By
From the * Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111; and the Department of
Microbiology, Arizona State University, Tempe, Arizona 85287
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Abstract |
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Here we show that suppression of VH-DJH rearrangement in mice bearing a µ heavy (H) chain transgene (µ-tg mice) is associated with an extended period of DH-JH rearrangement, the first step of Immunoglobulin H chain gene rearrangement. Whereas DH-JH rearrangement is normally initiated and completed at the pro-B cell stage, in µ-tg mice it continues beyond this stage and occurs most frequently at the small (late) pre-B stage. Despite ongoing DH-JH rearrangement in late pre-B cells of µ-tg mice, VH-DJH rearrangement is not detectable in these cells. We infer that the lack of VH-DJH rearrangement primarily reflects tg-induced acceleration of B cell differentiation past the stage at which rearrangement of VH elements is permissible. In support of this inference, we find that the normal representation of early B lineage subsets is markedly altered in µ-tg mice. We suggest that the effect of a productive VH-DJH rearrangement at an endogenous H chain allele may be similar to that of a µ-tg; i.e., cells that make a productive VH-DJH rearrangement on the first attempt rapidly progress to a developmental stage that precludes VH-DJH rearrangement at the other allele (allelic exclusion).
Key words: V(D)J rearrangement; immunoglobulin transgenic mice; B cell differentiation; allelic exclusion ![]() |
Introduction |
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In antibody-producing cells, only one of two immunoglobulin (Ig)1 heavy (H) chain alleles is normally expressed; the other allele is excluded (1, 2). Several models have been proposed to explain allelic exclusion in Ig-producing cells (3). The two models of particular interest here are the stochastic and regulatory models. According to the stochastic model (3), allelic exclusion reflects the imprecision of V(D)J rearrangement, the process responsible for rearranging V, D, and J elements to form contiguous VDJ or VJ coding segments for Ig variable regions (reviewed in 7). As this process is error prone, the chance is low that a cell will make in-frame (productive) rearrangements at both alleles of a given locus. Thus, in this model, each allele rearranges independently and has an equal but low chance of being rearranged productively. The regulatory model (4, 5) adds a regulatory rider to the stochastic model and states that rearrangement at one allele may affect rearrangement at the other allele. An example of apparent nonindependent rearrangement of allelic elements is seen at the H chain locus. B cell plasmacytomas with an incomplete (DJH) rearrangement at one allele generally show a productive, in-frame VDJH (VDJH+) rearrangement at the other allele (8). This implies that the product of a VDJH+ allele is able to prevent further VH-DJH rearrangement (8). In support of this idea, VH-DJH rearrangement is suppressed in µ H chain transgenic mice (9-11 and reviewed in 12).
It is still uncertain how the µ chain product of a VDJH+
allele serves to prevent further VH-DJH rearrangement.
What is clear is that expression of a µ chain, in the form of a
pre-B cell receptor (pre-BCR), results in progression of
pro-B cells to the pre-B stage and the cessation of VH-DJH
rearrangement (reviewed in 13, 14). It is not clear, however, to what extent cessation of VH-DJH rearrangement
may reflect (a) rapid differentiation of pro-B cells to a stage
(pre-B) at which such rearrangement can no longer occur, or (b) an ability of the pre-BCR to signal direct inhibition
of VH-DJH rearrangement in addition to the progression of
pro-B to pre-B cells. Similarly, it is not clear to what extent
cessation of H and L chain rearrangement after possible
premature expression of a BCR may reflect rapid differentiation to the recombinase-inactive B cell stage or direct
feedback inhibition. To gain further insight into these issues, we looked at initiation of VDJH rearrangement in scid
and scid/+ mice bearing a µ transgene (tg) or both a µ and
tg (µ/
-tg mice). Scid mice are homozygous for a mutation (scid) that severely impairs rearrangement of V, D, and
J elements (15). Thus, B cell differentiation in scid mice is arrested at the pro-B cell stage (16), the stage at which H chain
gene rearrangement is initiated (17, 18). In µ-tg scid mice,
however, B cell differentiation proceeds to the late pre-B cell
stage before being arrested (19), and in µ/
-tg scid mice,
differentiation can proceed to the B cell stage (20).
Here we report that tg-induced suppression of VH-DJH
rearrangement may primarily reflect accelerated B cell differentiation rather than direct feedback inhibition. In µ-tg
scid mice, initiation of DH-JH rearrangement was observed
to occur predominantly at the late pre-B stage rather than
at the pro-B stage, and in µ/-tg scid mice, initiation of
DH-JH rearrangement was significantly reduced compared
with non-tg scid mice. Similar results were obtained with
µ-tg and µ/
-tg scid/+ mice (heterozygous for the scid
mutation). We interpret these findings to reflect accelerated
development of tg-expressing B lineage cells such that
there is insufficient time to initiate or complete DH-JH rearrangement at both alleles in developing pro-B cells. The
idea that a µ tg might accelerate B cell development has
been proposed previously (21). In support of this notion,
and consistent with our earlier studies (22), we show that
developing pro-B cells in µ-tg scid mice appear to bypass
the late pro-B stage and progress directly into large (early) pre-B cells, which are known to be deficient in recombinase activation gene (RAG) expression (23). Similarly, in
µ/
-tg scid mice, we present evidence that developing B
lineage cells may transit the pro- and pre-B stages very rapidly during their progression to the RAG-inactive B cell
stage. We suggest that the effect of endogenously coded Ig
chains may be analogous; i.e., when a cell expresses a µ chain, it rapidly progresses to a developmental stage that
precludes further rearrangement of VH elements.
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Materials and Methods |
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Mice.
Ig transgenic lines of C.B-17 scid mice hemizygous for the H chain tgs, M54 (24), 3H9 (25), or the kappa L chain tg, VCells.
