By
From the Institute for Genetics, University of Cologne,Weyertal 121, 50931 Cologne, Germany
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Abstract |
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In mouse mutants incapable of expressing µ chains, VJ
joints are detected in the CD43+ B
cell progenitors. In agreement with these earlier results, we show by a molecular single cell
analysis that 4-7% of CD43+ B cell progenitors in wild-type mice rearrange immunoglobulin (Ig)
genes before the assembly of a productive VHDHJH joint. Thus, µ chain expression is not
a prerequisite to Ig
light chain gene rearrangements in normal development. Overall, ~15%
of the total CD43+ B cell progenitor population carry Ig
gene rearrangements in wild-type
mice. Together with the results obtained in the mouse mutants, these data fit a model in which
CD43+ progenitors rearrange IgH and Ig
loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors V
J
joining rapidly initiates
chain expression, irrespective of the presence of a µ chain.
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Introduction |
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During B cell development, genes encoding immunoglobulin V regions are generated by recombination of individual gene segments. Genes encoding Ig heavy chains (IgH genes) are formed by first rearranging a DH to a JH segment, followed by a VH to DHJH rearrangement. In the light chain (L) loci, a VL to JL recombination event generates an Ig light chain (IgL) gene. If the resulting joints are in a contiguous open reading frame, the rearrangements are referred to as "productive".
In regard to the relative order of VHDHJH and VL JL recombination events, two models have been proposed. According to the "ordered" model, expression of a µ heavy
chain from a productively rearranged IgH gene induces
light chain gene rearrangement. Evidence that formation of
VHDHJH complexes usually precedes light chain gene rearrangement comes from the analysis of Abelson murine leukemia virus-transformed pre-B cells in culture (1, 2) and
from studies of Ig gene rearrangements in B cell precursor
populations isolated ex vivo (3). Furthermore, the expression of a transfected membrane-bound µ chain as well as
cross-linking of pre-B cell receptor complexes (consisting
of membrane-bound µ chains and the products encoded by
the 5 and VpreB genes; reference 4) stimulated the rearrangement of endogenous
light chain genes in transformed pre-B cell lines (5). In addition, an increased number of V
J
rearrangements was observed in fetal livers
of heavy chain transgenic mice as compared with nontransgenic mice (8).
In contrast, the "stochastic" model of IgH and IgL gene
recombination states that µ chain expression and pre-B receptor signaling are not required for IgL gene rearrangement and suggests that IgH and IgL loci rearrange independently of each other (9, 10). This hypothesis is supported
by the analysis of Abelson murine leukemia virus-transformed murine pre-B cell lines derived from normal (11)
and scid mice (12). In both cases, some cells were shown to
rearrange Ig loci in the absence of a membrane-bound µ heavy chain. In vitro differentiation experiments using normal murine pre-B cell lines have also demonstrated that
protein could be expressed in the absence of a µ chain (13).
Moreover,
chain expression was detected in the absence
of productive VHDHJH rearrangements in immortalized B
cell precursors of human fetal bone marrow (14). Examination of transformed embryonic bursal cells showed that
during chicken B cell development, IgL genes can also be
rearranged before IgH gene rearrangement has been completed (15).
Although in vivo most Ig rearrangements occur in the
pre-B cell compartment into which progenitor cells are
driven upon pre-B cell receptor (i.e., µ chain) expression
(5, 16), evidence indicates that initially, when gene rearrangements in IgH are set in motion in CD43+ progenitors, V
J
rearrangements also occur, albeit at low frequency (17, 18). At this early stage of development,
rearrangements appear to be independent of µ chain expression and, indeed, any rearrangement in the IgH locus,
as they are also seen in mutant mice unable to either express membrane-bound µ chains (µMT mice; 19) or generate VHDHJH joints due to a targeted deletion of the JH
elements (20, 18). These data suggest that gene rearrangements in the Ig
locus occur at two stages of development: in early CD43+ progenitors at low frequency and independent of µ chain expression, and later on, at high frequency,
in pre-B cells upon pre-B cell receptor expression. However, one might argue that the analysis of the mutant mice
could be misleading because in these animals the progenitors do not develop beyond the CD43+ stage and therefore
persist in this cellular compartment for a prolonged time,
accumulating gene rearrangements that normally would
not have occurred. On the other hand, Ig
gene rearrangements seen in CD43+ B cell progenitors of wild-type mice
(17) could be derived from cells already expressing µ chains.
