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J. Exp. Med. Vol. 206 No. 8 1803-1816
B lymphocytes gain the potential to recognize
>108 antigens (Cobb et al., 2006) by using a
novel genetic mechanism called V(D)J recombi-
nation to generate a large repertoire of Ig heavy
chain (IgHC) and Ig light chain (IgLC) variable
domain exons (Brack et al., 1978; Tonegawa,
1983). Variable domain exons are composed of
V, D, and J gene segments (IgHC) or V and J
gene segments (IgLC). Successive stages of B
cell development are defined by the ordered as-
sembly of Ig genes; the IgHC locus rearranges in
pro–B cells, the IgLC locus rearranges in pre–B
cells, and the newly synthesized B cell receptor
(BCR) is first expressed on the cell surface in
immature B cells. V(D)J recombination begins
with recognition and cleavage of a pair of re-
combination signal sequences (RSSs) flanking
rearranging gene segments by the V(D)J recom-
binase composed of the lymphoid-restricted
RAG1 and RAG2 proteins (Schatz et al., 1989;
Oettinger et al., 1990). After RAG-mediated
cleavage, the nonhomologous end-joining ma-
chinery repairs the DNA breaks, forming cod-
ing joints between the gene segments and signal
joints between the two broken RSS ends
(Bassing et al., 2002).
Transcription of rearranging gene segments
correlates with their developmentally regulated
activation for rearrangement (Alt et al., 1987).
Mutations that disrupt this “germline” transcrip-
tion interfere with V(D)J recombination. This has
led various workers to examine specific transcrip-
tion factors for their ability to influence gene rear-
rangement and B cell development. One such
factor, NF-B, was initially discovered as a result
of its ability to bind to a sequence in the Ig
intronic enhancer (Sen and Baltimore, 1986).
NF-B is composed of homo- or heterodimers of
five rel family members: RelA (p65), RelB,
c-Rel, p50, and p52 (Hayden et al., 2006). Re-
cent evidence suggests that additional proteins
may associate with the rel proteins and influence
Mark S. Schlissel:
Abbreviations used: -gal, -
galactosidase; BCR, B cell re-
ceptor; cDNA, complementary
DNA; FDG, fluorescein di--
syl transferase; IgHC, Ig heavy
chain; IgLC, Ig light chain;
PCR; mRNA, messenger
RNA; RPS3, ribosomal protein
S3; RSS, recombination signal
R.H. Amin’s present address is Fred Hutchison Cancer Re-
search Center, Seattle, WA 98109.
NF-B activity marks cells engaged
in receptor editing
Emily J. Cadera,1 Fengyi Wan,2 Rupesh H. Amin,1 Hector Nolla,1
Michael J. Lenardo,2 and Mark S. Schlissel1
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
2Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD 20892
Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are
necessary to ensure that B cells do not respond to self-antigens. One such tolerance
mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell
recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene
rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a
heterozygous mutant mouse in which the NF-B–dependent IB gene was replaced with a
lacZ (-gal) reporter complementary DNA (cDNA; IB+/lacZ) suggests a potential role for
NF-B in receptor editing. Sorted -gal+ pre–B cells showed increased levels of various
markers of receptor editing. In IB+/lacZ reporter mice expressing either innocuous or self-
specific knocked in BCRs, -gal was preferentially expressed in pre–B cells from the mice
with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IB
superrepressor in primary bone marrow cultures resulted in diminished germline and
rearranged transcripts but similar levels of RAG expression as compared with controls.
We found that IRF4 transcripts were up-regulated in -gal+ pre–B cells. Because IRF4 is a
target of NF-B and is required for receptor editing, we suggest that NF-B could be
acting through IRF4 to regulate receptor editing.
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The Journal of Experimental Medicine
A ROLE FOR NF-B IN RECEPTOR EDITING | Cadera et al.
confirm that -gal activity correlates with IB expression in
IB+/lacZ reporter mice, IB transcripts were analyzed in
RNA purified from pre–B cells sorted for -gal activity as
assayed using the fluorescent substrate fluorescein di--d-
galactopyranoside (FDG). Using real-time PCR, we deter-
mined that the -gal+ pre–B cells express 13-fold more IB
messenger RNA (mRNA) as compared with -gal cells
(Fig. 1 A). The greater amounts of IB transcripts in -gal+
cells confirms that -gal detected using FDG is a suitable re-
porter for IB expression in IB+/lacZ mice.
Several groups have shown that IB transcription is reg-
ulated by NF-B activity (Gugasyan et al., 2000; Ghosh and
Karin, 2002; Hoffmann et al., 2002; Hayden et al., 2006;
Hayden and Ghosh, 2008). Therefore, we hypothesized that
-gal activity also reports NF-B activity in IB+/lacZ mice.
We confirmed this using an Ableson virus–transformed pro–
B cell line made from IB+/lacZ mice. After LPS treatment,
-gal was up-regulated (Fig. 1 B) and p65 was relocalized
from the cytoplasm to the nucleus in this cell line (Fig. 1 C).
-gal activity was observed in IB+/lacZ Ableson cells within
2 h of LPS treatment and the level of -gal activity increased
with longer treatment, reaching a plateau after 8 h (Fig. 1 B).
To determine the half-life of -gal in this system, we treated
the IB+/lacZ Ableson cell line with LPS for 9 h, washed out
the LPS, and recultured the cells in the presence of the pro-
tein synthesis inhibitor cycloheximide (Fig. 1 D). Using this
approach, we found the half-life of -gal to be 7.55 h. These
experiments demonstrate that known activators of NF-B
can also activate -gal in IB+/lacZ cells and that -gal is re-
porting recent NF-B activity.
