![DNA specificity of Ig H/L chain combinations. DNA specificity of secreted transfectoma IgM antibodies was evaluated in competition experiments, in which increasing amounts of M13 phage ssDNA (squares) or dsDNA (circles) were used as competitive inhibitors of E. coli [14C]dsDNA binding to the individual purified antibodies in the nitrocellulose filter assay. The amount of each antibody was adjusted to bind 50 percent of 25 ng (1,500 cpm) input [14C]dsDNA in the absence of inhibitor.](https://www.researchgate.net/profile/Nurit-Yachimovich-Cohen/publication/11432603/figure/fig5/AS:267814011404294@1440863244135/DNA-specificity-of-Ig-H-L-chain-combinations-DNA-specificity-of-secreted-transfectoma_Q320.jpg)
![Anti-DNA activity in the serum of Ig H/L chain targeted mice. Undiluted or sequentially diluted in borate-buffered saline, heat-inactivated (56°C, 30 min) serum samples (10 μl) were tested for dsDNA binding in the nitrocellulose filter assay. Input E. coli [14C]dsDNA was 25 ng (1,500 cpm). Background radioactivity (with no added serum) was subtracted from all binding values. Average binding values are indicated for each mouse strain.](https://www.researchgate.net/profile/Nurit-Yachimovich-Cohen/publication/11432603/figure/fig1/AS:267546590969866@1440799486091/Anti-DNA-activity-in-the-serum-of-Ig-H-L-chain-targeted-mice-Undiluted-or-sequentially_Q320.jpg)
Content uploaded by Nurit Yachimovich-Cohen
Author content
All content in this area was uploaded by Nurit Yachimovich-Cohen on Jul 27, 2018
Content may be subject to copyright.
0014-2980/02/0404-1164$17.50+ .50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2002
The efficiency of B cell receptor (BCR) editing is
dependent on BCR light chain rearrangement
status
Nurit Yachimovich, Gustavo Mostoslavsky, Yuval Yarkoni, Inna Verbovetski and
Dan Eilat
Department of Medicine, Hadassah University Hospital, Faculty of Medicine, Hebrew University,
Jerusalem, Israel
Anti-DNA knock-in mice serve as models for studying B cell tolerance mechanisms to a
ubiquitous antigen. We have constructed six strains of double transgenic (C57BL/6×BALB/
c)F1 mice, each expressing an unmutated or somatically mutated anti-DNA heavy (H) chain,
combined with one of three different light (L) chains, namely V
‹
1-J
‹
1, V
‹
4-J
‹
4andV
‹
8-J
‹
5. In
vitro analysis of the various Ig H/L chain combinations showed that all had a similar specific-
ity for single-stranded DNA and double-stranded DNA, but that antibodies encoded by the
mutated H chain had higher affinities for the autoantigen. None of the targeted mouse strains
exhibited significant levels of serum anti-DNA activity. However, while B cells from mice car-
rying the V
‹
1-J
‹
1 transgenic L chains were tolerized almost exclusively by L chain receptor
editing in an affinity-independent manner, the mice expressing V
‹
8-J
‹
5 L chains have utilized
affinity-dependent clonal anergy as their sole mechanism of B cell tolerance. V
‹
4-J
‹
4tar-
geted mice exhibited an intermediate phenotype with respect to these two mechanisms of B
cell tolerance. Our results suggest that receptor editing is the preferred mechanism of B cell
tolerance and that the efficiency of L chain editing is directly related to the number of avail-
able J
‹
segments on the expressed V
‹
allele.
Key words: Tolerance / Receptor editing / Anergy / Lupus / Transgenic mice
Received 14/9/01
Revised 13/12/01
Accepted 22/1/02
[I 22467]
Abbreviations: BCR: B cell receptor ds: Double stranded
ss: Single stranded NZB: New Zealand Black NZW: New
Zealand White ES: Embryonic stem CFSE: Carboxyfluo-
rescein diacetate succinimidyl ester RAG: Recombinase
activating gene
1 Introduction
B cell receptor (BCR) editing by secondary rearrange-
ment of immunoglobulin (Ig) genes has been identified in
recent years as a major mechanism of B cell tolerance to
self antigens [1–3]. However, its relation to other well-
known tolerance mechanisms, such as clonal deletion
[4, 5] and clonal anergy [6, 7] has not been well estab-
lished. For example, it is not known whether there is a
temporal relationship between receptor editing and other
tolerance mechanisms. Nor is it clear how the specificity
or affinity of the tolerogenic antigen influences the choice
of the appropriate tolerance mechanism.
We have previously established two transgenic mouse
lines, in which germ-line-encoded or somatically mu-
tated, rearranged heavy chain variable region genes,
derived from an anti-DNA hybridoma (D42) were targeted
to the IgH-chain locus of the mouse [8]. On a non-
autoimmune (C57BL/6×BALB/c)F1 mouse genetic back-
ground, these H-chain-only knock-in mice were shown
to be tolerant in that they did not produce significant
amounts of anti-DNA antibodies in their serum [8]. How-
ever, when transferred to the lupus-prone NZB/NZW F1
genetic background, the targeted D42H chain gave rise
to very high titers of DNA-binding antibodies due to
selection of particular Ig heavy/light chain combinations
with high affinity for double-stranded (ds) DNA [9]. In tol-
erant mice, a quantitative analysis of B cell populations
inthebonemarrow(BM)aswellasofJ
‹
utilization and
DNA binding of hybridoma Ab suggested that immature
B cell deletion and L chain editing were the major toler-
ance mechanisms. Surprisingly, these mechanisms were
less effective in targeted mice expressing the somatically
mutated anti-DNA H chain (D42H), than in mice express-
ing the germ-line-encoded H chain (glD42H), possibly
due to the greater abundance of high affinity, anti-DNA B
cells in mice with the mutated D42H. Autoreactive B cells
in the periphery of D42H targeted mice exhibited several
features of functional inactivation (clonal anergy) [8].
1164 N. Yachimovich et al. Eur. J. Immunol. 2002. 32: 1164–1174
Fig. 1. DNA specificity of Ig H/L chain combinations. DNA
specificity of secreted transfectoma IgM antibodies was
evaluated in competition experiments, in which increasing
amounts of M13 phage ssDNA (squares) or dsDNA (circles)
were used as competitive inhibitors of E. coli [14C]dsDNA
binding to the individual purified antibodies in the nitrocellu-
lose filter assay. The amount of each antibody was adjusted
tobind50percentof25ng(1,500cpm)input[
14C]dsDNA in
the absence of inhibitor.
To gain a better understanding of the inter-relationships
between different tolerance mechanisms and their de-
pendence on the specificity and affinity for the tolerogen,
we have now constructed several new knock-in (C57BL/
6×BALB/c)F1 mouse lines with discrete Ig H/L chain
combinations. Specifically, the D42H and glD42H trans-
genic mice were crossed with knock-in mice expressing
V
‹
1- J
‹
1, V
‹
4- J
‹
4 [10] and V
‹
8- J
‹
5 [11] L chains. None of
the ensuing six lines of H/L chain double transgenic mice
produced DNA binding antibodies in their serum. A
detailed analysis of the various mechanisms of B cell tol-
erance in these mouse lines shows a hierarchy in their
ability to edit their BCR and suggests that the L chain
rearrangement status, rather than BCR affinity for the self
antigen determines the efficiency of receptor editing. In
contrast, non-editing self-reactive B cells are subjected
to clonal anergy and the intensity of their functional inac-
tivation is dependent on their BCR affinity for the self
antigen.
