Loss of Precursor B Cell Expansion but Not Allelic Exclusion in VpreB1/VpreB2 Double-deficient Mice

Cornelia Mundta, Steve Licencea, Takeyuki Shimizub, Fritz Melchersb, and Inga-Lill Mårtenssona
a Developmental Immunology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom
b Basel Institute for Immunology, 4005 Basel, Switzerland

Correspondence to: Inga-Lill Mårtensson, Developmental Immunology, The Babraham Institute, Cambridge CB2 4AT, UK. Tel:44-1223-496469 Fax:44-1223-496022 E-mail:lill.martensson{at}bbsrc.ac.uk.


  Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

The pre-B cell receptor consists of immunoglobulin (Ig) µ heavy chains and surrogate light chain, i.e., the VpreB and {lambda}5 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.

Key Words: B cell development, surrogate light chain, pre-B cell receptor, B cell deficiency, B1-a B cells


  Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Mouse B cell development follows a sequence of cellular stages which are characterized by selective expression of cell surface receptors and stage-specific genes, and which can be ordered in development by their status of rearrangement in the IgH and L chain gene loci (1) (2) (3) (4) (5) (6). One selective marker of early B cell development is the surrogate light (SL)1 chain composed of the VpreB and {lambda}5 proteins (7) (8) (9) (10) (11).

Mice have two VpreB genes, VpreB1 and VpreB2, separated by an unknown distance on chromosome 16 (8). The two genes show 97% identity at the nucleotide and amino acid level, giving rise to a difference of four amino acids in the protein. The VpreB1 gene is expressed in all cells that express {lambda}5, whereas VpreB2 RNA is detected in ~30% of these same cells as determined by single cell reverse transcription (RT)-PCR (12). Both VpreB gene products are functional and can form an SL chain with {lambda}5 (12) (13).

The earliest identifiable B-lineage progenitor already transcribes, and probably also expresses as protein, the SL chain (14) (15). The cell population is in the process of DJH rearrangements, and is phenotypically characterized as B220+CD19-c-kit+flk-2+. B220+CD19+c-kit+flk-2- pre-BI cells follow, which have both IgH chain alleles DJH rearranged and express the SL chain as protein associated with gp130 (13) (16). The function of this protein complex is unknown, especially because pre-BI cells are generated in {lambda}5-deficient mice, even in elevated numbers (17).

At the transition of pre-BI to pre-BII cells, V to DJH rearrangements are initiated. Whenever they occur in-frame, a µH chain can be produced which is then probed for its capacity to associate with SL chain and form a pre-B cell receptor (pre-BCR) on the cell surface (18) (19). The pre-BCR expressing pre-BII cells are stimulated to enter the cell cycle and become large pre-BII cells. They downregulate the expression of c-kit, SL-chain, and of the rearrangement machinery, i.e., recombination activating gene (RAG)-1, RAG-2, and terminal deoxynucleotidyl transferase (TdT), and upregulate CD25 expression (2) (20). Allelic exclusion is established at the IgH chain alleles, either because the first allele was nonproductively VDJH rearranged, or because the cell is inhibited from recombining the second DJH rearranged allele. The latter is thought to be effected both by the immediate downregulation of the rearrangement machinery, as well as by a closing of the DJH rearranged allele, both signaled by the deposition of pre-BCR in the surface membrane of large pre-BII cells (21) (22) (23) (24). Hence, cells with productive VDJH rearrangements resulting in a µH chain which is incapable of forming a pre-BCR are not allelically excluded in wild-type B-lineage cells (25). Large pre-BII cells proliferate for two to five divisions, then come to rest as small pre-BII cells which continue to express CD25 (26). At the transition of large to small pre-BII cells, rearrangements at the IgL chain gene loci are initiated. Whenever they are productive and lead to the synthesis of a µH chain pairing IgL chain, immature sIgM+sIgD-AA4.1+ B cells are formed which become subject to selection by autoantigen (21). Negative selection deletes autoreactive B cells, whereas B-1 cells may be generated by positive selection (27). Finally, mature, conventional B cells appear to be generated in two steps, first by migration of immature cells from bone marrow to spleen, second by maturation of immature to mature, sIgM+sIgD+AA4.1- cells in spleen (28). SL chain becomes undetectable in small pre-BII cells, as large pre-BII cells loose pre-BCR surface expression. This appears to confine all functional influences of the SL chain to cells before the small pre-BII cell stage.

In mice lacking the transmembrane portion of µH chain (µmT) the development of B cells appears impeded at the transition from the pre-BI into large pre-BII stage, so that the cells can V to DJH rearrange the IgH locus but cannot express a membrane-bound pre-BCR and are unable to enter the cell cycle (29). In the µmT mice, the µH chains are allelically included, i.e., cells expressing two µH chain proteins from the two IgH chain alleles are found in 9% of all cells at that stage (30). Mice lacking {lambda}5 ({lambda}5-/-) are also impaired in B cell development at the transition from the pre-BI into the large pre-BII stage, again evident in a lack of proliferation of pre-BII cells (17) (31). However, {lambda}5 deficiency does not result in a complete block in B cell development, as low numbers of small pre-BII, immature, and mature B lymphocytes are produced. Surprisingly, IgH allelic exclusion is functioning in {lambda}5-/- mice (25). One hypothesis to explain this finding is that a pre-BII cell can make a modified pre-BCR consisting of µH chain and the VpreB polypeptide(s), unable to signal proliferation, but able to signal allelic exclusion.

We have recently described the analysis of mice lacking the VpreB1 (VpreB1-/-) gene (32). These mice use a SL chain composed of VpreB2 and {lambda}5 which appears to be sufficient, although not as efficient as VpreB1 (or VpreB1 plus VpreB2) to form a functional SL chain. A partial block at the transition from pre-BI to large pre-BII cells was found, possibly due to lowered efficiency of this SL chain in its capacity to pair with different µH chains. However, the cells that reach the large pre-BII stage are expanded in a normal fashion.

