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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.

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 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.

We describe the phenotypic and functional properties of B lineage cells developing in fetal liver organ cultures (FLOC) of mouse embryos at day 14 or 15 of gestation which contain pro/pre-B-I cells. FLOC B cell development proceeds to mature IgM+, IgD+ and CD23+ lipopolysaccharide-reactive B cells within a culture period of 5-6 days. The phenotypes and relative proportions of pro/pre-B-I, pre-B-II, immature and mature B cells from FLOC were similar to that seen in livers freshly isolated from age-matched, i.e. newborn, mice. More importantly, the numbers of cells recovered in the different B lineage subpopulations from FLOC were close to those developed in vivo. Hence, in contrast to single-cell suspension cultures of fetal liver, FLOC allow the proliferative expansion of pre-B cell receptor-expressing pre-B-II cells. FLOC from embryos of mice with targeted mutations in the RAG-2 and lambda5 genes, which cannot expand by proliferative expansion of their pre-B-II compartment in vivo because they cannot express a pre-B cell receptor on their surface, show this same defect in vitro. FLOC are accessible to the action of mAb and cytokines. Thus, addition of anti-IL-7 receptor mAb to FLOC of normal mice inhibits B cell development at the transition of pro/pre-B-I to pre-B-II cells. This inhibition is reversed by addition of excess rIL-7. Addition of IL-7 alone stimulates the proliferation of pro/pre-B-I cells and inhibits their differentiation to pre-B-II and immature B cells, as it does in single-cell suspension cultures. FLOC should be useful to study the effects of other mAb, cytokines, ligands and other molecules on early B cell development.

It has been known for some time that single mutant nude or CD40T mice have apparently normal numbers of cells in the precursor compartments of bone marrow and the mature B cell compartments of the periphery. X-linked immunodeficiency (XID) mice are deficient only in some of the sIgM+sIgD+ B cells. We have investigated further the contributions of the xid mutation, of the T cell deficiency of nude of the inability of CD40T cells to cooperate with T cells in the generation of the precursor and the mature B cell compartments in mice. Double mutant XID/nu and XID/CD40T mice have precursor B cell compartments that are no more deficient than the single mutant XID mice. However, the peripheral B cell compartments of both XID/mu and XID/CD40T are even more deficient than those of single mutant XID mice. While 10% of the peripheral B cells of wild-type or CD40T, one-third of XID and half of XID/nu mice turn over rapidly, as many as three-quarters of those in XID/CD40T are short-lived. Total numbers of sIgM+sIgD+ B cells in the spleen are at best 10-15% of normal mice at 6-8 weeks of age in XID, XID/nu and XID/CD40T mice. They remain that low at 3 months of age in XID/CD40T mice, while in XID mice these peripheral B cells slowly build up in numbers with age. As expected, double mutant XID/CD40T mice do not respond to the T-dependent antigen keyhole limpet hemocyanin. Only the responses to the T-independent type I antigen, TNP-lipopolysaccharide (LPS), appear to be normal. In vitro, their splenic B cells respond poorly to LPS or to IgM-specific antibody in either the absence or presence of cytokines. Most notably, serum IgM, IgG2b or IgG3 levels are severely depressed, while IgG1, IgG2a and IgA levels are < 10 micrograms/ml. These results suggest a model of mature B cell development in which the peripheral, mature B cell compartments are generated in two parallel, not tandemly organized pathways. They could be selected and/or stimulated at the transition from immature to mature B cells: in btk controlled or in CD40 controlled ways.

B cell development in RAG-2-deficient (RAG-2T) mice is impeded at an early stage, due to the inability of these animals to rearrange their endogenous ig gene loci. Expression of an E mu-bcl-2 transgene in these mice did not change this phenotype. However, stromal cell/IL-7-reactive B cell progenitors (pro-B cells) were found in fetal live and bone marrow of RAG-2T and RAG-2T/E mu-bcl-2 transgenic mice in numbers comparable to normal mice. Like cells from normal mice they are c-kit+, surrogate L chain+ and CD25-, and can proliferate in vitro for long periods of time. Upon IL-7 deprivation, they can be induced to differentiate into c-kit-, surrogate L chain- and CD25+ cells that are no longer clonable on stromal cells and IL-7. Furthermore, sterile transcription from the kappa L chain gene loci is induced. The latter was also observed with pro-B cells directly isolated ex vivo from the bone marrow of RAG-2-deficient animals. The results suggest that progenitor B cell differentiation can occur in cells from V(D)J recombinase-deficient mice to the stage where kL chain gene rearrangements would normally be initiated. It further indicates that some molecular programs of early B cell differentiation can take place in the absence of Ig gene rearrangements.