2 Results
2.1 Characterization of anti-DNA Ig H/L chain
combinations
Non-secreting NSO myeloma cells were stably co-
transfected with linearized plasmid vectors expressing
various combinations of the anti-DNA
?
HchainsD42Hor
glD42H [12] and the
‹
L chains V
‹
1-J
‹
1 targeting vector
(this report), V
‹
4- J
‹
4 [13] and V
‹
8-J
‹
5 [14]. G418-
selected, cloned transfectomas were grown in serum-
free medium and the secreted antibodies were purified
by column chromatography and evaluated for DNA
specificity. Fig. 1 shows that, within the limits of experi-
mental error, the different
?
/
‹
chain combinations had
the same specificity for single-stranded (ss) DNA and
dsDNA. In all cases, both ssDNA and dsDNA with the
same nucleotide sequence (M13 phage DNA) competed
with transfectoma antibodies for binding to E. coli
dsDNA in solution. These binding measurements in solu-
tion eliminate possible orientation effects that are likely
to occur when DNA is bound to solid-phase supports
(e.g. in ELISA). The results shown in Fig. 1 are consistent
with the notion [8] that the affinity and specificity of
D42H/L chain anti-DNA antibodies are primarily depen-
dent on the D42 heavy chain. Additionally, the various
?
‹
transfectoma antibodies showed a very similar speckled
pattern in the fluorescent anti-nuclear Ab (FANA) test
(data not shown; see also Fig. 5C in [9]).
The apparent affinities of the various Ig
?
/
‹
chain combi-
nations for dsDNA were measured by the nitrocellulose
filter assay and calculated from Scatchard plots [12].
Table 1 shows that the six transfected H/L chain combi-
nations may be divided into a lower affinity group com-
posed of the three L chains paired with the germ-line-
encoded D42H chain and a higher affinity group, com-
posed of the same L chains paired with mutated D42H.
Within each group, the difference in apparent affinity is
1.4–2.3-fold, while the two groups differ by a factor of
2–8. A similar H chain influence on antibody affinity is
evident in the
?
/
‹
transfectoma derived from the original
NZB/NZW anti-DNA hybridoma, where the D42H/V
‹
D42
H/L pair has about 12-fold higher affinity for dsDNA than
its unmutated counterpart (Table 1 and [12]). However,
the D42H/V
‹
D42 chain combination that is representa-
tive of anti-DNA antibodies produced by NZB/NZW H
chain-only transgenic B cells [9] has 50–90-fold higher
affinity for DNA than the combination of the same H
chain with the three transfected L chains. Thus, the H/L
pairs in this study represent low to medium affinity anti-
DNA antibodies that are typically produced by H chain-
only non-autoimmune mice [9].
2.2 Anti-DNA activity in the serum of Ig H/L
double-transgenic mice
Six lines of anti-DNA knock-in mice, heterozygous for
both targeted heavy and light chains were constructed
by crossing glD42H or D42H single transgenic C57BL/6
mice with BALB/c mice, targeted with V
‹
1-J
‹
1, V
‹
4-J
‹
4or
V
‹
8-J
‹
5 L chains. All of the double transgenic mice pro-
duced low serum titers of dsDNA binding antibodies
(Fig. 2) that did not increase with age and were not sig-
nificantly different from those of non-transgenic or D42H
chain-only transgenic (C57BL/6×BALB/c)F1 mice. For
Eur. J. Immunol. 2002. 32: 1164–1174 B cell receptor editing and clonal anergy in anti-DNA transgenic mice 1165
Fig. 2. Anti-DNA activity in the serum of Ig H/L chain tar-
geted mice. Undiluted or sequentially diluted in borate-
buffered saline, heat-inactivated (56°C, 30 min) serum sam-
ples (10
?
l) were tested for dsDNA binding in the nitro-
cellulose filter assay. Input E. coli [14C]dsDNA was 25 ng
(1,500 cpm). Background radioactivity (with no added
serum) was subtracted from all binding values. Average
binding values are indicated for each mouse strain.
comparison, D42H chain-only female NZB/NZW F1 mice
had serum anti-DNA titers that were several orders of
magnitude higher than those of all single- and double-
transgenic non-autoimmune mice (Fig. 2).
2.3 BCR editing in anti-DNA Ig H/L chain double-
transgenic mice
The very low titers of DNA binding antibodies in the sera
of H/L chain targeted mice could be due to the relatively
low affinity of the anti-DNA antibodies produced in these
mice (Table 1) or to the induction of tolerance mecha-
nisms that operate on higher affinity autoreactive Bcells.
To test the latter possibility, the mechanism of receptor
editing by secondary rearrangements in Ig L chains was
evaluated by direct sequencing of expressed L chains in
LPS hybridomas obtained from spleen cells of the vari-
ous double transgenic mouse lines. Fig. 3 shows the
rearrangement status of
‹
light chains obtained from
hybridomas produced from the different double trans-
genic mice. The vast majority of both low-affinity
glD42H/V
‹
1-J
‹
1 and medium-affinity D42H/V
‹
1-J
‹
1B
cells (100% and 89%, respectively) were shown to edit
Table 1. DNA binding affinity of Ig H/L chain combinations
H/L combination Apparent affinity (M–1/bp)
glD42H/V
‹
1-J
‹
14.6×10
6
D42H/V
‹
1-J
‹
11.1×10
7
glD42H/V
‹
4-J
‹
42.0×10
6
D42H/V
‹
4-J
‹
48.7×10
6
glD42H/V
‹
8-J
‹
53.3×10
6
D42H/V
‹
8-J
‹
51.6×10
7
glD42H/V
‹
D42-J
‹
56.5×10
7
D42H/V
‹
D42-J
‹
57.8×10
8
their
‹
L chains by secondary rearrangements to down-
stream J
‹
minigenes, particularly to J
‹
5 (87% and 56%
for glD42H/V
‹
1 and D42H/V
‹
1, respectively). These sec-
ondary rearrangements were not biased towards any
particular V
‹
segment and both J
‹
-proximal and J
‹
-distal
V
‹
genes were identified in L chain cDNA (data not
shown). PCR analysis of
‹
alleles in glD42H/V
‹
1-J
‹
1
hybridomas showed that 10 of 32 edited hybridomas
(31%) retained a germ-line configuration on the untar-
geted
‹
allele, suggesting that in most of these B cells, L
chain editing involved both the transgenic and endoge-
nous
‹
alleles (data not shown). Some of these hybrid-
omas (4/19) demonstrated a PCR product that could
reflect an RS-dependent C
‹
deletion [15] of the original
transgene (not shown). It should be pointed out that the
percentage of non-rearranged endogenous
‹
alleles is a
maximum estimate, since rearrangement by inversion to
any J
‹
segment except J
‹
1 could retain the PCR product
of the germ-line configuration.
