{lambda}5 is required for rearrangement of the Ig {kappa} light chain gene in pro-B cell lines

Takahiro Miyazaki1, Ibuki Kato1, Sunao Takeshita1, Hajime Karasuyama2 and Akira Kudo1

1 Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama226-8501, Japan
2 Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, 113–8613 Japan

Correspondence to: A. Kudo


    Abstract
 Top
 Abstract
 Introduction
 Methods
 References
 
{lambda}5 associates with Vpre-B to form the surrogate light (L) chain. The phenotype of {lambda}5 knockout mice showed severe impairment of B cell development from pro-B to immature B cell stages. To investigate the function of the surrogate L chain at this stage, we restored expression of {lambda}5 to {lambda}5-deficient pro-B cell lines which were established from bone marrow cells of {lambda}5 knockout mice in the presence of IL-7 and a stromal cell line. Some of these lines are severely impaired in B cell development from pro-B to immature B cell stages as is seen in vivo in {lambda}5 knockout mice. Restoration of {lambda}5 protein by retroviral-mediated gene transfer into established {lambda}5-deficient pro-B cell lines induced rearrangement of the Ig {kappa} L chain genes after removal of IL-7 from the culture. Immunoprecipitation revealed that the restored {lambda}5 in the cell line is coupled with Vpre-B to form the surrogate L chain. The results demonstrate that formation of a complete surrogate L chain, consisting of both {lambda}5 and Vpre-B, stimulates efficient rearrangement of the {kappa} L chain genes.

Keywords: {kappa} light chain, {lambda}5, rearrangement, surrogate light chain, Vpre-B


    Introduction
 Top
 Abstract
 Introduction
 Methods
 References
 
B cell differentiation from pluripotent stem cells to immature B cells in bone marrow is characterized by successive rearrangement of the gene segments of the Ig heavy (H) and light (L) chain gene loci (1). The Ig H chain locus is rearranged before the Ig L chain locus. When a functional VHDHJH rearrangement occurs in a pre-B cell, this cell will express the pre-B cell receptor formed by the membrane-bound µ H chain in complex with the surrogate L chain (2,3). In both mouse and human, the surrogate L chain is composed of two proteins encoded by the pre-B cell-specific genes, Vpre-B and {lambda}5 (47). The analyses of bone marrow cells from {lambda}5 knockout mice revealed that the number of CD43 small pre-B cells and of sIgM+ immature and mature B cells was drastically reduced, whereas that of CD43+ early precursor B cells was normal (8). Another analysis using c-kit, CD25 and the surrogate L (SL) chain as markers showed that c-kit+CD25 SL+ pro-B/pre-B I cells were produced in normal numbers, whereas c-kit CD25+ SL+ large pre-B-II cells and c-kit CD25+ SL large and small pre-B-II cells, as well as immature B cells, were at least 40-fold reduced (9). Mutation in the human {lambda}5 gene markedly reduces the number of CD19+ B cells in the peripheral blood and there were almost no mature B cells in bone marrow, indicating that a more severe B cell deficiency is caused by loss of {lambda}5 expression in humans than in mice (10). These results indicate that in {lambda}5 knockout mice and in humans with {lambda}5 mutation, B cell differentiation is impaired at the transition from the pro-B/pre-B-I to the pre-B-II cell stage in which Ig L chain rearrangement takes place.

Several hypotheses have been proposed regarding functions for the pre-B cell receptor, including allelic exclusion, proliferation and differentiation, and induction of {kappa} chain rearrangement. It was strongly suggested that the pre-B cell receptor functions in controlling rearrangement of H and L chain genes, possibly by affecting expression of Rag genes (11). Allelic exclusion at the IgH locus requires the expression of the pre-B cell receptor (12) and the L chain locus is efficiently rearranged following appropriate signals given by the pre-B cell receptor cross-linking (13). Although in {lambda}5 knockout mice, {kappa} chain rearrangement occurs in leaky B cells, the question remains whether the pre-B cell receptor actively up-regulates {kappa} chain rearrangement. We and others have speculated that when the pre-B cell receptor appears during B cell differentiation, one of the first signals stimulates {kappa} chain rearrangement. Several reports showing coincident effects of H chain expression on germline transcription of the Ig {kappa} locus and its rearrangement support this speculation (1418).

