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5 Triple-Deficient Mice Show Impaired B Cell Development but Functional Allelic Exclusion of the IgH Locus1
* Basel Institute for Immunology, Basel, Switzerland; and
Developmental Immunology, Babraham Institute, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5, to form the pre-B cell
receptor (pre-BCR). Surface expression of the pre-BCR is believed to
signal both proliferation and allelic exclusion of the
IgH locus. Mice which lack either
VpreB1/VpreB2 or 5 show
a lack of precursor B cell expansion but normal IgH
allelic exclusion. This would suggest that one of either 5 or VpreB
can make a pre-BCR-like complex which is still able to signal allelic
exclusion but not proliferation. To investigate this, we established
mice lacking all components of the SL chain. These mice showed severely
impaired B cell development which was similar to that previously found
in mice lacking either 5 or
VpreB1/VpreB2. Surprisingly, the
IgH locus was still allelically excluded and thus the SL
chain appears not to be involved in allelic
exclusion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
L chain loci (5, 6). It has been proposed that
the product of a successful rearrangement at one IgH allele
signals the developing B cell to turn off rearrangements at the other
allele of the same locus, thereby ensuring that one B cell produces
only one type of IgH (7, 8, 9). Precursor (pre-) B
lymphocytes first rearrange DH to
JH segments on both IgH alleles before
they enter VH to DJH rearrangements (3, 10). If the µH chain resulting from a productive
VH to DJH rearrangement can
pair with the surrogate L
(SL)4 chain, a pre-B
cell receptor (pre-BCR) is formed, in combination with SL chain, on the
cell surface. The SL chain is formed by the noncovalent association of
the VpreB and 5 proteins, and is assumed to form a L chain-like
structure capable of disulfide-bound association with a µH chain
(11, 12, 13, 14, 15). Approximately half of all productively
rearranged IgH loci have been found to produce µH chains
capable of combining with the SL chain (16). Nonproductive
VDJ rearrangements and productive rearrangements which result in
nonpairing µH chains appear to change neither the phenotype nor the
functional stage of the cell and they allow rearrangement at the second
IgH allele. In contrast, pairing µH chains cause allelic
exclusion by preventing the rearrangement of the second allele. Surface deposition of the pre-BCR allows the cell to be stimulated to between two and five rounds of cell division (17). Such proliferating large pre-BII cells thereby expand the number of cells which produce SL chain-pairing µH chains. All other cells, either nonproductively VDJH rearranged on both IgH alleles or producing only nonpairing µH chains, do not expand, and may even be induced to apoptose due to their lack of surface expression of the pre-BCR (18). A quantification by single-cell PCR analysis of the two µH chains in VDJH-rearranged cells in the pre-BII and mature B cell compartments and the repertoire suppression of the SL chain-nonpairing VH domains of µH chains produced from the productively rearranged alleles (and not seen in the nonproductively rearranged alleles) support such a scenario (16, 19).
Experimentally induced, as well as naturally occurring, mutations in
the genes encoding the SL chain support the view that the pre-BCR
signals proliferative expansion of pre-BII cells. Targeted disruption
of the 5 gene in mice as well as a naturally occurring
deletional mutation in humans both abolish proliferative expansion of
pre-BII cells and reduce the rate of B cell generation compared with
that expected from a normal number of
DJH/DJH-rearranged pre-BI
cells (20, 21, 22). In bone marrow without selective expansion
of SL chain-pairing µH chain-producing precursors, subsequent sites
of L chain rearrangements and production finally lead to selectable
surface IgM+ immature B cells. These results also
clearly indicate that an incomplete VpreB/µH chain pre-BCR-like
complex (23) is not sufficient to signal proliferative
expansion. Furthermore, targeted disruption of one of the two
VpreB genes (in mice two genes encode the VpreB1 and VpreB2
proteins which are 98% structurally identical (12, 24)),
i.e., VpreB1, is not sufficient to induce a marked defect in the
generation of B cells, demonstrating that VpreB2 alone, together with
5, is sufficient to generate a functional SL chain and pre-BCR
(25). However, the targeted disruption of VpreB1 and
VpreB2 on the same chromosome gave rise to a B cell
generation-defective phenotype which is apparently indistinguishable
from that obtained following disruption of the 5 gene
(26). This result suggests that there are no other
proteins which can substitute for VpreB1 and VpreB2.