Increased light chain gene rearrangements correlate
with IB expression in pre–B cells
We used multiparameter flow cytometry to analyze -gal ex-
pression in cells isolated from IB+/lacZ bone marrow to deter-
mine the pattern of IB transcription and, thus, NF-B activity
during B cell development. We observed stage-specific -gal
expression; it was low in pro–B cells, peaked at the pre–B stage,
remained high in immature B cells, and was low in mature B
cells (Fig. 2 A). The IB-positive and -negative subsets of pre–B
cells were chosen for further analysis. We used real-time RT-
PCR to quantify various light chain locus transcripts. A 10-fold
greater amount of V1,2-C–rearranged transcript was observed
in -gal–expressing pre–B cells compared with -gal–negative
pre–B cells (Fig. 2 B). Germline transcripts are transcripts
through the unrearranged locus and correlate with the accessi-
bility of the locus to the recombinase. There was a twofold in-
crease in both germline and rearranged transcripts in the pre–B
cells with -gal activity (Fig. 2). These data indicate that in-
creased accessibility and, presumably, increased light chain
gene rearrangement correlate with IB expression and NF-B
activity in pre–B cells.
We analyzed RAG1 and RAG2 transcripts in the -gal–
positive and –negative populations to assess whether differ-
ences in expression of these proteins might be responsible
for the differences observed in light chain rearrangements.
the affinity and specificity of binding (Wan et al., 2007). Inactive
NF-B is sequestered in the cytoplasm bound to an inhibitory
protein of the IB family. Various signaling pathways result in
the activation of a kinase that phosphorylates IB leading to its
degradation. Once released from IB, NF-B can translocate
to the nucleus, bind DNA sequences, and regulate transcription.
Remarkably, one of the transcriptional targets of NF-B is IB
itself, leading to negative-feedback regulation of NF-B activa-
tion (Chiao et al., 1994).
Previous work attempting to elucidate the role of NF-B
in B cell development has lead to contradictory conclusions.
Expression of a mutant IB “superrepressor” was reported to
prevent light chain gene rearrangements in a transformed cell
line (Scherer et al., 1996; O’Brien et al., 1997; Bendall et al.,
2001). Retrovirus-mediated expression of a similar IB su-
perrepressor in primary B cells, however, revealed a different
phenotype: a block at the pro–B stage of development as de-
fined by cell surface marker expression (Feng et al., 2004; Jimi
et al., 2005) or a complete lack of B cells (Igarashi et al., 2006).
This block could be overcome by expression of an antiapop-
tosis gene (Feng et al., 2004) or by neutralizing TNF-
(Igarashi et al., 2006). Adding to this confusion, targeted dis-
ruption NEMO, a protein required in some pathways leading
to IB degradation, did not seem to alter B cell development
until the mature stage (Sasaki et al., 2006).
A potential role for NF-B in the regulation of IgLC gene
rearrangement was reported by workers studying receptor ed-
iting, a process in which engagement of the BCR on an im-
mature B cell with self-antigen provokes further recombination
in an effort to replace the offending variable exon with an in-
nocuous one (Nemazee, 2006). At least 25% of the primary
B cell repertoire is thought to undergo editing (Casellas et al.,
2001). These workers found that in vitro cross-linking of BCR
on immature B cells leads to an increase in RAG expression
and IgLC gene rearrangement that correlates with NF-B ac-
tivation and the binding of NF-B to sites in the RAG locus
and the intronic enhancer (Verkoczy et al., 2005). Another
group, however, showed that targeted mutation of the NF-B
binding site in the intronic enhancer had little if any appar-
ent effect on V-to-J rearrangement (Inlay et al., 2004).
In an attempt to clarify the role of NF-B in B cell devel-
opment, we took advantage of a targeted mutant mouse that
expresses a lacZ complementary DNA (cDNA; encoding
-galactosidase [-gal]) from the IB locus (Beg et al., 1995).
Because IB is directly regulated by active nuclear NF-B,
we were able to analyze NF-B activity using a fluorescent
-gal substrate and multiparameter flow cytometry without
perturbing its endogenous activity. Our results point to a role
for NF-B in the regulation of receptor editing.
IB+/lacZ mice report IB and NF-B activity
In an attempt to assess NF-B activity at various stages of
B cell development, we analyzed IB+/lacZ mice. Although
these mice express less IB protein than wild-type mice,
they display no obvious phenotype (Beg et al., 1995). To
JEM VOL. 206, August 3, 2009
Figure 1. IB+/lacZ mouse reports IB transcription and nuclear NF-B activity. (A) Real-time RT-PCR analysis of IB expression in RNA puri-
fied from flow-sorted -gal+ (dark gray) and -gal (light gray) IB+/lacZ pre–B cells (B220+CD43lowsIgM/D). The ratio is shown between IB transcript
and a control transcript, HPRT, with error bars indicating the standard deviation of triplicate assays. (B) Flow cytometric analysis of -gal expression in
IB+/lacZ Ableson cells cultured in the absence (gray shading) or presence of LPS for the indicated lengths of time. (C) Anti-p65 immunofluorescence
microscopy was performed on IB+/lacZ Ableson cells cultured in the presence (right) or absence (left) of LPS for 2 h. Bar, 10 µm. (D) Flow cytometric
analysis of -gal expression in IB+/lacZ Ableson cells cultured in the absence (gray shading) or presence (thick black line) of LPS for 9 h, or after the
indicated times after the LPS had been washed out and the cells had been recultured with cycloheximide. Each of the experiments in A–C were repeated a
minimum of twice with similar results.