Here we have established mice lacking both VpreB1 and VpreB2 in the germline (VpreB1-/-VpreB2-/-) in order to analyze the combined effects of these two genes on B cell development and IgH allelic exclusion.


  Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Targeting Vectors.
Conventional DNA techniques were used to construct the targeting vectors (33). The VpreB1 targeting vector has been described previously (32). In brief, the VpreB1 vector contained 5.3 kb of genomic sequence from the VpreB1 locus and the neomycin resistance gene. In the VpreB2 targeting vector (129 DNA), the entire VpreB2 coding sequence was replaced by the 1.9-kb hygromycin resistance gene. The final VpreB2 targeting vector comprised from 5' to 3': 1.2 kb of 5' VpreB2 genomic sequence, the hygromycin resistance gene (opposite transcriptional orientation), and 7.2 kb of 3' VpreB2 genomic sequence (sequence data are available from GenBank/EMBL/DDBJ under accession no. AC005817). Restriction enzyme sites are indicated in Fig 1 A (SalI site originates from the vector). Homologous integration introduced two new EcoRI restriction enzyme sites.



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Figure 1. Homologous recombination of VpreB1 and VpreB2. (A) VpreB2 genomic locus before and after homologous integration. The targeting vector consists of 8.4 kb of VpreB2 genomic sequences and the VpreB2 coding sequence replaced by the hygromycin (hygro) resistance gene. Shown is the location of primers used for detection of homologous recombination (nos. 1 and 2), giving rise to a 1.8-kb PCR product. Offspring were later screened by PCR with primer nos. 6 and 7 and nos. 8 and 9, and the expected size of the respective product is indicated. The probe for Southern blotting and expected fragments are indicated. Restriction enzymes: R, EcoRI; H, HindIII; K, KpnI; S, SalI. (B) The arrow indicates the 1.8-kb PCR product (primer nos. 1 and 2) upon homologous recombination of the VpreB2 locus. E14, original nontargeted ES cells; Y2, VpreB1-targeted E14 cells; ES94 and ES163, VpreB2-targeted Y2 cells; ES94 and ES163 mice, mice established from these ES clones. (C) The VpreB1 genomic locus before and after homologous integration. The VpreB1 targeting vector consisted of 5.3 kb of VpreB1 genomic sequences and the neomycin (neo) resistance gene which replaced the VpreB1 coding sequence. Primer nos. 3, 4, and 5 were used to screen for genotype and the expected size of the respective product is shown. The probe used for Southern blotting is indicated and expected fragments. Restriction enzymes: R, EcoRI; X, XbaI. (D) Homologous recombination of the VpreB1 locus using the probe in C. Samples are as indicated in B, except ES65. The asterisk (*) shows the extra band detected in ES163 ES cells and in ES163 +/- and -/- mice.

ES Cells and Transfections.
Embryonic stem (ES) cells were grown on irradiated embryonic fibroblasts (resistant to G418 and hygromycin) in IMDM (Life Technologies) supplemented with MEM nonessential amino acids, 5 x 10-5 M 2-mercaptoethanol, penicillin, and streptomycin, 0.1% LIF, and 15% FCS (Boehringer). The VpreB1-targeted Y2 clone (32) was used as the recipient of the VpreB2 targeting vector. Resistant clones (150 µg/ml hygromycin) were picked 7–9 d after transfection, expanded, and frozen, and genomic DNA was prepared.

Screening of Targeted Clones.
ES clones were screened by PCR using primers no. 1 and no. 2 (Fig 1 A). Homologous recombination results in a product of 1.8 kb. Primer sequences: VpreB2 no. 1, 5'-ATGCCCACTTGTCTGCCTCTATC-3'; hygromycin: no. 2, 5'-ACCGATGGCTGTGTAGAAGTACT-3'. Conditions: 40 cycles of 94°C, 30 s; 60°C, 30 s; 72°C, 2.5 min. A total of 585 hygromycin-resistant clones were analyzed and 6 clones were targeted in the VpreB2 locus. Positive ES clones were also analyzed by Southern blotting. Several probes located outside the targeting vector were tested but on account of the high degree of repetitive sequences in the VpreB2 region these did not hybridize specifically. One probe (Fig 1 A) was better than the others; it also gave rise to a high background, but did confirm proper targeting of the VpreB2 locus (data not shown).

Establishing Mice Targeted in Both the VpreB1 and VpreB2 Loci.
The murine VpreB1 and VpreB2 genes are both located on chromosome 16 at an unknown distance from each other (8). As it was not possible to determine if the VpreB2 locus had been targeted on the same chromosome as the VpreB1-targeted locus, five ES clones were injected into blastocysts, chimeric mice established and bred with C57BL/6 females. The agouti offspring (all five clones gave rise to germ line transmission) were screened for recombination at the VpreB1 and VpreB2 loci. Targeting on the same chromosome gives rise to 50% of the pups being positive for both targeting events (ES94 and ES163), whereas targeting on separate chromosomes gives rise to 50% of the pups being positive for the VpreB1 and 50% for the VpreB2 event (ES47, ES65, and ES223). VpreB1+/-VpreB2+/- heterozygous mice from ES163 and ES94 were then inter-crossed to establish VpreB1-/- VpreB2-/- mice. The mice have been bred and investigated under proper Project Licenses (80/1143, 1263) approved by the Home Office, UK.