We therefore decided to verify the results obtained in
the mouse mutants by the analysis of IgH and Ig rearrangements in individual CD43+ B cell progenitors in
wild-type animals. This approach allows us to investigate
whether recombination of IgL loci can indeed precede the
generation of productive IgH gene rearrangements in the
course of B cell development under physiological conditions, and, if so, to evaluate the frequency of these events.
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Materials and Methods |
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Cell Sorting
Single cell suspensions were prepared from bone marrow by flushing femurs with DMEM (containing 5% FCS, 0.1% NaN3) or from splenic tissues of BALB/c mice (8-12 wk old; Bomholtgaard, Denmark). Cells were treated with Tris-buffered 0.165 M NH4Cl to eliminate erythrocytes and washed by centrifugation through FCS.
3-83i mice (21) were used at 8-12 wk of age. Wild-type
mice used in the staining shown in Fig. 1 were F1 at the age of 8-12 wk from a 129sv × BALB/c cross.
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Cell sorting was performed using a dual laser flow cytometer
(FACStar®). Single cells were directly deposited into 0.5-ml microtubes containing 20 µl 1× PCR buffer (GIBCO BRL, 2.5 mM
MgCl2) supplemented with 1 µg/ml rRNA from Escherichia coli
(Boehringer Mannheim), immediately frozen on dry ice, and
stored at 80°C. Single cells from the E14 embryonic stem cell
line (22) were isolated accordingly as negative controls for the
PCR. Depending on the set of the cytometer, up to 20% of the
tubes could be empty during a particular sorting procedure.
Isolation of Fraction B, C, and D Cells.
Fractions were classified according to Hardy et al. (23). Pooled bone marrow cells from two to six mice were depleted of MAC-1+ cells (and of IgM+ cells in the experiment with subsequent
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PCR and Sequence Analysis of Ig Gene Rearrangements
To prepare DNA for amplification, 1 µl of an aqueous solution of proteinase K (10 mg/ml; Boehringer Mannheim) was
added, samples were overlaid with paraffin oil, and were incubated for 30 min at 55°C. Subsequently, proteinase K was inactivated for 10 min at 95°C. PCR amplification was carried out in
two rounds: the first reaction contained all 5' primers, JH4E (29),
and J5E primers (2.5 pmol each; Table I). Amplification was
done over 30 cycles (1 min at 95°C, 1 min at 60°C, and 2.5 min
at 72°C). For the second PCR, 1.5-µl aliquots of the first round
amplification product were transferred into separate reactions (set
up in 96-well microtiter plates; Costar Corp.), each containing a
single 5' primer in combination with either the nested JH4A
(amplification of IgH genes; reference 29) or the J
5A primer
(amplification of Ig
genes) (7 pmol of each primer). 30 cycles
were performed (1 min at 95°C, 1 min at 63°C, and 1.5 min at
72°C). All PCRs contained dATP, dCTP, dGTP, dTTP (Pharmacia Biotech) at 200 µM each, PCR buffer (GIBCO BRL), 2.5 mM MgCl2, 5 U of Taq DNA polymerase (GIBCO BRL) in the
first round, and 3 U of Taq DNA polymerase in the second
round. The final volume of each reaction was 50 µl. Each PCR
was followed by a 5-10-min incubation at 72°C. 10 µl of the
second-round PCR product was analyzed on agarose gels. Before
sequencing, 1.5 µl of second-round product was reamplified for
20 cycles (30 s at 95°C, 1 min at 63°C, and 2 min at 72°C) using
appropriate 5' primers and nested 3' primers, DNA was isolated from preparative agarose gel using Spin-X columns (Costar
Corp.). Cycle sequencing was performed using the Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Applied
Biosystems) following the manufacturer's instructions and an ABI
373A sequencer (Applied Biosystems). Sequencing primers recognize sequences downstream of the respective rearranged J
genes (Table I).
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The primers used for amplification and sequencing of Ig heavy
chain genes have been described by Ehlich et al. (23) and Löffert
et al. (30). The VHH primer (30) was not used in the analyses of
fraction C. KGI (Table I) was used only in the analyses of fraction
B cells irrespective of protein staining.