Notably, both the 5-deficient as well as the VpreB1/VpreB2
double-deficient mice showed allelic exclusion of the IgH
locus in the immature/mature B cell compartments (19, 26, 27), questioning the idea that the pre-BCR-like complex,
composed of µH chain and one of the SL chain components, signals
allelic exclusion in pre-B cells.
To delete all genes encoding the SL chain, we have generated a
triple-deficient mouse strain in which both VpreB genes as
well as the 5 gene have been disrupted by targeted
integration of deleting elements on the same chromosome. Interestingly
the triple-mutant mice showed the same B cell generation defect as
reported for the 5- and the VpreB1/VpreB2-deficient mice.
Surprisingly, however, immature and mature B cells in these mice still
showed IgH allelic exclusion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A targeting vector for the VpreB1-5
locus was made as follows. A 5.5-kb XbaI-HindIII
fragment which contains the second and third exons and the 3' region of
the 5 gene was isolated from phage clone 7pB12-2
(28). This fragment was cloned between the
XbaI-HindIII sites of pBluescript II (Stratagene,
CA). The thymidine kinase gene cassette was inserted downstream of the
5 gene. In a separate vector, the neomycin (neo)
resistance gene cassette was inserted between the
SalI-XhoI sites of pBluescript II. A 1.6-kb
SacII-BglII fragment, which contains the region
5' of the VpreB1 gene, was isolated from pUCE5.6X
(24) and inserted between the
SacII-BamHI sites. The resulting 5'VpreB1-neo
gene hybrid was cut out using SacII-XhoI and
inserted into the blunt-ended XbaI site of the vector which
contained the 3' end of the 5 gene and the thymidine
kinase gene. As a result, the entire region of the VpreB1
gene and the first exon of the 5 gene were deleted in the
targeting construct. The neo gene was cloned in the opposite
transcriptional orientation to VpreB1 and 5. The targeting vector
for VpreB2 has been described previously and uses hygromycin as a
selection marker (26). In addition, the neo cassette has a
loxP site on either side whereas the hygromycin gene has a loxP site on
only one side (see Fig. 1
).
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E14 embryonic stem (ES) cells (strain 129) were cultured
on irradiated embryonic fibroblasts (Incyte Genomics, St. Louis, MO) in
DMEM (Life Technologies, Grand Island, NY) with 50 µM 2-ME
(Sigma-Aldrich, St. Louis, MO), 1000 U/ml ESGRO (Life Technologies),
gentamicin, and 15% FCS. The VpreB1-5 targeting vector was
linearized with NotI. The ES cells were transfected and
cultured with 300 µg/ml G418 (Life Technologies) and 2 µM
gancyclovir (Roche, Basel, Switzerland). Single colonies were screened
for VpreB1-5 homologous recombination by Southern blotting. Two
homologously targeted ES cell clones (522 and 515) were transfected
with the linearized VpreB2 targeting vector and selected in 300 µg/ml
hygromycin (Roche Molecular Biochemicals, Mannheim, Germany).
Single colonies were screened for VpreB2 homologous recombination by
PCR (26). Positive clones were confirmed by Southern
blotting.
Four ES clones targeted at both the VpreB1-5
and VpreB2 loci were used to establish chimeric mice. Two of
these (515-3H11 and 522-H2A3) had the two mutations integrated on the
same chromosome, whereas the other two ES clones (522-N3C4 and 515-4F4)
had integrated the two mutations on separate chromosomes. The agouti
mice (129 x C57BL6 background) with targeted mutations from
515-3H11 and 522-H2A3 ES clones were intercrossed to establish
homozygous SL chain-deficient mice. The genotypes of the mice were
determined by PCR using previously described primers (26)
and/or Southern blotting with probes A and B shown in Fig. 1
.
Homozygous SL chain-deficient mice were also crossed with mice
expressing cre recombinase ubiquitously. These were then intercrossed
to obtain homozygous SL chain-deficient mice with a deletion of the neo
cassette on both alleles (SL
neo-deficient) but with the hygromycin
cassette intact. These mice were screened by several PCRs to confirm
that the genotype was correct using a combination of primers detecting
VpreB1, VpreB2, 5, neo, and hygromycin. In addition, they were also
screened for the presence of an 
700-bp PCR product indicative of the
neo deletion using one primer located just upstream of the recombined
loxP site (5'-TTGTCTCATTATGTAGCCAAGGCC-3') and another primer
downstream of the XbaI site in 5 intron 1
(5'-CAAGGTCTTCTTGACTGGGT-3'). Conditions were as follows: 30 cycles
of 94°C for 30 s, 55°C for 20 s, and 72°C for 20
s. Mice were established and analyzed under PPL 80/1143 and PPL
80/1501.