Bone marrow cells were flushed from femurs with staining medium using a syringe and 22 gauge needle. The cells were then dispersed by gentle pipetting, treated with 0.165 M NH4CL, washed, and resuspended in staining medium and passed through a sterile nylon screen. B cell hybridomas were obtained by fusing unstimulated splenic cells from adult M54/VFlow Cytometric Analysis.
Bone marrow cell suspensions were analyzed for the presence of B lineage cells representing different stages of development (17). In brief, cell suspensions were stained with Cy5 (Biological Detection Systems, Inc.) or allophycocyanin (APC; PharMingen) conjugated anti-CD45(B220), FITC-conjugated anti-CD43, or biotinylated anti-IgM. Binding of biotinylated antibodies was revealed by Texas red conjugated Streptavidin (Southern Biotechnology). B220+CD43+IgMAnalysis of Genomic DNA.
Genomic DNA was prepared from sorted cell subsets (0.5-1.0 × 106 cells) as described previously (30) and dissolved in water at a concentration corresponding to 105 cell genome equivalents/µl. Ligation-mediated PCR (LM-PCR) (31) was used to assay DNA samples for double strand breaks (DSBs) resulting from the initiation of H chain gene rearrangement. Initiation of V(D)J rearrangement results in site-specific DSBs at the recombination signal/coding borders of V, D, and J elements: two kinds of broken DNA molecules are generated; covalently closed (hairpin) coding ends and blunt signal ends (34, 35). We assayed for broken molecules with signal ends; specifically, those with JH signal ends and those with 5' or 3' DHfl16.1 (DHfl) signal ends. We also assayed for signal joints (by inverse PCR), completed DH-to-JH rearrangements and unrearranged JH loci as scored by the retention of germline sequence immediately upstream of JH1. Assays were performed as follows. A double strand linker was ligated to DNA (equivalent to ~4 × 105 cell genomes). The linker was constructed according to Roth et al. (32) by annealing two oligonucleotides, DR19 (5'-CACGATTCCC-3') and DR20 (5'-GCTATGTACTACCCGGGAATTCGTG-3'). After ligation, different dilutions of the ligation reaction (input DNA) were used to perform PCR amplifications of one or more of the following: (a) linkered JH signal ends using DR20 and an oligonucleotide (MB221) complementary to a sequence immediately 5' of JH1 (5'-TCTCTTGTCACAGGTCTCACTATGC-3'); (b) linkered 5' DHfl signal ends using DR20 and an oligonucleotide (MB222) complementary to a sequence 5' of DHfl (5'-GCCTTCCACAAGAGGAGAAG-3'); (c) linkered 3' DHfl signal ends using DR20 and an oligonucleotide (MB241) complementary to a sequence 3' of DHfl (5'-TGGGTCAGTGGTCAAGACTCG-3'); (d) signal joints resulting from the joining of JH1, JH2, or JH3 signals to the 3' DHfl signal using MB221 and MB241; (e) DJH coding joints using a DHfl/DHsp primer MB109 (5'-CCGAATTCGTCCTCCAGAAACAGACC-3') and a primer (MB 92) complementary to a JH4 sequence (5'-GCCGGATCCCTTGACCCCAGTAGTCC-3'); (f) retained sequence immediately upstream of JH1 using MB221 and MB92; and (g)Probes.
Blots were hybridized with: (a) pJH6.3 (36) to reveal H chain gene rearrangements, DJH coding and signal joints, and unrearranged JH alleles; (b) a genomic fragment corresponding to DHfl and its surrounding region (amplified by PCR using MB222 and MB 241) to reveal LM-PCR-amplified 3' and 5' DHfl signal ends; and (c) pActin (17) to reveal PCR-amplified ![]() |
Results |
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Fig. 1 is a
schematic representation of the effects of µ and µ/ tgs on
scid B cell development. The different stages of B cell development are designated with the letter code of Hardy et al.
(17); the alternative nomenclature of Rolink and Melchers (37) is shown for comparison. As indicated, B cell development in scid bone marrow is blocked at stage C, shortly
after B lineage cells initiate H chain gene rearrangement.
Relief from this block can be achieved by introduction of a
µ tg into the scid genome. In µ-tg scid mice (22), developing pro-B cells appear to bypass stage C and develop directly into early pre-B cells, denoted as C'. Most cells in
subset C' are in cycle (17) and show downregulated RAG
expression (23). Cells of subset C' give rise to the D subset. At this late stage of pre-B cell differentiation, RAG expression is again upregulated (23) and L chain gene rearrangement is initiated (17, 18, 38). Differentiation does not proceed beyond stage D in µ-tg scid mice, presumably because
scid pre-B cells are unable to repair DNA DSBs resulting
from the initiation of
gene rearrangement (22, 30). Complete relief from the scid block can be achieved in double tg
scid mice, bearing both a µ and
tg (20). In µ/
-tg scid
bone marrow, B cell development proceeds to stage E and
appears to do so very rapidly, as evidenced by the near-normal percentage of B cells and virtual absence of pro- and
pre-B cells.
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Data supporting the above model are illustrated in Figs.
2 and 3. Fig. 2 shows the effect of two different µ tgs, M54
(24) and 3H9 (25), on scid B cell development before
the late pre-B cell stage (stage D). Members of subsets C
(BP1+HSAdull) and C' (BP1+HSAbright), both positive for the
early B lineage marker BP1 (29), are distinguished by their
level of staining for heat stable antigen (17). Note that subset C, which is present in scid mice, appears to be replaced by
subset C' in M54 and 3H9 scid mice. Note also that the
BP1HSA+ cell fraction, which consists exclusively of HSAdull
cells in scid mice, includes both HSAdull and HSAbright cells
in M54 and 3H9 scid mice. We designate HSAdull and
HSAbright cells in the BP1
HSA+ fraction as B and B', respectively. The upregulation of HSA when µ-tg expressing cells
transit from stage B to B' to C' presumably reflects µ chain-
dependent signaling. The effect of both µ and
chain tgs
on scid B cell development is shown in Fig. 3. As indicated,
scid mice bearing M54 (or 3H9) and the L chain tg, V
8
(26), have near-normal percentages of B (B220+IgM+) cells
in their bone marrow, but are severely deficient in early B
lineage (B220+CD43+ and B220+CD43
) cells comprising
subsets B-D.
