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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.

A series of checkpoints for antigen receptor fitness and specificity during B cell development ensures the elimination or anergy of primary, high-avidity-autoantigen-reactive B cells. Defects in genes encoding molecules with which this purging of the original B cell repertoires is achieved may break this B cell tolerance, allowing the development of B cell- and autoantibody-mediated immune diseases. Furthermore, whenever tolerance of helper T cells to a part of an autoantigen is broken, a T cell-dependent germinal center-type response of the remaining low--or no--autoreactive B cells is activated. It induces longevity of these B cells, and expression of AiD, which effects Ig class switching and IgV-region hypermutation. The development of V-region-mutant B cells and the selections of high-avidity-autoantigen-reactive antibodies producing B cells by autoantigens from them, again, can lead to the development and propagation of autoimmune diseases such as lupus erythematosus or chronic inflammatory rheumatoid arthritis by the autoantibody BcR-expressing B cells and their secreted autoantibodies.

In this Opinion article, I address the role of the pre-B-cell receptor (pre-BCR) in the development of antigen-specific B cells in terms of immunoglobulin heavy chain (IgH) variable-region repertoire selection, precursor B-cell differentiation and proliferation, and IgH allelic exclusion. Comparisons with the role of the pre-T-cell receptor (pre-TCR) in T-cell development raise provocative questions. Why do B- and T-cell lineages both use a surrogate chain - the surrogate light chain and the pre-TCR alpha-chain, respectively - as a step to develop their repertoires of antigen-recognizing cells? What are the functions of the pre-BCR and pre-TCR in lymphocyte differentiation and antigen-receptor allelic exclusion? This article, together with the accompanying article by Harald von Boehmer, hopes to answer some of these questions.

Following injection into recombinase-activating gene-deficient (Rag1(-/-)) mice, pro-B cells lacking the Pax5 transcription factor (Pax5(-/-)) develop into most major hematopoietic lineages, with the notable exception of B cells. We assessed whether Pax5(-/-) pro-B cells that were also rendered deficient for the linker for activation of T cells (LAT), an adaptor essential for T cell receptor signaling, can be used for the rapid in vivo analysis of mutant forms of LAT. We showed that Pax5(-/-) Lat(-/-) pro-B cell lines can be infected with recombinant retroviruses expressing a LAT cDNA and sorted for the expression of LAT. When injected into Rag1(-/-) mice, they restore normal intrathymic T cell development and give rise to functional peripheral T cells. Considering that the handling of Pax5(-/-) pro-B cell lines is easier than that of bone marrow hematopoietic precursors, we used them for the rapid functional analysis of a novel Lat allelic series. When compared to knock-in and transgenic approaches, a major advantage of our Pax5(-/-) pro-B cell-based experimental approach consists in the production of mice bearing a given mutation within 2-3 months. Therefore, it constitutes a powerful first-line screen for mutations worth fastidious knock-in approaches.

The gene encoding BILL-cadherin/cadherin-17, a nonclassical cadherin expressed on B lymphocytes in a stage-and-site-specific manner, was inactivated by targeted disruption of its transmembrane/cytoplasmic portion-encoding parts. BILL-cadherin deficiency caused a threefold proB cell accumulation, as well as a reduction to half of the numbers of immature B cells in bone marrow. In spleen, CD21(hi)CD23(lo) marginal zone B cells were found reduced and the structure of the marginal zone was impaired. In addition, the size and number of germinal center as well as the number of PNA(+) cells were significantly reduced in BILL-cadherin-deficient mice. In the peritoneal cavity of mutant mice IgM(+)Mac-1(+)CD5(+) B1a cell, that express high BILL-cadherin in wild-type mice, was also reduced in number. The IgG1 and IgG3 antibody response to the T-independent antigen, TNP-Ficoll, was impaired in the mutant mice. These results indicate that BILL-cadherin participates in B lymphocyte development at least at two stages, first at the transition of pro/preB-I cells to preB-II cells possibly in association with surrogate light chain in bone marrow, and later at the point of development, accumulation and reactiveness of immature B cells in spleen.

This chapter will review the scenario of normal B cell development--from the decision of a lymphoid progenitor to enter the B-lineage, over the stages of the generation of the repertoires of antigen-receptor (immunoglobulin)-expressing cells, to the response of mature B cells to develop memory and plasma cells--highlighting some of the cellular stages and the molecular mechanisms that can generate and select transformed states of cells. The scenarios for pre-B lymphoma (lymphocytic leukaemia) development are discussed in more detail.