Screening of VpreB1/VpreB2-targeted Mice.
After establishing the ES94 and ES163 lines, offspring were screened by PCR. For the VpreB1 locus, primer nos. 3, 4, and 5 were used (32). For the VpreB2 locus, two separate reactions were performed using primer nos. 6 and 7, primer nos. 8 and 9 (Fig 1): VpreB1: (no. 3) 5'-TGCTCATGCTGCTGGCCTAT-3'; VpreB1: (no. 4) 5'-CTCCGGAGCCCCACGGCA-3'; Neomycin: (no. 5) 5'-TTGGCTACCCGTGATATTGCTGA-3'; VpreB2: (no. 6) 5'-CTCATGCTGCTGGCCCAC-3'; VpreB2: (no. 7) 5'-TCGTGGCTCCGGAGCCCCACAGCG-3'; hygromycin: (no. 8) 5'-GGGTAAATAGCTGCGCCGATGG-3'; hygromycin: (no. 9) 5'-GCCGCAGCGATCGCATCCATGG-3'. Conditions: VpreB1, 30 cycles of 94°C, 20 s; 55–60°C, 20 s; 72°C, 30 s; VpreB2, 4 min at 94°C, then 30 cycles of 94°C, 30 s; 60°C, 20 s; 72°C, 15 s. Hygromycin: same conditions as VpreB2.

RT-PCR Analysis.
Total RNA was prepared from ~107 cells using RNAzol (Tel-Test Inc.) followed by cDNA reaction using random primers (Life Technologies). cDNAs were analyzed by PCR using primers specific for HGPRT, {lambda}5, VpreB1 (nos. 3 and 4), and VpreB2 (nos. 6 and 7) as described previously (12).

Analysis of VpreB and {lambda}5 Protein.
Bone marrow cells from young mice were cultured on irradiated stromal cells (S17) in the presence of IL-7 (10% supernatant) in IMDM supplemented with 5 x 10-5 M 2-mercaptoethanol, antibiotics, and 10% FCS. Pre-BI cells, which grow out after a week in culture (34), were analyzed on a FACSCaliburTM (Becton Dickinson) using mAbs VP245 recognizing VpreB1 and VpreB2, and LM34 recognizing {lambda}5 (12) (13).

FACS® Analysis of Lymphoid Organs.
Bone marrow cell suspensions were prepared by flushing out femurs using HBSS (Life Technologies) supplemented with 10 mM Hepes and 3% FCS. Spleen and thymus cell suspensions were prepared using conventional techniques. Peritoneal cells were prepared by lavage of the peritoneal cavity with 3–5 ml HBSS supplemented as above. Cells were stained in HBSS (supplemented as above) with either two or three mAbs in combination with propidium iodide (0.25 µg/ml PI, analyzed in FL3) to exclude dead cells and then analyzed on a FACSCaliburTM (Becton Dickinson). The following mAbs were used: FITC- and allophycocyanin (APC)-labeled anti-B220/CD45R (clone RA3-6B2); biotin- and FITC-labeled anti-CD19 (clone 1D3); biotin- and PE-labeled anti–c-kit/CD117 (clone ACK 45); biotin-labeled anti-CD43 (clone S7); biotin-labeled anti-CD25 (clone 7D4); biotin-labeled anti-IgM (clone R6-60.2); PE-labeled anti-IgMa (clone DS-1); FITC-labeled anti-IgMb (clone AF6-78); FITC-labeled anti-CD4 (clone RM4-5); biotin-labeled anti-CD8{alpha} (clone 53-6.7); biotin-labeled anti-CD5 (clone 53-7.3); biotin-labeled anti-Ig{kappa} (clone R5-240); and FITC-labeled anti-Ig{lambda} (clone R26-46; all from BD PharMingen). Biotin-labeled anti-IgD (clone 1.19), anti-{lambda}5 (clone LM34), and anti-VpreB (clone VP245) were purified and biotinylated in the laboratory. Polyclonal FITC-conjugated goat anti–mouse-IgM was from BD PharMingen. When biotinylated mAbs were used, positive cells were revealed using PE-labeled streptavidin (Southern Biotechnology Associates, Inc.). To determine IgM allotype, cells from both femurs were stained with APC-labeled anti-B220, biotinylated anti-IgD, and PE-labeled anti-IgMa in the presence of 5% rat serum for 30 min. The cells were washed twice and incubated with FITC-labeled anti-IgMb and SA-Tricolor (Caltag) for 30 min in the presence of 5% rat serum, then washed as before. This procedure was crucial to achieve proper staining of the cells.

Serum Titres.
5-mo-old wild-type, heterozygous, and homozygous VpreB1/VpreB2 double-mutant mice were bled and the sera analyzed for the presence of IgM. ELISA plates were coated with 2 µg/ml of an mAb recognizing mouse IgM (R6-60.2; BD PharMingen). The samples were serially diluted, added to the plates, and bound IgM was detected with a biotinylated goat anti–mouse IgM antiserum (Sigma-Aldrich) and streptavidin-biotinylated horseradish peroxidase (Amersham Pharmacia Biotech) using o-phenylene-diamine (Sigma-Aldrich) as substrate. The ELISA was measured in a Multiskan MCC/340 plate reader (Titertek) at 492 nm. The concentration of IgM was calculated using purified mouse IgM (Sigma-Aldrich) as standard.

Immunizations.
Wild-type, heterozygous, and homozygous VpreB1/VpreB2 double-mutant mice, at the age of ~4–5 mo, were immunized with 100 µg alumprecipitate of either phenyl-oxazolone coupled to OVA (Ox-OVA) or OVA, or 5 µg NP-Ficoll. 2 wk after immunization, the mice were bled and the sera analyzed for the presence of antigen-specific Abs (IgM, IgG). ELISA plates were coated with either Ox-BSA, OVA, or NP-OVA at a concentration of 10 µg/ml. Samples were serially diluted and added to the plates. Antigen-specific IgM was detected with a biotinylated goat anti–mouse IgM antiserum (Sigma-Aldrich), and IgG with a biotinylated goat anti–mouse IgG antiserum (Sigma-Aldrich). The detection was as described above.


  Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
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Establishment of VpreB1-/-VpreB2-/- Mice.
To analyze how the two VpreB genes, on chromosome 16 of the mouse, influence B cell development, the two genes were replaced by homologous integration of antibiotic resistance genes. Earlier we have described the targeting of the VpreB1 gene (32), which yielded the Y2 ES clone that is neomycin resistant and heterozygous for the targeting event. This ES clone was used as recipient for the VpreB2 targeting construct. The VpreB2 targeting vector was made by substituting the VpreB2 coding sequences for the gene encoding hygromycin resistance. It included ~9 kb of VpreB2 homologous sequences (Fig 1 A). As both VpreB genes are located on chromosome 16, and if the targeting of the VpreB2 construct occurs randomly and is not influenced by the first integration, half of all successfully targeted ES clones should have both targeting constructs (VpreB1 and VpreB2) integrated on the same chromosome. Out of five double VpreB1/VpreB2 targeted ES clones, two were found to have integrated the two vectors on the same chromosome (ES94 and ES163). The others, ES47, ES65, and ES223, were found to be targeted for VpreB1 on one, and for VpreB2 on the other chromosome 16.

Targeting of the VpreB2 locus was determined by PCR, in which homologous recombination gives rise to a 1.8-kb product (Fig 1 A, primer nos. 1 and 2). Fig 1 B shows that ES94 and ES163 both gave rise to a 1.8-kb PCR product, whereas the original E14 and the recipient Y2 cells did not. Heterozygous and homozygous VpreB1/VpreB2 double-mutant mice from ES94 and ES163 also produced the 1.8-kb PCR product whereas the respective wild-type mice did not, demonstrating that homologous recombination had occurred at the VpreB2 locus. The targeting of the VpreB2 locus was further confirmed by Southern blotting (see Materials and Methods, and data not shown). Further analysis of ES94 and ES163, using the hygromycin resistance gene as a probe, demonstrated that ES94 carried only one copy of the VpreB2 targeting vector, whereas ES163 had integrated at least one more copy elsewhere in the genome (data not shown). To determine the status of the VpreB1 locus, we used Southern blotting (32) and, as shown in Fig 1 D, the VpreB1 locus remained intact after the second targeting event. The deletion of the two VpreB genes was further confirmed by Southern blotting using VpreB1 cDNA as a probe (data not shown). Thus, ES94 and ES163 gave rise to mice lacking both VpreB1 and VpreB2. As the targeting of VpreB1 and VpreB2 was correct in both strains we analyzed mice derived from both ES94 and ES163 (VpreB1-/-VpreB2-/-).

Lack of VpreB RNA Expression in VpreB1-/-VpreB2-/- Mice.
After establishing that the mice were correctly targeted, we investigated whether any RNA encoding VpreB could be detected. For this purpose, we analyzed RNA from bone marrow cells by RT-PCR using primers that were specific for the respective VpreB gene (12). RNA encoding VpreB1 and VpreB2 was detected in both wild-type and heterozygous mice, whereas there was no message found in VpreB1-/-VpreB2-/- mice, as revealed for both ES163 and ES94 (Fig 2 A). The {lambda}5 gene, which encodes the other component of the SL chain, is located just downstream of the VpreB1 gene (Fig 1 C). To find out if the expression of this gene had been affected by the targeting events, expression of this gene was also analyzed. {lambda}5 RNA was present in all samples, independent of genotype, suggesting that its expression had not been altered. As a control for the RT-PCR assays, RNA representing a house keeping gene (HGPRT) was measured and was detected in all bone marrow samples independent of genotype. Thus, VpreB1-/-VpreB2-/- mice lacked VpreB1 and VpreB2 RNA but expressed {lambda}5 RNA at apparently normal levels.



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Figure 2. Lack of VpreB expression in VpreB1-/-VpreB2-/- pre-B cells. (A) Total RNA from bone marrow cells of the indicated mice. RT-PCR was performed for HGPRT, VpreB1, VpreB2, and {lambda}5. C indicates negative control. (B) In vitro cultured pre-BI cells were stained for B220 in combination with VP245 (anti-VpreB), LM34 (anti-{lambda}5), or c-kit and analyzed by FACS®. The histograms from the VP245, LM34, and CD117 stains are shown.

Lack of VpreB Protein Expression.
The lack of VpreB RNA implied that there should also be a lack of VpreB protein. To test this, we analyzed in vitro–cultured pre-BI cells which express the SL chain on the cell surface together with a set of glycoproteins, i.e., the pro-BCR (13) (34) (35). Pre-BI cells from heterozygous ES94 mice expressed VpreB on the cell surface (Fig 2 B), as demonstrated by staining with an mAb (VP245) recognizing both VpreB1 and VpreB2 (12) (13). In contrast, pre-BI cells from VpreB1-/-VpreB2-/- mice were not recognized by the VP245 Ab and, hence, did not express VpreB on the cell surface. The pre-BI cultures, independent of genotype, expressed both B220 (CD45R) and c-kit (CD117) on the cell surface (Fig 2 B, and data not shown). Thus, pre-BI cells from the VpreB1/VpreB2 double-deficient mice lacked surface VpreB expression.

As {lambda}5 is also part of the pro-BCR and was expressed as RNA in VpreB1-/-VpreB2-/- bone marrow cells, we analyzed the in vitro–cultured cells for the presence of this protein using the mAb LM34 (13). Pre-BI cells from heterozygous mice expressed {lambda}5 on the cell surface. In contrast, the cells from VpreB1-/-VpreB2-/- mice were not recognized by the LM34 Ab and thus did not express {lambda}5 on the cell surface (Fig 2 B). Analysis of cells from ES163 mice demonstrated that both VpreB and {lambda}5 could be detected on the surface of pre-BI cells derived from wild-type and heterozygous but not homozygous VpreB1/VpreB2 double-mutant mice (data not shown). Thus, the targeting of the two VpreB genes resulted in a lack of VpreB protein on the surface of pre-BI cells. In addition, although expressed as RNA, {lambda}5 protein was also not detected on the cell surface.

