International Immunology, Vol. 11, No. 8, pp. 1195–1201
© 1999 The Japanese Society for Immunology
λ5 is required for rearrangement of the Ig κ
light chain gene in pro-B cell lines
Takahiro Miyazaki1, Ibuki Kato1, Sunao Takeshita1, Hajime Karasuyama2 and
Akira Kudo1
1Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama
226-8501, Japan
2Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, 113–8613 Japan
Keywords: κ light chain, λ5, rearrangement, surrogate light chain, Vpre-B
Abstract
λ5 associates with Vpre-B to form the surrogate light (L) chain. The phenotype of λ5 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 λ5 to
λ5-deficient pro-B cell lines which were established from bone marrow cells of λ5 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 λ5 knockout mice.
Restoration of λ5 protein by retroviral-mediated gene transfer into established λ5-deficient pro-B
cell lines induced rearrangement of the Ig κ L chain genes after removal of IL-7 from the culture.
Immunoprecipitation revealed that the restored λ5 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 λ5 and Vpre-B, stimulates efficient rearrangement of the κ L chain genes.
Introduction
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
λ5 (4–7). The analyses of bone marrow cells from λ5 knockout
mice revealed that the number of CD43– small pre-B cells
and of sIgM1 immature and mature B cells was drastically
reduced, whereas that of CD431 early precursor B cells
was normal (8). Another analysis using c-kit, CD25 and the
surrogate L (SL) chain as markers showed that c-kit1
CD25– SL1 pro-B/pre-B I cells were produced in normal
numbers, whereas c-kit– CD251 SL1 large pre-B-II cells and
c-kit– CD251 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 λ5 gene markedly reduces the number of
CD191 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 λ5 expression
in humans than in mice (10). These results indicate that in λ5
knockout mice and in humans with λ5 mutation, B cell
differentiation is impaired at the transition from the pro-B/preB-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 κ 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 λ5
knockout mice, κ chain rearrangement occurs in leaky B
cells, the question remains whether the pre-B cell receptor
actively up-regulates κ 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
κ chain rearrangement. Several reports showing coincident
effects of H chain expression on germline transcription of the
Correspondence to: A. Kudo
Transmitting editor: D. Kitamura
Received 21 January 1999, accepted 8 April 1999
1196 Requisition of λ5 for κ chain rearrangement
Ig κ locus and its rearrangement support this speculation
(14–18).
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 λ5 knockout mice by this system. Compared to normal
pro-B cell lines, λ5-deficient pro-B cell lines differentiate
poorly in response to removal of IL-7 in vitro. To examine the
effect of λ5, λ5 was reconstituted by retroviral-mediated
gene transfer into λ5-deficient pro-B cell lines. λ5 expression
restored κ chain rearrangement.
Methods
Animals and cell lines
BDF1 mice were obtained from BRL (Fullinsdorf, Switzerland).
λ5 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 λ5 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 5310–5 M 2-mercaptoethanol, 13non-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 53105 to 13106 cells/
ml on mitomycin C-treated ST2 without IL-7.
Retrovirus-mediated gene transfer
The sense and anti-sense λ5 cDNAs were recloned into a
retroviral expression vector, pMX-puro, and λ5 constructs
were transfected into φNX-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 FITCconjugated goat anti-mouse IgM (µ chain specific) and antimouse κ chain were purchased from Southern Biotechnology
(Birmingham, AL). The rat mAb VP245 (anti-mouse Vpre-B)
(22), LM34 (anti-mouse λ5) (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 κ 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
κ) 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 κ, 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, 59-AGGTSMARCTGCAGSAGTCWGG-39; JH4, 59-AAAGACCTGCAGAGGCCATTCTTACC-39; and for Ig κ: Vκcon, 59-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-39; Jκ5, 59-TGCCACGTCAACTGATAATGAGCCCTCTC-39.