In a sharp contrast to the almost complete L chain
receptor editing in mice expressing D42H/V
‹
1BCR,the
overwhelming majority of B cells expressing glD42H/
V
‹
8-J
‹
5 and D42H/V
‹
8-J
‹
5 retained their original Ig H/L
chain combination (Fig. 3). PCR analysis of
‹
alleles in
glD42H/V
‹
8-J
‹
5 hybridoma DNA samples indicated that
all (18/18) had retained the germ-line configuration on
the untargeted
‹
allele (data not shown). None of these
hybridomas showed an RS-dependent C
‹
deletion. In
transgenic mice expressing glD42H/V
‹
4orD42H/V
‹
4
BCR, the level of L chain receptor editing was intermedi-
ate between the D42/V
‹
1 and D42V
‹
8 knock-in mice
(Fig. 3). Of the V
‹
4-J
‹
4-expressing hybridomas, 40–60%
retained their original H/L chain combination. The rest
have edited their L chains, mostly to J
‹
5. The great
majority of these secondary rearrangements appeared to
involve both
‹
alleles, since only 3 of 16 originally
1166 N. Yachimovich et al. Eur. J. Immunol. 2002. 32: 1164–1174
Fig. 3. J
‹
usage in hybridoma cells derived from Ig H/L chain
targeted mice. J
‹
usage was determined by direct sequenc-
ing of hybridoma RNA following reverse transcription-PCR
amplification. Empty bars represent expression of the origi-
nal V
‹
-J
‹
transgenic L chain.
Table 2.
Q
light chain and
?
aexpression in splenic B cells of Ig H/L chain targeted mice
Mouse B cells/spleen
(× 10–7)
Q
-expressing cells
(% of B cells)
?
a-expressing cells
(% of B cells)
glD42H/V
‹
1-J
‹
12.5±0.9(n=9) 5.1±1.2(n=7) 7.3±1.3(n=6)
D42H/V
‹
1-J
‹
11.4±0.7(n= 11) 6.9 ± 0.8 (n=8) 19.8±6.5(n=5)
glD42H/V
‹
4-J
‹
43.2±0.5(n=4) 3.2±1.0(n=4) 2.9±0.4(n=4)
D42H/V
‹
4-J
‹
41.7±0.7(n=7) 7.6±1.4(n=7) 14.9±2.7(n=5)
glD42H/V
‹
8-J
‹
55.0 ± 1.9 (n=12) 0.4 ± 0.2 (n=7) 1.4 ± 0.9 (n=9)
D42H/V
‹
8-J
‹
53.7 ± 2.1 (n=7) 0.2 ± 0.1 (n=4) 2.0 ± 1.6 (n=9)
expressing V
‹
4-J
‹
4 hybridomas have retained the
unrearranged germ-line configuration. Secondary
rearrangements apparently occurred also in V
‹
4-J
‹
4
hybridomas that continued to express the targeted
transgene, since 6/9 of these hybridomas had a non-
germ-line configuration on the unexpressed allele. As in
the case of V
‹
1-J
‹
1 hybridomas, no bias of particular V
‹
genes in L chain secondary rearrangements was evident
in V
‹
4-J
‹
4 hybridomas (data not shown).
To explore further the difference between the various Ig
H/L chain transgenic mice in their capacity to induce
BCR editing, the relative number of splenic B cells
expressing
Q
L chains was investigated by FACS analy-
sis. Table 2 shows that V
‹
1-J
‹
1 and V
‹
4-J
‹
4knock-in
mice had similar levels (3.2–7.6%) of
Q
-expressing
splenic B cells. If correction is made for the number of B
cells per spleen, and assuming that
Q
-expressing cells
are not deleted, then there is little difference in the levels
of
Q
expression between B cells harboring low-affinity
glD42H combinations with V
‹
1-J
‹
1orV
‹
4-J
‹
4 and their
higher affinity D42H/L chain counterparts. Strikingly,
however, the number of
Q
-expressing cells among B cells
from mice transgenic for glD42H and D42H chain combi-
nations with V
‹
8-J
‹
5 was extremely low (Table 2), further
indicating that these cells did not edit their BCR L chains.
In a previous study [12] we showed that the targeted, b-
allotype D42H chain could be inactivated by specific
rearrangements of DHor VH-DHinto the H chain leader
intron, thereby activating the non-transgenic, endoge-
nous H chain allele that expresses the a-allotype H
chains. Although these secondary rearrangements are
made possible due to the non-physiological retention of
the heavy chain D region on the targeted allele, they may
nevertheless represent an ongoing editing process.
Indeed, the relative number of
?
a-expressing B cells in
mice targeted with V
‹
8-J
‹
5 H/L chain combinations was
very low compared with mice having the V
‹
1-J
‹
1 and
V
‹
4-J
‹
4 H/L combinations (Table 2). This difference still
holds when one corrects for the number of B cells per
spleen and assumes that
?
a-expressing B cells are not
deleted. The same proportions of
?
aand
?
bchains were
seen in the various hybridoma populations derived from
the corresponding transgenic mice, suggesting that LPS
hybridomas truly represent the spectrum of B cell popu-
lations in vivo. The lack of H chain editing in V
‹
8-J
‹
5-
expressing B cells is compatible with the very low levels
of
‹
and
Q
L chain editing in mice harboring these trans-
genic B cells.
Finally, RAG mRNA levels were measured in bone mar-
row cells derived from the various Ig H/L chain knock-in
mice. RAG levels in targeted bone marrow B cells should
be roughly proportional to the rate of ongoing BCR edit-
ing, since both H and L chains were prerearranged in B
cells of these double-transgenic animals. Total RNA was
prepared, and levels of RAG-2 were determined by a
Eur. J. Immunol. 2002. 32: 1164–1174 B cell receptor editing and clonal anergy in anti-DNA transgenic mice 1167
Fig. 4. RAG-2 expression in bone marrow cells from Ig H/L
chain targeted mice. RAG-2 mRNA was measured by a
semiquantitative reverse transcription-PCR, followed by blot
hybridization with CD19 and RAG-2 DNA probes. The ratio
of CD19 to RAG-2 signal was determined by densitometry
after subtracting the individual backgrounds.
semiquantitative reverse transcription-PCR. As a control
for the B cell number and gel loading, samples were ana-
lyzed in parallel for CD19 expression (Fig. 4). Quanti-
tation of the radioactive signal revealed that RAG-2
expression levels were similar in non-transgenic (C57BL/
6×BALB/c)F1 mice and transgenic D42H/ V
‹
1-J
‹
1 and
D42H/ V
‹
4-J
‹
4 mice. In contrast, RAG-2 levels in D42H/
V
‹
8-J
‹
5 knock-in mice were tenfold lower (Fig. 4). These
results are consistent with the low rate of H and L chain
editing in V
‹
8-J
‹
5-expressing mice and suggest that V
‹
8-
targeted mice are not using receptor editing as a major
mechanism of B cell tolerance.