In mice, large numbers of immature B cells are continuously produced in the bone marrow. To enter the pools of mature B cells, these immature B cells have to pass two checkpoints. First, B cells have to migrate from the bone marrow to the spleen. The second checkpoint involves the immature B cells differentiating to mature B cells within the spleen. As the net result of this selection and maturation, only a fraction of the newly produced B cells enters the mature B-cell pool. Recent advances in the understanding of the molecular mechanisms that operate at these two checkpoints are described and discussed.

BAFF, a member of the family of tumour necrosis factor (TNF) ligands, is essential for the development of peripheral mature, long lived B lymphocytes. It binds to three different receptors, BCMA, TACI, and BAFF-R, which are all members of the family of TNF receptors. Defects in the genes encoding BAFF or BAFF-R abolish the generation of mature B cells. BAFF is made by myeloid cells whereas BAFF-R is expressed preferentially on B cells. BAFF induces polyclonal maturation of resting, short lived immature B cells to resting, long lived mature B cells without proliferation. Lupus erythematodes prone mice have elevated blood levels of BAFF, and treatment of these mice with the BAFF decoy receptor (BCMA-Ig) prevents the onset of this systemic autoimmune disease. Human lupus patients also have elevated blood levels of BAFF. Treatment with BAFF neutralising agents (decoy receptors, monoclonal antibodies) should prevent, delay, or, at least, slow down the disease.

The pre-B cell receptor (preBCR), composed of mu immunoglobulin (Ig) and surrogate light chains, signals large 'preB-II' cells to proliferate in the apparent absence of ligands or cooperating cells. We deleted the N-terminal, nonimmunoglobulin (nonlg) portion of lambda5, or mutated seven arginine residues in it to serine residues. PreBCRs with such mutant lambda5 proteins showed increased cell surface representation and a diminished rate of aggregation and internalization. Tyrosine phosphorylation of preBCR complexes containing mutant lambda5 proteins was abolished. These results indicate that the nonIg portion of lambda5, and the seven arginine residues in it, are needed for signal transduction, and that signaling could be cell autonomous. We propose two models to explain the apparently constitutive, ligand-independent signal-transducing capacity of the preBCR.

RNA expression profiles of consecutive stages of mouse and human B-cell precursors, and of mouse T-cell precursors, from DJ re-arranged states to mature lymphocytes have been generated using high-density oligonucleotide arrays. Approximately 10% of all genes present on the arrays are differentially expressed across all cellular stages of each of the lineages. Approximately half of these are differentially expressed during both T-cell and B-cell differentiation. Many of the genes that are shared are involved in cell cycle regulation and DNA replication, whereas most of the signaling and adhesion molecules and transcription factors are expressed in a lineage-specific manner. Comparable stages of the T-and B-lineage developmental programs share only a few genes. By contrast, homologous genes differentially expressed during both human and mouse B-cell development have very similar expression patterns.

In the thymi of WT1-transgenic (Tg) mice with the 17AA+/KTS- spliced form of the Wilms tumor gene WT1 driven by the lck promoter, the frequencies of CD4-CD8- double-negative (DN) thymocytes were significantly increased relative to those in normal littermates. Of the 4 subsets of CD4-CD8- DN thymocytes, the DN1 (CD44+CD25-) subset increased in both frequency and absolute cell number, whereas the DN2 (CD44+CD25+) and DN3 (CD44-CD25+) subsets decreased, indicating the blocking of thymocyte differentiation from the DN1 to the DN2 subsets. Furthermore, CD4-CD8+ T-cell receptor (TCR) -gammadelta T-cells increased in both frequency and absolute cell number in the spleen and peripheral blood of the WT1-Tg mice relative to those of normal littermates. The CD8 molecules of these CD4-CD8+ TCRgammadelta T-cells were CD8alphabeta, suggesting that they originated from the thymus. These results are the first direct evidence demonstrating that the WT1 gene is involved in the development and differentiation of T-lineage cells.

Ribonucleic acid expression profiles of seven consecutive stages of mouse thymocyte development were generated on high density oligonucleotide arrays. Previously known expression patterns of several genes were confirmed. Ten percent (1,304 of more than 13,000) of the monitored genes were found with 99% confidence to be differentially expressed across all T cell developmental stages. When compared with 1,204 genes differentially expressed in five consecutive B lineage developmental stages of bone marrow, >40% (546 genes) appeared to be shared by both lineages. However, when four pools of functionally distinct cell stages were compared between B and T cell development, DJ-rearranged precursor cells and resting immature precursor cells before and after surface Ag receptor expression shared less than 10%, mature resting lymphocytes between 15 and 20%, and only cycling precursors responding to precursor lymphocyte receptor deposition shared >50% of these differentially expressed genes. Three general rules emerge from these results: 1) proliferation of cells at comparable stages is in majority executed by the same genes; 2) intracellular signaling and intercellular communication are effected largely by different genes; and 3) most genes are not used strictly at comparable, but rather at several, stages, possibly in different functional contexts.