A ROLE FOR NF-B IN RECEPTOR EDITING | Cadera et al.
receptor editing. Rearrangements involving the RS element,
which lies 25 kb 3 of C, and either a V gene segment or the
IRS sequence in the J-C interval are considered hallmarks
of receptor editing (Fig. 3 A; Tiegs et al., 1993; Retter and
Nemazee, 1998; Vela et al., 2008). Using a PCR assay, we de-
tected more RS-IRS and RS-V rearrangements in the -gal+
than in the -gal pre–B cell population (Fig. 3 B). Thus, RS
rearrangements are enriched in pre–B cells expressing IB.
The relative amounts of primary and secondary rear-
rangement reaction intermediates can also help identify cell
populations undergoing receptor editing. Primary rearrange-
ments occur on germline alleles. After this initial V-to-J rear-
rangement, an upstream V can rearrange to a downstream J
if the primary rearrangement is nonproductive, if it is unable to
pair with the preexisting IgHC, or if it contributes to a self-spe-
cific BCR. These are termed secondary rearrangements and are
increased in cells undergoing receptor editing. Ligation-medi-
ated PCR (LM-PCR; Schlissel et al., 1993) was used to detect
reaction intermediates associated with primary and secondary
RAG levels were almost identical between the -gal–positive
and –negative pre–B cells (Fig. 2 B). This implies that a mecha-
nism other then differentially regulated RAG expression is
responsible for the differences observed in light chain rear-
rangements in the two pre–B cell subpopulations. To verify
that the sorted -gal pre–B cell population was not contami-
nated with pro–B cells, which could potentially account for
the observed differences in various transcripts, we analyzed
V(D)J-rearranged heavy chain gene transcripts in these popu-
lations. We found very similar levels of heavy chain tran-
scripts in these two populations (Fig. S2), confirming that
they were comparable B cell populations.
Receptor-editing markers correlate with IB expression
Considering what process might divide the pre–B cell com-
partment into nonequivalent subpopulations, we proceeded to
test the idea that NF-B might be specifically activated in cells
undergoing receptor editing. To test this idea, we examined
-gal–positive and –negative pre–B cells for various markers of
Figure 2. IB expression during B cell development. (A) -gal expression in an IB+/lacZ reporter mouse was analyzed at various stages of B cell
development. Cells were gated on forward and side scatter and divided into developmental stages using the cell surface markers B220, CD43, IgM, and
IgD (Hardy et al., 1991). The data are representative of three independent experiments as indicated by the percentages and standard deviations accompa-
nying each FACS histogram. (B) cDNA synthesized from sorted -gal–positive and –negative pre–B cell subpopulations was analyzed for the indicated
transcripts. Each transcript level was normalized to HPRT levels to control for any differences in cDNA template and data from each -gal sample was
arbitrarily set to 1, with error bars indicating range of triplicate assays. The cDNA analysis used pre B cells from a pool of five to six mice and was re-
peated in two independent experiments.
JEM VOL. 206, August 3, 2009
cells from unperturbed cultures (Fig. 4 A). The level of cyto-
plasmic Ig , however, is less than that found on sIgM+ cells
cultured in the absence of F(ab)2 fragments (Fig. 4 A).
We proceeded to analyze wild-type mice for cytoplasmic
expression in three different B cell populations: CD19+IgM,
IgMlow, and IgMhigh cells (Fig. 4 B). We found that only a small
percentage of CD19+IgM cells express cytoplasmic (13%;
Fig. 4 B). We suggest that this cytoplasmic Ig+ subpopulation
of sIgM cells is undergoing receptor editing. We then analyzed
IB+/lacZ bone marrow. In this same CD19+IgM population,
we detected cytoplasmic Ig in -gal+ cells but not in -gal
cells (Fig. 4 C). This result implies that, in the pro–/pre–B cell
compartment, cells undergoing receptor editing are -gal+. In
wild-type mice, the majority of IgMlow and IgMhigh cells express
cytoplasmic Ig (74 and 87%, respectively; Fig. 4 B). IgMlow
cells are either early immature cells (in the process of up-regu-
lating IgM expression) or cells undergoing receptor editing (in
the process of down-regulating surface IgM). Most IgMlow cells
Ig rearrangements in DNA purified from -gal–positive and
–negative pre–B cells. In both populations, similar amounts of
primary Ig rearrangement intermediates were detected, but
secondary rearrangement intermediates were highly enriched
in the -gal+ subpopulation (Fig. 3 C). Thus, secondary Ig re-
arrangements, which are enhanced in receptor editing, corre-
late with IB expression.
The BCR is down-regulated from the cell surface during
receptor editing (Tze et al., 2000). One feature that distinguishes
cells undergoing receptor editing from proper pre–B cells is cy-
toplasmic expression (Pelanda et al., 1997). To examine this
feature of receptor editing, we cultured bone marrow from an
innocuous BCR knockin mouse (B1-8 -HEL-, see subse-
quent section), in the presence or absence of anti-IgM F(ab)2
fragments, that mimic a signal inducing receptor editing (Hertz
and Nemazee, 1997). As expected, the cultures where IgM was
cross-linked down-regulated IgM off the cell surface, resulting
in levels of cytoplasmic Ig greater than those seen in sIgM
Figure 3. Markers of receptor editing are increased in IB-expressing cells. (A) The top diagram shows that the locus is in its germline con-
figuration with the primary break intermediate shown below. The bottom diagram is the locus after a V has rearranged to J1, with the secondary
break intermediate shown below. (B) Agarose gel analysis of PCR assays for RS rearrangements to either IRS (RS-IRS) or V (RS-V) in DNA from pre–B
cells sorted for -gal expression. PCR amplification of the APRT locus was used as a template control, and H20 indicates PCR reactions without template.