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Since pro-B cells (subsets
B and B') in µ-tg scid mice appear to differentiate directly
or very rapidly into early pre-B cells deficient in RAG expression (subset C'), we suspected that many developing B
lineage cells in these mice might not initiate DH-JH rearrangement until the late pre-B cell stage (D) when RAG
expression is again upregulated. To assay for the initiation
of DH-JH rearrangement, we tested for DSBs at JH recombination signal/coding borders in FACS®-sorted B220+
CD43+ (CD43+) and B220+CD43 (CD43
) bone marrow
cells (CD43+ cells would include stages B-C' and CD43
cells would correspond to stage D). Broken DNA molecules with JH signal ends were detected by LM-PCR (31,
33). We also tested for completed DH-to-JH rearrangements
and for retention of JH germline alleles (see Materials and
Methods for details).
As shown in Fig. 4 and Table I, JH signal ends resulting
from the initiation of DH-JH rearrangement in M54 scid
mice were much more abundant in the late pre-B (CD43)
cell fraction than in the CD43+ fraction containing pro-B
and early pre-B cells. In M54 scid/+ mice as well, JH signal ends were more abundant in the CD43
than CD43+
cell fraction (Fig. 4 and Table I). We conclude that initiation of DH-JH rearrangement in M54 mice occurs predominantly at the late pre-B cell stage. Despite the relatively
low abundance of JH signal ends in the CD43+ cell fraction
of M54 scid/+ mice, alleles with (completed) DH-JH rearrangements were readily detectable in this cell fraction (Fig. 4). This is not surprising as DH-JH rearrangements would be
expected to result in DJH complexes that have a much longer
half-life than JH signal ends, especially in µ-tg-expressing
pro-B cells that fail to rearrange their VH elements (20).
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Non-tg scid and scid/+ control mice showed widely
different levels of JH signal ends; i.e., JH signal ends were
more abundant in the CD43+ cell fraction of scid than
scid/+ bone marrow (Fig. 4 and Table I). As discussed
later, a possible explanation for this difference is that initiation of DH-JH rearrangement continues unabated in scid
mice, whereas in scid/+ mice, DH-JH rearrangement is
limited by the onset of VH-DJH rearrangement. JH signal
ends were in low abundance in the CD43 cell fraction of
scid/+ mice, consistent with a low retention of germline
JH alleles (Fig. 4) and the completion of H chain gene rearrangement. The high retention of germline JH alleles in the
CD43+ cell fraction of scid/+ (and scid) mice is presumed
to reflect in part the presence of early B lineage cells not yet
expressing RAG protein and the known contamination of
this fraction with non-B lineage cells (39).
To test for ongoing DH-JH and VH-DJH rearrangement
in the CD43+ and CD43 cell fractions of scid, scid/+,
and M54 scid/+ mice, we assayed for DSBs at both the 3'
and 5' signals of the DHfl16.1 (DHfl) element. DHfl is the
most upstream DH element (40) and is used in
50% of
DH-JH rearrangements (41). Broken DNA molecules
with 3' DHfl signal ends signify initiation of DH-JH rearrangement, whereas 5' DHfl signal ends can be taken to reflect initiation of VH-DJH rearrangement (33).
Scid and scid/+ mice showed striking differences in their
levels of 3' and 5' DHfl signal ends (Fig. 5). In the CD43+
cell fraction of scid mice, 3' but not 5' DHfl signal ends
were abundant, whereas, in the corresponding cell fraction
of scid/+ mice, 5' but not 3' DHfl ends were abundant.
Thus, in the CD43+ cell fraction of scid mice, initiation of
DH-JH rearrangement predominates over that of VH-DJH
rearrangement, whereas the reverse is true in the CD43+ cell
fraction of scid/+ mice. In the late pre-B (CD43) cell fraction of scid/+ mice, neither 3' nor 5' DHfl signal ends were
detectable, indicating that H chain gene rearrangement is
normally completed before this stage, which is in agreement with the results of Fig. 4. In contrast, in the CD43
cell fraction of M54 scid/+ mice, DH-JH rearrangement was ongoing, as indicated by the abundance of 3' DHfl signal ends
(Fig. 5). Note that 5' DHfl signal ends were not detectable in
the CD43
(or CD43+) cell fraction of M54 scid/+ mice.
Therefore, even though DH-JH rearrangement is ongoing in
late pre-B cells of M54 scid/+ mice, initiation of VH-DJH
rearrangement does not evidently occur in these cells. This
apparent inability of the V(D)J recombinase system to target
VH elements in late pre-B cells of µ-tg mice is consistent
with the early findings of Yancoupolus and Alt (45). These
investigators found that VH558 transcripts are detectable in
µ
but not µ+ lines of transformed pre-B cells and concluded that VH elements in µ+-transformed pre-B cells are
not accessible to the V(D)J recombinase system.
