Censoring of autoreactive B cell development by the pre-B cell receptor.

Antibody diversity occurs randomly as B cells recombine their immunoglobulin (Ig) heavy- and light-chain genes during development. This process inevitably generates reactivity against self structures, and several mechanisms prevent the development of autoreactive B cells. We report here a role for the pre-B cell receptor, composed of Ig heavy and surrogate light chains, in the negative selection of cells expressing Ig heavy chains with the potential to generate autoantibodies. Surrogate light-chain-deficient (SLC-/-) mice harbored elevated levels of antinuclear antibodies (ANAs) in their serum and showed evidence of escape of pre-B cells expressing prototypic autoantibody heavy chains from negative selection, leading to mature autoantibody secreting CD21-CD23- B cells in the periphery. Thus, the pre-B cell receptor appears to censor the development of certain autoantibody-secreting cells and may represent an important factor in multifactorial autoimmune diseases.

Transcription of productive and nonproductive VDJ-recombined alleles after IgH allelic exclusion.

The process of allelic exclusion ensures that each B cell expresses a B-cell receptor encoded by only one of its Ig heavy (IgH) and light (IgL) chain alleles. Although its precise mechanism is unknown, recruitment of the nonfunctional IgH allele to centromeric heterochromatin correlates with the establishment of allelic exclusion. Similarly, recruitment in activated splenic B cells correlates with cell division. In the latter, the recruited IgH allele was reported to be transcriptionally silent. However, it is not known whether monoallelic recruitment during establishment of allelic exclusion correlates with transcriptional silencing. To investigate this, we assessed the transcriptional status of both IgH alleles in single primary cells over the course of B-cell development, using RNA fluorescence in situ hybridization. Before allelic exclusion both alleles are transcribed. Thereafter, in pre-BII and subsequent developmental stages both functional and nonfunctional VDJ- and DJ-transcription is observed. Thus, after the establishment of IgH allelic exclusion, monoallelic recruitment to heterochromatin does not silence VDJ- or DJ-transcription, but serves another purpose.

The pre-B-cell receptor.

The pre-B-cell receptor (pre-BCR) is composed of two immunoglobulin mu heavy chains and two surrogate light chains, which associate with the signaling molecules Igalpha and Igbeta (Igalpha/beta). The production of a functional pre-BCR is the first checkpoint in the current model of B-cell development. The pre-BCR mediates signals resulting in heavy chain allelic exclusion, down-regulation of the recombination machinery, developmental progression, V(H) repertoire selection, proliferation and down-regulation of the surrogate light chain genes. Recent studies suggest that some of these processes could take place at an earlier stage in B-cell development than previously thought, and might not result from signals mediated by the pre-BCR.

Only VpreB1, but not VpreB2, is expressed at levels which allow normal development of B cells.

The surrogate light chain (SLC) consists of the polypeptides lambda5 and, in the mouse, either VpreB1 or VpreB2. SLC associates with BILL-Cadherin and other glycoproteins to form the pro-B cell receptor (pro-BCR) at the pre-BI cell stage, and with the immunoglobulin mu heavy chain to form the pre-BCR at the pre-BII cell stage. The function of the pro-BCR, if any, is unknown, whereas the pre-BCR is crucial for proliferative expansion of pre-BII cells. To shed light on the functional properties of VpreB1 and VpreB2 in vivo, mice with either one or two VpreB1, or one or two VpreB2, alleles have been investigated. We show that B cell development in mice with two VpreB1 alleles is indistinguishable from that of normal mice. In contrast, mice with two VpreB2 alleles show an approximately 1.6-fold increase in pre-BI and a 35% decrease in pre-BII cell numbers, while mice with only one VpreB2 allele show a reduction in B cell development manifested in a 2-fold enrichment in pre-BI cells and a 75% reduction in pre-BII cells. However, such a gene dosage effect is not observed for VpreB1. Our results suggest that the difference between VpreB1- and VpreB2-deficient mice is due to lower VpreB2 protein expression, thus limiting the formation of pre-BCRs and thereby the number of large, cycling pre-BII cells.

The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription.

The pre-B-cell receptor (pre-BCR), composed of Ig heavy and surrogate light chain (SLC), signals pre-BII-cell proliferative expansion. We have investigated whether the pre-BCR also signals downregulation of the SLC genes (VpreB and lambda5), thereby limiting this expansion. We demonstrate that, as BM cells progress from the pre-BI to large pre-BII-cell stage, there is a shift from bi- to mono-allelic lambda5 transcription, while the second allele is silenced in small pre-BII cells. A VpreB1-promoter-driven transgene shows the same pattern, therefore suggesting that VpreB1 is similarly regulated and thereby defines the promoter as a target for transcriptional silencing. Analyses of pre-BCR-deficient mice show a temporal delay in lambda5 downregulation, thereby demonstrating that the pre-BCR is essential for monoallelic silencing at the large pre-BII-cell stage. Our data also suggest that SLP-65 is one of the signaling components important for this process. Furthermore, the VpreB1/lambda5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing.