(C) LM-PCR assays to detect primary and secondary double-stranded DNA RSS break intermediates in DNA from sorted -gal–positive and –negative pre–
B cells. An ethidium-stained agarose gel analysis of LM-PCR products is shown. Pooled bone marrow from five to six mice was used for each of two rep-
etitions of this experiment yielding similar results.
A ROLE FOR NF-B IN RECEPTOR EDITING | Cadera et al.
Figure 4. Cytoplasmic expression correlates with IB expression. (A) B1-8 -HEL- bone marrow was cultured in IL-7 with or without cross-
linking with anti-IgM F(ab)2 to imitate a receptor-editing signal. Cells were surface stained with anti-CD19 and anti-IgM antibodies and a fourfold excess
of a biotinylated anti-Ig antibody to block surface Ig. Cells were fixed, permeabilized, and stained with anti-Ig antibody. The dot plots show CD19
versus IgM. The histogram displays cytoplasmic staining with the shaded gray representing isotype control staining, the thick black line representing
IgM cells without cross-linking, the dashed line representing cells cross-linked with anti-IgM F(ab)2, and the thin black line representing IgM+ cells
without cross-linking. This experiment was repeated three times. (B) Bone marrow from a wild-type mouse was stained as described in A. The dot plots
show CD19 and IgM staining with gates defining IgM, IgMlow, and IgMhigh populations. The histograms display cytoplasmic expression. The shaded gray
indicates isotype control, the thick black line indicates IgM, the dashed line indicates IgMlow, and the thin black line indicates IgMhigh. This experiment
was performed three times with bone marrow from different individual mice. (C) IB+/lacZ bone marrow was sorted for -gal expression and subse-
quently stained as described in A. The histograms display cytoplasmic expression in -gal–sorted cells gated on the IgM, IgMlow, and IgMhigh popula-
tions. -gal–positive cells are indicated by the black lines and -gal–negative cells are shown in gray shading. The numbers indicate the percentage of
cytoplasmic –positive -gal–positive cells, with mean values and standard error generated from analyses of three different mice.
JEM VOL. 206, August 3, 2009
undergoing receptor editing will be cytoplasmic Ig positive
because these cells once expressed this protein on the cell sur-
face and are now in the process of down-regulating it or delet-
ing the offending Ig variable exon. Newly generated innocuous
immature B cells, which are also IgMlow, will not have synthe-
sized high levels of Ig protein, so these cells do not necessarily
express high levels of cytoplasmic Ig. High levels of cytoplas-
mic Ig were expressed in the majority of the -gal+IgMlow
cells but were only expressed in 30% of -galIgMlow cells
(Fig. 4 C). This suggests that the -gal+IgMlow cells are recep-
tor-editing cells and -galIgMlow cells may be newly generated
immature B cells. The majority of IgMhigh cells express cytoplas-
mic Ig, regardless of -gal expression levels (Fig. 4 C). This is
not surprising, as cells with high IgM expression are continu-
ously synthesizing immunoglobulin, accounting for the cyto-
plasmic Ig expression. Thus, expression of cytoplasmic Ig is
associated with IB expression in populations of cells where
receptor-editing signals are present.
Developing B cells expressing an innocuous BCR do not
express appreciable levels of IB and have a reduction
in nuclear p65 and ribosomal protein S3 (RPS3)
To further test the idea that NF-B is involved in receptor
editing, IB+/lacZ mice were crossed to various BCR
knockin mice. The B1-8 heavy chain, when paired with the
anti–HEL- light chain (-HEL-), produces an innocuous
Figure 5. Cells undergoing receptor editing show increased IB levels as well as increased nuclear NF-B and RPS3. (A) Bone marrow was
isolated from IB+/lacZ B1-8 -HEL-, IB+/lacZB1-8high -HEL-, and IB+/lacZB1-8low -HEL- mice. Cells were loaded with FDG and stained with
anti-CD19, anti-CD43, anti-IgM, and anti-IgD antibodies. Cells were first gated on IgD. CD19 verses IgM staining is displayed. (B) -gal activity in the
CD19+IgD gate (pro–B, pre–B, and immature B cells) is displayed. IB+/lacZ B1-8 -HEL- is shaded gray, IB+/lacZB1-8low -HEL- is the thin black line,
and IB+/lacZB1-8high -HEL- the thick black line. A and B are representative of at least three different mice individually analyzed for each genotype.
(C) Immunofluorescence microscopy detecting p65 and RPS3 in B1-8 -HEL-, B1-8low -HEL-, and B1-8high -HEL- pro–B, pre–B, and immature
B cells. The data shown is representative of two independent sorts on each occasion scoring between 250 and 400 cells. Bar, 10 µm. Fig. S3 displays
quantitative data from a repetition of this experiment.
A ROLE FOR NF-B IN RECEPTOR EDITING | Cadera et al.
hibits activation of the classical NF-B pathway (Scherer et al.,
1996). Pro–B cells in IL-7–dependent wild-type bone marrow
cultures were infected with an IBN-expressing retrovirus
or an empty vector control. We failed to detect a block in
pro–/pre–B development in IBN-expressing cells using
the surface CD19 expression and cytoplasmic (Fig. 6 A).
There was, however, a twofold reduction in IgM+ cells among
those expressing IBN (Fig. 6 A). RNA was purified and
transcript levels in the IBN-infected pro–/pre–B cells
were compared with empty vector–infected pro–/pre–B cells.