To assess mutagenesis during early B-lymphocyte development in vitro, progenitor B cells (pre-B cells) were obtained from fetal livers of BALB/c mice and DBA/2N mice that harbored the transgenic shuttle vector, pUR288, with a lacZ reporter gene for the determination of mutant frequencies (MFs). Differentiation-arrested pre-B cells demonstrated a marked dose-dependent increase in lacZ mutant levels after exposure to gamma-irradiation with a peak MF of 250 x 10(-5) at 2.5 Gy. Without genotoxic treatment, pre-B cells undergoing spontaneous differentiation into surface IgM expressing immature B cells exhibited lacZ mutant levels of up to 95 x 10(-5). The mutational pattern was dominated in both experiments by illegitimate recombination mutations of lacZ, not point mutations. Likewise, in both experiments, the enforced expression of Bcl-2 resulted in a striking reduction of lacZ mutations. These findings indicated that mouse pre-B cells are prone to accumulate induced and self-inflicted mutations, particularly recombinations. Additionally, our studies revealed a heretofore unknown role of Bcl-2 in inhibiting mutagenesis during early B-cell development in mice., (Copyright 2002 Elsevier Science B.V.)

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.

In Pax5-deficient mice, B cell development is blocked at the pre-BI cell stage. Like wild-type, Pax5-/- pre-BI cells can be grown long-term in vitro in the presence of stromal cells and IL-7. However, unlike their wild-type in vitro-grown counterparts, Pax5-/- pre-BI cells possess an extraordinary developmental plasticity showing hematopoietic stem cell features such as multipotency and self renewing capacity. Here we review and discuss this in vitro and in vivo plasticity of Pax5-/- pre-BI cells.

Human B cell-activating factor (BAFF) induces mouse surface IgM+ B cells of the immature type from bone marrow and of the immature types 1 and 2 from spleen, as well as of the mature type from spleen to increased longevity in tissue culture. BAFF does so polyclonally and without inducing proliferation in any of these B cell subpopulations. BAFF induces phenotypic and functional maturation of immature to mature B cells so that all immature cells loose C1qRp (AA4.1, 493) expression and type 1 immature cells up-regulate IgD, CD21 and CD23. Immature B cells of types 1 and 2, upon pre-incubation with BAFF, change their reactiveness to Ig-specific antibodies so that they no longer enter apoptosis but now proliferate. However, BAFF does not seem to overcome negative selection of developing immature B cells in vitro.

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.

In mice deficient for the transcription factor Pax-5, B cell development is blocked at the pre-B I cell stage. Like wild type, Pax-5-/- pre-B I cells can be grown long-term in vitro in the presence of stromal cells and IL-7. However, unlike their wild type in vitro grown counterparts, Pax-5-/- pre-B I cells posses an extraordinary developmental plasticity showing hematopoeitic stem cell features such as multipotency and self renewing capacity. Here we review and discuss this in vitro and in vivo plasticity of Pax-5-/- pre-B I cells.

Self-renewal, pluripotency, and long-term reconstitution are defining characteristics of single hematopoietic stem cells. Pax5(-/-) precursor B cells apparently possess similar characteristics. Here, using serial transplantations, with in vitro recloning and growth of the bone marrow-homed donor cells occurring after all transplantations, we analyzed the extent of self-renewal and hematopoietic multipotency of Pax5(-/-) precursor B-cell clones. Moreover, telomere length and telomerase activity in these clones was analyzed at various time points. Thus far, 5 successive transplantations have been performed. Clones transplanted for the fifth time, which have proliferated for more than 150 cell divisions in vitro, still repopulate the bone marrow with precursor B cells and reconstitute these recipients with lymphoid and myeloid cells. During this extensive proliferation, Pax5(-/-) precursor B cells shorten their telomeres at 70 to 90 base pairs per division. Their telomerase activity remains at 3% of that of HEK293 cancer cells during all serial in vivo transplantations/in vitro expansions. Together, these data show that Pax5(-/-) precursor B-cell clones possess extensive in vivo self-renewal capacity, long-term reconstitution capacity, and hematopoietic multipotency, with their telomeres shortening at the normal rate.