B Cell Development Is Impaired in VpreB1-/-VpreB2-/- Mice at the Transition from Pre-BI to Pre-BII Cells.
After confirming that the targeting of both VpreB genes on the same chromosome resulted in a lack of VpreB RNA and protein expression we analyzed whether this had an effect on B cell development. We observed no differences between wild-type and heterozygous VpreB1/VpreB2 double-mutant mice (data not shown). The data from these mice were therefore pooled and represent control mice.

The number of cells in different bone marrow cell populations of control mice varied between two- and sevenfold depending on age. Therefore, the mice were separated into two age groups. Table 1 summarizes the data from mice aged 10 and 50–70 d, respectively. As shown in Table 1, the lack of both VpreB1 and VpreB2 caused a slight decrease in total number of nucleated bone marrow cells in young mice. This difference became more apparent in older mice (~60% of control). Furthermore, the numbers of B220+ cells in young and old VpreB1-/-VpreB2-/- mice were also lower, around half that of control mice. The number of CD19+ lymphocytes was similarly decreased (Table 1). It has been shown by Rolink et al. that most of the B220+CD19- cells in bone marrow not only include pro-B cells but also progenitors of other cell types, e.g., NK cells (36). However, the total number of B220+CD19- cells in VpreB1-/-VpreB2-/- mice was similar to that in control mice. Hence, it appears that the lack of both VpreB1 and VpreB2 has either no or very little effect on this cell population.


 
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Table 1. Impaired B Cell Development in VpreB1-/-VpreB2-/- Mice

To determine at which point of development the defect in both VpreB genes impedes B-lineage cell generation the sizes of cellular pools of pre-BI, large and small pre-BII, and immature and mature B cells in bone marrow were determined by FACS® analysis using B220, CD19, c-kit, CD25, and cell size as distinguishable markers (Fig 3 A and Table 1). VpreB1/VpreB2 double-deficient mice showed an about twofold increase in the number of B220+c-kit+ pre-BI cells compared with control mice. The number of CD19+c-kit+ cells was also increased about twofold (data not shown). By contrast, the number of B220+CD25+ pre-BII cells was severely reduced. In young mice, the difference in cell numbers, compared with control littermates, was ~40-fold, whereas it was ~10-fold in older mice. These results suggest that the lack of VpreB1 and VpreB2 expression result in a defect in the generation of pre-BII from pre-BI cells, i.e., at the stage of surface pre-BCR expression.




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Figure 3. Impaired B cell development in VpreB1-/- VpreB2-/- mice. (A) FACS® analysis of bone marrow cells from VpreB1+/-VpreB2+/- (top) and VpreB1-/-VpreB2-/- (bottom) ES94-derived 10-d-old littermates. Cells were stained for B220 (CD45R) in combination with either CD19, c-kit (CD117), CD25, IgM, or IgD. Cells falling within the lymphocyte gate are displayed. Numbers indicate percentage of cells within this quadrant. (B) FACS® analysis of spleen cells from the mice in A. Cells were stained for B220 in combination with CD19, IgM, IgD, or Ig{lambda}, and also with IgM in combination with IgD. Cells falling within the lymphocyte gate are displayed. Numbers indicate percentage of cells within this quadrant. (C) FACS® analysis of peritoneal cells from VpreB1+/+VpreB2+/+ and VpreB1-/-VpreB2-/- ES94 mice stained for B220 and CD5 aged 12 d and 12 wk, respectively. Cells falling within the lymphocyte gate are displayed. The numbers indicate the percentage of cells within these regions.

Lack of Proliferative Expansion at the Pre-BII Cell Stage.
Pre-BII cells (B220+CD25+) can be divided into large cycling and small resting cells (2). We determined the ratio between these subsequent stages of B cell development in VpreB1/VpreB2 double-deficient old mice. The number of B220+CD25+ cells in young VpreB1-/-VpreB2-/- mice was too low to give meaningful results (Fig 3 A; <1%). In control mice, the ratio of large versus small pre-BII cells was 1:4, whereas in VpreB1/VpreB2 double-mutant mice the ratio was 1:1–1.5. Thus, in VpreB1/VpreB2 double-deficient mice there was an ~5-fold reduction in large and an ~15-fold reduction in small pre-BII cells. Hence, the lack of both VpreB1 and VpreB2 proteins affected the production of both large and small pre-BII cells, with the greatest impact on small pre-BII cells. It suggests that, as in {lambda}5-/- mice, the proliferative expansion of pre-BII cells does not occur and/or that the progression from large to small cells is impaired.

Reduced Numbers of Immature and Mature B Cells in Bone Marrow of VpreB1/VpreB2 Double-mutant Mice.
In VpreB1-/- VpreB2-/- mice, the total numbers of sIgM+ immature and mature B cells were severely reduced, ~25-fold in young and 10-fold in old mice. Comparable reductions in numbers of sIgM+{kappa}L+ B cells were observed (data not shown). As shown in Fig 3 A, sIgM+sIgD+ mature B cells in bone marrow were reduced as much as the sIgM+ immature B cells.

As small pre-BII cells are the precursors of immature B cells, we analyzed the sizes of these two populations. Independent of genotype, the proportion of immature B cells was ~50% of the sIgM+ population. In control mice, ~5 x 106 small pre-BII cells and ~1.5 x 106 immature B cells were found, whereas in the VpreB1/VpreB2 double-mutant mice these values were ~0.4 x 106 and ~0.2 x 106, respectively. From these data it appears likely that the VpreB1/VpreB2 double defect does not significantly affect the transition from small pre-BII to immature B cells in the bone marrow.