Differential expression of c-kit, CD25 (TAC), surrogate L chain and cytoplasmic muH chain, and surface expression of IgM and IgD allows the separation of B220 (CD45+) B cell subpopulations. PCR analyses with DNA of single cells developed by others and by us have been used to monitor the conformation of the Ig H and L chain gene loci in these different B lineage subpopulations. The results of these analyses indicate that B220+/c-kit+/CD25- cells are the precursors of large B220+/CD25+/sIgM- which, in turn, are the precursors of small B220+/CD25+/sIgM- cells. The majority of B220+/c-kit+/CD25- cells are DHJH-rearranged, with L chain loci in germline configuration and are thus pre-B I cells. More than 90% of all large B220+/CD25+/sIgM- cells have at least one H chain locus VHDHJH rearranged; half of them have also the second locus VHDHJH rearranged and are thus large pre-B II cells. Rearrangements of at least one allele of the kappa L chain loci become detectable in 65% of the small B220+/CD25+/sIgM- cells, 67% of the immature B and > 75% of the mature B cells. The ratio of kappa L to lambda L gene rearrangements in all three subpopulations is approximately 10:1, indicating that the kappa L/lambda L ratio is established as soon as rearrangements are made.

The analysis of the expression of the alpha chain of the IL-2 receptor (CD25, TAC) on the surface of B lineage cells in mouse bone marrow reveals that it is a useful marker to distinguish pre-B-I from pre-B-II cells. CD25 is not expressed on CD45R(B220)+ c-kit+ CD43+ TdT+ lambda 5+ c mu- sIg-IgH chain locus DJH-rearranged pre-B-I cells of mouse bone marrow. It is expressed on large cycling CD45R(B220)+ c-kit- CD43+ TdT- lambda 5+ c mu+ sIg- and on small resting CD45R(B220)+ c-kit- CD43- TdT- lambda 5- c mu- sIg- IgH chain locus VHDJH-rearranged pre-B-II cells. Therefore, the transition from pre-B-I to large pre-B-II cells is marked by the downregulation of c-kit and terminal deoxynucleotidyl transferase (TdT), and by the upregulation of CD25. SCID, RAG-2T, microMT and lambda 5T mutant mice do have normal, if not elevated numbers of pre-B-I cells but lack all CD25+ pre-B-II cells in their bone marrow. The expression of a transgenic H chain under control of the microH chain enhancer in RAG-2T bone marrow B lineage precursors allows the development of large and small CD25+ pre-B-II cells. The results suggest that the differentiation of pre-B-I to pre-B-II cells in mouse bone marrow requires the expression of microH chains and surrogate L chains in membranes, probably on the surface of precursor B cells.

The lambda 5 gene is expressed specifically in pre-B cells; during B lymphocyte differentiation the expression of the lambda 5 gene is turned on in pre-B cells and turned off again at the mature B cell stage. No other cell type has yet been found to express the lambda 5 gene. The pre-B cell-specific expression of the lambda 5 gene is regulated at the level of transcription. We asked whether the region 5' of the lambda 5 gene (5' lambda 5) could explain the stage- and tissue-specific expression. The lambda 5 promoter lacks a TATA box and transcription is initiated at multiple sites. In the presence of a heterologous enhancer, 5' lambda 5 confers pre-B cell-specific expression on the reporter gene chloramphenicol acetyl-transferase. Deletion analysis of 5' lambda 5 defines two separate regions, referred to as A lambda 5 and B lambda 5. Region B lambda 5 suppresses the expression in non pre-B cells since deletion of B lambda 5 allows A lambda 5 to promote transcription of the reporter gene in in all cell types tested. In addition, B lambda 5 acts as an enhancer on the heterologous kappa light chain promoter in pre-B cells but not in B cells. Thus region A lambda 5 functions as a basal promoter in all cell types tested. Region B lambda 5, in concert with a heterologous enhancer, acts as a suppressing region in non-pre-B cells and therefore confers pre-B cell specificity on the expression of the lambda 5 gene.