SL chain expression
Total RNA was prepared from two million bone marrow cells from
3-wk-old mice using RNAzol (Tel-Test, Friendswood, TX). cDNA was
synthesized using SuperScript II reverse transcriptase-polymerase and
random primers (Life Technologies). PCR primers and reaction conditions
for the VpreB genes were described previously
(24). Primer sequences for 5 exon 2 were:
5'-GTTTTGGTATGTCTTTGGTGGTGGG-3' and 5 exon 3:
5'-GGTCTGTTTGGAGGGTTGGGTTG-3'. Conditions were as follows: 30 cycles of
94°C for 20 s, 60°C for 20 s, and 72°C for 30
s.
One million bone marrow cells from 3-wk-old mice were cultured on irradiated stromal cells (S17) in RPMI 1640 medium (Life Technologies) supplemented with 50 µM 2-ME, antibiotics, 10% FCS, and 10% IL-7. One week later, the cultured cells were collected and expression of the SL chain on the cell surface and in the cytoplasm was analyzed by FACS.
FACS analysis
Cell suspensions were prepared by conventional methods and stained in HBSS (Life Technologies) supplemented with 10 mM HEPES and 3% FCS. After staining, the cells were suspended in the same medium containing propidium iodide (0.25 µg/ml) to exclude dead cells. To analyze the allotype of IgM, bone marrow cells were stained with PE-labeled anti-IgMa, biotin-labeled anti-IgD, and allophycocyanin-labeled anti-B220 Abs in the presence of 5% rat serum. After washing, the cells were stained with FITC-labeled anti-IgMb Abs and tricolor-labeled streptavidin. Cytoplasmic staining (for detection of SL chain) was performed according to the manufacturer’s instructions (Caltag Laboratories, Burlingame, CA). The Abs used were as described previously (26). All data were acquired on a FACSCalibur and analyzed using the CellQuest program (BD Biosciences, Mountain View, CA).
Immunization and ELISA
Control and homozygous mutant littermates were immunized by i.p.
injection of either 100 µg of NP-chicken 
-globulin in alum or 5
µg of 4-hydroxy-3-nitrophenylacetyl (NP)-Ficoll. Two weeks after
immunization, the mice were bled and sera were prepared and analyzed by
ELISA as previously described (26). The sera were analyzed
as 5-fold serial dilutions. Immunizations were performed under PPL
80/1263.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The three genes encoding the SL chain (VpreB1,
VpreB2, and 5) are all located on mouse
chromosome 16 (29, 30); therefore, targeting of these
three genes in ES cells was performed in two steps. In the first step,
the VpreB1 gene and the first exon of the 5
gene were replaced with the gene encoding neomycin resistance (Fig. 1). Two correctly targeted ES cell clones
(clones 522 and 515, data not shown) were used for the second step in
which the VpreB2 gene was substituted by the hygromycin
resistance gene (Fig. 1). The double-targeted ES cell clones were
injected into blastocysts and four clones gave rise to germline
transmission. In two of these (522-H2A3, 515-3H11), the two targeting
events were found to segregate together, demonstrating that they were
on the same chromosome. The heterozygous mutant mice were bred for
homozygosity which was confirmed by Southern blotting as shown in Fig. 1. Both strains showed the same phenotype and the homozygous
VpreB1-/-VpreB2-/-5-/-
mice are hereafter called SL chain-deficient mice.
Lack of VpreB and 5 expression in SL chain-deficient mice
Total RNA from bone marrow cells of SL chain-deficient as well as
control mice was prepared and reverse transcribed. The existence of the
SL chain gene mRNA was first analyzed using primers specific for VpreB1
and VpreB2 (Fig. 2A). In
wild-type and heterozygous mutant mice, expression of both genes was
detected, while in the homozygous mutant mice, no message was found.
Since mRNA encoding a ubiquitously expressed gene (hypoxanthine guanine
phosphoribosyl transferase) was detected, we concluded that the
VpreB genes are not expressed in the SL chain-deficient
mice.