Interactions of BAFF (B-cell activating factor) with BAFF-R, one of three BAFF-binding receptors that are preferentially expressed on B cells, are essential for B-cell development, because defects in either the ligand or the receptor arrest progression from immature type-1 B cells to type-2 cells and mature cells; B1 B cells are unaffected. Transgenic BAFF overexpression leads to B-cell hyperplasia and autoimmune disease. In vitro, BAFF increases survival of immature and mature B cells; immature B cells also mature polyclonally to mature B cells, without proliferation. Upon BAFF-influenced differentiation, immature B cells change their surface-IgM signal transduction machinery and proliferate rather than undergoing apoptosis.

Pax5-deficient pre-B I-cell clones, transplanted into natural killer (NK)-cell-deficient RAG2(-/-) IL-2Rgamma(-/-) hosts, populate the NK-cell compartment with functional NK cells. NK-cell generation from Pax5(-/-) pre-B I cells is also observed in NK-cell-proficient Balb/c RAG2(-/-) hosts. In the same Balb/c RAG2(-/-) hosts, Pax5(-/-) pre-B I-cell clones not only populate the pre-B I-cell compartment and fill the deficient T-cell-lineage compartment in the thymus and the periphery of all hosts, as shown before, they also generate CD8alpha(-) and CD8alpha(+) dendritic cells (DCs), macrophages, and granulocytes in vivo in approximately half the hosts. In some recipients, practically all the mature myeloid cells are of Pax5(-/-) origin, indicating the effectiveness by which Pax5(-/-) pre-B I cells can compete with endogenous myeloid precursors. In a smaller percentage of hosts, the generation of Pax5(-/-) pre-B I-cell-derived erythrocytes is observed 4 to 6 months after transplantation. The results indicate that Pax5(-/-) pre-B I cells can develop in vivo in hosts that have undergone transplantation to erythroid, myeloid, and lymphoid cell lineages. Hence, the Pax5(-/-) mutation introduces an unusual instability of differentiation in pre-B I cells so that they appear to dedifferentiate as far back as the pluripotent hematopoietic stem cell.

Nineteen different mu heavy-chains, seven of them not capable of forming a pre B cell receptor were expressed in Drosophila melanogaster Schneider cells together with either VpreB1, VpreB2, lambda5, or the complete surrogate light-chain to study their interactions in the formation of the pre B cell receptor. The lambda5 protein alone was unable to bind properly to any of the mu heavy-chains, while the VpreB proteins alone formed complexes with five of the mu heavy-chains. All mu heavy-chains incapable of forming a pre B cell receptor with surrogate light-chain were also incapable of complex formation with VpreB. The possible role of the VpreB/mu heavy-chain in allelic exclusion of the heavy-chain locus during B cell development is discussed.

B cells have to progress through various checkpoints during their process of development. The three transcription factors E2A, EBF (early B cell factor) and Pax5 play essential roles in B cell commitment checkpoints. The various forms of the BCR and their downstream signaling molecules, which are expressed at different stages of B cell development, act as critical checkpoint guards allowing (positive selection) or preventing (negative selection) developmental progression. The recent advances on the molecular mechanisms operating at these various checkpoints are here summarized and discussed.

The pre-B cell receptor consists of immunoglobulin (Ig) mu heavy chains and surrogate light chain, i.e., the VpreB and lambda5 proteins. To analyze the role of the two VpreB proteins, mice lacking the VpreB1 and VpreB2 genes were generated. VpreB1(-/-) VpreB2(-/-) mice were impaired in their B cell development at the transition from pre-BI to large pre-BII cells. Pre-BII cells did not expand by proliferation, consequently 40-fold less small pre-BII and immature B cells were found in bone marrow, and the generation of immature and mature conventional B cells in spleen appeared reduced. In addition, only low numbers of B-1a cells were detected in the peritoneum. Surprisingly, Ig heavy chain allelic exclusion was still active, apparently ruling out a signaling role of a VpreB1/VpreB2-containing receptor in this process.

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.

