NF-kappaB activity marks cells engaged in receptor editing.

Abstract

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-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

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ARTICLE
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 206 No. 8 1803-1816
www.jem.org/cgi/doi/10.1084/jem.20082815
1803
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
CORRESPONDENCE
Mark S. Schlissel:
mss@berkeley.edu
Abbreviations used: -gal, -
galactosidase; BCR, B cell re-
ceptor; cDNA, complementary
DNA; FDG, fluorescein di--
d-galactopyranoside; HPRT,
hypoxanthine-guanine phosphoribo-
syl transferase; IgHC, Ig heavy
chain; IgLC, Ig light chain;
LM-PCR, ligation-mediated
PCR; mRNA, messenger
RNA; RPS3, ribosomal protein
S3; RSS, recombination signal
sequence.
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 IB gene was replaced with a
lacZ (-gal) reporter complementary DNA (cDNA; IB+/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 IB+/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 IB
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.
© 2009 Cadera et al. This article is distributed under the terms of an Attribu-
tion–Noncommercial–Share Alike–No Mirror Sites license for the first six months
after the publication date (see http://www.jem.org/misc/terms.shtml). After six
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The Journal of Experimental Medicine

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A ROLE FOR NF-B IN RECEPTOR EDITING | Cadera et al.
confirm that -gal activity correlates with IB expression in
IB+/lacZ reporter mice, IB 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 IB
messenger RNA (mRNA) as compared with -gal cells
(Fig. 1 A). The greater amounts of IB transcripts in -gal+
cells confirms that -gal detected using FDG is a suitable re-
porter for IB expression in IB+/lacZ mice.
Several groups have shown that IB 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 IB+/lacZ mice.
We confirmed this using an Ableson virus–transformed pro–
B cell line made from IB+/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 IB+/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 IB+/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 IB+/lacZ cells and that -gal is re-
porting recent NF-B activity.
Increased light chain gene rearrangements correlate
with IB expression in pre–B cells
We used multiparameter flow cytometry to analyze -gal ex-
pression in cells isolated from IB+/lacZ bone marrow to deter-
mine the pattern of IB 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 IB-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 V1,2-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 IB 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 IB family. Various signaling pathways result in
the activation of a kinase that phosphorylates IB leading to its
degradation. Once released from IB, NF-B can translocate
to the nucleus, bind DNA sequences, and regulate transcription.
Remarkably, one of the transcriptional targets of NF-B is IB
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 IB “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 IB 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 IB 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 IB locus (Beg et al., 1995).
Because IB 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.
RESULTS
IB+/lacZ mice report IB and NF-B activity
In an attempt to assess NF-B activity at various stages of
B cell development, we analyzed IB+/lacZ mice. Although
these mice express less IB protein than wild-type mice,
they display no obvious phenotype (Beg et al., 1995). To

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Figure 1. IB+/lacZ mouse reports IB transcription and nuclear NF-B activity. (A) Real-time RT-PCR analysis of IB expression in RNA puri-
fied from flow-sorted -gal+ (dark gray) and -gal (light gray) IB+/lacZ pre–B cells (B220+CD43lowsIgM/D). The ratio is shown between IB transcript
and a control transcript, HPRT, with error bars indicating the standard deviation of triplicate assays. (B) Flow cytometric analysis of -gal expression in
IB+/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 IB+/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 IB+/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.

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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 IB.
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 IB 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. IB expression during B cell development. (A) -gal expression in an IB+/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.

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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
IB+/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 IB 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 IB-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 J1, 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.