An in vitro B cell differentiation system has been established (19); pro-B cell lines from fetal liver or bone marrow cells are cultured and cloned in the presence of the stromal cell line and IL-7, and then removal of IL-7 from the culture induces differentiation from pro-B to immature B cells. Consequently, we have established pro-B cell lines from bone marrow cells of {lambda}5 knockout mice by this system. Compared to normal pro-B cell lines, {lambda}5-deficient pro-B cell lines differentiate poorly in response to removal of IL-7 in vitro. To examine the effect of {lambda}5, {lambda}5 was reconstituted by retroviral-mediated gene transfer into {lambda}5-deficient pro-B cell lines. {lambda}5 expression restored {kappa} chain rearrangement.


    Methods
 Top
 Abstract
 Introduction
 Methods
 References
 
Animals and cell lines
BDF1 mice were obtained from BRL (Fullinsdorf, Switzerland). {lambda}5 knockout mice were bred in the breeding facilities of the Basel Institute for Immunology.

The stromal cell line, ST2, was obtained from Dr Nishikawa (Kyoto University, Kyoto) (20). Pro-B cell lines were established from bone marrow cells of BDF1 mice or {lambda}5 knockout mice cultured with ST2 in the presence of IL-7. Pro-B cell lines were cultured on mitomycin C (Kyowa Hakko, Tokyo, Japan)-treated ST2 in SF-O3 medium (Sanko Jyunyaku, Kyoto, Japan) containing 5x10–5 M 2-mercaptoethanol, 1xnon-essential amino acids (Gibco/BRL, Gaithersburg, MD), 0.03% primatone (Quest International, Naarden, Netherlands), 2% FCS and 100 U/ml of recombinant IL-7 (a gift of Dr Sudo, Toray, Tokyo). In in vitro differentiation, cells were washed to remove IL-7 and were cultured for 2–3 days at 5x105 to 1x106 cells/ml on mitomycin C-treated ST2 without IL-7.

Retrovirus-mediated gene transfer
The sense and anti-sense {lambda}5 cDNAs were recloned into a retroviral expression vector, pMX-puro, and {lambda}5 constructs were transfected into {phi}NX-Eco packaging cells (21), and cells were subsequently selected with 2 µg/ml of puromycin (Nacalai Tesque, Tokyo, Japan). The virus infection was performed with co-culture of pro-B cell lines, packaging cells and ST2. The stable transfectants were established by the selection of 1 µg/ml of puromycin in the presence of IL-7.

Antibodies
The FITC-conjugated mAb RA3-6B2 (anti-B220) was purchased from PharMingen (San Diego, CA). The FITC-conjugated goat anti-mouse IgM (µ chain specific) and anti-mouse {kappa} chain were purchased from Southern Biotechnology (Birmingham, AL). The rat mAb VP245 (anti-mouse Vpre-B) (22), LM34 (anti-mouse {lambda}5) (22) and A7R34 [anti-mouse IL-7 receptor (IL-7R); a gift of Dr Nishikawa, Kyoto University, Kyoto] were purified by Protein G–Sepharose (Pharmacia, Uppsala, Sweden). The FITC-conjugated goat anti-rat IgG was purchased from Cappel (Organon Technika, Durham, NC). Flow cytometric analyses using the FACSCalibur (Becton Dickinson, Mountain View, CA) were performed as described (22).

