By
From the * National Jewish Medical and Research Center, Division of Basic Sciences, Department of
Pediatrics, Denver, Colorado 80206; and the University of Colorado Health Science Center,
Department of Immunology, Denver, Colorado 80220
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Abstract |
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Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression
sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of
immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is
transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that light chain genes
usually rearrange after
genes. This allowed us to analyze
loci in IgM
+ cells to determine
how frequently in-frame
genes fail to suppress
gene rearrangements. To do this, we analyzed recombined V
J
genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the
locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS
recombination is frequently induced by, and inactivates, functionally rearranged
loci, as
nearly half (47%) of the RS-inactivated V
J
joins were in-frame. These findings suggest that
receptor editing occurs at a surprisingly high frequency in normal B cells.
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Introduction |
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The fact that virtually all B cells express a single H and L
chain prompted many studies to elucidate the underlying mechanism. One process that clearly contributes to
allelic exclusion is the imprecision of V(D)J rearrangement
that generates a maximum of one in-frame rearrangement
per three attempts (1), but more active feedback processes
are also involved. Classic studies showing the ability of a H
chain transgene (2, 3) or an L chain transgene (4, and for
review see reference 5) to mediate feedback suppression of
H and L chain rearrangements, respectively, established important paradigms that have been widely accepted. But in
the case of L chain allelic exclusion, this paradigm was
weakened by an increasing number of "exceptions", in
which ongoing L chain rearrangement occurred despite expression of functional chain (6). Studies with autoantibody transgenic (Tg)1 mice suggested that many of the exceptions to the feedback regulation model of L chain allelic
exclusion could be explained by postulating self-tolerance-
induced receptor editing (10). In addition, recent in
vitro studies (16) and analyses of autoantibody Ig knock-in mice (19, 20) have shown that L chain gene receptor editing can be an important mechanism of B cell
tolerance. Despite these findings, it is unclear how frequently receptor editing is used for tolerance induction in
normal, non-Ig Tg autoreactive B cells, in part because the
extent of autoreactivity in the preselected B cell repertoire
is unknown.
The organization of the locus, with arrangement of V
genes in both sense and antisense transcriptional orientations, the absence of D region gene segments, and the presence of several J
gene segments facilitates sequential, nested
V
-to-J
rearrangement attempts (for review see reference
21). In developing B cells, these secondary rearrangements
can both rescue receptor expression in cells that fail to
assemble in-frame L chains (1, 22) and rescue autoreactive B
cells from tolerance elimination by replacing rearranged
genes with new ones that alter specificity (for review see
reference 23). Another way that the organization of the
locus promotes receptor editing is suggested by the existence of the conserved element known as recombining sequence (RS) in the mouse (or the homologous "
deleting
element" in humans; reference 24). RS is located ~25 kb
downstream of the C
exon (25) and has no coding function (26), but undergoes V(D)J recombinase-dependent rearrangement that inactivates the
locus by deletional rearrangements in cis (26) (see Fig. 1). In an autoantibody
knock-in model system, RS rearrangements can inactivate
functional
genes (20), but the extent of RS-mediated receptor editing in normal B cells remains unknown.
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One approach to estimate the extent of receptor editing
in normal B cells is to analyze V(D)J recombinational remnants that are the predicted residue of editing. In mouse B
cells, which contain both and
L chain loci,
gene rearrangement almost always occurs after
rearrangement (for
review see references 29, 30). Thus, if an appropriate
gene
is not assembled, rearrangement at the
locus often follows.
In
+ B cells, RS rearrangements usually have deleted the
C
loci (27, 28, 31) either by recombining to V
s, through
the well characterized heptamer-nonamer recombination
signal sequences (Fig. 1 B), or to heptamer sites in the
J
-C
intron (Fig. 1 C) (27, 28, 32). Besides destroying the
function of the
locus, this latter mode of RS recombination has two important effects: first, unlike nested V
J
recombinations, it eliminates the C
-associated cis-acting enhancer elements that are critical for V
J
expression and
rearrangement (33), and second, it retains any V
J
join
that was previously adjacent to C
. This physiological
knockout of regulatory sequences required for
gene rearrangement thus "freezes" the locus, allowing an analysis of
the V
J
gene that was assembled adjacent to the C
exon
just before RS and
gene rearrangement.