2.4 B cell clonal anergy in anti-DNA Ig H/L chain
double-transgenic mice
Since Ig H/L chain targeted mice carrying the V
‹
8-J
‹
5
transgenic L chain did not seem to edit their BCR, other
mechanisms of immunological tolerance should be
responsible for the very low anti-DNA levels in their
serum. We, therefore, looked at different parameters of
clonal anergy in these knock-in mice. In particular, BCR
density [7, 8, 16] as well as B cell proliferation [17] follow-
ing LPS [7, 8] or anti-CD40 [18] stimulation were exam-
ined in these mice. Fig. 5A shows that the mean fluores-
cence intensity (MFI) of IgMb, which is a measure of BCR
density on the surface of B cells from transgenic mice
carrying the V
‹
8-J
‹
5 L chain, was reduced compared
with the IgMbsurface density of non-transgenic C57BL/6
B cells. However, the induction of clonal anergy, as rep-
resented by BCR density, was dependent on BCR affini-
ty for antigen. The MFI of IgMbon D42H/V
‹
8-J
‹
5-
expressing B cells was drastically reduced (
˚
sevenfold),
while the MFI of IgMbon glD42H/ V
‹
8-J
‹
5wasonly
slightly reduced (
˚
twofold), as compared with non-
transgenic C57BL/6 mice (Fig. 5A). Presumably, a rela-
tively small decrease in receptor density is sufficient to
anergize B cells that express low-affinity BCR. This situa-
tion is different from that of L chain editing (Fig. 3), where
secondary rearrangements were independent of BCR
affinity for antigen within the range of affinities measured
for anti-DNA antibodies in this study. The BCR densities
of D42H/V
‹
4-J
‹
4 and D42H/V
‹
1-J
‹
1 transgenic B cells
were also reduced (
˚
fourfold), although to a lesser
extent than that of D42H/V
‹
8-J
‹
5 (Fig. 5A), probably
because some of their edited H/L chain combinations
(estimated at
˚
30%, [8]) are still capable of binding DNA,
due to the dominance of the targeted H chain (Indeed,
the BCR densities of the editing H/L combinations were
similar to that of D42H chain-only transgenic mice, data
not shown). However, the difference between BCR den-
sities of mutated and non-mutated H chains in combina-
tion with editing V
‹
4-J
‹
4andV
‹
1-J
‹
1 was narrowed as
compared with the difference between D42H/V
‹
8-J
‹
5
and glD42H/V
‹
8-J
‹
5 (Fig. 5A). This is apparently because
the latter, non-editing combinations are fixed in their
anti-DNA affinities, while most of the editing L chains (in
combination with either glD42H or D42H) have given rise
to similar, non-DNA binding H/L chain combinations.
To further demonstrate features of clonal anergy and
BCR affinity dependence of anergy induction, splenic B
cells from the H/L chain knock-in mice were stimulated
in vitro for 3–4 days with either 1
?
g/ml LPS or anti-CD40
antibody and measured for proliferation as compared
with non-transgenic C57BL/6 B cells. Fig. 5B shows by
CFSE dilution [17] that the highest inhibition of cell divi-
sion was apparent in LPS-stimulated D42H/V
‹
8-J
‹
5
transgenic B cells, but not in glD42H/V
‹
8-J
‹
5 B cells.
Substantial differences in B cell proliferation were also
noted between the mutated and non-mutated D42H
chain combinations with V
‹
4-J
‹
4andV
‹
1-J
‹
1. Virtually
identical results of B cell proliferation were obtained fol-
lowing anti-CD40 antibody stimulation in vitro (data not
shown). Collectively, these data indicate that the medium
affinity D42H/V
‹
8-J
‹
5-expressing B cells are tolerized by
functional silencing (clonal anergy), while the effect of
this tolerance mechanism on the lower affinity glD42H/
V
‹
8-J
‹
5ismuchsmaller,suggestingthatthestrengthof
the inactivation signal is critically dependent on BCR
affinity for the tolerizing antigen.
2.5 Bone marrow B cell compartments in anti-
DNA Ig H/L chain double-transgenic mice
Table 3 shows the relative sizes of the major develop-
mental B cell compartments in the bone marrow in the
various strains of Ig H/L chain knock-in mice. It is evident
that the pro-B cell compartment has about the same rel-
ative size in all the transgenic mice tested. However, the
mice expressing H/L chain combinations with V
‹
8-J
‹
5
targeted L chain have substantially different proportions
1168 N. Yachimovich et al. Eur. J. Immunol. 2002. 32: 1164–1174
Fig. 5. B cell anergy in Ig H/L chain targeted mice. Splenic B cells from mice transgenic for V
‹
1-J
‹
1, V
‹
4-J
‹
4orV
‹
8- J
‹
5 L chains,
in combination with glD42H (thin line) or D42H (thick line) H chains were measured for BCR density (A), using a heavy chain anti-
mouse
?
bantibody in flow cytometric analysis. For comparison, non-transgenic
?
b-expressing C57BL/6 B cells were also ana-
lyzed. Splenic (B220+) B cells from the H/L double-transgenic mice were also studied for LPS-induced proliferation, as analyzed
by CFSE dilution (B). The MFI of
?
bBCR density (A) and the MFI of R1-gated, dividing cells and their percentage of total trans-
genic B cells (B) are given in the accompanying table. The results represent several experiments in which the same relative MFI
were obtained.
Table 3. Bone marrow B cell compartments in Ig H/L chain targeted mice
Pro-B Pre-B Immature B Mature B
Mouse n%a) %a) %a) %a)
B220lo CD43+B220lo CD43–B220lo CD43–B220hi CD43–
IgM–IgM–IgM+IgM+
Non-transgenic 10 3.2 ± 2.1 66.2 ± 7.0 19.1 ± 7.3 11.5 ± 4.3
glD42H/V
‹
1-J
‹
1 6 5.3 ± 2.6 71.1 ± 11.9 10.4 ± 4.3 13.1 ± 10.3
D42H/V
‹
1-J
‹
1 6 3.0 ± 1.9 75.0 ± 11.1 13.1 ± 4.3 8.7 ± 16.0
glD42H/V
‹
4-J
‹
4 5 12.2 ± 3.6 63.4 ± 11.9 7.8 ± 2.9 16.5 ± 8.2
D42H/V
‹
4-J
‹
4 6 2.6 ± 0.6 76.2 ± 13.9 12.0 ± 5.9 9.0 ± 5.1
glD42H/V
‹
8-J
‹
5 10 3.2 ± 1.2 43.0 ± 7.9 21.4 ± 8.9 32.4 ± 13.0
D42H/V
‹
8-J
‹
5 6 2.9 ± 0.7 39.4 ± 6.2 18.3 ± 9.2 39.3 ± 12.2
a) Percent of B cells
Eur. J. Immunol. 2002. 32: 1164–1174 B cell receptor editing and clonal anergy in anti-DNA transgenic mice 1169
of pre-B, immature B and mature B cell compartments in
the bone marrow. In V
‹
1-J
‹
1- and V
‹
4-J
‹
4-expressing B
cells, there is a very high ratio of IgM-“pre-B” cells to
IgM+immature/mature B cells. This probably reflects the
fact that most of the cells that initially express the tar-
geted autoreactive BCR are induced to modulate their
BCR as a result of contact with antigen and assume a
pre-B-like phenotype to edit their receptors [19, 20]. It is
also consistent with the finding that B cells undergoing
editing are specifically delayed in the small pre-BII cell
compartment [21]. In contrast, the ratio of immature/
mature B cells to pre-B cells is reversed in the bone mar-
row of Ig H/L chain knock-in mice that express the V
‹
8-
J
‹
5 transgenic L chain. These anergized B cells would
readily enter the immature and mature developmental
stage and leave the bone marrow to enter peripheral
organs such as the spleen. Indeed, the total number of
splenic B lymphocytes, known to consist of immature
and mature B cells, was found to be higher in V
‹
8-J
‹
5-
expressing H/L chain mice, than in the corresponding
V
‹
1-J
‹
1orV
‹
4-J
‹
4 transgenic mice (Table 2). This sug-
gests that many of the latter B cells are lost through non-
productive secondary rearrangements.