Cell surface labeling and immunoprecipitation
For surface labeling, cells were washed twice with biotinylation buffer (50 mM NaCl, 0.1 M HEPES, pH 8.0, 1 mM PMSF and 2 µg/ml leupeptin) and were incubated with 1.0 mg/ml of sulfo-NHS-biotin (Pierce, Rockford, IL) for 20 min at 4°C. After washing with cold PBS, aliquots of 107 cells were lysed in 300 µl of NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 50 mM iodoacetamide, 0.02% NaN3, 1 mM PMSF, 2 µg/ml aprotinin and 1 µg/ml pepstatin) for 30 min on ice. Precleared lysate was incubated with a mAb at 4°C for 1 h followed by binding with a rabbit anti-rat antibody for 30 min and then incubated with Protein A–Sepharose beads (Amersham Pharmacia, Little Chalfont, UK). Western blot analyses were performed by a standard protocol, and then surface biotinylated proteins were reacted with streptavidin–horseradish peroxidase and detected by using the chemiluminescence ECL kit (Amersham Pharmacia).

PCR analyses of IgH and Ig {kappa} gene rearrangement
Genomic DNAs were extracted from cultured cells. PCR was performed as described (23). Briefly, reaction mixtures for PCR amplification consisted of 100–200 ng of genomic DNAs, 200 nM dNTPs, 500 nM each oligonucleotide, 10 mM Tris–HCl, pH. 8.3, 50 mM KCl, 1.8 mM (for IgH) or 1.5 mM (for Ig {kappa}) MgCl2 and 2.5 U of Taq polymerase (Takara, Shiga, Japan) in 50 µl. Reactions were cycled as follows: Ig H, 30 cycles of 94°C for 30 s, 58°C for 1 min and 72°C for 2 min; Ig {kappa}, 28 cycles of 94°C for 30 s, 60°C for 1 min 30 s and 72°C for 1 min.

PCR primers were used for IgH: VH all, 5'-AGGTSMARCTGCAGSAGTCWGG-3'; JH4, 5'-AAAGACCTGCAGAGGCCATTCTTACC-3'; and for Ig {kappa}: V{kappa}con, 5'-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3'; J{kappa}5, 5'-TGCCACGTCAACTGATAATGAGCCCTCTC-3'.

Results
Properties of {lambda}5-deficient pro-B cell lines
Bone marrow cells from a normal BDF1 mouse and {lambda}5 knockout mice were cultured on the stromal cell line, ST2, in the presence of IL-7, and one clonal cell line, BPB, from a BDF1 and three clonal cell lines, clone 1, clone 2 and clone 3, from {lambda}5 knockout mice were established (Table 1Go). All cell lines showed pro-B cell phenotypes, such as B220+ and IL-7R+. Representative FACS profiles of BPB, clone 1 and clone 3 are shown in Fig. 1Go. The normal pro-B clone, BPB, expressed B220, Vpre-B, {lambda}5 and IL-7R, but not µ H and {kappa} L. Clone 1 and clone 3 expressed only B220 and IL-7R, and other markers were negative. Northern blot analyses revealed that all {lambda}5-deficient pro-B cell lines expressed Vpre-B and a truncated form of {lambda}5 (data not shown) that did not produce {lambda}5 protein (8). The H chain configuration was determined by PCR analyses by using common PCR primers originating upstream of D and down-stream of JH4. The clone 1 and the clone 3 were DJH1/DJH3 and DJH2 configuration respectively, as analyzed by PCR, and another allele of clone 3 was DJ, which was determined by Southern blot analyses (data not shown). In clone 2, one allele, DJH1, was assessed by PCR and cytoplasmic µ H chain expression was detected by FACS analysis (data not shown), therefore the H chain configuration must be DJH1/VDJ. The majority of established pro-B cell lines from normal mice, which have a DJ/DJ configuration at the H chain loci, are able to differentiate into immature B cells after removal of IL-7 (19). The normal pro-B cell line, BPB, became IgM+ (11% of µ H and {kappa}+) after removal of IL-7, demonstrating that DJ to VDJ rearrangement took place at the H chain locus and also the {kappa} L chain gene was rearranged (Fig. 2Go). The remaining 89% of cells failed to make productive rearrangements and were destined to die by apoptosis (19). Clone 1 and clone 3 showed little differentiation into surface IgM+ immature B cells 3 days after removal of IL-7 from the culture and thus <0.5% of clone 1 and clone 3 were IgM+ immature B cells, which may reflect the presence of a minor population of leaky B cells in {lambda}5 knockout mice. These results demonstrate that B cell differentiation in vitro from pro-B to immature B cells is suppressed in {lambda}5-deficient pro-B cell lines.