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To test whether initiation of
DH-JH rearrangement occurs at a normal frequency in µ/-tg mice, we sorted B220+IgM
bone marrow cells from
scid, 3H9/V
8 scid, and 3H9/V
8 scid/+ mice, and then
assayed for the level of JH signal ends. The B220+ IgM
cell population would include B lineage subsets (B-D) before the immature B cell stage (E). We also assayed for circular DNA molecules with signal joints resulting from the
joining of the JH1, JH2, or JH3 signals with the 3' DHfl signal (see Materials and Methods). Signal joint formation, in
contrast to coding joint formation, is not impaired in scid
mice (46, 47). Also, we would expect circular DNA molecules to have a longer half-life than broken molecules with
JH signal ends, thus making signal joint formation a sensitive
assay for attempted DH-JH rearrangement in scid mice.
As shown in Fig. 6, JH signal ends were more abundant
in the B220+IgM cell fraction of non-tg scid mice than in
the corresponding cell fraction of 3H9/V
8 scid and 3H9/
V
8 scid/+ mice. Thus, the initiation of DH-JH rearrangement is clearly reduced in the presence of these tgs.
This is also apparent from the reduced level of signal joints in 3H9/V
8 mice compared with control non-tg scid mice
(Fig. 6). The level of JH2 signal joints in 3H9/V
8 scid
and 3H9/V
8 scid/+ mice was estimated to be ~10 and
60%, respectively, the level in non-tg scid mice (see Fig. 6,
legend). We suggest later that the lower level of signal joints
in 3H9/V
8 scid than 3H9/V
8 scid/+ mice may be attributable to premature death of developing scid B cells
resulting from persisting DSBs at DH and JH coding elements.
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Given that most
developing scid B cells fail to rearrange their D and J elements successfully (15, 48, 49) and die with persisting DSBs
(33, 35), the cells most favored to become B cells in µ/-tg
scid mice would be those in which DH-JH rearrangement is not attempted. To test this prediction, we generated and
cloned B cell hybridomas from the spleen of M54/V
8 and
3H9/V
8 scid mice, and then examined these hybridomas
for the status of their H chain alleles. Control hybridomas
were obtained from M54/V
8 and 3H9/V
8 scid/+ mice.
Representative results are illustrated in Fig. 7 for 9 3H9/
V
8 scid/+ hybridomas and 10 3H9/V
8 scid hybridomas. Note that one or two H chain gene rearrangements
were clearly evident in all but one of the scid/+ hybridomas. In contrast, none of the scid hybridomas showed a
rearranged allele.
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44 hybridomas from µ/-tg mice were analyzed and the
results are summarized in Table II. 10 scid/+ hybridomas
showed one allele to be rearranged with the other allele in
germline configuration; 11 (10 of which came from 3H9/
V
8 scid/+ mice) showed both alleles to be rearranged
and 5 showed a single rearrangement with the other allele
missing or undetectable. Two scid/+ hybridomas showed germline H chain alleles only. As normal B cells and their
precursors show H chain rearrangements at both alleles (3,
8, 50), the retention of at least one germline H chain allele
in ~40% of the scid/+ hybridomas demonstrates significant
transgene-mediated reduction of DH-JH rearrangement.
These results are in agreement with the results of Fig. 6 and
with previous reports showing that the frequency of endogenous H chain rearrangement is reduced in B lineage
cells of M54 (10, 51) and 3H9/V
8 (25) wild-type mice.
In contrast to the scid/+ results, all M54/V
8 and 3H9/ V
8 scid hybridomas showed germline H chain alleles only
(Table II). The absence of detectable H chain gene rearrangement in the scid hybridomas indicates that cells that
succeed in becoming B cells in µ/
-tg scid mice do not attempt DH-JH rearrangement.
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Discussion |
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The preceding results show a striking alteration in the
representation of B lineage subsets and duration of DH-JH
rearrangement in bone marrow of µ-tg and µ/-tg scid
mice compared with non-tg scid and scid/+ control mice.
Specifically, late pro-B cells (subset C) appear to be missing in
µ-tg scid mice and DH-JH rearrangement occurs predominantly at stage D, the late pre-B cell stage. In µ/
-tg scid
mice, early B lineage subsets (B-D) are grossly under represented and initiation of DH-JH rearrangement is less frequent than in non-tg control mice. Further, pro-B cells that succeed in reaching the B cell stage in µ/
-tg scid mice do not attempt DH-JH rearrangement. Interestingly, in non-tg control mice, we found initiation of DH-JH rearrangement to be
greater or more sustained in scid mice than in scid/+ mice.
The implications of these findings are discussed below.
In µ-tg mice initiation of DH-JH rearrangement
was found to occur most frequently at the late pre-B cell
stage (stage D). As DH-JH rearrangement is normally completed before this stage (17, 18), initiation of H chain gene
rearrangement appears to be somewhat delayed in µ-tg
mice. To explain this result, we suggest the following model:
µ-tg- and RAG-expressing pro-B cells (subsets B and B')
rapidly differentiate into recombinase-deficient early pre-B
cells (subset C'), such that many cells do not have time to
initiate or complete DH-JH rearrangement at both alleles until the late pre-B stage (subset D) when RAG expression
is again upregulated.2 Rapid progression of pro-B cells to
the C' stage would presumably result from premature expression of a pre-BCR containing a tg-encoded µ chain,
surrogate light (SL) chain and the signal transducing chains,
Ig and Ig
(reviewed in 13, 52). Consistent with this
model is the known early expression of µ tgs (30) and the genes for SL chain (55), the apparent absence of subset
C in µ-tg scid mice (see Fig. 2), the shortened duration of
the pro-B stage in µ-tg mice (57a), and the finding that the
majority of cells corresponding to subset C in non-tg mice
contain nonproductive VDJH (VDJH
) rearrangements (58).
The latter finding has been interpreted to suggest that
pro-B cells containing a VDJH+ rearrangement quickly
exit the subset C compartment (58).