3 Discussion
This study extends our investigation of B cell tolerance
mechanisms to a ubiquitous antigen (DNA) in normal and
autoimmune-prone animals. The anti-DNA H-chain-only
mice [8, 9] have the advantage that their targeted H
chains were allowed to combine with the complete,
native L chain repertoire of the mouse, presumably giv-
ing rise to a wide spectrum of anti-DNA antibody affini-
ties. However, one has to make several assumptions to
interpret the experimental results. These include [8] (i)
that the targeted H chains will combine with a large vari-
ety of L chains to give DNA binding BCR, and (ii) that the
dominance of the H chain in determining affinity for DNA
can result in higher affinity anti-DNA BCR for most H/L
chain combinations containing the mutated D42 H chain,
as compared with those having the germ-line-encoded
H chain. None of these assumptions have to be made in
the present study, where the targeted H/L pairs were
compared for their affinity and anti-DNA specificity. The
limitation of this study, however, is that very high affinity
anti-DNA H/L combinations, that are typical of lupus-
prone NZB/NZW F1 mice (Table 1) and are relatively rare
for a given anti-DNA H chain [9], were not included. This
would explain why clonal deletion, a B cell tolerance
mechanism that is probably responsible for direct elimi-
nation of the very-high-affinity anti-DNA B cells in the
normal animal [4, 8] is not demonstrated in this study,
except as an indirect consequence of receptor editing. In
contrast, the low- and medium-affinity anti-DNA trans-
genic mice that we have generated permit us to study
several important characteristics of two other well-
documented B cell tolerance mechanisms, clonal anergy
and receptor editing.
The relationship between editing and anergy comes to
focus in this study, because of the similarities in affinity
and specificity and the differences in rearrangement sta-
tus within the different H/L-targeted mice. Most striking
is the difference in the choice of tolerance mechanism by
anti-DNA transgenic mice carrying the V
‹
1-J
‹
1 and V
‹
8-
J
‹
5 L chains. The V
‹
1-J
‹
1 mouse strains edit the majority
of their L chains in an affinity-independent manner within
the range of DNA binding affinities measured in this
study. Receptor editing has been previously found to be
exquisitely sensitive to ultralow affinity by Lang et al. [22],
who studied anti-class I transgenic mice. The L chain
editing demonstrated by the anti-DNA V
‹
1-J
‹
1trans-
genic B cells is not limited to the targeted
‹
allele which
can obviously utilize its remaining available J
‹
segments
for secondary rearrangements, but is also evident on the
second
‹
allele as well as on
Q
alleles.
In contrast to V
‹
1-J
‹
1 H/L chain double-transgenic mice,
the V
‹
8-J
‹
5 B cells of anti-DNA knock-in mice do not
show any tendency to edit their L chains, either at the
non-transgenic
‹
or the
Q
alleles. Instead, clonal anergy
is the tolerance mechanism of choice, and unlike recep-
tor editing, this mechanism is affinity-dependent, as
demonstrated by the difference in the anergy parameters
that were measured (Fig. 5), between the higher affinity
D42H/V
‹
8-J
‹
5 and lower affinity glD42H/V
‹
8-J
‹
5 trans-
genic B cells. These results confirm our earlier work with
anti-DNA H chain-only knock-in mice [8], where D42H
transgenic B cells had lower receptor densities and were
less responsive to mitogenic stimulation than glD42H B
cells. The difference in the fate of V
‹
8-J
‹
5 and V
‹
1-J
‹
1
targeted B cells in this study is also reflected in the level
of RAG expression in the bone marrow (Fig. 4), and in the
much higher ratio of bone marrow immature/mature cells
to pre-B cells in the former B cell population (Table 3),
resulting in higher number of B cells in the spleen of mice
expressing the V
‹
8-J
‹
5 transgenic L chain (Table 2). The
higher number of splenic B cells may also reflect previ-
ous reports indicating that anergized anti-DNA B cells
are long lived [16, 23].
Casellas et al. [21], who studied knock-in mice express-
ing the same V
‹
8-J
‹
5 L chain and a different anti-DNA H
chain (3H9), observed little editing, if any, of the targeted
L chain allele, in full agreement with the results reported
here. However, when the same V
‹
8-J
‹
5 L chain was
expressed in combination with the native repertoire of
the mouse H chains, about 18% of B cells replaced the
targeted L chain. This may reflect a strong selection for a
1170 N. Yachimovich et al. Eur. J. Immunol. 2002. 32: 1164–1174
dominant, endogenous H chain which results in an H/L
pair with very high-affinity autoreactive specificity.
The anti-DNA Ig H/L chain mice expressing the V
‹
4-J
‹
4
transgenic L chain were perfectly capable of inducing
Q
L
chain rearrangement (Table 2), but their LPS hybridomas
showed an intermediate
‹
chain editing efficiency
between V
‹
1-J
‹
1 and V
‹
8-J
‹
5-expressing anti-DNA
knock-in mice. This is evident in Fig. 3, where 40% and
60% of the glD42H/V
‹
4-J
‹
4 and D42H/V
‹
4-J
‹
4, respec-
tively, expressed their original Ig H/L chain combination,
as compared with only 10% or less of the V
‹
1-J
‹
1-
expressing hybridomas. Moreover, the great majority
(13/16) of the editing V
‹
4-J
‹
4 transgenic hybridomas
seem to have edited both their transgenic and endoge-
nous
‹
allele. Interestingly, of the “non-edited” V
‹
4-J
‹
4-
expressing hybridomas (Fig. 3), the majority (6/9) had a
non-germ-line configuration on the unexpressed allele,
suggesting that secondary rearrangements may occur in
editing B cells, while the original H/L combination is still
expressed (see below).
The clear distinction between the two routes of tolerance
in V
‹
1-J
‹
1- and V
‹
8-J
‹
5-expressing anti-DNA knock-in
mice raises the question of what determines the choice
of B cell tolerance mechanism. It is clear from Table 1
and Fig. 1 that there is no fundamental difference in the
DNA affinity or specificity of Ig D42H or glD42H combi-
nations comprising the two L chains. Therefore,the DNA
antigen is not likely to make the choice between anergy
and editing. On the other hand, the two L chains differ in
their V-gene family and rearrangement status (J
‹
1vs.J
‹
5
rearrangement). It is hard to see why the difference in
V-gene family would determine the route of tolerance,
unless a tolerizing antigen other than DNA reacts differ-
ently with the two H/L chain combinations (see below).