Sequences were analyzed using DNAPLOT at ik.
uni-koeln.de/dnaplot/. The database used consists of mouse V
gene sequences from an EMBL/GenBank/DDBJ nucleotide
sequence database, a Kabat database (31), and the V sequence list
compiled by Kofler et al. (32).
Control Experiment to Confirm the Isolation of Single Cells by FACS®
We chose two mutant mouse strains in which a rearranged Ig
heavy chain variable region gene was introduced by gene targeting into the heavy chain locus, replacing the JH elements (T15i
mice, reference 28, and B1-8i mice, reference 33, containing a
rearranged VH186.2 gene isolated from the hybridoma B1-8; reference 34). From each of the two mouse strains, which were homozygous for the introduced heavy chain, 4 × 105 light chain-
positive splenic B cells were isolated by FACS® and subsequently
pooled to yield a 1:1 mixture. Of this mixture single cells as well
as two cells were sorted directly into microtubes containing PCR
buffer. The inserted VHDHJH complexes of these splenic B cells
were amplified in a semi-nested PCR approach analogous to the
one described above. The B1-8 (T15i) gene was amplified by the
5' primer VHA (VHT15) and the 3' primers JH2E and JH2
(JH1E and JH1) in the first and second rounds of amplification,
respectively. The primers used in this experiment have been described elsewhere (30).
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Results |
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We extended
our previously described single cell PCR system for the
analysis of IgH genes (29) to simultaneously examine Ig
genes. For this purpose, seven Ig
gene-specific oligonucleotides were included to detect rearranged V
J
complexes as well as Ig
loci in germline configuration (Table I).
To estimate the efficiency of the amplification of Ig loci
rearrangements, we used splenic, surface Ig
-positive B
cells. 197 V
J
joints were amplified from 210 single B cells (none, one, or two per cell). Assuming that ~30% of all
splenic B cells carry two V
J
complexes (9, 16), this corresponds to a V
J
rearrangement detection efficiency of
~70%. To determine the detection efficiency of IgH gene
joints, VHDHJH and DHJH gene rearrangements were amplified from 311 B cell precursors of the CD43+ fraction C
(reference 23; excluding fraction C' cells) in the presence
of Ig
locus-specific oligonucleotides. Two IgH gene PCR products were obtained from 41% of cells. In the remaining cells, either one (51%) or no (7%) IgH gene PCR
products were amplified. Thus, the efficiency of the amplification was sufficient to allow the simultaneous analysis of
heavy and light chain loci.
When the interdependence of rearrangements of the
various Ig loci is investigated by single cell analysis, it is essential to demonstrate that the amplification products are
indeed derived from the same cell, and that the samples do
not occasionally contain more than one cell. Therefore, a
control experiment similar to the one described by Löffert
et al. (30) was performed using two mutant mouse strains
in which different heavy chain transgenes were inserted
into the heavy chain locus, replacing the JH elements (T15i
mice, reference 28, and B1-8i mice, reference 33). Cell suspension containing equal proportions of Ig-positive
splenic B cells from both strains was prepared. From this,
either "one cell" or "two cell" samples were deposited into
microtubes using the FACS®. Subsequently, the IgH transgenes were amplified from these cells, using appropriate
PCR primers (30).
127 "one cell" samples yielded indeed only one PCR product (Table II). In the case of the "two cell" samples, 50% of the tubes would be expected to contain two cells from the same mouse strain that would not be identified as "two cells" because both have given rise to identical PCR products. Two different PCR products were obtained in 53% of the "two cell" samples (Table II). The rare cases in which no PCR product was obtained (Table II) may be explained by a relatively poor amplification efficiency using this particular primer set, or, alternatively, these tubes may not have contained a cell. These results indicate that the direct deposition of cells by FACS® used in the experiments described below represents a reliable method for obtaining samples containing single cells.
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To investigate whether IgL gene rearrangements in B cell precursors can occur before µ chain expression, we had to
look into the compartment of early B cell progenitors,
where cells both with and without productively rearranged
heavy chain genes are present. To classify different stages of
B cell development in the bone marrow, we used the system developed by Hardy et al. (23), which divides B220+,
surface Ig cells into five cellular fractions according to
their differential expression of CD43, heat stable antigen
(HSA), and BP-1. For initial studies, we chose fraction C
(excluding fraction C'; references 17, 23) of early B cell
progenitors in which V
J
rearrangements are six to seven
times less frequent than in
+ splenic B cells (17).