Results
Properties of λ5-deficient pro-B cell lines
Bone marrow cells from a normal BDF1 mouse and λ5
knockout mice were cultured on the stromal cell line, ST2, in
Table 1. Surface phenotype and Ig rearrangements in the
λ5-deficient pro-B cell clones
B220a
BPB
1
Clone 1 1
Clone 2 1
Clone 3 1
aDetected
IL-7Ra
1
1
1
1
Surrogate L chainb
Vpre-B
λ5
cµa
H
L
1
1
1
1
1
2
2
2
2
2
1
2
DJH1/DJH3
DJH1/DJH3
DJH1/VDJ
DJH2/DJ
G
G
G
G
by FACS analyses.
by Northern blot analyses.
by PCR and Southern blot analyses.
bDetermined
cDetermined
Ig genec
Requisition of λ5 for κ chain rearrangement 1197
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 λ5 knockout mice were established (Table 1). All
cell lines showed pro-B cell phenotypes, such as B2201 and
IL-7R1. Representative FACS profiles of BPB, clone 1 and
clone 3 are shown in Fig. 1. The normal pro-B clone, BPB,
expressed B220, Vpre-B, λ5 and IL-7R, but not µ H and κ L.
Clone 1 and clone 3 expressed only B220 and IL-7R, and
other markers were negative. Northern blot analyses revealed
that all λ5-deficient pro-B cell lines expressed Vpre-B and a
truncated form of λ5 (data not shown) that did not produce
λ5 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 IgM1 (11% of µ H and κ1) after removal of IL-7,
demonstrating that DJ to VDJ rearrangement took place at
the H chain locus and also the κ L chain gene was rearranged
(Fig. 2). 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
IgM1 immature B cells 3 days after removal of IL-7 from the
culture and thus ,0.5% of clone 1 and clone 3 were IgM1
immature B cells, which may reflect the presence of a minor
population of leaky B cells in λ5 knockout mice. These results
demonstrate that B cell differentiation in vitro from pro-B to
immature B cells is suppressed in λ5-deficient pro-B cell lines.
Restoration of λ5 to the λ5-deficient pro-B cell line using
retrovirus transfection
To examine the role of λ5 in B cell differentiation, restoration
of λ5 expression to λ5-deficient pro-B cell lines, clone 1
and clone 3, was performed. Both sense and anti-sense
orientations of a mouse λ5 cDNA were inserted into an
expression vector, and the λ5 expression constructs were
transfected into the φNX-Eco cells. The resulting retrovirus
particles were infected into λ5-deficient pro-B clones, and
stable tansfectants, clone 1-λ5 and clone 3-λ5 for sense and
clone 1-Rλ5 and clone 3-Rλ5 for anti-sense, were established.
Clone 1-λ5 and clone 3-λ5 developed expression of Vpre-B
and λ5 on the surface as shown in Fig. 3, presumably due to
enforced expression of λ5 protein. In contrast, clones 1-Rλ5
and 3-Rλ5 showed identical phenotypes with original clones,
1 and 3. To investigate the association of Vpre-B and λ5
molecules, the surrogate L chain in a representative clone,
1-λ5, was examined. Surface biotinylation of clone 1-λ5 cells
followed by immunoprecipitation using Vpre-B or λ5 antibodies
revealed correct formation of and cell surface expression of
the surrogate L chain consisting of λ5 (22 kDa) and Vpre-B
(16 kDa) proteins as described previously (2,24) (Fig. 4). The
result showed that the introduced λ5 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.
Fig. 1. Phenotype of pro-B cell lines from a λ5 knockout mouse. The phenotype of pro-B cell lines, clone 1 and clone 3 from a λ5 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, λ5, IL-7R, µ H and κ L. Clone 1 and
clone 3 are negative for Vpre-B, λ5, µ H and κ L on the surface. The negative control is indicated by the dotted line.
1198 Requisition of λ5 for κ chain rearrangement
Discussion
Fig. 2. Induction of B cell differentiation in vitro using the λ5-deficient
pro-B cell lines. The pro-B cell lines, clone 1 and clone 3 from bone
marrow of a λ5 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 κ
L were detected by FITC-conjugated goat antibodies respectively,
which are indicated by the percent.