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5 message using primers specific for
exon 2 and exon 3, since these are still present after the targeting
event. As shown in Fig. 2B, there is a properly spliced
message, as judged by the size of the band, present in the homozygous
mutant mice, with levels around 10-fold decreased as compared with that
of the control. Northern blot analysis on total RNA prepared from in
vitro-cultured pre-BI cells (the same cultures as were used for protein
analysis, see below) was then performed. A 5 cDNA probe gave rise to
a signal in RNA from control but not homozygous mutant cells, whereas
an actin probe gave a signal of the same strength in both RNA
preparations (data not shown). Thus, the truncated 5 mRNA levels
were too low to be detected by Northern blotting.
The 5 exon 2/3 mRNA in the homozygous mutant mice is probably the
result of cryptic transcription initiation sites just upstream of exon
2 in intron 1: we did not detect any transcripts starting further
upstream in the intron (data not shown). The level of exon 2/3
expression might be due to sequences in the neo cassette. Especially
since this was cloned in the opposite direction, positioning the
enhancer in the cassette just upstream of the potential cryptic
initiation sites in the 5 intron. To test whether the enhancer in
the neo cassette had any effect on the 5 exon 2/3 transcripts, we
deleted the neo cassette by crossing SL chain-deficient mice with
transgenic mice expressing cre recombinase ubiquitously. The offspring
were intercrossed resulting in SL chain-deficient mice with the neo
cassette deleted on both alleles. These mice are hereafter termed
SLneo-deficient mice. We analyzed bone marrow RNA from these mice by
RT-PCR, again using primers for 5 exon 2/3. As shown in Fig. 2B (Neo), by deleting the enhancer in the neo cassette
the transcripts are no longer detectable in the homozygous mutant mice.
Thus, the enhancer in the neo cassette was the cause of the truncated
5 transcripts.
We also searched for possible translation starts sites and open reading
frames to determine whether a truncated 5 protein could be
translated from the exon 2/3 transcripts. Even if the transcript starts
in intron 1, it would have to be closer than 159 bp from the splice
site as determined by RT-PCR assays (data not shown). There are no
translation start sites with an open reading frame that could encode a
protein in this region. The few ATG present all result in a stop codon
after a few amino acids. Thus, even though the SL chain-deficient mice
transcribe exon 2/3, this could not give rise to a protein.
We then tested for the presence/absence of SL chain protein in the SL
chain-deficient mice. Bone marrow cells from wild-type, heterozygous,
and homozygous SL chain-deficient mice were cultured in vitro for 1 wk
on irradiated stromal cells in the presence of IL-7. Under these
conditions, it is possible to expand and maintain pre-BI cells which
express the SL chain on the cell surface along with BILL-cadherin and
other proteins (31, 32, 33). Surface expression of the SL
chain was analyzed using mAbs specific for VpreB and 5 (Fig. 2C). Pre-BI cells from wild-type and heterozygous mutant
mice expressed both 5 and VpreB on the surface, while cells from
homozygous mutant mice failed to express these proteins. Cells from all
cultures expressed B220, CD19, and c-kit (data not shown),
confirming that the cells were indeed pre-BI cells. These results
showed that in SL chain-deficient mice, none of the SL chain components
are present on the cell surface.
We also analyzed the same cultures for expression of SL chain proteins
in the cytoplasm. As shown in Fig. 2D, whereas both VpreB
and 5 were readily detectable in the cytoplasm of cultured wild-type
cells, none of these proteins was found in the cytoplasm of cells from
homozygous mutant mice. In addition, we analyzed ex vivo cells from
homozygous SLNeo-deficient and control mice by staining the cell
surface with B220 and c-kit followed by staining of the
cytoplasm with either the VP245 or the LM34 Ab in combination with Abs
recognizing the µH chain. By gating on the
B220+c-kit+
(pro-/pre-BI) cells, it was clear that the control cells stained
positive for VpreB and 5, whereas the homozygous SLNeo-deficient
cells were negative (data not shown). Thus, pre-B cells from SL
chain-deficient as well as SLNeo-deficient mice do not express any
detectable SL chain proteins.