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Table 1. Surface phenotype and Ig rearrangements in the {lambda}5-deficient pro-B cell clones
 


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Fig. 1. Phenotype of pro-B cell lines from a {lambda}5 knockout mouse. The phenotype of pro-B cell lines, clone 1 and clone 3 from a {lambda}5 knockout mouse, and BPB from a BDF1 mouse, established from bone marrow cells cultured with the stromal cell line, ST2, in the presence of IL- 7 was examined by FACS analyses (FACSCalibur, Becton Dickinson) using antibodies against B220, Vpre-B, {lambda}5, IL-7R, µ H and {kappa} L. Clone 1 and clone 3 are negative for Vpre-B, {lambda}5, µ H and {kappa} L on the surface. The negative control is indicated by the dotted line.

 


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Fig. 2. Induction of B cell differentiation in vitro using the {lambda}5-deficient pro-B cell lines. The pro-B cell lines, clone 1 and clone 3 from bone marrow of a {lambda}5 knockout mouse and BPB from a BDF1 mouse, were cultured on ST2 in the presence of IL-7. IL-7 was removed from the culture and FACS analyses were performed after 3 days. µ H and {kappa} L were detected by FITC-conjugated goat antibodies respectively, which are indicated by the percent.

 
Restoration of {lambda}5 to the {lambda}5-deficient pro-B cell line using retrovirus transfection
To examine the role of {lambda}5 in B cell differentiation, restoration of {lambda}5 expression to {lambda}5-deficient pro-B cell lines, clone 1 and clone 3, was performed. Both sense and anti-sense orientations of a mouse {lambda}5 cDNA were inserted into an expression vector, and the {lambda}5 expression constructs were transfected into the {phi}NX-Eco cells. The resulting retrovirus particles were infected into {lambda}5-deficient pro-B clones, and stable tansfectants, clone 1-{lambda}5 and clone 3-{lambda}5 for sense and clone 1-R{lambda}5 and clone 3-R{lambda}5 for anti-sense, were established. Clone 1-{lambda}5 and clone 3-{lambda}5 developed expression of Vpre-B and {lambda}5 on the surface as shown in Fig. 3Go, presumably due to enforced expression of {lambda}5 protein. In contrast, clones 1-R{lambda}5 and 3-R{lambda}5 showed identical phenotypes with original clones, 1 and 3. To investigate the association of Vpre-B and {lambda}5 molecules, the surrogate L chain in a representative clone, 1-{lambda}5, was examined. Surface biotinylation of clone 1-{lambda}5 cells followed by immunoprecipitation using Vpre-B or {lambda}5 antibodies revealed correct formation of and cell surface expression of the surrogate L chain consisting of {lambda}5 (22 kDa) and Vpre-B (16 kDa) proteins as described previously (2,24) (Fig. 4Go). The result showed that the introduced {lambda}5 was coupled with Vpre-B to form the surrogate L chain expressed on the surface, although Vpre-B alone did not express on the surface.



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Fig. 3. Surface phenotype of {lambda}5-restored {lambda}5-deficient pro-B cell lines. The sense and anti-sense constructs of {lambda}5 were introduced into the {lambda}5-deficient pre-B cell lines, clone 1 and clone 3, resulting in stable transfectants, clone 1-{lambda}5 and clone 3-{lambda}5 (sense) and clone 1-R{lambda}5 and clone 3-R{lambda}5 (anti-sense). Surface expression was examined by FACSCalibur using rat mAb against B220, Vpre-B, {lambda}5 and IL-7R, and goat antibodies against µ H and {kappa} L. Vpre-B and {lambda}5 expression became positive on clone 1-{lambda}5 and clone 3-{lambda}5. The negative control is indicated by the dotted line.