RAG expression was comparable in these two populations
(Fig. 6 B). In the IBN-infected pro–/pre–B cells, a large
decrease in V1,2-C–rearranged transcripts was observed
(7-fold), as well as a decrease in germline transcripts (10-fold;
Fig. 6 B). These results indicate that B cells with diminished
NF-B activity have diminished light chain gene rearrange-
ments and accessibility. This confirms our results from the
IB+/lacZ reporter mice that NF-B influences light chain status
without changing RAG expression.
IB expression correlates with IRF4 expression but not
with expression of Bcl-2 family members
To investigate potential targets of regulation by NF-B that
might play a role in receptor editing, transcript levels of mul-
tiple NF-B target genes were analyzed in RNA purified
from sorted -gal–positive and –negative pre–B cells. Bcl-2
family members are antiapoptosis genes and targets of NF-B
(Catz and Johnson, 2001). Mice that contain self-specific
BCR transgenes, as well as a Bcl-2 transgene, display en-
hanced receptor editing (Lang et al., 1997), suggesting that
survival regulated by this gene may be involved in receptor
editing. We used RT-PCR to compare levels of transcripts
from the antiapoptosis genes Bcl-2, Bcl-X, and Mcl-1 in
-gal–positive and –negative pre–B cell cDNA. There was no
increase in these antiapoptosis genes in the -gal–positive
cells (Fig. S1). These data indicate that increased survival,
mediated by these antiapoptosis genes, is unlikely to be re-
sponsible for the increased receptor editing observed in the
IRF4 is a target of NF-B that has been previously shown
to be important in both pre–B cell development and receptor
editing. (Lu et al., 2003; Muljo and Schlissel, 2003; Ma et al.,
2006; Saito et al., 2007; Johnson et al., 2008; Pathak et al.,
2008). We used real-time RT-PCR to quantify IRF4 tran-
scripts in pro–/pre–B cells sorted based on -gal activity.
The signal was normalized to hypoxanthine-guanine phosphori-
bosyl transferase (HPRT) to account for differences in template
quality. We observed a fourfold increase in IRF4 transcripts
in -gal+ pre–B cells (Fig. 7 A). A twofold increase was ob-
served in IRF4 expression in B1-8low -HEL- CD19+IgD
cells compared with B1-8 -HEL- CD19+IgD cells (Fig. 7 B).
IRF4 has binding sites in the 3 enhancer as well as in both
enhancers (Pongubala et al., 1992; Eisenbeis et al.,
1995). This increase in IRF4 in the -gal–positive popula-
tion implies that NF-B could be acting through IRF4 to
regulate receptor editing.
BCR (Casellas et al., 2001). Specific mutations in the B1-8
heavy chain result in autoreactivity when paired with
-HEL-. Thus, B1-8high -HEL- and B1-8low -HEL- mice
express mutated B1-8 heavy chains and self-specific BCRs
(Casellas et al., 2001). There are few pro–/pre–B cells in B1-8
-HEL- because these mice express an innocuous BCR and
rapidly generate immature and mature B cells (Fig. 5 A).
B1-8low -HEL- and B1-8high -HEL- B cells also rush through
pro– and pre–B cell development, but because the knocked-
in BCR in these cells is self-specific, it is down-regulated at
the immature stage during receptor editing. Therefore, the
cells in the pro–/pre–B gate in B1-8low -HEL- and B1-8high
-HEL- mice are almost all undergoing receptor editing.
IB+/lacZ B1-8 -HEL- mice, with an innocuous BCR,
express almost no -gal in pro–B, pre–B, and immature B
cells (CD19+IgD gate). In contrast, 70% of either IB+/lacZ
B1-8low -HEL- and IB+/lacZ B1-8high -HEL- mice
express -gal in the pro–B, pre–B, and immature B cell gate
(Fig. 5 B). Thus, when cells do not undergo receptor editing,
the IB reporter is not expressed. However, in two differ-
ent mice where a majority of B cells undergo receptor edit-
ing, the majority of B cells express the IB reporter.
RPS3, a component of the 40S ribosome subunit, was
recently found to interact with p65, increase the activity and
binding affinity of NF-B, and influence the binding site
preferences of the NF-B complex (Wan et al., 2007). Vari-
ous data implicate RPS3 in the regulation of IB and the
locus. Chromatin immunoprecipitation assays show that this
ribosomal protein binds to the IB promoter. In addition, a
small interfering RNA directed against RPS3 inhibits IB
expression and a RPS3-specific short hairpin RNA reduces
expression. As with p65, RPS3 is localized in the cytoplasm
but enters the nucleus when it is participating in transcrip-
tional regulation (Wan et al., 2007). We used immunofluo-
rescence microscopy to determine the localization of RPS3
and p65 in a mixture of pro–B, pre–B, and immature B cells
(B220+IgD) from B1-8 -HEL- and B1-8low -HEL-
bone marrow. Interestingly, the B1-8 -HEL- cells con-
tained mostly cytoplasmic RPS3 expression (only 17% of
these cells scored positive for nuclear RPS3), whereas the
majority (68% of the cells scored) of B1-8low -HEL- cells
contained nuclear RPS3 expression (Fig. 5 C and Fig. S3).