In this study, we have isolated such VJ
joins from a
large number of individual IgM+
+ B cells and determined
their nucleotide sequences in order to ascertain the extent
to which RS inactivates functional
genes in a normal,
non-Ig Tg immune system. The results indicate that in
normal IgM+ B cells RS-mediated receptor editing is induced by and frequently inactivates functionally rearranged
genes, probably because of immune tolerance.
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Materials and Methods |
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Mice.
Mice homozygous for the targeted deletion of the JCell Sorting and Genomic DNA Isolation.
Splenic cells from JCAnalysis of Direct PCR Amplified Ig Rearrangements.
Genomic DNA from sorted cells was used as a template to amplify VProduction of + Hybridomas.
Analysis of Hybridoma Ig Rearrangements.
Hybridoma genomic DNA was digested with EcoRI or BamHI, then fractionated on 0.8% agarose gels, blotted to nylon membrane, and hybridized with the RS 0.8 (27) or IVS probes (Fig. 1 A). VCloning and Expression of V(D)J Rearrangements for Analysis of H/L
Chain Pairing.
IgM ELISA for Analysis of H/
L Pairing.
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Results |
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To determine
the extent to which RS recombination inactivates functional, in-frame VJ
joins in the preimmune B cell repertoire, IgM+
+ splenic B cells were isolated by fluorescence
activated cell sorting and their genomic DNA was analyzed
by the PCR strategy outlined in Fig. 1 C. This cell sorting
strategy should exclude from the template pool cells that
are
+, H chain isotype switched, surface (s)Ig
, or cells of
a sIglo, germinal center phenotype. In a second series of experiments, IgM
secreting hybridomas were isolated and
their
loci analyzed in detail. To simplify these analyses, all
B cells analyzed were heterozygous for a targeted deletion
of the J
-C
locus (JC
D/+), in which only a single
locus and RS allele could rearrange (33). The potential
gene and RS element rearrangements are depicted in Fig. 1.
Genomic DNA from sorted IgM cells was used as template
for a PCR using a panspecific V
FWR 3 oligonucleotide
primer, which recognizes ~80% of V
genes (37), together
with an RS-specific primer to amplify V
J
-intron-RS rearrangements (Fig. 1 C, primers A and B). V
J
-intron-RS
rearrangements containing V
genes rearranged to each of
the four functional J
genes were detected by PCR amplification and Southern blotting (data not shown), but V
J
5-intron-RS rearrangements were most abundant, in part
because their smaller size promoted preferential amplification. Amplified V
J
5-intron-RS rearrangements were gel-purified and cloned, and a total of 52 clones were sequenced across both the V
J
and the J
-intron-RS joins (Fig. 2).
These two different recombination joins, present on each
PCR product analyzed, provided markers for uniqueness.
PCR products that were identical to one another, or that
differed by just one nucleotide, were assumed to represent
repeated isolates derived from the same initial template (i.e.,
derived from a single B cell clone). This represents an underestimate because the single base changes could have reflected
real differences and because it was possible that some of the
apparent repeats were independent events that happened to
have identity in the portions of the genes studied, but not in
upstream portions of the V genes. In this sample, at least 37 of the 52 clones represented independent events. Analysis of the V
J
join sequences allowed an assessment of the potential prior functionality of the V
J
5 joins just upstream of
intron-RS rearrangements. Surprisingly, 15 of the 37 clones
(41%) contained V
J
joins that were in-frame (Fig. 2), and
if the apparent repeats were not excluded 23 out of 52 (44%)
were in-frame.