 


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Fig. 4. Surface biotinylation and immunoprecipitation of Vpre-B and {lambda}5 using the restored cell line, clone 1-{lambda}5. The pro-B cell line, clone 1-{lambda}5, was surface biotinylated and immunoprecipitated by using a control antibody, an anti-Vpre-B antibody (Vpre-B Ab) and an anti-{lambda}5 antibody ({lambda}5 Ab). Vpre-B (16 kDa) and {lambda}5 (22 kDa) were detected with both anti-Vpre-B and anti-{lambda}5 antibodies.

 
Differentiation of the {lambda}5-reconstituted {lambda}5-deficient pro B cell lines
In order to determine if transfected clones, 1-{lambda}5, 1-R{lambda}5, 3-{lambda}5 and 3-R{lambda}5, could differentiate in vitro, IL-7 was removed from the culture to induce differentiation and FACS analyses were performed after 3 days. The results shown in Fig. 5Go indicated that clone 1-{lambda}5 and clone 3-{lambda}5 differentiated to become ~8 and 7% surface IgM+ B cells, respectively, whereas clones 1-R{lambda}5 and 3-R{lambda}5 differentiated very little (<1.0% ). To confirm the induction of differentiation from pro-B to immature B cell, the rearrangements of H and {kappa} chains of BPB, clone 1 and clone 1-{lambda}5 were examined by PCR analyses (Fig. 6Go). The H chain configuration of all these lines was DJH1/DJH3 and the {kappa} chain was in germline as described in Table 1Go. By removal of IL-7, the IgH chain genes of these clones were rearranged from DJ to VDJ. In both clone 1 and clone 1-{lambda}5, the major three bands, VDJH1, VDJH2 and VDJH3, were found from the upper to the lower bands. Additional rearrangement must have occurred at the allele of DJH1 to form DJH2, proceeding to VDJH2 rearrangement. In the case of BPB, preferential rearrangement occurred at the allele of DJH1 to form VDJH1. The {kappa} chain genes were rearranged in BPB and clone 1-{lambda}5, and barely in clone 1. Major two bands detected at the {kappa} chain loci of BPB and clone 1-{lambda}5 corresponded to VJ{kappa}1 and VJ{kappa}2 rearrangements. This result indicated that the surrogate L chain is necessary to induce {kappa} chain rearrangement.



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Fig. 5. In vitro differentiation of the {lambda}5-restored pro-B cell lines. The stable transfectants, clone 1-{lambda}5, clone 3-{lambda}5, clone 1-R{lambda}5 and clone 3-R{lambda}5, were cultured on ST2 in the presence of IL-7. IL-7 was removed from the culture and FACS analyses were performed after 3 days. µ H and {kappa} L were detected by FITC-conjugated goat antibodies respectively, which are indicated by the percent.

 


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Fig. 6. Analysis of H chain and {kappa} chain gene rearrangement by PCR. Genomic DNAs were isolated from pro-B cell lines, clone 1, clone 1-{lambda}5 and BPB cultured in the presence of ST2 and IL-7 (Diff. –), and then IL-7 was removed from the culture to induce differentiation (Diff. +). PCR was performed to detect H chain rearrangement from DJ to VDJ (VHDHJH) and {kappa} chain rearrangement (V{kappa}J{kappa}). Southern blot analysis was carried out to detect the rearranged bands using probes, a 2.0 kb BamHI–EcoRI fragment containing JH3 and JH4 for VDJ rearrangement, and a 2.6 kb HindIII fragment containing J{kappa}1–5 for {kappa} chain rearrangement respectively.