Differentiation of the λ5-reconstituted λ5-deficient pro B cell
lines
In order to determine if transfected clones, 1-λ5, 1-Rλ5, 3-λ5
and 3-Rλ5, 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. 5 indicated
that clone 1-λ5 and clone 3-λ5 differentiated to become ~8
and 7% surface IgM1 B cells, respectively, whereas clones
1-Rλ5 and 3-Rλ5 differentiated very little (,1.0% ). To confirm
the induction of differentiation from pro-B to immature B cell,
the rearrangements of H and κ chains of BPB, clone 1 and
clone 1-λ5 were examined by PCR analyses (Fig. 6). The H
chain configuration of all these lines was DJH1/DJH3 and the
κ chain was in germline as described in Table 1. 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-λ5, 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 κ chain genes were rearranged in BPB and clone 1-λ5,
and barely in clone 1. Major two bands detected at the κ
chain loci of BPB and clone 1-λ5 corresponded to VJκ1 and
VJκ2 rearrangements. This result indicated that the surrogate
L chain is necessary to induce κ chain rearrangement.
We have demonstrated that the surrogate L chain is required
for κ chain rearrangement by using retrovirus infection to
restore λ5 protein expression to λ5-deficient pro-B cells. In
our established system of in vitro B cell differentiation, the
pro-B cell lines differentiate to immature IgM1 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 IgM1 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 IgM1 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 λ5-deficient pro-B cell lines, H chain
rearrangement was induced from DJ to VDJ, but the efficiency
of κ chain rearrangement was extremely low, suggesting that
an incomplete pre-B cell receptor does not activate κ 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 λ5 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 virustransformed pre-B cell lines induced the activation of κ
chain rearrangement (13). However, no previous evidence
addressed whether the surrogate L chain itself is directly
involved in the regulation of κ chain rearrangement. Our
results described here are the first to demonstrate that a
complete surrogate L chain, consisting of Vpre-B and λ5, is
required for the activation of κ 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
Requisition of λ5 for κ chain rearrangement 1199
Fig. 3. Surface phenotype of λ5-restored λ5-deficient pro-B cell lines. The sense and anti-sense constructs of λ5 were introduced into the λ5deficient pre-B cell lines, clone 1 and clone 3, resulting in stable transfectants, clone 1-λ5 and clone 3-λ5 (sense) and clone 1-Rλ5 and clone
3-Rλ5 (anti-sense). Surface expression was examined by FACSCalibur using rat mAb against B220, Vpre-B, λ5 and IL-7R, and goat antibodies
against µ H and κ L. Vpre-B and λ5 expression became positive on clone 1-λ5 and clone 3-λ5. The negative control is indicated by the dotted line.
Fig. 4. Surface biotinylation and immunoprecipitation of Vpre-B and
λ5 using the restored cell line, clone 1-λ5. The pro-B cell line, clone
1-λ5, was surface biotinylated and immunoprecipitated by using a
control antibody, an anti-Vpre-B antibody (Vpre-B Ab) and an anti-λ5
antibody (λ5 Ab). Vpre-B (16 kDa) and λ5 (22 kDa) were detected
with both anti-Vpre-B and anti-λ5 antibodies.
induced immediately after the pre-B cell receptor is formed
and that the surrogate L chain is rapidly replaced by κ 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 κ chain was not strictly regulated in our system in vitro
and, thus, in λ5-deficient pro-B cell lines, a minor level of κ
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 λ5 (29).
The molecular mechanism of how the pre-B cell receptor
functions in κ chain rearrangement is the next question to be
answered. One of the potential factors implicated in κ chain
rearrangement as a target of the signals from the pre-B cell
receptor is Pax-5, although the relationship between the preB cell receptor and Pax-5 is unclear. The deletion of KI and
KII sites located upstream of Jκ in knockout mice severely
impaired κ 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 κ chain rearrangement. We are examining
further how the signal of the pre-B cell receptor regulates
κ chain rearrangement using pre-B cell receptor-positive
cell lines.
Acknowledgment
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
1200 Requisition of λ5 for κ chain rearrangement
Fig. 6. Analysis of H chain and κ chain gene rearrangement by
PCR. Genomic DNAs were isolated from pro-B cell lines, clone 1,
clone 1-λ5 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. 1). PCR was performed to detect H chain
rearrangement from DJ to VDJ (VHDHJH) and κ chain rearrangement
(VκJκ). 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 Jκ1–5 for κ chain rearrangement
respectively.
5
6
Fig. 5. In vitro differentiation of the λ5-restored pro-B cell lines. The
stable transfectants, clone 1-λ5, clone 3-λ5, clone 1-Rλ5 and clone
3-Rλ5, 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 κ L were detected by FITC-conjugated goat
antibodies respectively, which are indicated by the percent.