Reduced numbers of pre-BII cells in the bone marrow of SL chain-deficient mice
To investigate the effect of SL chain deficiency on B cell
development, we analyzed the different bone marrow B cell compartments
by FACS (Fig. 3A). As there
was no obvious difference between wild-type and heterozygous mutant
mice (data not shown), data from these genotypes were pooled and are
shown as controls in the figures and tables. Table I summarizes the number of B lineage
cells. Compared with control mice, the total number of nucleated cells
in bone marrow was decreased in SL chain-deficient mice (80%) as
were B220+ cells (50%). The
B220+ B lineage cells were thereafter analyzed in
detail. The pro-/pre-BI cell population
(B220+c-kit+) was
increased 2-fold compared with control mice, whereas the pre-BII
cell population
(B220+CD25+) was severely
decreased in SL chain-deficient mice. The SL chain-mutant mice showed a
25-fold decrease in young (3 wk) and a 12-fold reduction in 8- to
9-wk-old mice as compared with control mice. In wild-type mice, pre-BII
cells have productively rearranged the IgH loci and they
express cytoplasmic µH chain, some of which can pair with the SL
chain to form a pre-BCR and signal cell proliferation. This population
can be divided into large (cycling) and small (resting) cells. By
collecting a large number of bone marrow cells from 8- to 9-wk-old
mice, we determined that the proportion of large cells among the
B220+CD25+ cells was 26%
in control and 41% in SL chain-deficient mice (data not shown). From
this, we calculated the number of large and small pre-BII cells.
Compared with control mice, SL chain-deficient mice showed an 7-fold
decrease in the large pre-BII cell population and an 15-fold
decrease in the small pre-BII population. These data suggested that in
SL chain-deficient mice the generation of pre-BII cells and/or the
proliferative expansion of large pre-BII cells is impaired.
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The number of both immature
(IgM+IgD-) and mature
(IgM+IgD+) B cells in bone
marrow was markedly reduced in SL chain-deficient mice. Depending on
age, the difference in IgM+ cells was 20- and
9-fold in young (3 wk) and old (8–9 wk) mice, respectively. However,
the ratio of small pre-BII to immature B cells, as analyzed in 8- to
9-wk-old animals, was 2:1 in control and 1:1 in SL chain-deficient mice
(Table I). This suggested that the absence of the SL chain, while
impairing the transition from pre-BI to pre-BII cells, does not
influence the transition from pre-BII to immature B cells.
Immature B cells generated in the bone marrow migrate to the spleen
where they mature. Therefore, spleen cells of SL chain-deficient mice
were analyzed to study the effect of the mutations on the peripheral B
cell populations (Fig. 3B and Table I). The total number of
nucleated cells was 4- and 2-fold decreased in young and old SL
chain-deficient mice, respectively. The B220+
cell population was also affected. In young mice, an almost 15- to
20-fold reduction was observed whereas in older mice the difference was
less pronounced (5-fold). The majority of IgM+
cells were also IgD+ (mature B cells) independent
of genotype. The number of mature B cells was also decreased 20- to
25-fold in 3-wk-old and 5-fold in 8- to 9-wk-old SL chain-deficient
mice. This suggested that the SL chain does not affect the
differentiation from immature to mature B cells. The ratio of Ig vs
Ig did not vary between the genotypes.
SLNeo-deficient mice
The data presented in Fig. 2B demonstrate the presence
of a truncated 5 transcript (exon 2/3) in the bone marrow of
homozygous SL chain-deficient mice, but this was not detectable in bone
marrow from SLNeo-deficient mice. The sequence data indicated that
it would not be possible to make a truncated protein from this RNA. In
addition, the FACS analyses in Fig. 2 showed that no 5 protein was
detected in the homozygous SL chain-deficient pre-BI cells. Even so, it
may be argued that a truncated 5 protein is produced that is not
recognized by the LM34 Ab. We therefore analyzed the SLNeo-deficient
mice, where we performed the same analyses as above both on bone marrow
and spleen cells from homozygous mutant and wild-type mice. We did not
detect any obvious difference in any of these analyses when compared
with those described above for the SL chain-deficient mice (data not
shown). We therefore conclude that the SL chain-deficient mice are
"true" knockouts and show the phenotype of mice lacking the entire
SL chain, i.e., VpreB1, VpreB2, and 5. Therefore, with the exception
of IgH allelic exclusion, the following experiments were
performed only on the SL chain-deficient mice.