A 130-kDa glycoprotein (p130) has been found to be associated with surrogate light chain on pro- and pre-B I cells. Using peptide sequences obtained from purified p130 we have cloned its gene. The gene encodes a typical cadherin type 1 membrane protein with six extracellular cadherin domains (one pseudo domain) but lacking the catenin-binding site in its cytoplasmic part. Even without this catenin-binding site, p130 mediates Ca(2+)-dependent homotypic adhesion of cells. The interaction of p130 with surrogate light chain is confirmed by co-transfection and co-immunoprecipitation experiments. The expression of p130 is biphasic during the B cell development. Reverse transcriptase-polymerase chain reaction and flow cytometric analyses revealed that it is expressed on B220(+)c-Kit(+) pro-B and pre-B-I cells as well as on B220(+)CD25(-)IgM(+) immature and mature B cells but not on B220(+)CD25(+) pre-B-II cells. It is also expressed in fetal liver, at low levels in myeloid cells, and strongly in intestinal epithelial cells. In the spleen, p130-expressing cells are mainly localized in the marginal zone. We call this B lineage-, intestine-, liver- and leukocyte-expressed gene BILL-cadherin. The possible functions of BILL-cadherin in B cell development are discussed.

During B-lymphocyte development in mouse fetal liver and bone marrow, a pre-B I cell stage is reached in which the cells express B-lineage-specific genes, such as CD19, Ig alpha and Igbeta and VpreB and lambda5, which encode the surrogate light (SL) chain. In these pre-B I cells both alleles of the immunoglobulin heavy (IgH) chain locus are D(H)J(H) rearranged. Transplantation of pre-B I cells from wild-type (e.g. C57Bl/6) mice in histocompatible RAG-deficient hosts leads to long-term reconstitution of some of the mature B-cell compartments and to the establishment of normal IgM levels, a third of the normal serum IgA levels, and IgG levels below the detection limit. Neither T-lineage nor myeloid cells of donor origin can be detected in the transplanted hosts, indicating that the pre-B I cells are committed to B-lineage differentiation. Consequently, the B-cell-reconstituted hosts respond to T-cell-independent antigens but not to T-cell-dependent antigens. Responses to T-cell-dependent antigens can be restored in the pre-B I-cell-transplanted, RAG-deficient hosts by the concomitant transplantation of mature CD4+ T cells. The transplanted wild-type pre-B I cells do not home back to the bone marrow and become undetectable shortly after transplantation. B-lymphocyte development in Pax-5-deficient mice becomes arrested at the transition of pre-B I to pre-B II cells i.e. at the stage when V(H) to D(H)J(H) rearrangements occur and when the pre-B-cell receptor, complete with muH chains and SL chains, is normally formed. T-lineage and myeloid cell development in these mice is normal. Pre-B I cells of Pax-5-deficient mice have a wild-type pre-B I-cell-like phenotype: while they do not express Pax-5-controlled CD19 gene, and express Ig alpha to a lesser extent, they express Igbeta, VpreB and lambda5, and proliferate normally in vitro on stromal cells in the presence of interleukin (IL)-7. Clones of these pre-B I cells carry characteristic D(H)J(H) rearrangements on both IgH chain alleles. However, removal of IL-7 from the tissue cultures, unlike wild-type pre-B I cells, does not induce B-cell differentiation to surface IgM-expressing B cells, but induces macrophage differentiation. This differentiation into macrophages requires either the presence of stromal cells or addition of macrophage colony-stimulating factor (M-CSF). Addition of M-CSF followed by granulocyte-macrophage colony-stimulating factor induces the differentiation to MHC class II-expressing, antigen-presenting dendritic cells. In vitro differentiation to granulocytes and osteoclasts can also be observed in the presence of the appropriate cytokines. Moreover, transplantation of Pax-5-deficient pre-B I clones into RAG-deficient hosts, while not allowing B-cell differentiation, leads to the full reconstitution of the thymus with all stages of CD4-CD8- and CD4+CD8+ thymocytes, to normal positive and negative selection of thymocytes in the thymus, and to the development of normal, reactive mature CD4+ and CD8+ T-cell compartments in the peripheral lymphoid tissues, all carrying the clone-specific D(H)J(H) rearrangements. On the other hand, Ig alpha, Igbeta, VpreB and lambda5 are turned off in the thymocytes, demonstrating that the expression of these genes does not commit cells irreversibly to the B lineage. Further more, Pax-5-deficient pre-B I cells are long-term reconstituting cells. They home back to the bone marrow of the RAG-deficient host, can be reisolated and regrown in tissue culture, and can be retransplanted into a secondary RAG-deficient host. This again develops thymocytes and mature T cells and allows the transplanted clonal pre-B I cells to home to the bone marrow.