Other considerations that might, in principle, affect the
efficiency of receptor editing, such as the number, dis-
tance and orientation of potentially rearranging V
‹
genes
are not applicable in the targeted locus. This is because
the targeting procedure was identical for the two trans-
genic L chains and the full repertoire of endogenous V
‹
genes remains intact in both models. The targeting pro-
cedure is also not likely to play a role since conventional
(non-targeted) transgenic B cells carrying the same V
‹
8-
J
‹
5 L chain also become anergic [16, 23]. Additionally,
we have searched the V
‹
1andV
‹
8 genes for cryptic
”heptamer-like” sequences that could potentially facili-
tate L chain inactivation through V-gene replacement
[24, 25]. A higher number of heptamer-like sequences
were identified in V
‹
8 than in V
‹
1, suggesting that such
mechanism is not likely to account for the difference in
editing between the two L chains. Also, we have not
identified in our sequenced hybridomas any functional
“hybrid” V
‹
sequences, and this putative replacement
mechanism has not been identified in mouse L chains
[10, 11].
Our data, therefore, suggest that the choice between
BCR editing and clonal anergy (or deletion) is determined
by the rearrangement status of the
‹
L chain. The
sequence of events that is consistent with the data
described in this report suggests the following princi-
ples: (i) receptor editing is the preferred mechanism of
tolerance due to its greater sensitivity to low affinity tole-
rogens. Autoreactive B cells that are incapable of recep-
tor editing would likely resort to other mechanisms of tol-
erance, such as clonal anergy. (ii) Allelic and isotype
exclusion is fully maintained during the process of recep-
tor editing, at least under the conditions (i.e. range of
antigen affinities) described in this study. Consequently,
the tolerized cell would express an edited L chain allele
only if the original
‹
-expressing gene has been replaced
by a productive secondary rearrangement. Alternatively,
the endogenous
‹
or
Q
alleles would be activated when
secondary rearrangement on the active allele is non-
productive, or due to RS-dependent C
‹
deletion [15, 26]
or somatic mutation [27]. (iii) Secondary rearrangements
on the active
‹
allele is the rate-limiting step in L chain
editing. The efficiency of rearrangement within a given
“window of opportunity”, should be directly proportional
to the number of chromatin accessible recombination
signal sequences (RSS) [28]. This number is dependent
on the available J
‹
segments on the active
‹
allele. Thus,
editing V
‹
1-J
‹
1 L chain has the maximal opportunity to
rearrange or inactivate the targeted allele within a given
time frame, resulting in over 90% of edited BCR in these
mice. V
‹
4-J
‹
4-targeted mice can only utilize J
‹
5forL
chain secondary rearrangement on the active allele; con-
sequently, only half of the V
‹
4-J
‹
4 B cells express an
edited BCR. In the case of V
‹
8-J
‹
5 L chain, only RS-
dependent C
‹
deletion would permit the expression of
edited
‹
(or
Q
) alleles and this may be a very slow pro-
cess.
As discussed above, allelic and isotype exclusion is
maintained during the editing process, although second-
ary rearrangements may occur on both alleles. Indeed,
we found no evidence for the expression of more than
one H and one
‹
chain in all of our sequenced hybrid-
omas. Likewise, in FACS analysis, no
‹
/
Q
double produc-
ing splenic B cells could be identified. Allelic exclusion
has been reported to be violated in other anti-DNA trans-
genic mouse models that include (i) transgenic mice in
which conventional rather than targeted Ig H/L chains
were employed [13], (ii) transgenic Ig chains that were
backcrossed onto an autoimmune genetic background
[29], (iii) transgenic Ig chains with very high affinity for
DNA (e.g. 3H9 56R; [30]).
Eur. J. Immunol. 2002. 32: 1164–1174 B cell receptor editing and clonal anergy in anti-DNA transgenic mice 1171
There may be alternative explanations for the relative
inefficiency of autoreactive B cells expressing V
‹
8-J
‹
5in
editing their Ig chains. These include: (i) the possibility
that a productive rearrangement to J
‹
5 may slow down
editing by limiting accessibility of the other L chain
alleles to the rearrangement machinery and by down
regulation of RAG enzymes [31]. (ii) A V
‹
-J
‹
5 productive
rearrangement may accelerate the transition of B cells to
a mature phenotype that is not amenable to induction of
receptor editing [1, 3]. Indeed, conventional transgenic
3H9H/V
‹
8-J
‹
5 anergic B cells were shown to be pheno-
typically mature and long-lived [23]. (iii) One has to con-
sider the possibility that D42H/V
‹
8-J
‹
5 B cells are anerg-
ized first by the tolerizing antigen and anergic cells are
refractory to editing. This possibility seems unlikely,
however, because receptor editing has been shown in
this study to be more sensitive to low-affinity tolerogens
than clonal anergy. Also, it has been reported recently
that anergic B cells in hen egg lysozyme Ig (Ig-HEL)/
soluble HEL double-transgenic mice show evidence of
receptor editing in vivo [32].
One remaining possibility is that one or more of the self
antigens that induce immunological tolerance in vivo are
different from DNA. To be consistent with the results
reported here, this would require that V
‹
1-J
‹
1-expressing
B cells be tolerized by one cross-reacting antigen, V
‹
8-
J
‹
5-expressing cells by a second antigen, and V
‹
4-J
‹
4-
expressing cells which show features of editing and
anergy be tolerized by both antigens. This is a rather
unlikely scenario, because all three H/L chain combina-
tions were shown to bind DNA with comparable specific-
ities, and at least in the case of V
‹
8-J
‹
5-expressing B
cells, the degree of tolerance induction can be directly
related to their affinity for DNA. The weight of the evi-
dence, therefore, suggests that L chain receptor editing
is the preferred mechanism of B cell tolerance and its
efficiency is dependent on the rearrangement status of
the expressed L chain. It should be noted, however, that
although receptor editing is clearly the preferred mecha-
nism of tolerance in these anti-DNA transgenic mice, this
study focuses on B cells specific for one particular auto-
antigen and with a rather limited window of affinities.
Studies of B cells specific for other self antigens are
needed to support a general conclusion regarding the
preferred use of receptor editing for induction of B cell
tolerance.
4 Materials and methods
4.1 Knock-in mice
C57BL/6 transgenic mice, heterozygous for D42H and
glD42H targeted heavy chains, were constructed by homol-
ogous recombination in ES cells as described previously [8].