627 fraction C cells were examined. For 14 out of 50 cells bearing VJ
rearrangements, the configurations of both IgH alleles were determined (Tables III and VI).
Seven cells are potentially able to express µ chains because
they harbor functional VHDHJH rearrangements. However,
seven other cells contain an Ig
gene rearrangement in the
absence of a functional VHDHJH complex. Two of these
cells carry nonfunctional VHDHJH rearrangements at both
IgH alleles, and four carry a nonproductive VHDHJH rearrangement together with a DHJH joint. Two nonproductive
VHDHJH joints (in cells 298 and 717) comprise DH elements
rearranged in reading frame 2 (in the nomenclature of Ichihara et al.; reference 35). Thus, these cells could have expressed a truncated heavy chain (Dµ protein; reference 36)
before VHDHJH complex formation. The remaining cell
harbors a rearranged Ig
allele and contains only DHJH
complexes (cell 352). The DH elements in this cell are rearranged in reading frames other than reading frame 2.
It has been suggested that cells incapable of expressing a
pre-B cell receptor accumulate in fraction C (29). Thus, at
least some of the cells carrying VJ
joints observed in fraction C could represent dead-end cells that cannot mature
further and may have persisted for a prolonged time in fraction C. Such prolonged persistence may increase the probability to rearrange Ig
genes. Therefore, we decided to
also analyze fraction B, the earliest stage at which VHDHJH
rearrangements are detected, for the presence of cells containing rearranged
genes in the absence of productive
VHDHJH complexes. According to our previous analysis, V
J
rearrangements are 14 times less frequent in this cell
population than in
-positive splenic B cells (17).
To enrich for cells bearing Ig rearrangements, we isolated cells that stained for
chains intracellularly. 88 single
fraction B cells positive for intracellular
chains were analyzed. Ig
gene rearrangements were amplified (either one
or two per cell) from 47 cells. For 15 of these we were able
to determine the configuration of both heavy chain alleles.
8 out of 15 cells bearing V
J
rearrangements contained a
productive VHDHJH joint. Seven cells were found to harbor
either DHJH joints on both heavy chain alleles (five cells) or
a nonproductive VHDHJH joint on one allele and a DHJH
joint on the other (two cells) (Tables IV and VI). Reading frame 2, which encodes Dµ protein, appeared on one or
both alleles in all five cells that bear only DHJH rearrangements and in one of the two cells containing a DHJH joint
together with a nonproductive VHDHJH. Only one V
J
-bearing cell (cell 62, Tables IV and VI) that is unable to
produce a (truncated) heavy chain was found in this experiment. It carried a nonproductive VHDHJH rearrangement
(with the DH element in reading frame 3) on one allele and
a DHJH joint in reading frame 1 on the other.
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However, in order to maximally enrich for chain producers we had isolated only cells that displayed high levels
of
protein. These cells might be already selected for µ chain or Dµ protein expression, considering that the stability of the
protein could depend upon the presence of a
(truncated) heavy chain in the cell. For this reason we decided to look again in cells from fraction B, this time not
selected by intracellular staining for
protein, but randomly selected by PCR for the presence of V
J
rearrangements.
373 single cells sorted from fraction B were analyzed. In
32 cells we detected one or two rearrangements at the locus. In 11 of these cells we were also able to amplify and
sequence rearrangements of both heavy chain alleles. In
four cells no productive VHDHJH joint was present (Tables
V and VI). One cell contained two nonproductive VHDHJH
joints (one of which comprises a DH element in reading frame 2), and three cells carried DHJH rearrangements on
both heavy chain alleles. None of these DHJH/DHJH cells
harbored DH elements rearranged in reading frame 2.
Given the
efficiency of VJ
joint detection of ~70% and the fact that
single cell sorting procedure will leave up to 20% of the
tubes empty, the overall frequency of cells bearing V
J
rearrangements (either productive or nonproductive) in both
fractions B and C is in the range of ~11-16%.