DHJH rearrangements start in progenitor and precursor B cells and occur in three reading frames (rf). A strong bias for rf I has been noticed in murine and chicken antibodies, while the representation of rf II has been found suppressed both in peripheral as well as in precursor B cells. H chain gene loci DHJH rearranged in rf II are potentially capable of expressing a truncated DHJHC mu protein on the cell surface. Mice incapable of expressing this protein on the surface have previously been shown to have all reading frames represented in near equal frequency, suggesting that membrane-bound DHJHC mu protein is involved in the suppression of rf II. In this paper we show that suppression of rf II is not yet established in c-kit+ CD43+ IL-7/stromal cell-reactive pre-B I cells of fetal liver at day 15 of gestation, but becomes established when such precursor cell populations are expanded in vitro on stromal cells in the presence of IL-7. H chain gene loci using the DQ52 segment for rearrangements (which contains a stop codon in rf II, thus being unable to make DHJHC mu protein) do not show rf II suppression under these conditions. The same type of fetal liver-derived pre B-I cells from lambda 5 deficient mice also do not show rf II suppression after in vitro expansion. Bone marrow-derived pre B-I cells from normal mice assayed ex vivo and expanded in vivo show rf II suppression, while the corresponding pre-B I cells from lambda 5T mice do not. Collectively these experiments suggest that surrogate L chain is involved in rf II suppression. This may happen by inhibition of proliferation of pre-B cells expressing a complex of DHJHC mu protein and surrogate L chain.

The kinetics of kappa light (kappa L) chain gene rearrangement and expression on mRNA and protein level has been studied with four stromal cell/IL-7 reactive, long-term in vitro proliferating pre-B cell lines and clones, two from fetal liver of normal mice and two from fetal liver of E microH-bcl-2 transgenic (bcl-2-tg) mice. These pre-B cell lines and clones are DJH-rearranged on both H chain alleles. Two of the clones harbor H chain rearrangements which do not allow the expression of VHDJH rearranged H chain genes as microH chain proteins. Upon removal of IL-7 from the pre-B cell cultures all four cell lines rearrange VH-DJH and VL-JL gene segments, loose the surface expression of c-kit, CD43, and surrogate light chain, as well as the capacity to be clonable on stromal cells in the presence of IL-7. Pre-B cells from normal mice die by apoptosis during differentiation, while those from bcl-2-tg mice do not. All four lines and clones express comparable levels of mRNA for microH and kappa L chains with the same time kinetics during 3 days of differentiation. However, only two of the four pre-B cell lines and clones express microH chain protein, whereas all four pre-B cell lines and clones express kappa L chain protein at comparable levels between 2 x 10(5) and 1.4 x 10(6) kappa L chain molecules per cell. These results suggest that microH chain expression is not mandatory for rearrangement and normal expression of kappa L chain genes when pre-B cells differentiate to B cells.

The expression of RNA transcripts from two pre B lymphocyte related genes, VpreB and lambda 5, has been studied in a series of transformed cell lines which appear frozen at different states of B lineage differentiation, from early progenitors to surface Ig positive B cells. In the HAFTL-1 cell line, which arose from fetal liver by transformation with a retrovirus containing the Hras oncogene, Northern analysis of poly A+ mRNA as well as in situ hybridization of RNA in single cells revealed that lambda 5 and VpreB are already expressed at the progenitor stage and increase in expression as the progenitors differentiate to precursor (preB) cells, or are turned off as the progenitors differentiate to myeloid cells. Continued rearrangements of Ig genes in pre B cell lines leading to Ig expression on the surface of NFS-5 pre B cells do not influence the continued expression of VpreB and lambda 5. Surface Ig-positive B lineage cell lines also express the pre B-related genes. Both Ly1+ as well as Ly1- pre B cells are VpreB- and lambda 5-positive. Lipopolysaccharide (LPS) stimulation of 70Z/3 pre B cells does not turn off lambda 5 expression. It therefore appears that, at least in transformed cell lines, the expression of VpreB and lambda 5 is not directly regulated by the expression of microH, kappa L, or lambda L chains, LPS reactivity, or the Ly1 surface antigen. Fusion of plasmacytoma cells with normal pre B cells to generate pre B hybridomas leads to down-regulation of VpreB/lambda 5 expression.(ABSTRACT TRUNCATED AT 250 WORDS)