 
Discussion
We have demonstrated that the surrogate L chain is required for {kappa} chain rearrangement by using retrovirus infection to restore {lambda}5 protein expression to {lambda}5-deficient pro-B cells. In our established system of in vitro B cell differentiation, the pro-B cell lines differentiate to immature IgM+ B cells 3 days after removal of IL-7 from the culture. During differentiation from pro-B to immature B cell, two rearrangement events occur; the rearrangement from DJ to VDJ on the H chain locus and rearrangement on the L chain locus (19). Most established pro-B cell lines have the structures of DJ/DJ configuration at the H chain locus and of the germline configuration at the L chain locus. Thus, relatively low numbers of IgM+ cells are expected to arise from the pro-B cell line, if it is considered that the probability of in-frame joining of H and L chain gene rearrangements is one-third respectively, and that significant numbers of V gene segments carry stop codons. Actually, in the previous study, <10% of fetal liver derived early pre-B cells mature to IgM+ B cells with in-frame structures on both H and L chains alleles (19). During the differentiation, a productive rearrangement gives rise to a µ H chain molecule, which is able to associate with the surrogate L chain to form the pre-B cell receptor. Here we have shown that, in the culture of {lambda}5-deficient pro-B cell lines, H chain rearrangement was induced from DJ to VDJ, but the efficiency of {kappa} chain rearrangement was extremely low, suggesting that an incomplete pre-B cell receptor does not activate {kappa} chain rearrangement. Interestingly, our results also suggest that signals from the IL-7R, caused by removal of IL-7, affect H chain rearrangement. Recently, Corcoran et al. also reported the involvement of IL-7 in rearrangement (25); rearrangement of the H chain from DJ to VDJ is impaired in mice lacking the IL-7R although D–J joining is normal in the same mice.

The studies suggesting the involvement of the pre-B cell receptor in B cell development or rearrangement have been reported. Two reports (17,26) described that conventional L chains could rescue B cell development in {lambda}5 knockout mice on supposed behalf of the surrogate L chain, which required the µ H chain to activate the pre-B cell transition. Constantinescu and Schlissel reported (27) that locus-specific recombinase activity at the L chain loci was induced by the µ H chain protein, suggesting that the pre-B cell receptor activates L chain rearrangement. Tsubata et al. also reported that cross-linking of the pre-B cell receptor in abelson virus-transformed pre-B cell lines induced the activation of {kappa} chain rearrangement (13). However, no previous evidence addressed whether the surrogate L chain itself is directly involved in the regulation of {kappa} chain rearrangement. Our results described here are the first to demonstrate that a complete surrogate L chain, consisting of Vpre-B and {lambda}5, is required for the activation of {kappa} chain rearrangement in an established in vitro B cell differentiation system.

The next question is whether the surrogate L chain serves as the pre-B cell receptor. It was difficult to detect the formation of the pre-B cell receptor during the in vitro differentiation from pro-B to immature B cells. This is consistent with observations that the pre-B cell receptor-positive pre-B cells were not detected in bone marrow cells by FACS analyses (28). One possible explanation for this is that rapid differentiation is induced immediately after the pre-B cell receptor is formed and that the surrogate L chain is rapidly replaced by {kappa} L chain to form the IgM receptor. However, we have recently established the pre-B cell receptor-positive pre-B cell lines in the presence of IL-7 without a stromal cell line, and these cell lines expressed the surrogate L chain coupled with µ chain on the surface as revealed by FACS and immunoprecipitation analyses (I. Kato et al., manuscript in preparation), indicating the possible involvement of the pre-B cell receptor in the differentiation from pro-B to immature B cells. The rearrangement of {kappa} chain was not strictly regulated in our system in vitro and, thus, in {lambda}5-deficient pro-B cell lines, a minor level of {kappa} chain rearrangement (<0.5%) occurred after removal of IL-7. It may be explained that an incomplete receptor complex of µ chain and Vpre-B located in cytoplasm weakly functions since µ chain could bind to Vpre-B without {lambda}5 (29).

The molecular mechanism of how the pre-B cell receptor functions in {kappa} chain rearrangement is the next question to be answered. One of the potential factors implicated in {kappa} chain rearrangement as a target of the signals from the pre-B cell receptor is Pax-5, although the relationship between the pre-B cell receptor and Pax-5 is unclear. The deletion of KI and KII sites located upstream of J{kappa} in knockout mice severely impaired {kappa} chain rearrangement (30), while we found that Pax-5 binds the KI and KII sites (31), suggesting that Pax-5 may play a role in {kappa} chain rearrangement. We are examining further how the signal of the pre-B cell receptor regulates {kappa} chain rearrangement using pre-B cell receptor-positive cell lines.