Applying the above model to non-tg mice, we suggest
that pro-B cells that make a VDJH+ rearrangement on the
first attempt may exclude VH-DJH rearrangement at the
other allele by rapidly progressing to the RAG-deficient C'
stage, and then to stage D, at which rearrangement of VH
elements is no longer permissible. For allelic exclusion to
occur in this model, a pre-BCR need only signal developmental progression. This notion is consistent with previous
reports showing that exclusion of VH-DJH rearrangement
is tightly linked with progression of pro-B cells to the
pre-B stage (14, 59). Such linkage is even observed in µ-tg
mice that express a truncated µ chain, which results in a
pre-BCR complex lacking (specificity) a µ variable region
and surrogate light chain (62, 63). However, pro- to pre-B
progression and VH-DJH rearrangement are both blocked
in µ-tg mice that express a mutated µ chain that precludes
assembly of a pre-BCR complex with the signal transducing
Ig /
chains (64). Interestingly, the few B lineage cells
that reportedly escape the above developmental block show
allelic exclusion (67), consistent with our proposed model.
Ongoing initiation of DH-JH (Fig. 4) and V-J
rearrangement (30) in late pre-B cells of M54 scid mice could
help explain why these mice uniformly lack B cells (20) and
appear no more leaky than non-tg scid mice (68). If attempted rearrangement of DH and JH elements in developing M54 scid pre-B cells is initiated before that of V
and J
elements, some cells might be expected to succeed in making a DH-JH rearrangement. Indeed, DH-JH rearrangements were recovered from late pre-B cells of M54 scid mice
(see Fig. 4). However, the chance of a scid cell surviving attempted rearrangements at both H and L chain loci would
seem unlikely, consistent with the absence of detectable VJ
coding joints in late pre-B cells of M54 scid mice (22, 30).
In µ/-tg mice, we found initiation of DH-JH rearrangement was less frequent than in non-tg scid mice. Signal joints
resulting from the initiation of DHfl to JH rearrangement in
3H9/V
8 scid and 3H9/V
8 scid/+ mice were estimated
to be present at ~10 and 60%, respectively, the level observed in non-tg scid mice. Based on the difference in level
of recovered signal joints in 3H9/V
8 scid/+ and non-tg
scid mice, we estimate that initiation of DH-JH is ~40% less
frequent in µ/
-tg than non-tg mice. This estimate agrees
favorably with the observed frequency of germline H chain alleles in B cell hybridomas from 3H9/V
8 (6/35 alleles) and
M54/V
8 (6/14 alleles) scid/+ mice (Table II). The much
lower level of signal joints in 3H9/V
8 scid than 3H9/V
8
scid/+ mice is taken to reflect loss (death) of scid cells that
attempt DH-JH rearrangement. This could account for the
absence of rearranged H chain alleles in B cell hybridomas
recovered from µ/
-tg scid mice (Table II).
To explain the reduced level of DH-JH rearrangement
in µ/-tg mice, we suggest that expression of a tg-coded
BCR in early pro-B cells promotes very rapid progression
of these cells to the B cell stage, such that there is little time
to initiate DH-JH rearrangement. Consistent with this notion, (a) µ and V
8 tgs are known to be expressed early in
B cell development (30), (b) µ/
-tg scid/+ mice contain
near-normal percentages of B cells in bone marrow but markedly reduced percentages of pro- and pre-B cells (20), and
(c) µ/
-tg scid mice show near-normal percentages of B
cells in bone marrow but sharply reduced percentages of
pro-B cells compared with non-tg scid controls and virtually no pre-B cells (
1%; Fig. 3).
In non-tg control mice, we found that early B lineage cells in the CD43+ cell fraction from scid mice showed a much higher level of JH signal ends than the corresponding cell fraction from scid/+ mice. As scid does not impair the joining of signal ends (46, 47), one cannot attribute the relatively high level of JH signal ends in scid mice to a blockage in signal joint formation. What scid does impair, however, is the processing of coding ends before their being joined (33, 35). Thus, developing B lineage cells in scid mice do not often succeed in joining DH and JH coding ends (48, 49) and would not be expected to initiate the second step of H chain gene rearrangement (VH-DJH rearrangement). Indeed, 5' DHfl signal ends, signifying the initiation of VH-DJH rearrangement, were not detectable in CD43+ scid cells (Fig. 5). We suggest that in the absence of VH-DJH rearrangement, initiation of DH-JH rearrangement continues unabated in CD43+ cells, resulting in a high level of JH signal ends. On the other hand, in the CD43+ cell fraction of scid/+ mice, initiation of VH-DJH rearrangement was prominent and that of DH-JH rearrangement barely evident (Fig. 5). This implies that initiation of DH-JH rearrangement in scid/+ mice may be limited to the earliest stage of pro-B cell development, consistent with the idea discussed below, that onset of VH-DJH rearrangement may preclude further DH-JH rearrangement.
In wild-type or scid/+ cells, a DH-JH rearrangement may be followed by rearrangement of a VH element to the resulting DJH complex or the complex may be replaced by the joining of an upstream DH element to a downstream JH element (41). The latter event, DJH replacement would seem counterproductive to efficient assembly of VH, DH, and JH elements. Thus, it makes sense, as originally postulated by Alt et al. (69), that after DH-JH rearrangement VH rather than DH elements are preferentially rearranged. How might this happen? Recent evidence suggests that initiation of VH-DJH rearrangement is associated with a shift in the targeting of the V(D)J recombinase activity from the 3' to the 5' side of DH elements (70). Targeting of the recombinase to signals on the 5' side of DH elements would minimize DJH replacement and limit the duration of DH-JH rearrangement to the earliest stage of pro-B cell development. Although DJH complexes can be readily detected in late pro-B cells (subset C) (17, 18, 38), this does not necessarily reflect ongoing DH-JH rearrangement at this stage; the observed DJH complexes could have been formed earlier in cells of subset B.