Although there was more nuclear p65 in the B1-8low -HEL-
cells compared with the B1-8 -HEL- cells, the differ-
ence is not as striking as RPS3 (Fig. S3). These data imply
that RPS3 could be acting to regulate NF-B during re-
Inhibition of NF-B inhibits light chain rearrangements
To determine if the differences observed between light chain
locus transcription and rearrangement in the IB-positive
and -negative populations were dependent on NF-B activity,
we took advantage of a retrovirus expressing an IB super-
repressor (IBN). IBN is missing amino acids 1–36
of the N terminus and cannot be phosphorylated or ubiqui-
tinated. Therefore, it remains bound to NF-B dimers and in-
JEM VOL. 206, August 3, 2009
Figure 6. B cell development in IBN-infected IL-7 bone marrow cultures. (A) Bone marrow IL-7 cultures were infected with an IBN ret-
rovirus or an empty vector control. 3 d after infection, cells were surface stained with Thy1.1 (a marker of viral infection), CD19, and IgM antibodies. The
cells were fixed, permeabilized, stained with IgM antibody, and then analyzed by flow cytometry. The top shows the empty vector control infection and
the bottom shows IBN-infected cells. The left displays CD19 versus IgM gated on Thy1.1+ cells. The right displays CD19 verses cytoplasmic gated
on CD19+IgM. (B) Real-time RT-PCR analysis of the indicated transcripts using RNA purified from Thy1.1-sorted IBN or empty vector retrovirally
transduced pro–/pre–B cells. All transcripts were normalized to HPRT transcription and data from each IBN sample was arbitrarily set to 1, with error
bars indicating range of triplicate assays. These experiments were repeated twice using bone marrow pooled from six mice.
A ROLE FOR NF-B IN RECEPTOR EDITING | Cadera et al.
Receptor editing is stimulated in immature B cells by BCR
recognition of autoantigen, which results in down-modula-
tion of BCR from the cell surface. BCR engagement is
known to activate NF-B. Using an IB-lacZ knockin re-
porter allele, we detected a subpopulation of pre–B cells that
contains active nuclear NF-B and found that this same pop-
ulation expresses markers of receptor editing. We propose
that this correlation between NF-B activity and receptor
editing may indicate a functional role for this transcription
factor in a key mechanism of self-tolerance.
Previous results have suggested various roles for NF-B
during B cell development, ranging from involvement in light
chain gene rearrangements (Scherer et al., 1996; O’Brien et al.,
1997; Bendall et al., 2001) to being dispensable for B cell de-
velopment (Igarashi et al., 2006; Sasaki et al., 2006). Because
receptor editing involves a specific subset of light chain gene
rearrangements, our data imply that NF-B could be involved
in light chain rearrangements without being required for B cell
development, which is consistent with seemingly contradic-
tory results. In addition, the role of NF-B in receptor editing
could be either direct or indirect, and its activation in pre–B
cells might be via the classical or nonclassical pathway or, per-
haps, even a novel pathway. Suggestive of the latter possibility,
Derudder et al. (2009) have found that NEMO, IKK1, and
IKK2 are all dispensable for receptor editing. Further experi-
ments will be required to determine whether alternative path-
ways of NF-B activation exist in developing B cells.
Much of the data presented in this paper are correlative;
nuclear NF-B activity correlates with -gal expression,
which in turn correlates with numerous markers of receptor
editing in phenotypic pre–B cells including elevated levels of
Ig rearrangement and transcription, increased cytoplasmic
Ig expression, increased J locus RS rearrangements, and
increased J RSS replacement breaks. We also observed a sev-
eral-fold increase in IRF-4 mRNA levels, which is consistent
with prior studies showing that IRF-4 plays a role in Ig and
Ig locus activation enhancers (Pongubala et al., 1992; Eisenbeis
et al., 1995) and is involved in receptor editing (Pathak
et al., 2008). We provided a critical test of these correlations
by examining NF-B activity in two knockin models of re-
ceptor editing, in each case finding that nuclear NF-B activ-
ity is elevated as compared with cells expressing a knocked-in
In addition, we found that overexpression of a cDNA en-
coding an IBN mutant suppressed the generation of sIgM+
immature B cells and diminished levels of germline Ig and re-
arranged Ig transcripts in a short-term primary cell culture
system. The results of this perturbation of NF-B activity are
consistent with a causal role for this factor in receptor editing.
It is possible that the activation of NF-B in a population
of cells undergoing receptor editing is serving a purpose other
than the induction of editing. For example, NF-B is known
to regulate the expression of various antiapoptotic genes in-
cluding Bcl-2, Bcl-xL, and Mcl-1. Thus, it is possible that NF-B’s
role in editing may be to promote a sufficient period of
cell survival to allow for ongoing V(D)J recombination activ-
ity to result in the generation of a self-tolerant BCR. To
test this idea, we compared the levels of antiapoptosis gene
mRNA in sorted -gal+ and -gal pre–B cells. We found
no significant differences in expression levels between these
cell populations (Fig. S1).
A series of elegant studies from the Behrens laboratory has
led to the suggestion that receptor editing is associated with the
loss of IgM from the surface of editing cells (Schram et al.,
2008). Others have found that cytoplasmic Ig expression is el-
evated after BCR down-regulation in cells undergoing recep-
tor editing (Pelanda et al., 1997). These results are consistent
with our observation that a high fraction of -gal+ pre–B cells
express cytoplasmic Ig chain (Fig. 4). We found that cytoplas-
mic Ig levels increase in the population of CD19+IgM cells
that results when immature B cells are treated with cross-link-
ing anti-IgM antibodies, an in vitro mimic of the receptor-edit-
ing signal. Schram et al. (2008) showed that this loss of surface
BCR in editing cells was associated with increased levels of
transcription of both IB and Bcl-x, which are known NF-B
RS rearrangements are considered a hallmark of receptor
editing (Retter and Nemazee, 1998; Vela et al., 2008). Large
increases in these rearrangements are observed in receptor-
editing mouse models (Chen et al., 1997; Pelanda et al., 1997;
Ait-Azzouzene et al., 2005). Mice lacking the RS sequence
display decreased receptor editing and fewer B cells express-
ing a light chain (Vela et al., 2008). An increase in RS rear-
rangement to both IRS and V gene segments correlates with
-gal expression in our experiments. The increase in RS re-
arrangement indicates that a substantial number of cells in the
-gal–positive pre–B cell population have inactivated the
locus, providing further evidence that this population is un-
dergoing receptor editing.