In conclusion, in the absence of VpreB1 and VpreB2 protein, bone marrow was enriched in pre-BI cells, decreased in large, and decreased even more in small pre-BII cells, whereas the differentiation of the latter population into immature B cells was unaffected. These data suggest a defect at the transition from pre-BI to large pre-BII cells, due to either a lack of proliferative expansion at, or an impairment of the progression through the pre-BII stage, resulting in the production of much lower numbers of pre-BII and immature B cells.

Decreased Numbers of B Cells in the Spleen of VpreB1-/- VpreB2-/- Mice.
Bone marrow is a primary lymphoid organ which generates B lymphocytes that migrate as immature B cells into the spleen where they mature. Therefore, we analyzed the effect of the VpreB1/VpreB2 double mutation on the generation of splenic B cells. Data shown in Fig 3 B and Table 2 demonstrate that the lack of both VpreB1 and VpreB2 caused a strong reduction in the number of B220+CD19+ B cells. The number of cells defined as either B220+ or CD19+ cells decreased ~10-fold in young and ~5-fold in older mutant mice. Total nucleated cells were ~70% of normal in young and ~45% in older mice.


 
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Table 2. Decreased Numbers of B Cells in the Spleen of VpreB1-/-VpreB2-/- Mice

Only half of the B220+ cells were also sIgM+ in double-mutant mice, whereas 80–90% were sIgM+ in control littermates. In the sIgM+ B cell pool, an ~15- and 5-fold reduction was seen in VpreB1-/-VpreB2-/- young versus old mice, respectively. Further determinations of the numbers of sIgD-expressing cells showed that 60–70% of the sIgM+ cells were also IgD+ in young double-mutant mice, compared with ~90% in control littermates. However, in old double-mutant mice this percentage had reached normal levels (90–100%). In actual cell numbers, ~5-fold fewer immature (sIgM+sIgD-) and ~20-fold fewer mature (sIgM+sIgD+) B cells were found in young, and ~5-fold fewer mature B cells in old double-mutant mice. Thus, with age, the number of B cells increased in VpreB1-/-VpreB2-/- mice, and most of these cells were of a mature phenotype.

Furthermore, the ratio of Ig {kappa}L versus {lambda}L chain expression in splenic B cells was found to be similar (15:1 versus 30:1) as determined in old control and double-mutant mice, respectively. This suggests that the lack of VpreB1 and VpreB2 expression did not affect the rearrangements at the IgL chain gene loci, and the repertoire selection of {kappa}L+ versus {lambda}L+ sIgM+ B cells. Comparable results were obtained with the second strain of VpreB1-/-VpreB2-/- mice (ES163, data not shown). In addition, no alterations were found in the numbers of thymocytes or in CD4+ and CD8+ T cells in the periphery of double-mutant mice (data not shown).

In conclusion, in the absence of VpreB1 and VpreB2, the number of B-lineage cells in the spleen of young mice was greatly decreased with a large proportion of these cells expressing an immature phenotype. However, in older mice, most of the B cells showed a mature phenotype although their numbers had not fully recovered at 70 d of age.

Impaired Development of B-1a Cells in the Peritoneum of VpreB1-/-VpreB2-/- Mice.
Although a large proportion of splenic B cells are expected to belong to the conventional B cell compartment, B cells in the peritoneum appear enriched for B-1 type B cells (37). B-1a cells are characterized as B220+CD5+sIgMhighsIgDdull. Fig 3 C and Table 3 show that the number of B-1a cells was reduced eightfold in young (10–12-d-old) mice, whereas at 9–13 wk of age the B-1a compartment in the peritoneum of double-mutant mice had reached the size of that in wild-type littermates. Heterozygous VpreB1/VpreB2 mice were comparable in their B-1a development to wild-type mice. The B220+CD5+ B-1a cells of double-mutant mice were also analyzed for other markers, such as sIgMhigh and sIgDdull, and were found to be normal (data not shown).


 
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Table 3. Reduction in B-1a B Cells in VpreB1-/-VpreB2-/- Mice

The analyses of peritoneal cells also demonstrated that the percentages and absolute numbers of other B-lineage cells, i.e., of B220+CD5- cells were reduced ~15-fold in 10–12-d-old VpreB1/VpreB2 double-mutant mice (Fig 3 C and Table 3). Independent of genotype, almost all these cells were IgM+ with varying IgD levels, demonstrating that they were B-lineage cells. In contrast to the B220+CD5+ B-1a cells, the B220+CD5- B cells had not reached normal levels by 9–13 wk of age. In contrast, both to B-1a and B220+CD5- cells, the B220-CD5+ non–B-lineage compartment was normal both in young and old double-mutant mice (Fig 3 C and Table 3). We conclude that the generation of the B-1a cell pool in peritoneum is initially affected by the lack of VpreB1 and VpreB2. With age this pool fills to normal size even in VpreB1-/-VpreB2-/- mice. However, as with B cells in spleen of double-mutant mice, the remaining peritoneal B cell populations, which would be either B-1b or conventional B cells, have not yet fully recovered at 70 d of age.

Normal Levels of IgM in Serum.
Sera from 5-mo-old VpreB1/VpreB2 double-mutant mice were analyzed by ELISA for the amounts of IgM and found to be ~0.8–1 mg/ml in VpreB1-/-VpreB2-/- mice, compared with ~0.8–0.9 mg/ml in wild-type and heterozygous littermates. Thus, serum IgM levels appeared not to be negatively affected by the lack of VpreB1 and VpreB2.

T Cell–dependent and –Independent Immune Responses.
To investigate whether B cells in VpreB1-/-VpreB2-/- mice were able to mount an immune response, we immunized mice 4–5 mo of age with T cell–dependent antigens (oxazolone coupled to OVA or OVA without hapten) as well as with a T cell–independent antigen (NP-Ficoll). After 2 wk, the mice were bled and the antigen-specific Ab responses analyzed. The results in Fig 4 demonstrate that VpreB1-/-VpreB2-/- mice were able to respond to both types of antigens. In terms of IgM and IgG responses against NP-Ficoll, VpreB1-/-VpreB2-/- mice mounted an as good immune response as wild-type and heterozygous control mice (Fig 4, and data not shown). In the case of T cell–dependent antigens, VpreB1-/-VpreB2-/- mice showed slightly lowered antigen-specific IgG responses compared with control mice (Fig 4). Thus, the emerging B cells in VpreB1-/-VpreB2-/- mice are functional in terms of immune responses.