In sum, DH-JH rearrangement in non-tg mice is normally
initiated and completed at the early pro-B stage. In µ-tg
mice, DH-JH rearrangement may begin at the pro-B stage,
but it appears to continue and occur most frequently at the
late pre-B stage. Based on the altered representation of
pro-B subsets in µ-tg scid mice, we suggest that the extended
period of DH-JH rearrangement in these mice primarily
reflects rapid progression of µ-tg-expressing pro-B cells to
the recombinase-deficient early pre-B cell stage. Thus, many
cells may not have time to initiate DH-JH rearrangement until the late pre-B stage when RAG expression is again
upregulated. In addition, ongoing DH-JH rearrangement
(including DJH replacement) at the late pre-B stage would
not be limited by initiation of VH-DJH rearrangement, as
the latter does not apparently occur in late pre-B cells of
µ-tg mice. Finally, rapid progression of µ/-tg-expressing
pro-B cells to the recombinase-inactive B cell stage could
explain why in µ/
-tg mice we find a reduced initiation of
DH-JH rearrangement compared with non-tg mice and a
striking deficiency of pro- and pre-B cells despite near-normal numbers of B cells.
![]() |
Footnotes |
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Address correspondence to Melvin J. Bosma, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. Phone: 215-728-3630; Fax: 215-728-2412; E-mail: mj_bosma{at}fccc.edu
Received for publication 11 December 1998.
Dr. Chang's current address is Arizona State University, Department of Microbiology, Tempe, AZ 85287.We thank Norman Ruetsch for help with Fig. 1 and Randy Hardy and Susan Shinton for providing anti- HSA and anti-BP1 reagents for the FACS® analyses in Fig. 2. We also thank David Allman, Randy Hardy, Dietmar Kappes, N. Klinman, Pamela Nakajima, Norman Ruetsch, David Wiest, and Martin Weigert for review of the manuscript, and Roseanne Diehl for assistance in typing the manuscript.
This work was supported by grants from the National Institutes of Health (CA06927 and CA04946) and an appropriation from the Commonwealth of Pennsylvania.
Abbreviations used in this paper BCR, B cell receptor; DSB, double strand break; HSA, heat stable antigen; LM-PCR, ligation-mediated PCR; RAG, recombinase activation gene; tg, transgene.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Weiler, E..
1965.
Differential activity of allelic ![]() |
2. | Pernis, B.G., A. Chiappino, S. Kelus, and P.G.H. Gell. 1965. Cellular localization of immunoglobulin with different allotype specificities in rabbit lymphoid tissues. J. Exp. Med. 122: 853-875 [Medline]. |
3. | Coleclough, C.R., R.P. Perry, K. Karjalainen, and M. Weigert. 1981. Aberrant rearrangements contribute significantly to the allelic exclusion of immunoglobulin gene expression. Nature. 290: 372-377 [Medline]. |
4. | Alt, F.W., N. Rosenberg, S. Lewis, E. Thomas, and D. Baltimore. 1981. Organization and reorganization of immunoglobulin genes in A-MuLV-transformed cells: rearrangement of heavy but not light chain genes. Cell. 27: 381-390 [Medline]. |
5. | Alt, F.W., N. Rosenberg, V. Enea, E. Siden, and D. Baltimore. 1982. Multiple immunoglobulin heavy-chain gene transcripts in Abelson murine leukemia virus-transformed cell lines. Mol. Cell. Biol. 2: 386-400 [Medline]. |
6. | Wabl, M., and C. Steinberg. 1982. A theory of allelic and isotypic exclusion for immunoglobulin genes. Proc. Natl. Acad. Sci. USA. 79: 6976-6978 [Abstract]. |
7. | Lewis, S.M.. 1994. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv. Immunol. 56: 27-150 [Medline]. |
8. | Alt, F.W., G.D. Yancopoulos, T.K. Blackwell, C. Wood, E. Thomas, M. Boss, R. Coffman, N. Rosenberg, S. Tonegawa, and D. Baltimore. 1984. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO (Eur. Mol. Biol. Organ.) J. 3: 1209-1219 [Abstract]. |
9. |
Rusconi, S., and
G. Kohler.
1985.