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To verify the analysis of the PCR-amplified VJ
-intron-RS rearrangements and to increase the sample size, an independent sampling of V
J
-intron-RS rearrangements
was derived from JC
D/+ splenocytes in the form of B
cell hybridomas. A total of 133 IgM
-expressing hybrids
were obtained from five separate fusions and their
locus rearrangements were analyzed. Genomic Southern blot and
PCR analysis revealed that at least 74% of the
+ hybrids
(99 out of 133) had inactivated the wild-type
locus by
RS rearrangements (Table 1), a value in accord with previous estimates (31, 34). Two hybridomas apparently had undergone inversional V
-RS rearrangements, as they had
unique restriction fragments that retained the C
locus as
revealed by the intron (IVS) probe (data not shown), but
scored positive in a V
-RS PCR (Fig. 1 B). Approximately
25% (26 out of 99) of the hybridomas with RS rearrangements had J
-intron-RS joins (Table 1), as detected with
primers B and C (Fig. 1 C). Genomic Southern blot analysis of 18 out of 20 hybrids scoring PCR positive for J
-
intron-RS rearrangements demonstrated that the RS rearrangements colocalized with EcoRI restriction fragments
hybridizing with the IVS probe (data not shown), thus independently confirming the V
J
-intron-RS rearrangement phenotype. V
J
-intron-RS rearrangements from
individual hybridomas were PCR amplified and directly
sequenced, rather than cloned, a procedure that diminishes
potential Taq polymerase-generated mutations. Like the
V
J
-intron-RS PCR clones, most of the V
J
-intron-RS loci from hybridomas used J
5, although four hybridomas had rearrangements to upstream J
s, including one to
J
2 and three to J
4, suggesting that developing B cells do
not frequently undergo RS rearrangement until all of the
J
s are rearranged. Sequence analysis over both the V
J
and intron-RS joins clearly showed that each cell line had a
unique sequence at the V
J
join and that, remarkably, 12 of 20 (60%) of the V
J
joins were in-frame (Fig. 3 A).
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VJ
-intron-RS rearrangements were clearly diverse because at least 32 different V
genes representing 11 of the 19 V
families were identified
among the 57 independent V
J
-intron-RS loci analyzed (data not shown). This value of V
gene representation in
the V
J
-intron-RS loci analyzed is most likely an underestimate of the diversity because the 5' PCR primer lies in
FWR3 and yields only a short stretch of V
gene sequence
for interclonal comparison. Despite this limitation, multiple
genes were observed within particular V
gene families.
For example, within the V
4/5 family, at least 11 different
genes were represented among the 14 in-frame and 7 out-of-frame joins (data not shown). In addition, V
genes were sometimes found repeatedly in independent V
J
-intron-RS rearrangements and there was considerable overlap in
usage among hybridoma and PCR clone sequences. 13 of
the hybridomas used V
genes that were observed in the
direct PCR-derived clones, whereas 6 hybridomas expressed distinct V
genes that were members of families
observed in the PCR clone sample and 1 hybridoma expressed a V
32 gene, a V
family not seen in the PCR
clone samples (Figs. 2 and 3 and data not shown).
The sequences of the J-intron-RS
joins in both the PCR clones (Fig. 2) and hybridomas (Fig.
3 B) were quite varied and were dominated by deletions at
both sides of the joins, as up to nine nucleotides were missing from either the J
-intron or RS heptamer-flanking sequences. There did appear to be a bias for a particular join (e.g., clone 4, Fig. 2 A), which was observed to be associated with 13 independent V
J
rearrangements. Two of
the intron-RS joins contained P nucleotides and one contained N-region addition nucleotides, consistent with findings described previously (7, 43).
To determine if the high frequency
of in-frame VJ
rearrangements silenced by intron-RS recombination was due to the inability of H chains to pair with
their
L chains, the V(D)J and V
J
rearrangements from
hybridomas 2H11 and 15E11 were cloned into Cµ and C
expression vectors, respectively. These H and L chain constructs were cotransfected into SP2/0 myeloma cells to generate transfectoma clones. Analysis of transfectoma supernatants by IgM
sandwich ELISA revealed that the in-frame
L chains were able to pair with their hybridoma H chains
(Fig. 4), suggesting that ongoing RS rearrangement was not
due to the inability of H/L chain pairing. The specificity of
the µ
transfectoma antibodies remains unknown, however.
Attempts in flow cytometry assays to detect recombinant antibody binding to the surfaces of bone marrow cells were
unsuccessful (data not shown).
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![]() |
Discussion |
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In this report we examined the DNA sequences of VJ
joins located upstream of intronic-RS rearrangements in
normal, non-Ig Tg B cells to determine the extent to
which RS-mediated recombination silences functionally rearranged
genes. Nearly half of all V
J
joins inactivated by
RS recombination were in-frame (27 out of 57). This high
frequency is clearly incompatible with a strict feedback suppression model of L chain allelic exclusion, which predicts
no in-frame V
J
joins upstream of the RS rearrangements. More strikingly, this high frequency is also significantly
higher than 33%, the percentage of in-frame joins expected
from random V
J
rearrangement, indicating that productive V
J
rearrangements actively induce intron-RS rearrangements. The data also demonstrate a physiological role
for the RS element in normal B cell development
the inactivation of functionally rearranged
genes.