BALB/c mice, heterozygous for the targeted L chains V
‹
4-
J
‹
4andV
‹
8-J
‹
5wereproducedinasimilarway[10].Togen-
erate V
‹
1-J
‹
1-targeted mice, a 10.5-kb BamHI fragment
containing the rearranged gene was cloned into a targeting
vector designed to allow the insertion of rearranged
‹
genes
upstream of the J
‹
region ([10], and M. Shannon, S. Ibrahim,
E. Luning Prak and M. Weigert, unpublished data). E14–1 ES
cells were transfected with the SalI linearized targeting vec-
tor. ES cells were subjected to positive-negative selection
using G418 and gancyclovir, respectively, and tested for
homologous recombination by PCR [10]. Targeted ES cells
were injected into blastocysts obtained from C57BL/6 preg-
nant females and these blastocysts were implanted into
pseudopregnant females. Chimeric animals were identified
by coat color. These chimeric mice were then mated with
C57BL/6 mice and agouti-colored offspring were screened
for the L-chain replacement by V
‹
1-J
‹
1 and gene targeting
PCR assays. Analysis of LPS hybridomas from heterozy-
gous V
‹
1-J
‹
1-targeted mice demonstrated that 82% of the
hybridoma clones retained the V
‹
1-J
‹
1 knock-in, 9% deleted
the knock-in, and 9% inverted the knock-in. In the course of
this analysis, a duplicated germ-line
‹
locus was identified
by PCR and Southern blotting in the V
‹
1-J
‹
1 knock-in mice,
in addition to the targeted allele. The additional
‹
germ-line
gene (
G
20 kb) does not interfere with the properly targeted
V
‹
1-J
‹
1 allele with respect to allelic exclusion (95%) or
receptor editing (M. Shannon and M. Weigert, unpublished
results). The presence of D42H, V
‹
1L, V
‹
4L and V
‹
8L trans-
genesintailDNAwastestedbyPCR,usingDNAprimers
described previously [8, 10]. All transgenic and control mice
were bred and maintained at the SPF animal facility of the
Hebrew University Faculty of Medicine.
4.2 Transfection of myeloma cells with Ig H/L chain
combinations and purification of IgM antibodies
Linearized expression vectors containing D42H or glD42H
?
heavy chains [12] and V
‹
1-J
‹
1, V
‹
4-J
‹
4[13]orV
‹
8-J
‹
5[14]
‹
light chains were used in different combinations to transfect
non-producer NSO myeloma cells, essentially as described
previously [12]. Following selection in 1.5 mg/ml active G418
and cell cloning, IgM-secreting transfectomas were grown in
serum-free, protein-free medium (Sigma Chemical Co., St.
Louis, MO; cat. no. S2897) and antibodies were isolated by
elution from a DE-52 column (Whatman, Maidstone, GB)
with 0.5 M NaCl in 0.05 M phosphate buffer pH 8.
4.3 Measurement of antibody binding affinity and
specificity
Antibody binding to DNA was assayed in solution by the
nitrocellulose filter assay [12, 32]. This method also mea-
sured DNA binding affinity at equilibrium, using 32P-labeled
plasmid DNA [12]. The relative specificities of anti-DNA anti-
bodies to dsDNA and ssDNA antigens were evaluated
1172 N. Yachimovich et al. Eur. J. Immunol. 2002. 32: 1164–1174
by competition experiments, in which single-stranded or
double-stranded M13 mp18 circular DNA species (New
England Biolabs, Beverly, MA) were used as competitive
inhibitors of antibody binding to E. coli [14C]dsDNA in the
nitrocellulose filter assay [33].
4.4 Flow cytometric analysis
Single-cell suspensions from bone marrow or spleen of
transgenic mice were stained with monoclonal or polyclonal
antibodies and analyzed by FACScan (Becton Dickinson,
San Jose, CA) using the “Lysis II” program. The antibodies
used for staining were the same as described previously [8].
Additional antibodies included biotin-conjugated goat anti-
mouse
‹
chain (clone R33–18–10.1, obtained from Dr. Klaus
Rajewsky, Cologne, Germany), and FITC-conjugated goat
anti-
Q
(clone Ls 136, Southern Biotechnology, Birmingham,
AL).
4.5 PCR and sequence analysis of hybridoma
antibodies
Hybridomas were produced by fusion of BALB/c NSO mye-
loma cells with spleen cells from wild-type or transgenic
mice following a 3-day incubation with 40
?
g/ml LPS
(Sigma). Secretion of IgMaand IgMbantibodies in hybridoma
supernatants was measured with allotype-specific anti-
bodies by ELISA [8]. cDNA preparation and direct sequenc-
ing of hybridoma DNA was carried out as described previ-
ously [8]. Germ-line configuration of the untargeted
‹
chain
allele was assayed by PCR, using a sense strand primer
upstream of J
‹
1, 5’-TGTGACATGCTTCTAAAGCAAAAGA-3’
and an antisense strand primer downstream of J
‹
1, 5’-CAA-
GATCTACCCTGCTTCTTTGAGCAT-3’. RS1-RS-dependent
C
‹
deletion was determined by PCR using primers b and c
in [26].
4.6 Measurement of B cell proliferation
B cell proliferation following LPS stimulation was evaluated
by CFSE (Molecular probes, Leiden, The Netherlands) dilu-
tion, measured by flow cytometry [17].
4.7 Measurement of RAG-2 expression in bone marrow
cells
Total RNA was extracted from bone marrow cells and
reverse transcribed to cDNA using random primers and M-
MLV reverse transcriptase (Promega). RAG-2 mRNA levels
were quantitated as described previously [34], using CD19
as an internal control.
Acknowledgements: This study was supported by the
United-States Israel Binational Science Foundation, by the
Israel Science Foundation administered by the Israeli Acad-
emy of Science and Humanities and by the German-Israeli
Foundation for Scientific Research and Development. We
thank Drs. Martin Weigert and Eline Luning Prak for provid-
ing the L chain targeted mice and for their critical reading of
the manuscript, and Dr. Doron Melamed for his helpful com-
ments and his procedure for measurement of RAG expres-
sion.
References
1Nemazee, D. and Weigert, M., Revising B cell receptors. J. Exp.
Med. 2000. 5: 1813–1817.
2Nemazee, D., ReceptorselectioninBandTlymphocytes.Annu.
Rev. Immunol. 2000. 18: 19–51.
3Meffre, E., Casellas, R. and Nussenzweig, M. C., Antibody reg-
ulation of B cell development. Nat. Immunol. 2000. 1: 379–385.
4Chen, C., Nagy, Z., Radic, M. Z., Hardy, R. R., Huszar, D.,
Camper,S.A.andWeigert,M.,Thesiteandstageofanti-DNA
B cell deletion. Nature 1995. 373: 252–255.
5Nemazee, D. and Buerki, K., Clonal deletion of B lymphocytes
in a transgenic mouse bearing anti-MHC class I antibody genes.
Nature 1989. 337: 562–566.
6Nossal,G.J.V.,Clonal anergy of B cells: a flexible, reversible,
and quantitative concept. J. Exp. Med. 1996. 183: 1953–1956.
7Goodnow, C. C., Crosbie, J., Adelstein, S., Lavoie, T. B.,
Smith-Gill, S. J., Brink, R. A., Pritchard-Briscoe, H., Wother-
spoon. J. S., Loblay, R. H., Raphael, K., Trent, R. J. and Bas-
ten, A., Altered immunoglobulin expression and functional silenc-
ing of self-reactive B lymphocytes in transgenic mice. Nature
1988. 334: 676–682.