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Figure 4. T cell–dependent and –independent immune responses. 4 to 5-mo-old mice of the indicated genotypes, were immunized with either NP-Ficoll or alum-precipitates of either oxazolone-ovalbumin (Ox) or ovalbumin (Ov). 2 wk later, the mice were bled and the sera analyzed in ELISA. The data are represented as titers. Preimmune sera were <1. wt, wild-type; het, heterozygous; homo, homozygous.

Allelic Exclusion in Immature B Cells.
It has been proposed that the pre-BCR signals allelic exclusion of the IgH chain. As {lambda}5-/- mice still display allelic exclusion, it has been hypothesized that VpreB alone, in the absence of {lambda}5, can pair with µH chain and cause IgH allelic exclusion (25). This would imply that the lack of VpreB1 and VpreB2 would cause IgH allelic inclusion. We have shown previously that the single VpreB1 deficiency does not abolish allelic exclusion (32). Hence, VpreB2 could still function as a component of a pre-BCR signaling allelic exclusion. This should no longer be possible in VpreB1-/-VpreB2-/- mice. To test this, IgMa/IgMb heterozygous bone marrow cells from VpreB1/VpreB2 double mutant and control ES94 mice were analyzed for expression of IgMa and IgMb allotypes on single B cells by FACS®. Upon allelic inclusion, the calculated theoretical value of double producers (IgMaIgMb) would be 12% (30). Due to the very low number of immature B cells (B220+IgM+IgD-) in the bone marrow of double-mutant mice, all cells from both femurs were collected and analyzed. As shown in Fig 5, in wild-type mice, ~55% of the IgM+ cells expressed only IgMa and 44% only IgMb on their surface, whereas 1.2% of the cells stained positive for both markers. In the VpreB1-/-VpreB2-/- mice, the percentages of cells expressing either IgMa or IgMb only were very similar to that of control mice. Most importantly, the percentage of cells expressing both IgMa and IgMb was only 2%. This demonstrates that in the absence of VpreB1 and VpreB2, immature B cells in the bone marrow were still allelically excluded at the IgH locus.



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Figure 5. Allelic exclusion of Ig µH chain expression on IgMaIgMb F1 B cells. FACS® analysis of bone marrow cells from ES94-derived VpreB1/VpreB2 double-mutant littermates of the indicated genotype at the age of 74 d. All cells from both femurs were stained for B220 in combination with IgD, IgMa, and IgMb. Cells falling both within the lymphocyte and the B220+IgD- gate were analyzed for expression of IgMa and IgMb. The numbers indicate the percentage of cells within these regions. Only 10% of the gated cells are shown for wild-type.


  Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

The production and surface deposition of the pre-BCR on pre-BII cells has been shown to signal these cells to enter two to five rounds of cell division, expanding the individual µH chain repertoire within the developing B-lineage cells between 2- and 30-fold (26). Whenever the pre-BCR cannot be deposited on the surface, be it that the DJH/DJH-rearranged pre-BI cell has not entered an in-frame VH to DJH rearrangement preventing the expression of a µH chain, that the µH chain made from a productive rearrangement cannot pair with SL chain (25), that the µH chain cannot be deposited in the surface membrane because of a lack of its transmembrane portion (30), that no Ig rearrangement can occur in pre-BI cells as is the case in SCID and RAG-deficient mice (38) (39) (40), or that production of the {lambda}5-component of the pre-BCR is defective as in {lambda}5-/- mice (17) (31), then this proliferation of pre-BII cells does not occur either in vivo or in vitro (26) (41). The analyses of the precursor B cell compartments in the bone marrow of VpreB1-/-VpreB2-/- mice presented here allow us now to add one more important deficiency of the pre-BCR to this list, which abolishes the proliferative expansion of precursor B cells at the transition from pre-BI to pre-BII cells. Although we have shown previously that the single deficiency of one of the VpreB genes of the mouse, i.e., the VpreB1 gene, does not impede in a major way this proliferative expansion of pre-BII cells (32), the double deficiency of both known VpreB genes does. This result then establishes that proliferative expansion of large pre-BII cells is controlled by surface deposition of a pre-BCR composed of µH chain, {lambda}5-protein, and either VpreB1 or VpreB2 protein, and that no other VpreB-like protein can assume the role of the two VpreB proteins for this cellular function. Hence, VpreB3, the protein encoded by 8HS20, also expressed in early B cell development, does not replace VpreB1 and VpreB2 in this signaling function (42).

VpreB1-/-VpreB2-/- B-lineage cells show strikingly similar impairment in B cell development to {lambda}5-defective cells (17) (31). Because proliferative expansion of pre-BII cells is abolished, the generation of B-lineage cells from DJH/DJH-rearranged pre-BI cells becomes less efficient (31). For {lambda}5-/- pre-BI cells it has been shown that the kinetics and rates of formation of sIgM+ and sIg- B cells is indistinguishable from wild-type pre-BI cells. This argues that it is not efficiency of progression in differentiation through the pre-BI to pre-BII to immature B cell stages, but rather the lack of proliferation of {lambda}5-/- pre-BII cells that accounts for the overall B cell developmental defect (31). Thus, in vitro studies of the kinetics and rates of differentiation have yet to be done with VpreB1-/-VpreB2-/- pre-BI cells, in order to further investigate the similarity or possible difference in the differentiation capacities of wild-type, {lambda}5-/-, and VpreB1-/-VpreB2-/- pre-BI cells.