B-1a B cells in the peritoneum of SL chain-deficient mice
In the peritoneum, the B-1a B cells can be distinguished from
conventional B cells by the expression of CD5. To investigate the
potential effect on the B-1a B cells, peritoneal cells were isolated
from SL chain-deficient and control mice and analyzed by FACS. The
absolute number of cells is summarized in Table II. In very young mice (10 days), the
number of B-1a B cells (B220+
CD5+) was reduced 12-fold, while at 8 to 9 wk
of age, SL chain-deficient mice had comparable numbers of B-1a B cells
to control mice. The number of conventional B cells was still reduced
in 8- to 9-wk-old SL chain-deficient mice. In contrast, non B lineage
B220-CD5+ cells were not
affected either in young or in older SL chain-deficient mice. Thus, the
number of B-1a B cells is also affected by the lack of SL chain.
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To investigate whether the decreased number of B lymphocytes in the periphery had any effect on the level of IgM in the serum, mice of different genotypes were bled and the sera analyzed by ELISA to determine the amount of IgM. Wild-type and heterozygous mutant mice both demonstrated levels of 1.4 mg/ml and homozygous mutant mice reached levels of 1.6 mg/ml (three animals per group). Thus, there was no significant difference in the total IgM concentration when comparing control and homozygous mutant mice.
The responses against T cell-dependent (TD) and -independent (TI) Ags
were also analyzed. Eight-week-old mice were immunized with either
NP-chicken -globulin (TD Ag) or NP-Ficoll (TI Ag). Two weeks later,
serum samples were prepared and Ag-specific Abs were measured by ELISA.
Immune responses to TD Ags, both against the hapten and the carrier,
were similar in SL chain-deficient and control mice (Fig. 4 and data not shown). The Ab response
against the TI Ag was reduced 5- to 10-fold in mice lacking the SL
chain as compared with control mice (Fig. 4). Thus, despite reduced
numbers of B lymphocytes in the periphery, the SL chain-deficient mice
were able to mount an immune response against both T cell-dependent and
-independent Ags, although not at the same level as the control,
implying that the peripheral B cells are functionally normal.
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To investigate whether allelic exclusion of the IgH
locus was functioning in the absence of the SL chain, the frequency of
single immature and mature B cells expressing two IgH
allotypes on the cell surface was analyzed. SL chain-deficient and
control mice heterozygous for the IgMa and
IgMb alleles were selected. Bone marrow
cells from these mice were stained with Abs recognizing B220, IgD,
IgMa, and IgMb and analyzed
by FACS (Fig. 5). Both
IgD- and IgD+ cells were
analyzed and no difference was observed (data not shown). In both
control and SL chain-deficient mice, 55–60% of the
IgM+ cells expressed IgMa
and 40–45% expressed IgMb. In control mice,
0.7% of the IgM+ cells expressed both
IgMa and IgMb and in SL
chain-deficient mice this percentage was 0.5%. In a separate
experiment, bone marrow cells from these mice were stained for
cytoplasmic IgMa and IgMb.
The results showed IgH allelic exclusion independent of the
genotype (data not shown). This demonstrated that in the absence of SL
chain allelic exclusion of IgH remains intact in bone marrow
immature and mature B cells. We also analyzed the SLneo-deficient
mice in the same way and similar results were obtained. Thus, the lack
of the entire SL chain does not affect the mechanism of IgH
allelic exclusion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-/- and
VpreB1-/-VpreB2-/- mice
(17, 21, 26), the SL chain-deficient,
VpreB1-/-VpreB2-/-5-/-
mice also lack pre-BII cell proliferative expansion and show a B cell
immunodeficiency (Fig. 3 and Table I). Since the SL chain- and the
SLneo-deficient mice show the same phenotype, they are throughout
the discussion termed SL chain-deficient mice. In terms of kinetics of
development, defective cellularity of the immature and mature B cell
compartments (Table I) and response to T cell-dependent Ags (Fig. 4),
all seem indistinguishable in the single
5-/-, the double
VpreB1-/-VpreB2-/-
(20, 26), and the SL chain-deficient mice. The pre-BI
population, as in the other SL chain-mutant mice, is 2- to 3-fold
larger than in wild-type littermates, probably because the cells cannot
exit this compartment by further B cell differentiation (Table I). The
ratio of the sizes of the small pre-BII compared with the immature B
cell compartment, as well as that of the small pre-BII compared with
the mature B cell compartment is similar in wild-type,
5-/-,
VpreB1-/-VpreB2-/-,
and SL chain-deficient mice (Table I) (21, 26). All these
observations suggest that the major measurable defect introduced by
mutations of the SL chain subunits is the lack of proliferative
expansion at the transition from pre-BI to pre-BII cells.