8Pewzner-Jung, Y., Friedmann, D., Sonoda, E., Jung. S.,
Rajewsky. K. and Eilat, D., B cell deletion, anergy, and receptor
editing in “knock-in” mice targeted with a germline-encoded or
somatically mutated anti-DNA heavy chain. J. Immunol. 1998.
161: 4634–4645.
9Friedmann, D., Yachimovich, N., Mostoslavsky, G., Pewzner-
Jung, Y., Ben-Yehuda, A., Rajewsky, K. and Eilat, D., Produc-
tion of high affinity autoantibodies in autoimmune New Zealand
black/New Zealand white F1 mice targeted with an anti-DNA
heavy chain. J. Immunol. 1999. 162: 4406–4416.
10 Luning Prak, E. and Weigert, M., Light chain replacement: a
newmodelforantibodygenerearrangement.J. Exp. Med. 1995.
182: 541–548.
11 Chen, C., Luning Prak, E. and Weigert, M., Editing disease-
associated autoantibodies. Immunity 1997. 6: 97–105.
12 Pewzner-Jung, Y., Simon, T. and Eilat, D., Structural elements
controlling anti-DNA antibody affinity and their relationship
to anti-phosphorylcholine activity. J. Immunol. 1996. 156:
3065–3073.
13 Gay, D., Saunders, T., Camper, S. and Weigert, M., Receptor
editing: An approach by autoreactive B cells to escape tolerance.
J. Exp. Med. 1993.177: 999–1008.
14 Carmack, C. E., Camper, S. A., Mackle, J. J., Gerhard, W. U.
and Weigert, M., Influence of a V
‹
8Lchaintransgeneonen-
dogenous rearrangements and the immune response to the HA
(SB) determinant on influenza virus. J. Immunol. 1991. 147:
2024–2033.
Eur. J. Immunol. 2002. 32: 1164–1174 B cell receptor editing and clonal anergy in anti-DNA transgenic mice 1173
15 Moore, M., Durdik, W. J., Persiani, D. M. and Selsing, E., Dele-
tion of
‹
chain constant region genes in mouse
Q
chain-producing
B cells involve intrachromosomal DNA recombinations similar to
V-J joining. Proc. Natl. Acad. Sci. USA 1985. 82: 6211–6215.
16 Nguyen, K., Mandik, L., Bui, A., Kavaler, J., Norvell, A., Mon-
roe, J., Roark, J. and Erikson, J., Characterization of anti-
single-stranded DNA B cells in a non-autoimmune background.
J. Immunol. 1997. 159: 2633–2644.
17 Lyons, A. B. and Parish, C. R., Determination of lymphocyte
division by flow cytometry. J. Immunol. Methods 1994. 171:
131–137.
18 Rolink, A. G., Andersson, J. and Melchers, F., Characterization
of immature B cells by a novel monoclonal antibody, by turnover
and by mitogen reactivity. Eur. J. Immunol. 1998. 28: 3738–3748.
19 Pelanda, R., Schwers, S., Sonoda, E., Torres R. M., Nemazee,
D. and Rajewsky, K., Receptor editing in a transgenic mouse
model: site, efficiency and role in B cell tolerance and antibody
diversification. Immunity 1997. 7: 765–775.
20 Yamagami, T., Boekel, E. T., Andersson, J., Rolink, A. and
Melchers, F., Frequencies of multiple IgL chain gene rearrange-
ments in single normal or
‹
L chain-deficient B lineage cells.
Immunity 1999.11: 317–327.
21 Casellas, R., Yang Shih, T.A., Kleinwietfeld, M., Rakonjac, J.,
Nemazee, D., Rajewsky, K. and Nussenzweig, M. C., Contri-
bution of receptor editing to the antibody repertoire. Science
2001. 291: 1541–1544.
22 Lang, J., Jackson, M., Teyton, L., Brunmark, A., Kane, K. and
Nemazee, D., B cells are exquisitely sensitive to central toler-
ance and receptor editing induced by ultralow affinity, membrane
bound antigen. J. Exp. Med. 1996. 184: 1685–1697.
23 Noorchashm, H., Bui, A., Li, H. L., Eaton, A., Mandik-Nayak,
L., Sokol, C., Potts, K. M., Pur ´e, E. and Erikson, J., Character-
ization of anergic anti-DNA B cells: B cell anergy is a T cell-
independent and potentially reversible process. Int. Immunol.
1999. 11: 765–776.
24 Chen, C., Nagy, Z., Luning Prak, E. and Weigert, M., Immuno-
globulin heavy chain gene replacement: A mechanism for recep-
tor editing. Immunity 1995. 3: 747–755.
25 Wilson, P. C., Wilson, K., Liu, Y-J., Banchereau, J., Pascual, V.
and Capra, J. D., Receptor revision of immunoglobulin heavy
chain variable region genes in normal human B lymphocytes.
J. Exp. Med. 2000. 191: 1881–1894.
26 Retter,M.W.andNemazee,D.,Receptor editing occurs fre-
quently during normal B cell development. J. Exp. Med. 1998.
188: 1231–1238.
27 Brard, F., Shanon, M., Luning Prak, E., Litwin, S. and Weigert,
M., Somatic mutation and light chain rearrangement generate
autoimmunity in anti-single-stranded DNA transgenic MRL/lpr
mice. J. Exp. Med. 1999. 190: 691–704.
28 Roth, D. B. and Roth, S. Y., Unequal access: regulating V(D)J
recombination through chromatin remodeling. Cell 2000. 133:
699–702.
29 Spatz, L., Saenko V., Iliev, A., Jones, L., Geskin, L. and Dia-
mond, B., Light chain usage in anti-double-stranded DNA B cell
subsets: role in cell fate determination. J. Exp. Med. 1997. 185:
1317–1326.
30 Li, Y., Li, H. and Weigert, M., Autoreactive B cells in the marginal
zone that express dual receptors. J. Exp. Med.. 2002. 195:
181–188.
31 Schlissel, M. S. and Stanhope-Baker, P., Accessibility and the
developmental regulation of V(D)J recombination. Semin.
Immunol. 1997. 9: 161–170.
32 Tze, L. E., Baness, E. A., Hippen, K. L. and Behrens, T. W., Ig
light chain receptor editing in anergic B cells. J. Immunol. 2000.
165: 6796–6802.
33 Eilat, D. and Fischel, R., Recurrent utilization of genetic ele-
ments in V regions of anti nucleic acid antibodies from autoim-
mune mice. J. Immunol. 1991. 147: 361–368.
34 Melamed, D., Kench, J. A., Grabstein, K., Rolink, A. and
Nemazee, D., A functional B cell receptor transgene allows effi-
cient IL-7-Independent maturation of B cell precursors. J. Immu-
nol. 1997. 159: 1233–1239.
Correspondence: Dan Eilat, Department of Medicine,
Hadassah University Hospital, P.O. Box 12000, Jerusalem
91120, Israel
Fax: +972-2-6437775
e-mail: eilatd
—
md2.huji.ac.il
1174 N. Yachimovich et al. Eur. J. Immunol. 2002. 32: 1164–1174