Beyond the pre-BII stage of B cell development, no major effect was observed in VpreB1-/-VpreB2-/- mice, a situation very similar to that in {lambda}5-/- mice. This is illustrated by the B-1a cell compartment and the IgM levels in serum, which become normalized soon after birth, resembling those of wild-type mice, whereas the defect in the accumulation and proper filling of the conventional B cell compartment remains visible for several months after birth. Also, the decreased cellularity of the peripheral, conventional B cell compartment does not impede T cell–independent or T cell–dependent responses of the VpreB1-/-VpreB2-/- mice.

Major changes in VH-repertoires of µH chain originally expressed in VH to DJH-rearranging precursor B cells are observed in wild-type mice as the pre-BCRs form (18). In fact, approximately half of all originally generated µH chains cannot pair with SL chain to form and deposit a pre-BCR on the cell surface. Hence, these are excluded from the proliferative expansion of large pre-BII cells and consequently are infrequent or absent in the repertoire of small pre-BII, immature, and mature B cells. In {lambda}5-/- mice, IgL chains appear to take the functional role of SL chain. Therefore, a very similar change in the VH repertoire of µH chains produced occurs in {lambda}5-/- mice at the transition of precursor B into immature B cells (18). It remains to be investigated whether a similar change in the VH repertoire of µH chains can also be observed in VpreB1-/-VpreB2-/- mice which, again, would possibly be mediated by IgL chains.

It has been proposed that surface deposition of pre-BCRs on precursor B cells, at the transition from pre-BI to pre-BII cells, also signal the cells to allelically exclude the VH to DJH rearrangement at the DJH-rearranged second IgH chain allele. This, in fact should be the case in half of all developing B-lineage cells in which the second allele remains DJH rearranged (5). The signal should cause a rapid downregulation of the expression of the rearrangement machinery, i.e., RAG-1, RAG-2, and TdT transcripts and proteins. It should also signal a closure of the DJH-rearranged IgH chain allele for further VH to DJH rearrangements, especially in cells at later stages of development, i.e., in small pre-BII cells which reactivate the recombination machinery and VL to JL rearrange the {kappa}L and {lambda}L chain gene loci (20).

It was already surprising that {lambda}5-deficient mice still showed allelically excluded B cell compartments (25). To explain allelic exclusion in {lambda}5-/- mice, it was proposed that a modified pre-BCR consisting of µH chain and VpreB protein might act as signaling receptor (25). In line with this suggestion, it was observed that µH chains could bind to VpreB1 and VpreB2 proteins in the absence of {lambda}5 protein, and that SL chain pairing µH chains could do so, whereas nonpairing µH chains could not (unpublished observations). In VpreB1-/-VpreB2-/- precursor B cells, such modified pre-BCRs should not be made. Further, it has been shown that {lambda}5 alone cannot form a {lambda}5-µH chain modified pre-BCR, as in the absence of VpreB protein, {lambda}5 appears not to bind to a whole series of SL chain pairing as well as nonpairing µH chains (unpublished observations). All the more surprising, therefore, is our present finding that allelic exclusion of the µH chain does still function in the double-defective VpreB1-/-VpreB2-/- mice.

How, then, can allelic exclusion happen? It appears from studies with µH chain transmembrane region–deficient mice (30) and from studies of double µH chain–producing B cells of wild-type mice, in which only one µH chain can pair with SL chain, that membrane insertion of the µH chain is required for allelic exclusion. In the absence of both VpreB1 and VpreB2, two other candidates could possibly be part of a modified pre-BCR. One is the chaperone BiP, which is known to associate with µH chains before SL chain association (43). The other is the 8HS20-encoded VpreB3 protein (42). We will have to combine defects in these genes with the VpreB1, VpreB2, and {lambda}5 defects to investigate these possible pre-BCR–like associations and their roles in allelic exclusion. This is experimentally complicated, as VpreB1, VpreB2, {lambda}5, and VpreB3 all appear located on the same chromosome (44).

Yet another scenario for allelic exclusion at the µH chain loci is suggested from the finding that in {lambda}5-/- mice IgL chains take the place of SL chain to change the repertoire of VH-representation during B cell development. In the absence of proliferative expansion of pre-BII cells in these {lambda}5-/- mice, differentiation from pre-BI to small pre-BII cells might occur much more rapidly than in wild-type mice, so that VL to JL rearrangements might occur immediately following VH to DJH-rearrangements, so that a BCR could signal the cell to turn off the recombination machinery and close the IgH chain loci. In line with such functional roles of IgL chains in precursor B cells, it has been observed that the abnormally early expression of transgenic IgL can repair the {lambda}5 defect in B cell development, whereas in the absence of both {lambda}5 and IgL there is a complete block in B-lymphocyte production (45) (46). Early expression of L chain would also predict that signaling by the pre-BCR of proliferative expansion of large pre-BII cells would, in fact, also signal inhibition of IgL chain gene expression in these wild-type cells. If such a scenario were correct, we would predict that a VpreB1-, VpreB2-, VpreB3-, and {lambda}5-deficient mouse would still show allelic exclusion at the IgH chain loci.


  Footnotes

1 Abbreviations used in this paper: BCR, B cell receptor; ES, embryonic stem; RAG, recombination activating gene; RT, reverse transcription; SL, surrogate light.


  Acknowledgements
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

We thank our colleagues for critical reading of the manuscript. We also thank Dr. E. Källberg for Ox-Ova antigen, M. Turner for fibroblasts, and M. Sällström, T. Saunders, and M. George for blastocyst injections.

C. Mundt, S. Licence, and I.-L. Mårtensson are supported by the BBSRC (UK). The Basel Institute for Immunology was founded and supported by F. Hoffmann-La Roche Ltd., Basel.

Submitted: 6 December 2000
Revised: 28 December 2000
Accepted: 9 January 2001


  References
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

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