In both experimental systems used to study NF-B activ-
ity, analysis of IB+/lacZ pre–B cells, and retroviral infection
of cultured B cells with IBN, the change in light chain
status was not accompanied by a change in RAG expression.
This data implies that an increase in RAGs is not responsible
for receptor-editing rearrangements. There are two hypoth-
eses regarding RAG expression during receptor editing:
either an increase or a persistence of RAG expression upon
recognition of an autoantigen (Jankovic et al., 2004; Verkoczy
et al., 2005). We interpret our data as being consistent with
the idea that RAG expression is maintained, not necessarily
increased, during receptor editing.
RPS3 interacts with p65, increases the activity and bind-
ing affinity of NF-B, and influences the binding sites of the
NF-B complex (Wan et al., 2007). Using an immunofluo-
rescence microscopy assay, we found increased nuclear RPS3
in pro–B, pre–B, and immature B cells from receptor-editing
model mice (B1-8low -HEL-) compared with the same
population of cells from mice with innocuous BCRs (B1-8
-HEL-). Preliminary data suggests that knocking down
RPS3 causes a defect in receptor editing (unpublished data).
We infer from this data that RPS3 is influencing binding sites
JEM VOL. 206, August 3, 2009
Figure 7. Expression of NF-B target genes in IB-transduced pre–B cells. (A) cDNA from sorted -gal–positive and –negative pre–B cells
was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT transcripts to control for any differences in cDNA template.
-gal–negative cell result was arbitrarily set to 1, with error bars indicating range of triplicate assays. (B) cDNA from CD19+IgD cells from B1-8 -HEL-
and B1-8low -HEL- mice was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT, with error bars indicating range
of triplicate assays. These results are representative of two independent repetitions of this experiment using pools of sorted bone marrow cells from five
to six mice.
A ROLE FOR NF-B IN RECEPTOR EDITING | Cadera et al.
of NF-B during receptor editing and we are currently ex-
amining this issue in greater detail.
Our results implicate NF-B in the regulation of receptor
editing. These data imply that NF-B is not required for B cell
development to take place but, instead, contributes to a key
process that prevents the expression of self specific BCRs.
Materials and Methods
Mice. B1-8, B1-8high, B1-8low, and Ig -HEL mice were a gift from M.
Nussenzweig (Rockefeller University, New York, NY). IB+/lacZ mice
were a gift from B. Sha (University of California, Berkeley, Berkeley, CA).
All wild-type mice used were C57BL/6 (The Jackson Laboratory). Animal
experimentation was approved by the University of California, Berkeley
Animal Care and Use Committee.
Cell culture. Cells were cultured in RPMI 1640 medium supplemented
with 10% (vol/vol) fetal calf serum, 100 µg/ml penicillin, 100 µg/ml strep-
tomycin, and 50 µM -mercaptoethanol and grown at 37°C in 5% CO2.
10 µg/ml LPS was added to specified cultures for various lengths of time. For
the LPS washout experiments, cells were washed twice in PBS and then re-
cultured in RPMI as before with the addition of 50 µg/ml cycloheximide.
For primary cell cultures or retroviral infection, bone marrow was iso-
lated from 4–6-wk-old mice. The cells were passed through a 40-µm filter to
create a single cell suspension. Red blood cells were depleted by ACK lysis
and cells were filtered again. Cells were overlayed on irradiated S17 stromal
cells that were plated 1 d prior. 100 U/ml rIL-7 (R&D Systems) was added
to the culture. Goat anti–mouse IgM F(ab)2 fragments (Jackson Immuno-
Research Laboratories) were used at a final concentration of 10 µg/ml.
Retroviral infection of bone marrow cultures. Retroviral plasmids
were incubated with lipofectamine 2000 (Invitrogen) and added to Phoenix
cells. 5 h after transfection, 10% fetal calf serum was added to the Opti-MEM
media. 2 d after culture, the viral supernatant was removed and put through
a 0.45-µM syringe filter and added directly to the bone marrow cells supple-
mented with 100 U/ml IL-7 and 4 µg/ml polybrene. The cells and viral su-
pernatant were spun at 2,400 rpm for 90 min at room temperature. After 4–6 h,
the cells were pelleted and resuspended in media and overlayed on irradiated
S17 cells that had been plated 1 d prior.
FDG loading. Cells were resuspended at a concentration of three million
cells/50 µl RPMI media. FDG (Invitrogen) was diluted to 2 mM in H2O.
50 L of 2-mM FDG was added to 50 µl of cells and incubated at 37°C for
exactly 1 min. 1.5 ml RPMI media was added to stop the reaction.