7
8
9
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.
Abbreviation
IL-7R
SL
IL-7 receptor
surrogate light chain
10
11
12
References
1 Tonegawa, S. 1983. Somatic generation of antibody diversity.
Nature 302:575.
2 Karasuyama, H., Kudo, A. and Melchers, F. 1990. The proteins
encoded by the Vpre-B and λ5 pre-B cell-specific genes can
associate with each other and with µ heavy chain. J. Exp.
Med. 172:969.
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.
4 Kudo, A. and Melchers, F. 1987. A second gene, Vpre-B in the λ5
13
14
15
locus of the mouse, which appears to be selectively expressed
in pre-B lymphocytes. EMBO J. 6:2267.
Sakaguchi, N. and Melchers, F. 1986. λ5, a new light-chainrelated locus selectively expressed in pre-B lymphocytes.
Nature 324:579.
Kudo, A., Sakaguchi, N. and Melchers, F. 1987. Organization of
the murine Ig-related λ5 gene transcribed selectively in pre-B
lymphocytes. EMBO J. 6:103.
Bauer, S. R., Kudo, A. and Melchers, F. 1988. Structure and preB lymphocyte restricted expression of the Vpre-B gene in humans
and conservation of its structure in other mammalian species.
EMBO J. 7:111.
Kitamura, D., Kudo, A., Schaal, S., Muller, W., Melchers, F. and
Rajewsky, K. 1992. A critical role of λ5 protein in B cell
development. Cell 69:823.
Rolink, A., Grawunder, U., Winkler, T. H., Karasuyama, H. and
Melchers, F.1994. IL-2 receptor α chain (CD25, TAC) expression
defines a crucial stage in pre-B cell development. Int. Immunol.
6:1257.
Minegishi, Y., Coustan-Smith, E., Wang Y-H., Cooper, M. D.,
Campana, D. and Conley, M. E. 1998. Mutations in the human
λ5/14.1 gene result in B cell deficiency and agammaglobulinemia.
J. Exp. Med. 187:71.
Bauer, S. R. and Scheuermann, R. H. 1993. Expression of the
Vpre-B/λ5/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.
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.
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 κ locus. Int. Immunol. 4:637.
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.
Reth, M., Petrac, E., Wiese, P., Lobel, L. and Alt, F.W. 1987.
Activation of Vκ gene rearrangement in pre-B cells follows the
Requisition of λ5 for κ chain rearrangement 1201
16
17
18
19
20
21
22
23
expression of membrane-bound immunoglobulin heavy chains.
EMBO J. 6:3299.
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.
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.
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.
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.
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.
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.
Rolink, A., Karasuyama, H., Grawunder, U., Haasner, D., Kudo,
A. and Melchers, F. 1993. B cell development in mice with a
defective λ5 gene. Eur. J. Immunol. 23:1284.
Pennycook, J. L. M. H., Marshall, A. J. and Wu, G. E. 1997. PCR
assays for endogenous Ig gene rearrangement. In Lefkovits, I.,
24
25
26
27
28
29
30
31
ed., Immunology Methods Manual, vol. 1, p. 237. Academic
Press, London.
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 λ5. Prog. Immunol. 7: 339.
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.
Pelanda, R., Schaal, S., Torres, R. M. and Rajewsky, K. 1996. A
prematurely expressed Igκ transgene, but not a VκJκ gene
segment targeted into the Igκ locus, can rescue B cell
development in λ5-deficient mice. Immunity 5:229.
Constantinescu, A. and Schlissel, M. S. 1997. Changes in locusspecific V(D)J recombinase activity induced by immunoglobulin
gene products during B cell development. J. Exp. Med. 185:609.
Karasuyama, H., Rolink, A., Shinkai, Y., Young, F., Alt, F. W. and
Melchers, F. 1994. The expression of Vpre-B/λ5 surrogate light
chain in early bone marrow precursor B cells of normal and B
cell-deficient mutant mice. Cell 77:133.
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.
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.
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 λ5
promoters as well as the KI and KII sites upstream of the Jκ
genes. Eur. J. Immunol. 27:750.