Although we have not yet measured the kinetics of in vitro
differentiation of surface IgM+ B cells from SL
chain-deficient pre-BI cells, we would predict from our observations of
the B cell developmental defects that these kinetics, as in
5-/- pre-BI cells (21), would
also be indistinguishable between SL chain-deficient and wild-type
pre-BI cells. Hence, expression of the SL chain before expression of
the µH chain in pre-BI cells (35, 36), possibly with
BILL-cadherin (32, 33) and other associated proteins,
appears not to influence the capacity of these cells to enter
VH to DJH rearrangements
and later VL to JL
rearrangement and differentiate to pre-BII, immature, and mature B
cells. From an unaltered differentiation capacity of the
5-/- pre-BI cells in vitro
(21), we conclude that they have the same unaltered
capacity to differentiate in vivo. Hence, the developmental defect of
SL chain-mutant pre-BII cells is most likely their inability to
proliferate, leading to a reduction in the number of µH
chain-producing pre-BII cells in which subsequent L chain gene
rearrangement can take place. Furthermore, those cells producing µH
chains which best fit the SL chain are expanded most, and these µH
chains should also fit best to the conventional L chain which is
generated later (37, 38). Therefore, in SL chain-mutant
mice, many more pre-BI cells would have to enter
VH to DJH rearrangements to
produce an equal number of cells (as compared with wild-type mice)
producing well-fitting µH chains in a given period of time. According
to this view, B cell differentiation from the SL chain-mutant cells is
not leaky, but simply inefficient.
The original repertoire of VH domains expressed
in µH chains as a consequence of VH to
DJH rearrangements at the transition of pre-BI to
pre-BII cells in the bone marrow is influenced in a major way by the SL
chain (16). The VH domains that
cannot pair with SL chains, notably VH81X within
the VH7183 family, the
VHQ52 domains, and nearly half of all
VHJ558 domains are suppressed from the µH chain
repertoire of pre-BII and all subsequent B lineage cells; the cells
that express them cannot form a pre-BCR and therefore are not expanded
in the same way as those µH chain producers that contain pairing
VH domains. In 5-/-
mice, the pre-BII compartment still contains all of these nonpairing
µH chain producers. We predict that future single-cell PCR/pairing
analyses of the µH chain repertoires expressed in single pre-BII
cells of the SL chain-deficient mice will also show the same
nonsuppressed VH repertoire as that observed in
5-/- pre-BII cells. As L chain becomes
expressed at the transition from pre-BII cells to mature B
cells, the VH repertoire of µH chain-producing
cells becomes suppressed in 5-/- cells, as
it is in wild-type cells when the SL chain is testing the µH chain at
the transition from pre-BI to pre-BII cells. A future single-cell
PCR/pairing analysis is likely to show the same
VH repertoire change for SL chain-deficient cells
at the transition from pre-BII to mature B cells.
A second functional role has been proposed for the pre-BCR, namely, to signal a pre-BII cell, which has made one productive VDJH rearrangement and produced a µH chain that can pair with the SL chain and form a pre-BCR on the surface, to turn off the rearrangement machinery (recombination-activating gene 1, recombination-activating gene 2, and TdT), and to close the second DJH-rearranged allele (39). Thereby, the resulting VDJ+/DJH pre-BII cell is allelically excluded and prevented from possible VDJ rearrangement at the second allele. Since VH to DJH rearrangements occur randomly in- and out-of-frame, two-thirds of all original rearrangements are out-of-frame and unable to produce a µH chain. Consequently, no pre-BCR is made and the cell rearranges the second allele. Of these, one-third will be productive, thus generating a VDJ-/VDJ+-rearranged cell. This cell is allelically excluded without the need of suppression of any further VDJ rearrangement, but might have to be suppressed for secondary VH replacements (40). Therefore, it cannot be excluded that the machinery involved in VH replacements should be turned off in these cells, and the VDJ-rearranged loci should become inaccessible for VH replacements.