To understand why we conclude that the RS rearrangements were actively induced by functional L chains, consider the extreme hypothetical cases of mice in which all
gene rearrangements result in either autoreactive B cell receptors or nonproductive
chains (Table 2). If V
-to-J
and RS rearrangements proceed randomly, albeit with different relative frequencies, then in either case V
J
joins
located upstream of intronic-RS rearrangements should be
in-frame at a maximum frequency of one out of three. To
significantly exceed this frequency, in-frame V
J
joins
must stimulate the relative rate of (intronic) RS rearrangement. This argument applies to our data because the observed frequency of in-frame joins, 47.4%, is significantly
higher than one out of three (P < 0.04, single sample test
of a proportion based on a normal approximation). Since it
is exceedingly unlikely that the stimulus for increased in-frame rearrangements is mediated by anything other than
protein, and because
chains can probably only be perceived by the signaling machinery of B cells through their
association with H chains, we conclude that functional
chains actively stimulate the rate of RS rearrangement
based on B cell receptor antigenic specificity. These data
also predict that in mice in which the C
exon is inactivated, but surrounding cis-acting elements are left intact,
V
J
rearrangement should be extensive, whereas RS rearrangement should be reduced. This is in fact the experimental observation (44).
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The statistical argument also excludes the possibility that
a high frequency of rearrangeable V pseudogenes, L
chains that fail to pair with H chains, or a role for positive
selection is responsible for our results. Furthermore, complete sequencing of the coding regions from all the in-frame V
J
rearrangements derived from
+ hybridomas
revealed no stop codons or other obvious defects that would have precluded function (Fig. 3 A). It is also unlikely that frequent aberrant H/L chain pairing is responsible for the high frequency of in-frame V
J
joins in the
V
J
-intron-RS rearrangements, as demonstrated by the
ability of H/
L chains from two hybridomas to pair (Fig.
4). Moreover, there are few examples of L chains that fail
to pair with H chains and most experiments suggest that virtually all random H/L pairs can associate (40, 45).
Finally, if a lack of positive selection of surface Ig was responsible for the high frequency of in-frame joins, this
would predict that B cells should frequently express two
chains, a result that has not been observed.
The receptor editing events documented in this study
probably do not represent renewed V(D)J recombination
in mature B cells, such as has recently been described in the
germinal center (49), because the cells analyzed expressed high levels of IgM and chain and because they
were isolated and, in the case of the hybridomas, stimulated
in a manner that should not have induced V(D)J recombination. Another indication that receptor editing in mature
B cells is unlikely to explain our results is that the fraction
of
+ cells in newly formed and mature splenic B cells is
nearly identical, suggesting that in unmanipulated mice
mature
+ cells rarely give rise to
+ B cells (44). Overall,
it would appear from our data that the RS rearrangements
that we studied were actually stimulated, rather than inhibited, by productive
gene rearrangements, probably as the
result of immune tolerance-mediated receptor editing in immature B cells. To definitively test the prediction that
the
chains of the cells that we have analyzed generate autoantibodies in association with the same cell's heavy chain,
it will be necessary to generate mice transgenic for these
genes.
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Footnotes |
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Address correspondence to David Nemazee, The Scripps Research Institute, Mail drop IMM-29, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: 619-784-9528; Fax: 619-784-8805; E-mail: nemazee{at}scripps.edu
Received for publication 20 February 1998 and in revised form 22 July 1998.
We thank D. Huszar for providing JCD/JC
D mice; B. Diamond (Albert Einstein College of Medicine,
Bronx, NY) for providing the NSO-bcl2 myeloma; S. Sobus for cell sorting; D. Norsworthy for cycle sequence analyses; D. Iklé of the Biostatistics Department for statistical analyses; K. Karjalainen and L. Wysocki
for discussions; and M. Hertz, D. Melamed, V. Kouskoff, and other members of the lab for critical reading of
the manuscript.
This work was supported by grants from the National Institutes of Health and the Arthritis Foundation.
Abbreviations used in this paper
FWR, framework region;
JCD/+, heterozygous
deficient germline genotype;
RS, recombining sequence;
Tg, transgenic.
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