda 5-encoded molecules form membrane complexes with mu or D mu proteins in association with an additional protein specifically expressed in immature B cells that is encoded by the Vpre-B gene. We have generated mice in which the lambda 5 gene is inactivated by targeted gene disruption in embryonic stem cells. In these mice, B cell development in the bone marrow is blocked at the pre-B cell stage. However, the blockade is leaky, allowing B cells to populate the peripheral immune system at a low rate. These cells are allelically excluded and able to respond to antigen.     

Regulation of early precursor B cell proliferation in mouse bone marrow: stimulation by exogenous agents mediated by macrophages in the spleen.

An increase in pre-B cell proliferation and B lymphocyte production in mouse bone marrow has previously been shown to follow the administration of various foreign agents in vivo. The responses of early precursor B cells before the expression of mu chains (pro-B cells) have now been examined, using double immunofluorescence labeling for terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein as detected by monoclonal antibody 14.8. A single injection of sheep red blood cells (SRBC) was followed by an increase in the number of cells in three defined populations of early precursor B cells lacking mu chains (TdT+ 14.8- cells, TdT+ 14.8+ cells, and 14.8+ mu- cells) as well as cytoplasmic mu-bearing pre-B cells and surface mu-bearing B lymphocytes. An accompanying increase in proliferative activity was indicated by the numbers of 14.8+ mu- cells and pre-B cells which accumulated in metaphase after inducing mitotic arrest with vincristine. These effects were all abrogated either by treating mice with silica to depress macrophage function or by splenectomy. In mice given multiple injections of SRBC for 4 weeks the elevated levels of early precursor B cell production and B cell genesis were sustained. The work demonstrates that the in vivo production of early precursor B cells, putatively including those at the stage of Ig heavy chain gene rearrangement, can be stimulated by exposure to external agents acting indirectly by a silica-sensitive, spleen-dependent mechanism. The findings suggest that the level of pro-B cell proliferation and primary B cell genesis normally taking place in mouse bone marrow may reflect the level of exposure to potential stimulants in the external environment mediated by activation of splenic macrophages. The possibility that abnormally high levels of macrophage activation could predispose to dysregulations of the B cell lineage is raised.     

Interaction of murine precursor B cell receptor with stroma cells is controlled by the unique tail of lambda 5 and stroma cell-associated heparan sulfate

Efficient clonal expansion of early precursor B (pre-B) cells requires signals delivered by an Ig-like integral membrane complex, the so-called pre-B cell receptor (pre-BCR). A pre-BCR consists of two membrane micro H chains, two covalently associated surrogate L chains, and the heterodimeric signaling transducer Igalphabeta. In contrast to a conventional Ig L chain, the surrogate L chain is a heterodimer composed of the invariant polypeptides VpreB and lambda5. Although it is still unclear how pre-BCR signals are initiated, two recent findings support a ligand-dependent initiation of pre-BCR signals: 1) a pre-BCR/galectin-1 interaction is required to induce phosphorylation of Igalphabeta in a human precursor B line, and 2) soluble murine as well as human pre-BCR molecules bind to stroma and other adherent cells. In this study, we show that efficient binding of a soluble murine pre-BCR to stroma cells requires the non-Ig-like unique tail of lambda5. Surprisingly however, a murine pre-BCR, in contrast to its human counterpart, does not interact with galectin-1, as revealed by lactose blocking, RNA interference, and immunoprecipitation assays. Finally, the binding of a murine pre-BCR to stroma cells can be blocked either with heparin or by pretreatment of stroma cells with heparitinase or a sulfation inhibitor. Hence, efficient binding of a murine pre-BCR to stroma cells requires the unique tail of lambda5 and stroma cell-associated heparan sulfate. These findings not only identified heparan sulfate as potential pre-BCR ligands, but will also facilitate the development of appropriate animal models to determine whether a pre-BCR/heparan sulfate interaction is involved in early B cell maturation.     

Binding of free immunoglobulin light chains to VpreB3 inhibits their maturation and secretion in chicken B cells

The VpreB3 gene product was first characterized as an immunoglobulin (Ig) mu heavy chain-binding protein in mouse precursor B (pre-B) cells. Although its function is unknown, it has been proposed to participate in the assembly and transport of the pre-B cell receptor. We have identified a VpreB3 orthologous gene in chicken that is located close to the immunoglobulin light chain (LC) gene cluster and specifically expressed in the bursa of Fabricius. By overexpressing VpreB3 in the DT40 IgM(+) immature chicken B cell line, we have characterized VpreB3 as an endoplasmic reticulum-resident glycoprotein that binds preferentially to free IgLC. However, binding to IgHC is observed in IgLC-deficient DT40 cells. Interaction of VpreB3 with free IgLC is partly covalent and induces retention of free IgLC in the endoplasmic reticulum, preventing their secretion without affecting IgM surface expression. Our results demonstrate that this evolutionarily conserved molecule may play a role in the regulation of the maturation and secretion of free IgLC in B cells. We discuss possible implications in the regulation of the immune response.     

Organization of the murine Ig-related lambda 5 gene transcribed selectively in pre-B lymphocytes.

lambda 5 is an immunoglobulin lambda light chain-related gene which is selectively transcribed in murine pre-B lymphocytes to yield a 1.2 kb poly(A)+ mRNA. Comparison of the nucleotide sequence of a 1 kb cDNA clone with the sequence of a genomic clone isolated from 70Z/3 murine pre-B lymphoma cells shows lambda 5 is composed of three exons spanning a 3.75 kb DNA segment. Conserved splice signal sequences at all exon/intron boundaries and the presence of a long open reading frame indicate that a functional mRNA molecule can be made. Exon I contains a cap-site and a potential ATG start codon as well as sequences encoding a signal peptide. This gene could encode a lambda 5 protein of 209 amino acids which has, however, not yet been identified. The 3' portion of exon II and all of exon III shows strong sequence homologies to J lambda L and C lambda L exons. Homology to the lambda L chain genes is lost in the 5' portion of exon II and throughout exon I. In exon I short homologies to leader sequences and to VH framework 1 sequences are seen.     

Localization of the murine lambda 5 gene on chromosome 16

The chromosomal location of the murine lambda 5 gene was analyzed by Southern hybridization using restriction enzyme-digested DNA from a panel of 15 mouse X hamster somatic cell hybrids. Sequences homologous with those of lambda 5 DNA were detected in DNA of 5 hybrids. In all 5 hybrids lambda 5 was contained in restriction fragments of equal sizes, the lengths of which indicated that the germline configuration of lambda 5 with three exons and the restriction sites expected from its genomic structure were present. Southern hybridization with the murine lambda 1 gene as a probe detected the same 5 hybrids as positive. The only mouse chromosome present on all of the positive hybrids, and absent from negative ones, was number 16. We conclude that lambda 5 is situated on the same chromosome as lambda 1, i.e., on the murine chromosome 16.