Transmission and expression of a specific pair of rearranged immunoglobulin µ and ![]() |
10. | Weaver, D., F. Constantini, T. Imanishi-Kari, and D. Baltimore. 1985. A transgenic immunoglobulin mu gene prevents rearrangement of endogenous genes. Cell. 42: 117-127 [Medline]. |
11. | Nussenzweig, M.C., A.C. Shaw, E. Sinn, D.B. Danner, K.L. Holmes, H.C. Morse, and P. Leder. 1987. Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin µ. Science. 236: 816-819 [Medline]. |
12. | Storb, U.. 1987. Transgenic mice with immunoglobulin genes. Annu. Rev. Immunol. 5: 151-174 [Medline]. |
13. | Melchers, F., A. Rolink, U. Grawunder, T.H. Winkler, H. Karasuyama, P. Ghia, and J. Andersson. 1995. Positive and negative selection events during B lymphopoiesis. Curr. Opin. Immunol. 7: 214-227 [Medline]. |
14. | Rajewsky, K.. 1996. Clonal selection and learning in the antibody system. Nature. 381: 751-758 [Medline]. |
15. | Schuler, W., I.J. Weiler, A. Schuler, R.A. Phillips, N. Rosenberg, T.W. Mak, J.F. Kearney, R.P. Perry, and M.J. Bosma. 1986. Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency. Cell. 46: 963-972 [Medline]. |
16. | Bosma, M.J., and A.C. Carroll. 1991. The scid mouse mutant: definition, characterization and potential uses. Annu. Rev. Immunol 9: 323-350 [Medline]. |
17. | Hardy, R.R., C.E. Carmack, S.A. Shinton, J.D. Kemp, and K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173: 1213-1225 [Abstract]. |
18. | Ehlich, A., S. Schaal, H. Gu, D. Kitamura, W. Muller, and K. Rajewsky. 1993. Immunoglobulin heavy and light chain genes rearrange independently at early stages of B cell development. Cell. 72: 695-704 [Medline]. |
19. | Reichman-Fried, M., R.R. Hardy, and M.J. Bosma. 1990. Development of B-lineage cells in the bone marrow of scid/ scid mice following the introduction of functionally rearranged transgenes. Proc. Natl. Acad. Sci. USA. 87: 2730-2734 [Abstract]. |
20. | Chang, Y., G.C. Bosma, and M.J. Bosma. 1995. Development of B cells in scid mice with immunoglobulin transgenes: implications for the control of V(D)J recombination. Immunity. 2: 607-616 [Medline]. |
21. | Nussenzweig, M.C., E.V. Schmidt, A.C. Shaw, E. Sinn, J. Campos-Torres, B. Mathey-Prevot, P.K. Pattengale, and P. Leder. 1988. A human immunoglobulin gene reduces the incidence of lymphomas in c-Myc-bearing transgenic mice. Nature. 336: 446-450 [Medline]. |
22. | Reichman-Fried, M., M.J. Bosma, and R.R. Hardy. 1993. B-lineage cells in µ-transgenic scid mice proliferate in response to IL-7 but fail to show evidence of immunoglobulin light chain gene rearrangement. Int. Immunol. 5: 303-310 [Abstract]. |
23. | Grawunder, U., T.M. Leu, D.G. Schatz, A.G. Werner, A.G. Rolink, F. Melchers, and T.H. Winkler. 1995. Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement. Immunity. 3: 601-608 [Medline]. |
24. | Grosschedl, R., D. Weaver, D. Baltimore, and F. Costantini. 1984. Introduction of a µ immunoglobulin gene into the mouse germ line: specific expression in lymphoid cells and synthesis of functional antibody. Cell. 38: 647-658 [Medline]. |
25. | Erikson, J., M.Z. Radic, S.A. Camper, R.R. Hardy, C. Carmack, and M. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature. 349: 331-334 [Medline]. |
26. |
Carmack, C.E.,
S.A. Camper,
J.J. Mackle,
W.U. Gerhard, and
M.G. Weigert.
1991.
Influence of a V![]() |
27. | Kotloff, D.B., M.J. Bosma, and N.R. Ruetsch. 1993. Scid mouse pre-B cells with intracellular µ chains: analysis of recombinase activity and IgH gene rearrangements. Int. Immunol. 5: 383-391 [Abstract]. |
28. | Kearney, J.F., A. Radbruch, B. Liesegang, and K. Rajewsky. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123: 1548-1550 [Abstract]. |
29. | Cooper, M.D., D. Mulvaney, A. Coutinho, and P.A. Cazenave. 1986. A novel cell surface molecule on early B-lineage cells. Nature. 321: 616-618 [Medline]. |
30. | Chang, Y., and M.J. Bosma. 1996. Effect of different immunoglobulin transgenes on B cell differentiation in scid mice. Int. Immunol 9: 373-380 [Abstract]. |
31. | Mueller, P.R., and B. Wold. 1989. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 246: 780-786 [Medline]. |
32. | Roth, D.B., C. Zhu, and M. Gellert. 1993. Characterization of broken DNA molecules associated with V(D)J recombination. Proc. Natl. Acad. Sci. USA. 90: 10788-10792 [Abstract]. |
33. | Schlissel, M., A. Constantinescu, T. Morrow, M. Baxter, and A. Peng. 1993. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 7: 2520-2532 [Abstract]. |
34. |
Roth, D.B.,
P.B. Nakajima,
J.P. Menetski,
M.J. Bosma, and
M. Gellert.
1992.
V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor ![]() |
35. | Roth, D.B., J.P. Menetski, P.B. Nakajima, M.J. Bosma, and M. Gellert. 1992. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell. 70: 983-991 [Medline]. |
36. | Sakano, H., Y. Kurosawa, M. Weigert, and S. Tonegawa. 1981. Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy chain genes. Nature. 290: 562-565 [Medline]. |
37. | Rolink, A., and F. Melchers. 1991. Molecular and cellular origins of B lymphocyte diversity. Cell. 66: 1081-1094 [Medline]. |
38. | Li, Y.-S., K. Hayakawa, and R.R. Hardy. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178: 951-960 [Abstract]. |
39. | Li, Y.-S., R. Wasserman, K. Hayakawa, and R.R. Hardy. 1996. Identification of the earliest B lineage stage in mouse bone marrow. Immunity. 5: 527-535 [Medline]. |