During B-cell development the surrogate light (SL) chain is selectively expressed in progenitor and precursor B cells during the developmental stages of D(H) to J(H) and V(H) to D(H)J(H) rearrangements. Approximately half of all muH chains produced by these rearrangements cannot pair with SL chains and cannot form a pre-B-cell receptor (pre-BCR). A spectrum of affinities between VpreB and individual V(H) domains generates preB cells with pre-BCR of different fitness which, in turn, determines the extent of the pre-B II-cell proliferation and the fidelity of allelic exclusion of the H chain locus. Once pre-BCR is expressed, SL chain expression is turned off. As pre-B II cells proliferate, SL is diluted out, thus limiting pre-BCR formation. As a consequence, pre-B II cells stop proliferating, become small and resting and begin to rearrange the L chain loci. Multiple rearrangements of the kappaL chain alleles are often detected in wild-type small pre-B II cells. Around 20% of the muH chain-expressing small pre-B II cells also express L chains but do not display the Ig on the surface. Hence, it is likely that not all L chains originally generated in resting pre-B II cells can pair with the muH chain previously present in that cell. The best fitting ones are selected preferentially to generate sIg+ B cells. Furthermore, the transition of immature B cells from the bone marrow to spleen and their development to mature cells appear as two separate steps controlled by different genes.

In recent years, detailed analyses of B cell development in both humans and mice have revealed similar subsets of precursors along the same pathway of differentiation. From these studies it also became clear that both species undergo age related changes in this B lymphocyte development program. In this review we summarize these findings and discuss, potential mechanisms underlying these age related changes, and possible causative correlations between these changes and age related B cell abnormalities.

B cell development in organ cultures of fetal liver from mice at day 14 of gestation resembles in kinetics and cell numbers generated the one observed in vivo. This development in vitro can be blocked by an IL-7 receptor-specific monoclonal antibody. Monoclonal antibodies specific for the pre-B cell receptor, i. e. for VpreB, lambda5, or muH chains, do not perturb B cell development in these organ cultures up to and including the CD25+ small pre-BII cell stage. However, muH chain-specific antibodies inhibit the appearance of the subsequent surface IgM+ immature B cells. In organ cultures of muH chain allotype heterozygous (muHa x muHb)F1 fetal livers a dose-dependent inhibition by allotype-specific monoclonal antibodies of sIgM+ immature B cells expressing the corresponding, but not the other, allotype was observed. By combining cell sorting with limiting dilution analysis of lipopolysaccharide-reactive cells, the probable target cell of this muH chain-specific inhibition was identified as an IgM+, CD23-immature B cell. Hence, engagement of the pre-B cell receptor by specific antibodies does not influence B cell development, while engagement of the B cell receptor on immature B cells does.

The combined analysis of the expression of receptor tyrosine kinases c-Kit and Flt3/Flk-2 and of the human CD25 gene expressed as a transgene under the regulation of the mouse lambda5 promoter in the bone marrow of 1-week-old mice allows us to identify three stages of B lymphocyte development before the CD19(+)c-Kit(+) pre-B-I cells. Single-cell PCR analysis of the rearrangement status of the Ig heavy chain alleles allows us to order these early stages of B cell development as follows: (i) B220(+)CD19(-)c-Kit(lo)Flt3/Flk-2(hi)lambda5(-), (ii) B220(+)CD19(-)c-Kit(lo)Flt3/Flk-2(hi)lambda5(+) and (iii) B220(+)CD19(+)c-Kit(lo)Flt3/Flk-2(lo)lambda5(+) before B220(+)CD19(+)c-Kit(lo)Flt3/Flk-2(-)lambda5(+) pre-B-I cells. All these progenitors are clonable on stromal cells in the presence of IL-7 and can differentiate to CD19(+)c-Kit(-) B-lineage cells. A combination of stem cell factor, Flt3 ligand and IL-7 was also able to support the proliferation and differentiation of the progenitors in a suspension culture. Furthermore, the analyses indicate that the onset of D(H)J(H) rearrangements precedes the expression of the lambda5 gene. These progenitor populations were characteristic of juvenile mice and could not be detected in the bone marrow of adult mice. Hence the expression pattern, and probably the function, of the receptor tyrosine kinases in early B cell differentiation appears to be different in juvenile and adult mice.

The capacity of precursor B (pre-B) I cells from fetal liver and bone marrow to proliferate and differentiate into surface immunoglobulin-positive immature B cells in vitro was analyzed. Both fetal liver- and bone marrow-derived progenitors do so in a pre-B cell receptor (pre-BCR)-dependent manner in tissue culture medium alone, without addition of other cells or cytokines. Approximately 20% of the initial pre-B I cells enter more than one division. Analyses at the single-cell level show that approximately 15% divide two to five times. Coculture of pre-B I cells with stromal cells did not enhance proliferation or differentiation, whereas the presence of interleukin 7, especially in combination with stromal cells, resulted mainly in the expansion of pre-B I cells and prevented their further differentiation. Thus, the environment of fetal liver or bone marrow is not required for the pre-BCR to exert its function, which is to select and expand cells that have undergone an inframe V(H)-D(H)J(H) rearrangement that produces a pre-BCR-compatible muH chain. It appears unlikely that a ligand for the pre-BCR drives this pre-B cell proliferation.