To estimate the frequency of cells that are able to express
chain at the early stages of B cell development, we
stained fraction B cells for intracellular
protein. We used
wild-type mice and the 3-83
i mouse mutant in which a
productive V
J
gene segment encoding the VL region of
antibody 3-83 (37) was inserted by gene targeting into its
natural genomic localization so that its expression is controlled by the endogenous regulatory elements (21). Due to
the fact that in wild-type mice two-thirds of the V
J
rearrangements are out of frame, 3-83
i mice should show a
threefold increase in the number of
chain-expressing cells in fraction B. The result of this experiment is shown
in Fig. 1: ~7% of cells in fraction B in wild-type mice were
found to express
chains, whereas this value was 24% in
the 3-83
i mutant, yielding almost exactly the expected
1:3 ratio.
These data are in agreement with the frequency of V J
rearrangements in cells from fraction B estimated by PCR
analyses. Together, these results suggest that Ig
gene rearrangement and expression follow each other rapidly.
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Discussion |
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A control experiment in
which either one or two cells were deflected into each reaction tube (Table II) confirmed that the method to isolate
single cells by using the FACS® is highly reliable and that
the PCR products obtained from one sample are indeed
derived from a single cell. This is further supported by the
fact that PCR amplification of one sample never generated
more than four products (two from heavy chain loci and two from light chain loci; data not shown). There was
also no indication for the presence of contaminating DNA
molecules in the PCR, because rearranged Ig genes were
never amplified from control samples containing embryonic stem cells and the sequences of all rearrangements
were different. Therefore, it is unlikely that in the cases
where rearranged
genes were observed in the absence of
productive VHDHJH complexes, the IgH gene rearrangements amplified were derived from a second cell present in
the sample or from foreign DNA. The extent of a possible
contamination in fractions B and C by CD43
pre-B or B
cells due to inaccurate cell separation during FACS® sorting is discussed in the Materials and Methods section.
However, the presence of such contaminating cells (all
bearing productive VHDHJH joints) would result in an underestimation of the percentage of cells bearing V
J
joints
but no productive VHDHJH rearrangements in the early
fractions of B cell progenitors.
Although most Ig genes present in the germline are recognized by the collection of the primers used, certain combinations of gene rearrangements in a cell could not be detected. In particular, all DH elements (except the DHQ52
element) are recognized by the same primer and the primers specific for V genes are highly homologous in structure
(Table I). Therefore, most of the DHJH joints using the
same JH genes on both chromosomes or distinct V
J
rearrangements with the same J
segment could not be resolved. For these reasons the number of cells with DHJH
joints at both IgH loci and the number of cells bearing two V
J
joints could be underestimated.
The
question of whether expression of a productive VHDHJH
rearrangement is a prerequisite for light chain gene rearrangement during B cell development or whether Ig gene
rearrangement can take place also in the absence of a membrane-bound µ chain has been discussed controversially.
The analysis of Ig gene rearrangements of single B cell progenitors isolated ex vivo from wild-type mice addresses this
issue directly.
Cells of the earliest B cell progenitor fractions in which
VJ
rearrangements are detectable, namely, cells of the
CD43+ fractions B and C (17), were chosen for analysis.
The results obtained are summarized in Table VI. Overall,
18 cells were found to carry Ig
rearrangements in the absence of a productive VHDHJH joint. However, six of these
contained DHJH rearrangements in reading frame 2, and
thus were able to express Dµ proteins. Like a µ chain, the
Dµ protein could associate with the products of the
5 and
VpreB genes to form a pre-B cell receptor-like complex (7).
It has been suggested that Dµ protein expression, similar to
µ chain expression, provided a stimulatory signal for Ig
gene rearrangements (5, 38, 39). Among the other cells analyzed, three (cells 298, 717, and s147; Tables III, V, and
VI) had nonproductive VHDHJH joints in which DH elements were rearranged in reading frame 2, and thus could
have expressed a Dµ protein earlier. For these cells, as well
as for the ones containing Ig
rearrangements and productive VHDHJH joints, we can neither deduce the order of rearrangements at heavy and light chain loci nor state their interdependence. However, the remaining nine cells have
either nonproductive VHDHJH rearrangements (with the
DH elements in reading frames 1 or 3) and/or DHJH joints
in reading frames 1 or 3 (Table VI) and are thus unable to
express µ or Dµ chains.