    Acknowledgments
 
We thank Dr Testuo Sudo for providing IL-7, Dr Shinichi Nishikawa for an antibody against IL-7 receptor, Dr Antonius Rolink and Ms Monika Fluri for technical help in establishing in vitro culture and Dr Dirk Haasner for PCR analyses of clones. We also thank Dr Garry P. Nolan for providing the retro-viral infection system and Dr Steven R. Bauer for critical reading of the manuscript. This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


    Abbreviations
 
IL-7RIL-7 receptor
SLsurrogate light chain

    Notes
 
Transmitting editor: D. Kitamura

Received 21 January 1999, accepted 8 April 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 References
 

  1. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:575.[ISI][Medline]
  2. Karasuyama, H., Kudo, A. and Melchers, F. 1990. The proteins encoded by the Vpre-B and {lambda}5 pre-B cell-specific genes can associate with each other and with µ heavy chain. J. Exp. Med. 172:969.[Abstract]
  3. Melchers, F., Karasuyama, H., Haasner, D., Bauer, S., Kudo, A., Sakaguchi, N., Jameson, B. and Rolink, A. 1993. The surrogate light chain in B-cell development. Immunol. Today 14:60.[ISI][Medline]
  4. Kudo, A. and Melchers, F. 1987. A second gene, Vpre-B in the {lambda}5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes. EMBO J. 6:2267.[Abstract]
  5. Sakaguchi, N. and Melchers, F. 1986. {lambda}5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. Nature 324:579.[ISI][Medline]
  6. Kudo, A., Sakaguchi, N. and Melchers, F. 1987. Organization of the murine Ig-related {lambda}5 gene transcribed selectively in pre-B lymphocytes. EMBO J. 6:103.[Abstract]
  7. Bauer, S. R., Kudo, A. and Melchers, F. 1988. Structure and pre-B lymphocyte restricted expression of the Vpre-B gene in humans and conservation of its structure in other mammalian species. EMBO J. 7:111.[Abstract]
  8. Kitamura, D., Kudo, A., Schaal, S., Muller, W., Melchers, F. and Rajewsky, K. 1992. A critical role of {lambda}5 protein in B cell development. Cell 69:823.[ISI][Medline]
  9. Rolink, A., Grawunder, U., Winkler, T. H., Karasuyama, H. and Melchers, F.1994. IL-2 receptor {alpha} chain (CD25, TAC) expression defines a crucial stage in pre-B cell development. Int. Immunol. 6:1257.[Abstract]
  10. Minegishi, Y., Coustan-Smith, E., Wang Y-H., Cooper, M. D., Campana, D. and Conley, M. E. 1998. Mutations in the human {lambda}5/14.1 gene result in B cell deficiency and agammaglobulinemia. J. Exp. Med. 187:71.[Abstract/Free Full Text]
  11. Bauer, S. R. and Scheuermann, R. H. 1993. Expression of the Vpre-B/{lambda}5/µ pseudo-Ig complex correlates with downregulated RAG-1 expression and V(D)J type recombination: a mechanism for allelic exclusion at IgH locus. Transgene 1:33.
  12. Grawunder, U., Leu, T.M.J., Schatz, D.G., Werner, A., Rolink, A.G., Melchers, F. 1995. Downregulation of RAG-1 and RAG-2 gene expression in pre-B cells after functional immunoglobulin heavy chain rearrangement. Immunity 3:601.[ISI][Medline]
  13. Tsubata, T., Tsubata, R. and Reth, M. 1992. Crosslinking of the cell surface immunoglobulin (µ-surrogate light chains complex) on pre-B cells induces activation of V gene rearrangements at the immunoglobulin {kappa} locus. Int. Immunol. 4:637.[Abstract]
  14. Stanhope-Baker, P., Hudson, K.M., Shaffer, A.L., Constantinescu, A. and Schlissel, M.S. 1996. Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell 85:887.[ISI][Medline]