The mechanisms controlling the commitment of haematopoietic progenitors to the B-lymphoid lineage are poorly understood. The observations that mice deficient in E2A and EBF lack B-lineage cells have implicated these two transcription factors in the commitment process. Moreover, the expression of genes encoding components of the rearrangement machinery (RAG1, RAG2, TdT) or pre-B-cell receptor (lambda5, VpreB, Igalpha, Igbeta) has been considered to indicate B-lineage commitment. All these genes including E2A and EBF are expressed in pro-B cells lacking the transcription factor Pax5. Here we show that cloned Pax5-deficient pro-B cells transferred into RAG2-deficient mice provide long-term reconstitution of the thymus and give rise to mature T cells expressing alpha/beta-T-cell receptors. The bone marrow of these mice contains a population of cells of Pax5-/- origin with the same phenotype as the donor pro-B cells. When transferred into secondary recipients, these pro-B cells again home to the bone marrow and reconstitute the thymus. Hence, B-lineage commitment is determined neither by immunoglobulin DJ rearrangement nor by the expression of E2A, EBF, lambda5, VpreB, Igalpha and Igbeta. Instead, our data implicate Pax5 in the control of B-lineage commitment.

Membrane-bound preBCR of wild-type mice, and probably also preBCR-like V(preB) muH chain complexes in lambda5-deficient mice, signal allelic exclusion so that < 0.1% of all preB-II cells and all subsequent B lineage cells express two muH chains on their surface. On the other hand a large number of muH chains which are originally generated at the transition of preB-I to preB-II cells cannot pair with surrogate L chains, cannot form a preBCR on the surface and, hence, allow two H chain alleles to be productively rearranged in one B-lineage cell. By contrast membrane-bound BCR on immature B cells does not signal allelic or isotypic exclusion Of Ig kappaL and lambdaL chain gene loci. This allows the rearrangement machinery to remain active, and secondary L chain rearrangements on one kappaL chain allele are frequently observed. Rapid selection of fitting H/L chain pairs, forming BCR on the surface, allows B-lineage cells to enter the mature B cell pool where the rearrangement machinery is shut off, securing allelic exclusion of L chain loci in most B cells.

Deregulated overexpression of IL-7 under the control of the promoter of the Ealpha gene of MHC class II in IL-7-transgenic mice changes B cell development in wild-type mice and in mutants which limit B cell development at various cellular stages. While the introduction of deregulated IL-7 production does not change the size of the pro-B and pre-B I compartments in the bone marrow of wild-type and lambda5-/- mice, it increases these compartments 2.5- to fivefold in mice which cannot make immature and mature B cells, i. e. in RAG-2-/-, tmmuH-/-, and RAG-2-/- mice expressing a transgenic muH chain. Excessive IL-7 production also increases four- to fivefold the pre-B II compartment in all those mouse strains where it can be formed (i. e. in wild-type, lambda5-/- and muH chain-transgenic RAG-2-/- mice), while no pre-B- II-like cells appear in excessively IL-7-stimulated bone marrow of mice devoid of pre-B II cells (i. e. in tmmuH-/- and RAG-2-/- mice). In the spleen of all IL-7-transgenic mice significant numbers of both pro-B and pre-B I cells are detectable and increased numbers of pre-B II and immature B cells appear in the spleen of mouse strains which are capable of making them. The capacity of the spleen to accommodate expanded numbers of these B-lineage cells as well as mature B cells is much larger than that of the bone marrow of the IL-7-transgenic mice probably because the bone limits cellular expansion and provokes spillover into the peripheral lymphoid organs.

Three clustered mouse chemokine genes, ABCD-1, -2 and -3, are all expressed highly in dendritic cells and, at various levels, in activated B cells. T cell-independently activated B cells express ABCD-1 and -2, but not -3. T cell-dependently activated B cells express all three. ABCD-1 attracts activated CD8+ cytotoxic T cells and CD4+ helper T cells of type 1 and 2. ABCD-2 preferentially attracts type 2 helper T cells, while ABCD-3 does not attract T cells at all. Both ABCD-1 and ABCD-2 bind to the same receptor (CCR4). In addition, ABCD-1 binds to a second, unknown, receptor on a separate T cell population. The three chemokines might guide T cell-independent as well as -dependent responses with two types of CD4+ T cells.