We cannot rule out the possibility that some of the
heavy chain gene joints detected were formed by secondary
rearrangement events; specifically, previously productive
VHDHJH rearrangements could have been rendered nonproductive by VH gene replacement (40), and DHJH
joints could also have been substituted by recombining upstream D and downstream J elements with possible
changes to the reading frame (5, 40, 42, 43). However, it
has been implied that expression of the recombination activating genes RAG1 and RAG2 is downregulated upon
pre-B cell receptor expression, suggesting that recombination of heavy chain genes is terminated once a µ chain is
expressed (44). Furthermore, Dµ protein expression has
also been suggested to prevent further IgH gene rearrangements (20, 30). In line with this idea, recent data have
shown that Dµ protein transgene expression leads to a partial block in VH to DHJH rearrangements (39). For these
reasons, it is unlikely that a major fraction of cells carrying
VJ
joints but no DHJH rearrangement using reading frame
2 or no productive VHDHJH rearrangement had assembled
their IgL genes while expressing Dµ or µ chains, respectively, and altered their IgH gene complexes during subsequent rearrangements.
The data presented here are consistent with the earlier
detection of VJ
joints in B cell progenitors of mouse mutants unable to express µ chains (17) and support the
view that Ig gene rearrangements in CD43+ B cell progenitors of the mouse follow the "stochastic" model.
If rearrangements of IgH and IgL loci indeed occur independently in CD43+ B cell progenitors, productive and
nonproductive VHDHJH joints should distribute randomly
in cells bearing VJ
rearrangements. Although this is true
insofar as the ratio of productive to nonproductive joints is
similar in
chain+ V
J
rearrangement-containing and in
total CD43+ cells (~50%; Table VI and reference 29), it is
also obvious that, overall, the CD43+ progenitor population is selected for productive VHDHJH joints, as their frequency would be only 24% in a random distribution (considering that one-third of the joints are in-frame and that ~80% of the D elements in reading frame 3 contain stop
codons). An over-representation of productive versus nonproductive VHDHJH joints in these early progenitors has
been repeatedly observed in other experiments: 0.6 (reference 45), 0.6 (reference 30), and 0.8 (reference 46). How
can this selection be explained? Two possibilities can be
considered: either the bias is introduced by the expansion of pre-B cell receptor-expressing (and therefore µ+)
CD43+ progenitors that have downregulated RAG-1 and -2 expression (44, 47), or the CD43-expressing progenitors
that we have analyzed contain a subset of classical pre-B
cells in which RAG-1 and -2 are re-induced to mediate
gene rearrangement in IgL loci, but surface CD43 expression is (still) retained. The existence of such cells could explain the finding of Pelanda et al. (21) that in surface (s)Ig
,
CD43+, HSA+ B cell precursors, the frequency of cells expressing
light chains intracellularly is reduced approximately fourfold in the absence of the
5 gene product.
However, it is also possible that in the absence of
5,
and
µ chain-expressing progenitors transit more rapidly into
the compartment of sIgM+ B cells than in the wild-type.
That CD43+ B cell precursors are in principle able to express sIg has been shown in mice containing productively
rearranged heavy and light chain genes targeted into the
corresponding Ig loci (Lam, K.-P., personal communication).
Given those complexities, we cannot exclude that some
of the VJ
rearrangements that we have found in the
CD43+ B cell progenitors were induced upon pre-B cell
receptor expression, although we consider this unlikely.
However, the finding that about half of the CD43+ cells
bearing V
J
joints have yet to undergo IgH gene rearrangements for µ chain expression supports the concept originally developed from the analyses of mutant mice unable to express IgH chains (17), namely that in CD43+ B
cell progenitors, rearrangements of heavy and light chain
loci are initiated "stochastically", with an approximately
seven times higher frequency of rearrangements at the IgH
than at the Ig
loci (see below).
The order of the rearrangements at the IgH and IgL loci
determines the subsequent developmental route of the cell.
If a µ chain is assembled first, a pre-B cell receptor will be
expressed. The pre-B cell receptor gives a proliferative signal and directs the development of the cell to the CD43
pre-B cell compartment, where most IgL chain genes are
rearranged (16, 48). However, if an IgL chain is expressed
before or simultaneously with a µ chain, the cell is no
longer dependent on the pre-B cell receptor to enter the B
cell pool: as shown by Pelanda et al. (21), at least some
conventional
chains can substitute for the surrogate light
chain and promote the development of progenitor B cells.
Since we do not see any obvious bias towards some particular V
gene families among the
chain sequences derived
from CD43+ B cell progenitors, it seems that a large repertoire of V
J
joints can be generated in this compartment.