Both the pre-BCR and the IL-7Ralpha are essential for expansion at the pre-BII cell stage in vivo.

During B cell development, proliferative expansion takes place after expression of the pre-BCR. At this pre-BII cell stage, the IL-7Ralpha is also expressed. Some in vitro studies suggest that pre-BCR-dependent expansion relies on the IL-7Ralpha, and others that it does not. It has also been suggested that the pre-BCR mediates down-regulation of the IL-7Ralpha. However, the in vivo relationship between the pre-BCR and the IL-7Ralpha has not been previously examined. Here, we have investigated this by establishing mice lacking both receptors. Our results show that in the absence of the IL-7Ralpha, the pre-BII population is reduced, as previously seen in mice lacking the pre-BCR, demonstrating that the IL-7Ralpha is important at this stage. A deficiency in both receptors results in a further reduction of the pre-BII cell population. We conclude that both the IL-7Ralpha and the pre-BCR are required for optimal pre-BII cell expansion. Furthermore, IL-7Ralpha expression levels are normal in pre-BCR-deficient mice, suggesting that the pre-BCR does not mediate its down-regulation. As a consequence of the absence of both receptors, the peripheral B cell pool is severely depleted, resulting in atypical splenic B cell structures and reduced serum Ig levels.

Impaired B-1 and B-2 B cell development and atypical splenic B cell structures in IL-7 receptor-deficient mice.

The cytokine IL-7 and its receptor are essential for normal B and T lymphopoiesis. We have analyzed the role of this receptor in B cell development throughout ontogeny in IL-7 receptor alpha-deficient mice. We demonstrate that the IL-7 receptor becomes progressively more important with age. B lymphopoiesis takes place, albeit at reduced levels, in fetal liver and bone marrow of young mice, but is arrested in adults. The outcome is a severe reduction, from an early age, in peripheral B cells including follicular, marginal zone and B-1 B cells as well as perturbed splenic B cell structures, which are restored after adoptive transfer of normal spleen cells. We conclude that in the absence of the IL-7 receptor, the residual B lymphopoiesis occurring early in ontogeny must be facilitated by another component, whereas the IL-7 receptor is the key factor in adults. The impairment of marginal zone and B-1 B cells in IL-7 receptor- but not IL-7-deficient mice suggests non-redundant functions for the IL-7 receptor ligands, IL-7 and thymic stromal lymphopoietin.

The VpreB1 enhancer drives developmental stage-specific gene expression in vivo.

In adult mice, the VpreB genes are expressed in bone marrow progenitor (pro-) and precursor (pre-) B cells. As part of the pre-B cell receptor, the proteins are crucial for the proliferation of these cells and consequently normal B lymphocyte development. Using cell lines, we identified a lineage- and developmental-stage-specific VpreB1 enhancer. Here, we analyze its specificity in vivo by generating transgenic mice in which expression of a reporter gene (human CD122) is regulated by the VpreB1 enhancer in the context of its own promoter. All transgenic lines expressed the reporter gene in the bone marrow in a copy number-independent manner, whereas expression levels were integration site-dependent. While the enhancer is not tissue specific, within the B cell lineage the expression pattern of human CD122 mimicked that of endogenous VpreB1. Thus, low levels were detected in pro-B cells, high levels in pre-BI and slightly lower levels in pre-BII cells; no expression was detected in immature/mature B cells. Furthermore, when in vitro cultured transgenic pre-B cells differentiated into immature B cells there was concomitant down-regulation of human CD122 and endogenous VpreB1. Thus the VpreB1 enhancer is sufficient to ensure developmental stage-specific expression of a reporter gene in B lymphocytes in vivo.

The pre-B cell receptor and its role in proliferation and Ig heavy chain allelic exclusion.

The pre-B cell receptor (pre-BCR) is composed of the immunoglobulin (Ig) heavy (microH) chain and the surrogate light chain encoded by VpreB and lambda5. The pre-BCR has been implicated in precursor B cell proliferation, differentiation and IgH chain allelic exclusion. B cell development in mice lacking the transmembrane form of microH chain is blocked at the precursor B cell stage: the cells cannot proliferate or differentiate further and the IgH locus is allelically included. In mice lacking lambda5, the precursor B cells, although unable to proliferate, can nonetheless differentiate, whereas the IgH locus is allelically excluded. It was, therefore, postulated that microH chain together with VpreB could form a pre-BCR-like receptor that would allow IgH allelic exclusion but not proliferation. In mice lacking both VpreB genes, precursor B cells do not proliferate but are able to differentiate. Surprisingly, the IgH locus is allelically excluded. This suggests that microH chains find other partner proteins to signal allelic exclusion.

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.