PCR analyses of the kappaL chain locus in single B-lineage cells of wild-type, Ckappa-, or JCkappa-deficient homozygous or heterozygous mice often detect multiple in- and out-of-frame rearrangements at the kappaL and lambdaL loci. They are most frequent in small pre-BII cells and equally so in wild-type and kappaL chain-deficient cells. Hence, kappaL chain production appears not to inhibit secondary rearrangements. Around 20% of all small preBII cells express IgL chains in their cytoplasm. Cells with a first productive rearrangement on one allele are favored to enter the immature B cell compartment. Thus, allelic exclusion might be secured by control of accessibility of IgL chain loci for rearrangement and by rapid selection of cells with a fitting over those with a nonfitting IgL chain.

Single cell PCR assays have been further developed that detect over 80% of all VkappaJkappa, VkappaRS, and VlambdaJlambda rearrangements at efficiencies between 70% and 90%. These IgL chain gene rearrangement assays were used with small pre-BII cells that develop in comparably high numbers in the bone marrow of wild-type, Ckappa-deficient, and JCkappa-deficient homozygous and heterozygous mice. In all of these mice, only 15%-25% of all small pre-BII cells carry VlambdaJlambda rearrangements. These results confirm that lambdaL chain gene rearrangements occur independently of kappaL chain gene rearrangement and expression. They also show that a large part of the small pre-BII cells that express the rearrangement machinery can develop without IgL chain gene rearrangements.

Models of B-cell development in the immune system suggest that only those immature B cells in the bone marrow that undergo receptor editing express V(D)J-recombination-activating genes (RAGs). Here we investigate the regulation of RAG expression in transgenic mice carrying a bacterial artificial chromosome that encodes a green fluorescent protein reporter instead of RAG2. We find that the reporter is expressed in all immature B cells in the bone marrow and spleen. Endogenous RAG messenger RNA is expressed in immature B cells in bone marrow and spleen and decreases by two orders of magnitude as they acquire higher levels of surface immunoglobulin M (IgM). Once RAG expression is stopped it is not re-induced during immune responses. Our findings may help to reconcile a series of apparently contradictory observations, and suggest a new model for the mechanisms that regulate allelic exclusion, receptor editing and tolerance.

The mature B cell compartment of MHC class II-deficient B6 I-Aalpha(-/-) and the btk-defective CBA/N mouse strain is 4- to 5-fold smaller than in wild-type B6 mice. The defect in B6 I-Aalpha(-/-) mice is intrinsic to B cells and due to a 4- to 5-fold reduced lifespan, which however can be normalized by an I-Ealpha(d) transgene, but only when expressed early during B cell development. The reduced number of mature B cells in the btk-defective CBA/N mouse is due to a 4- to 5-fold lower number of immature splenic B cells entering the mature compartment. The combined defects of reduced lifespan and impaired generation in double mutant mice result in a severe deficiency in the mature B cell pool.

The surrogate light chain (SL) is composed of two polypeptides, Vpre-B and lambda5. In large pre-BII cells the SL chain associates with Ig mu heavy chain (muH) to form the pre-B cell receptor (pre-BCR). In mice there are two Vpre-B genes which are 98% identical within the coding regions. The two genes are co-expressed at the RNA level and encode functional proteins that can assemble with lambda5. However, it is not known whether both gene products serve the same function in vivo. Here we have established mice that lack the Vpre-B1 gene (VpreB1(-/-)), but still express the Vpre-B2 gene, both as RNA and protein. In Vpre-B1(-/-) mice, the bone marrow cellularity and the percentage of B220+ cells is normal. However, among the B220+ cells, the percentage of pre-BI cells is increased, and the percentage of pre-BII and immature B cells is slightly decreased, suggesting that the lack of Vpre-B1 causes a partial block at the transition from pre-BI to pre-BII cells, i.e. into the pre-BCR stage. The number of cells that produce a functional pre-BCR is thus lower, but the cells that reach this stage are normal as they can be expanded by proliferation and then differentiate into more mature cells. The spleens of Vpre-B1 homozygous mutant mice show normal numbers of B and T lymphocytes. Moreover, the Ig loci are allelicly excluded and the homozygous mutant mice respond with normal levels of antigen-specific antibodies to T-dependent antigens. These results demonstrate that VpreB2 alone is capable of supporting B lymphocyte development in the bone marrow and can give rise to immuno-competent cells in the periphery.