Real-time PCR. All taqman primers used fam/tamra chemistry. The
primers used were the following: HPRT forward, 5-CTGGTGAA-
AAGGACCTCTCG-3; HPRT reverse, 5-TGAAGTACTCATTAT-
AGTCAAGGGCA-3; HPRT TM probe, 5-TGTTGGATACAGGC-
CAGACTTTGTTGGAT-3; GT 5, 5-GGACGTTCGGTGGAGGC-
3; GT 3, 5-GGAAGATGGATACAGTTGGTGCA-3; GT probe,
5-CCAAGCTGGAAATCAAACGCTGAT-3; Irf4 sense, 5-GAAG-
CCTTGGCGCTCTCA-3; Irf4 antisense, 5-TCACGAGGATGTCCC-
GGTAA-3; Irf4 probe, 5-CTGCCGGCTGCATATCTGCCTGT-3;
RAG1 sense, 5-CATTCTAGCACTCTGGCCGG-3; RAG1 antisense,
5-TCATCGGGGCAGAACTGAA-3; RAG1 probe, 5-AAGGTAG-
CTTAGCCAACATGGCTGCCTC-3; RAG2 sense, 5-TTAATTCCT-
GGCTTGGCCG-3; RAG2 antisense, 5-TTCCTGCTTGTGGATGT-
GAAAT-3; and RAG2 probe, 5-AGGGATAAGCAGCCCCTCTG-
GCC-3. Real time PCR was also performed using Evergreen chemistry.
The following primers were used: V1,2, 5-TGGAGACAAGGCTGC-
CCTCACCATCACAG-3; V3, 5-TGGTGCTGATCGCTACCT-
TAGCATTTCCA-3; C RT, 5-GAGCTCYTCAGRGGAAGGT-
GGAAACABGGT-3; IB intron forward, 5-GCAATCATCCACG-
AAGAGAAGC-3; IB intron reverse, 5-CGTTGACATCAGCACC-
CAAAG-3; C, 5-GTCCTGATCAGTCCAACTGTTCAGG-3; and
PCR primers. PCR primers used were the following: RS reverse #1,
5-GGACATCTACTGACAGGTTATCACAGGTC-3; IRS forward #1,
5-ATGACTGCTTGCCATGTAGATACCATGG-3; VS, 5-CCGA-
ATTCGSTTCAGTGGCAGTGGRTCRGGRAC-3; APRT 747 for-
ward, 5-TGCTAGACCAACCCGCACCCAGAAG-3; APRT 964
reverse, 5-TCGTGACCGCACCTGAACAGCAC-3; CH, 5-ATG-
CAGATCTCTGTTTTTGCCTCC-3; VH558, 5-CGAGCTCTC-
CARCACAGCCTWCATGCARCTCARC-3; VH7183, 5-CGGT-
ACCAAGAASAMCCTGTWCCTGCAAATGASC-3; and VHQ52,
LM-PCR for primary and secondary break intermediates. LM-
PCR was performed as previously described (Schlissel et al., 1993). Linker-
ligated genomic DNA was analyzed by PCR using the linker primer BW-H
(5-CCGGGAGATCTGAATTCCAC-3) and the ko5 primer (5-GCCC-
AAGCGCTTCCACGCATGCTTGGAG-3) to assay for primary breaks
or a degenerate V primer (5-CCGAATTCGSTTCAGTGGCAGTG-
GRTCRGGRAC-3) to detect secondary breaks. A touchdown PCR pro-
gram was used: 94°C for 1 min, 19 cycles of 92°C for 30 s, and then 70°C
for 40 s, with the temperature dropped by 0.5°C for each successive cycle.
This was followed by 19 cycles of 92°C for 30 s and then 60°C for 40 s with
1 s added each successive cycle.
Immunofluorescence microscopy. In the experiment shown in Fig. 1,
cells were resuspended in PBS and spun onto a slide using a cytospin centri-
fuge. Cells were fixed in 4% paraformaldehyde, washed in PBS, and then in-
cubated in blocking solution (PBS, 0.5% fetal calf serum, 0.5% normal rat
serum, 0.2% Triton X-100, and 3% bovine serum albumin). The blocking
buffer was removed and the cells were incubated with the primary antibody
p65 (sc-372; Santa Cruz Biotechnology, Inc.). The slides were washed in PBS
containing 0.1% Triton X-100 and then PBS. Cells were next incubated with
secondary antibody, –rabbit Cy3 (Jackson ImmunoResearch Laboratories),
and then washed as for the primary antibody. Antifading solution containing
500 ng/ml DAPI was place on each cell spot. In the experiment shown in
Fig. 5, staining was performed as previously described (Wan et al., 2007).
Surface and cytoplasmic staining for flow cytometry. Antibodies used
to stain early B cell development in conjunction with FDG loading were the
following: IgM-PE (Southern Biotechnology), B220-PE Texas red (Invitro-
gen), CD43-biotin (clone S7; BD), and streptavidin-Cy5 (BD). Alternative
antibodies used to stain for B cell development were the following: IgD-PE
(Southern Biotechnology), CD43-biotin (clone S7; BD), streptavidin-
Red613 (Invitrogen), IgM-Cy5 (eBioscience), andCD19-Cy7 (BD).
For cytoplasmic Ig staining, cells were first surface stained using CD19-
Cy7 (BD), IgM-Cy5 (eBioscience), IgD-PE (Southern Biotechnology), and
a fourfold excess of -biotin (BD), and then fixed, permeabilized in BSA/
PBS with 0.1% saponin, and resuspended in BSA/PBS with 0.1% saponin
with -FITC (BD).
Online supplemental material. Fig. S1 shows the lack of correlation be-
tween NF-B activity and antiapoptosis gene expression. Fig. S2 is an analy-
sis of rearranged heavy chain gene transcripts in -gal–sorted pre–B cells.
Fig. S3 is a quantitative analysis of the immunofluorescence microscopy pre-
sented in Fig. 5 C. Online supplemental material is available at http://www
The authors wish to acknowledge Bill Sha (University of California, Berkeley, CA)
for providing the IB+/lacZ mice, as well as important advice during the conduct of
this project, and Michel Nussenzweig (Rockefeller University) for sharing various Ig
This work was supported by a grant from the National Institutes of Health
(RO1 HL48702) to M.S. Schlissel.
The authors have no conflicting financial interests.
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