A defect in signaling for allelic exclusion should be readily visible
as an increase in the number of "double producers" which are
immature and mature B cells which deposit IgM with µH chain produced
from both alleles on a single cell. If the defect is complete, the
VDJ+/DJ-rearranged cells should continue to
rearrange the second allele. Hence, they should disappear from the
repertoire of pre-BII and all subsequent cells. It was already
surprising that 5-/- pre-B and B cells had a
ratio of VDJ/DJ to
VDJ+/VDJ- cells which was
indistinguishable from that in wild-type B cells (19).
Furthermore, newly generated B cells of F1
IgMa/IgMb mice of wild-type
as well as 5-/- mice had <0.5% double
producers (27). It should be noted that both wild-type and
5-/- pre-BII and mature B cells contained
comparable numbers (between 4 and 8% of all VDJ/VDJ cells) of
VDJ+/VDJ+ double µH
chain-producing cells. However, in these cells it is always the case
that only one of the two µH chains produced could be deposited on the
surface as a pre-BCR (19). This might be expected from the
previous finding that although approximately half of all originally
produced µH chains can pair with the SL chain, the other half cannot
(16). This suggests that nonpairing µH chains which
cannot form a pre-BCR are also unable to turn off the rearrangement
machinery and close the second allele.
Although it was still conceivable that a partially defective pre-BCR, composed of VpreB and µH chain, might signal allelic exclusion (23), the results obtained from VpreB1-/-VpreB2-/- double-mutant mice now rule out this possibility. These double-mutant B cells still allelically exclude the IgH locus as well as wild-type B cells (26).
Although it has been observed previously that 5 protein cannot bind
µH chain in the absence of VpreB protein, as it appears incapable of
forming a disulfide-bonded complex (23), the possibility
existed that a noncovalent, weak interaction would suffice to form a
pre-BCR-like complex with the capacity to signal allelic exclusion. The
results reported here for SL chain-deficient mice now rule this out:
immature and mature B cells are still allelically excluded in
F1
IgMa/IgMb SL
chain-deficient mice. Although analysis of the VH
repertoire in the developing B cells and the contribution of pairing
and nonpairing µH chains in these repertoires has yet to be
conducted, we conclude from the results presented in this paper that
the SL chain, and hence the pre-BCR, is not required for allelic
exclusion of the IgH locus.
Allelic inclusion is observed in B cells, in which one IgH
allele has been mutated in the transmembrane portion of the µH chain
to prevent its membrane deposition (9). Hence,
membrane-bound µH chain signals allelic exclusion, but not together
with the SL chain. Three other possible partners have been suggested:
the heat shock protein 70 chaperon H chain binding protein
(41), the 8HS20-encoded VpreB3 (42), and
prematurely expressed L chains (43, 44, 45). VpreB3 has 36%
amino acid homology with VpreB1 and is associated with µH chain in
pre-B cell lines. The function of VpreB3 is still unknown. However, as
the VpreB3 gene is located on mouse chromosome 10
(46), it would be both possible and interesting to cross
VpreB3-deficient mice with the SL chain-deficient mice. We have not
observed N region insertions in V-J joints of L chains from mature B
cells of 5-/- mice (47). These
should have been detected if the L chain genes had been rearranged
before the IgH genes, since IgH from wild-type as
well as 5-/- mice have N regions inserted at
VDJ joints (47). In addition, we have not observed
-chain expression in pre-BI cells of 5-/-
mice expressing µH chain in the cytoplasm, nor have we detected
VJ-rearranged L chain loci in such cells by single-cell PCR.
Therefore, we do not favor the BCR as signal transducers for allelic
exclusion in SL chain-mutant mice.
In the search for genes encoding the signal transduction pathway for allelic exclusion at the IgH locus of pre-B cells, the SL chain-deficient ES cells and SL chain-deficient pre-BI cells should be valuable tools for additional mutations which might affect allelic exclusion.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takeyuki Shimizu at the current address: Division of Biosignaling, Research Institute for Biological Science, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan. E-mail address: shimizu{at}rs.noda.tus.ac.jp
3 Current address: Department of Cell Biology, Biozentrum of the University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
4 Abbreviations used in this paper: SL, surrogate L; neo, neomycin; NP, 4-hydroxy-3-nitrophenylacetyl; BCR, B cell receptor; TD, T cell dependent; TI, T cell independent; ES, embryonic stem.
Received for publication November 13, 2001. Accepted for publication April 3, 2002.
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References |
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