40. | Kurosawa, Y., and S. Tonegawa. 1982. Organization, structure, and assembly of immunoglobulin heavy chain diversity DNA segments. J. Exp. Med. 155: 201-218 [Abstract]. |
41. | Reth, M.G., S. Jackson, and F.W. Alt. 1986. VHDJH formation and DJH replacement during pre-B differentiation: non-random usage of gene segments. EMBO (Eur. Mol. Biol. Organ.) J. 5: 2131-2138 [Abstract]. |
42. | Ichihara, Y., H. Hayashida, S. Miyazawa, and Y. Kurosawa. 1989. Only Dfl16, Dsp2, and Dq52 gene families exist in mouse immunoglobulin heavy chain diversity gene loci, of which Dfl16 and Dsp2 originate from the same primordial DH gene. Eur. J. Immunol. 19: 1849-1854 [Medline]. |
43. | Feeney, A.J.. 1990. Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences. J. Exp. Med. 172: 1377-1390 [Abstract]. |
44. | Chang, Y., C.J. Paige, and G.E. Wu. 1992. Enumeration and characterization of DJH structures in mouse fetal liver. EMBO (Eur. Mol. Biol. Organ.) J. 11: 1891-1899 [Abstract]. |
45. | Yancopoulos, G.D., and F.W. Alt. 1985. Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell. 40: 271-281 [Medline]. |
46. | Lieber, M.R., J.E. Hessie, S. Lewis, G.C. Bosma, K. Mizuuchi, M.J. Bosma, and M. Gellert. 1988. The defect in murine severe combined immune deficiency: joining of signal segments but not coding segments in V(D)J recombination. Cell. 55: 7-16 [Medline]. |
47. | Blackwell, T.K., B.A. Malynn, R.R. Pollock, P. Ferrier, L.R. Covey, G.M. Fulop, R.A. Phillips, G.D. Yancopoulos, and F.W. Alt. 1989. Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins. EMBO (Eur. Mol. Biol. Organ.) J. 8: 735-742 [Abstract]. |
48. | Pennycook, J.L., Y. Chang, J. Celler, R.A. Phillips, and G.E. Wu. 1993. High frequency of normal DJH joints in B cell progenitors in severe combined immunodeficiency mice. J. Exp. Med. 178: 1007-1016 [Abstract]. |
49. | Araki, R., M. Itoh, K. Hamatani, and M. Abe. 1996. Normal D-JH rearranged products of the Ig H gene in SCID mouse bone marrow. Int. Immunol. 8: 1045-1053 [Abstract]. |
50. | Nottenburg, C., and I. Weissman. 1981. Cµ gene rearrangement of mouse immunoglobulin genes in normal B cells occurs on both the expressed and nonexpressed chromosomes. Proc. Natl. Acad. Sci. USA. 78: 484-488 [Abstract]. |
51. | Iacomini, J., N. Yannoutsos, S. Bandyopadhay, and T. Imanishi-Kari. 1991. Endogenous immunoglobulin expression in mu transgenic mice. Int. Immunol. 3: 185-196 [Abstract]. |
52. | Melchers, F., D. Haasner, U. Grawunder, C. Kalberer, H. Karasuyama, T. Winkler, and A.R. Rolink. 1994. Roles of IgH and L chains and of surrogate H and L chains in the development of cells of the B lymphocyte lineage. Annu. Rev. Immunol. 12: 209-225 [Medline]. |
53. | Reth, M.. 1992. Antigen receptors on B lymphocytes. Annu. Rev. Immunol. 10: 97-121 [Medline]. |
54. | Cambier, J.C., C.M. Pleiman, and M.R. Clark. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12: 457-486 [Medline]. |
55. |
Sakaguchi, N., and
F. Melchers.
1988.
![]() |
56. |
Kudo, A., and
F. Melchers.
1987.
A second gene, VpreB in
the ![]() |
57. |
Pillai, S., and
D. Baltimore.
1987.
Formation of disulphide-linked µ2![]() ![]() |
57a. | Arakawa, H., and S. Takeda. 1996. Early expression of Ig µ chain from a transgene significantly reduces the duration of the pro-B stage but does not affect the small pre-B stage. Int. Immunol. 8: 1319-1328 [Abstract]. |
58. | Ehlich, A., V. Martin, W. Muller, and K. Rajewsky. 1994. Analysis of the B cell progenitor compartment at the level of single cells. Curr. Biol 4: 573-583 [Medline]. |
59. | Loffert, D., A. Ehlich, W. Muller, and K. Rajewsky. 1996. Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. Immunity. 4: 133-144 [Medline]. |
60. |
Gong, S., and
M.C. Nussenzweig.
1996.
Regulation of an
early developmental checkpoint in the B cell pathway by
Ig![]() |
61. | Torres, R.M., H. Flaswinkel, M. Reth, and K. Rajewsky. 1996. Aberrant B cell development and immune response in mice with a compromised BCR complex. Science. 272: 1804-1808 [Abstract]. |
62. |
Corcos, D.,
O. Dunda,
C. Butor,
J.-Y. Cesbron,
P. Lores,
D. Bucchini, and
J. Jami.
1995.
Pre-B-cell development in the
absence of ![]() |
63. | Shaffer, A.L., and M.S. Schlissel. 1997. A truncated heavy chain protein relieves the requirement for surrogate light chains in early B cell development. J. Immunol. 159: 1265-1275 [Abstract]. |
64. |
Papavasiliou, F.,
Z. Misulovin,
H. Suh, and
M.C. Nussenzweig.
1995.
The role of Ig![]() |
65. |
Papavasiliou, F.,
M. Jankovic,
H. Suh, and
M.C. Nussenzweig.
1995.
The cytoplasmic domains of immunoglobulin
(Ig) ![]() ![]() |
66. | Papavasiliou, F., M. Jankovic, and M. Nussenzweig. 1996. Surrogate or conventional light chains are required for membrane immunoglobulin mu to activate the precursor B cell transition. J. Exp. Med. 184: 2025-2030 [Abstract]. |
67. |
Cronin, F.E.,
M. Jiang,
A.K. Abbas, and
S.A. Grupp.
1998.
Role of µ heavy chain in B cell development. I. Blocked B
cell maturation but complete allelic exclusion in the absence
of Ig![]() ![]() |
68. | Fried, M., R.R. Hardy, and M.J. Bosma. 1989. Transgenic scid mice with a functionally rearranged immunoglobulin heavy chain gene. Curr. Top. Microbiol. Immunol 152: 107-114 [Medline]. |
69. | Alt, F.W., T.K. Blackwell, and G.D. Yancopoulos. 1987. Development of the primary antibody repertoire. Science. 238: 1079-1088 [Medline]. |
70. | Van Dyk, L.F., T.W. Wise, B.B. Moore, and K. Meek. 1996. Immunoglobulin DH recombination signal sequence targeting. J. Immunol. 157: 4005-4015 [Abstract]. |