  15. Reth, M., Petrac, E., Wiese, P., Lobel, L. and Alt, F.W. 1987. Activation of V{kappa} gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J. 6:3299.[Abstract]
  16. Iglesias, A., Kopf, M., Williams, G.S. Buhler, B. and Kohler, G. 1991. Molecular requirements for the µ-induced light chain gene rearrangement in pre-B cells. EMBO J. 10:2147.[Abstract]
  17. Papavasiliou, F., Jankovic, M. and Nussenzweig M. C. 1996. Surrogate or conventional light chains are required for membrane immunoglobulin mu to activate the precursor B cell transition. J. Exp. Med. 184:2025.[Abstract]
  18. Young, F., Ardman B., Shinkai, Y., Lansford, R., Blackwell, T.K., Mendelson, M., Rolink, A., Melchers, F. and Alt, F. W. 1994. Influence of immunoglobulin heavy and light-chain expression on B-cell differentiation. Gene Dev. 8:1043.[Abstract]
  19. Rolink, A., Kudo, A., Karasuyama, H., Kikuchi, Y. and Melchers, F. 1991. Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo. EMBO J. 10:327.[Abstract]
  20. Sudo, T., Ito, M., Ogawa, W., Iizuka, M., Kodama, H., Kunisada, T., Hayashi, S.-I., Ogawa, M., Sakai, K. and Nishikawa, S.-I. 1989. Interleukin 7 production and function in stromal cell-dependent B cell development. J. Exp. Med. 170:333.[Abstract]
  21. Hofmann, A., Nolan, G. P. and Blau, H. M. 1996. Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette. Proc. Natl Acad. Sci. USA 93:5185.[Abstract/Free Full Text]
  22. Rolink, A., Karasuyama, H., Grawunder, U., Haasner, D., Kudo, A. and Melchers, F. 1993. B cell development in mice with a defective {lambda}5 gene. Eur. J. Immunol. 23:1284.[ISI][Medline]
  23. Pennycook, J. L. M. H., Marshall, A. J. and Wu, G. E. 1997. PCR assays for endogenous Ig gene rearrangement. In Lefkovits, I., ed., Immunology Methods Manual, vol. 1, p. 237. Academic Press, London.
  24. Kudo, A., Bauer, S. and Melchers, F. 1989. Structure, control of expression and putative function of the pre-B cell-specific genes Vpre-B and {lambda}5. Prog. Immunol. 7: 339.
  25. Corcoran, A.E., Riddle, A., Krooshoop, D. and Venkitaraman, A. R. 1998. Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 391:904.[ISI][Medline]
  26. Pelanda, R., Schaal, S., Torres, R. M. and Rajewsky, K. 1996. A prematurely expressed Ig{kappa} transgene, but not a V{kappa}J{kappa} gene segment targeted into the Ig{kappa} locus, can rescue B cell development in {lambda}5-deficient mice. Immunity 5:229.[ISI][Medline]
  27. Constantinescu, A. and Schlissel, M. S. 1997. Changes in locus-specific V(D)J recombinase activity induced by immunoglobulin gene products during B cell development. J. Exp. Med. 185:609.[Abstract/Free Full Text]
  28. Karasuyama, H., Rolink, A., Shinkai, Y., Young, F., Alt, F. W. and Melchers, F. 1994. The expression of Vpre-B/{lambda}5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice. Cell 77:133.[ISI][Medline]
  29. Hirabayashi, Y., Lecerf, J.-M., Dong, Z. and Stollar, D. 1995. Kinetic analysis of the interactions of recombinant human Vpre-B and Ig V domains. J. Immunol.155:1218.[Abstract]
  30. Ferradini, L., Gu, H., Smet, A. D., Rajewsky, K., Reynaud, C.-A. and Weil, J.-C. 1996. Rearrangement-enhancing element upstream of the mouse immunoglobulin kappa chain J cluster. Science 271:1416.[Abstract]
  31. Tian, J., Okabe, T., Miyazaki, T., Takeshita, S. and Kudo, A. 1997. Pax-5 is identical to EBB-1/KLP and binds to the Vpre-B and {lambda}5 promoters as well as the KI and KII sites upstream of the J{kappa} genes. Eur. J. Immunol. 27:750.[ISI][Medline]