The existence of a pre-B cell receptor-independent developmental pathway that may be evolutionary more ancient than the pre-B cell receptor-driven pathway (17) may allow the generation of B cells whose µ chains are incapable of pairing with the surrogate light chain and thus are bound to die unless rescued by a conventional IgL chain. For example, a fraction of VH81X-bearing heavy chains does not associate with the surrogate light chain (49), and thus these VH81X-expressing B cells must be generated via the pre-B cell receptor-independent pathway. ten Boekel et al. (49) found that ~50% of heavy chains of early B cell progenitors using VH elements of the VHQ52 or VHJ558 families are unable to pair with the surrogate light chain. IgH chain-independent recombination of IgL chain genes thus might add antigen receptor specificities to the B cell repertoire that would not arise via the pre-B cell receptor- driven pathway.
Frequency of IgWe found 50 out of 627 fraction C cells and 32 out of 373 fraction B cells harboring
VJ
rearrangements. (We disregard the data obtained from
sorted
chain expressing cells from fraction B, because this
cell population was selected for high levels of
chain expression; see Results.) Taking into account the detection efficiency of the assay (70%) and the proportions of cells
bearing V
J
joints in the absence of productive VHDHJH
rearrangements (7 out of 14 in fraction C and 4 out of 11 in fraction B; Table VI), we estimate that 4-7% of cells in fractions B and C carry V
J
joints in the absence of a productive VHDHJH joint, and a similar proportion of cells contains both V
J
rearrangement(s) and a productive VHDHJH joint. Overall, the frequency of the cells carrying Ig
gene rearrangements is ~15% of the total CD43+ B cell
progenitor population in wild-type mice. This value correlates well with B cell production observed in
5-deficient
animals, which is reduced by ~95% (17, 50) and is dependent on the generation of Ig light chains in the absence of
pre-B cell receptor function. To obtain 5% of B cells generated in wild-type mice, Ig
genes must be rearranged in
15% of the B cell progenitors, assuming that one-third of
the joints are in-frame and that the B cell receptor induces
a similar extent of proliferation in the progenitor compartment as does the pre-B cell receptor.
The results reported here are in a good agreement with
previous data based on quantitative PCR analyses, in which
VJ
rearrangements represented ~7 and 15% in fractions B
and C, respectively, taking the level of V
J
rearrangements
in splenic B cells as 100% (17). Our results do not contradict the experiments of ten Boekel et al. (16), who did not
detect V
J
rearrangements among 24 cells of early progenitor B cell phenotype (c-kit+, CD25
, B220+). Since this
population includes fractions A, B, and C (according to
Hardy's classification, reference 23), and no V
J
rearrangements are detectable in fraction A (17), the frequency of
cells bearing V
J
joints in the population analyzed by ten
Boekel and colleagues is expected to be lower than 1 in 24 in these cells.
Immunoglobulin gene transcription and rearrangements
are coordinately regulated during B cell development (for
review see reference 51). It has been suggested that transcription of unrearranged genes is required for the initiation
of the V(D)J joining process. We have observed that in
3-83i mice carrying a productively rearranged V
J
joint in
the germline there are approximately three times more cells
expressing
light chains in fraction B compared with the
wild-type situation (Fig. 1). This difference is quantitatively
accounted for by the fact that two-thirds of the newly
formed rearrangements in the wild-type cells are nonproductive. Therefore, this result suggests that the "opening"
of the Ig
locus for transcription and for recombination occurs simultaneously and may thus be controlled by the
same factor(s). Moreover, this result shows that at this early
developmental stage wild-type cells rearranging Ig
genes
express the recombinatorial products at the protein level.
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Footnotes |
---|
Address correspondence to Tatiana I. Novobrantseva, Institute for Genetics, University of Cologne, Weyertal 121, 50931 Cologne, Germany. Phone: 49-221-470-52-91; Fax: 49-221-470-51-85; E-mail: ntanya{at}mac.genetik.uni-koeln.de
Received for publication 25 August 1998 and in revised form 19 October 1998.
We thank G. Zöbelein, C. Göttlinger and B. Hampel for their excellent technical help. We are grateful to Dr. L. Pao for critical reading of the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft through SFB 243, the Land Nordrhein-Westfalen, the EU Biotechnology (B104-CT96-0037), and the Human Frontier Science Program.
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