Content uploaded by Tri Giang Phan
Author content
All content in this area was uploaded by Tri Giang Phan
Content may be subject to copyright.
Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
The Journal of Experimental Medicine
J. Exp. Med.
The Rockefeller University Press • 0022-1007/2003/04/845/16 $8.00
Volume 197, Number 7, April 7, 2003 845–860
http://www.jem.org/cgi/doi/10.1084/jem.20022144
845
B Cell Receptor–independent Stimuli Trigger
Immunoglobulin (Ig) Class Switch Recombination and
Production of IgG Autoantibodies by Anergic Self-Reactive
B Cells
Tri Giang Phan,
1
Michelle Amesbury,
1
Sandra Gardam,
1
Jeffrey Crosbie,
1
Jhagvaral Hasbold,
2
Philip D. Hodgkin,
2
Antony Basten,
1
and Robert Brink
1
1
Centenary Institute of Cancer Medicine and Cell Biology, Newtown NSW 2042, Australia
2
Walter and Eliza Hall Institute for Medical Research, Parkville VIC 3050, Australia
Abstract
In both humans and animals, immunoglobulin (Ig)G autoantibodies are less frequent but more
pathogenic than IgM autoantibodies, suggesting that controls over Ig isotype switching are re-
quired to reinforce B cell self-tolerance. We have used gene targeting to produce mice in
which hen egg lysozyme (HEL)-specific B cells can switch to all Ig isotypes (SW
HEL
mice).
When crossed with soluble HEL transgenic (Tg) mice, self-reactive SW
HEL
B cells became an-
ergic. However, in contrast to anergic B cells from the original nonswitching anti-HEL
sol-
uble HEL double Tg model, self-reactive SW
HEL
B cells also displayed an immature phenotype,
reduced lifespan, and exclusion from the splenic follicle. These differences were not related to
their ability to Ig class switch, but instead to competition with non-HEL–binding B cells gen-
erated by V
H
gene replacement in SW
HEL
mice. When activated in vitro with B cell receptor
(BCR)-independent stimuli such as anti-CD40 monoclonal antibody plus interleukin 4 or li-
popolysaccharide (LPS), anergic SW
HEL
double Tg B cells proliferated and produced IgG anti-
HEL antibodies as efficiently as naive HEL-binding B cells from SW
HEL
Ig Tg mice. These re-
sults demonstrate that no intrinsic constraints to isotype switching exist in anergic self-reactive
B cells. Instead, production of IgG autoantibodies is prevented by separate controls that reduce
the likelihood of anergic B cells encountering BCR-independent stimuli. That bacteria-
derived LPS could circumvent these controls may explain the well-known association between
autoantibody-mediated diseases and episodes of systemic infection.
Key words: self-tolerance • autoimmunity • LPS • CD40 • hen egg lysozyme
Introduction
Ig transgenic (Tg)
*
mice expressing BCRs directed against
endogenous or transgene-encoded self-antigens provide the
opportunity to track the development of self-reactive B
cells in vivo. Studies using such mice have revealed that
self-tolerance in the B cell compartment can be maintained
by silencing self-reactive B cells in several different ways.
These include clonal deletion (1, 2), receptor editing (3, 4),
and anergy (5) depending on the valency of the self-antigen
and the site of its encounter with differentiating B cells.
Anergic self-reactive B cells have been characterized pri-
marily using the anti–hen egg lysozyme (HEL)
soluble
HEL double Tg model (MD4 Ig Tg
ML5 soluble HEL
Tg mice). In this model, the induction of self-tolerance is
accompanied by down-regulation of surface IgM (5), fail-
ure of B cells to colonize the splenic marginal zone (MZ;
reference 6), inactivation of BCR signaling (7–9), and al-
terations in gene expression (10). Further analysis of the
anti-HEL double Tg system has revealed an additional dis-
tinct fate for anergic B cells. This is evident in mixed bone
marrow chimeras in which self-reactive MD4 B cells com-
Address correspondence to Robert Brink, Centenary Institute of Cancer
Medicine and Cell Biology, Locked Bag Number 6, Newtown NSW
2042, Australia. Phone: 61-2-95656-136; Fax: 61-2-95656-105; E-mail:
r.brink@centenary.usyd.edu.au
*
Abbreviations used in this paper:
BCM, B cell medium; BrdU, bro-
modeoxyuridine; CFSE, 5-(and 6-)carboxyfluorescein diacetate succin-
imidyl ester; CSR, class switch recombination; ES, embryonic stem;
HEL, hen egg lysozyme; m.f.i., mean fluorescence intensity; MZ, mar-
ginal zone; NB6, non-transgenic C57BL/6; PALS, periarteriolar lym-
phoid sheath; SA, streptavidin; Tg, transgenic.
The Journal of Experimental Medicine
846
Production of IgG Autoantibodies by Anergic Self-Reactive B Cells
prise
20% of the B cell repertoire, instead of making up
90–95% of the repertoire as occurs in MD4
ML5 double
Tg mice (11). In these chimeras, anergic B cells do not en-
ter the splenic follicle but accumulate at the follicular junc-
tion with the T cell–rich periarteriolar lymphoid sheath
(PALS). Furthermore, the half-life of these B cells is re-
duced to
3 d (11). Two theories have been put forward
to explain the altered migration and reduced lifespan of an-
ergic B cells under these conditions. According to the first,
anergic B cells cannot compete efficiently with nonself-
reactive B cells for limited “niches” within the follicle that
confer longer term survival (11, 12). However, significant
reductions in the frequency of HEL-binding B cells can
shift the antigen/BCR equilibrium toward higher levels of
antigen binding by individual B cells, which raises the al-
ternative possibility that more intense signaling by soluble
self-antigen may cause the outer PALS arrest and reduced
lifespan of anergic B cells (13). Because the interaction with
self-antigen and frequency of HEL-binding B cells were
not measured simultaneously in these studies (11–13), it re-
mains to be resolved which of these potential mechanisms
accounts for the altered behavior of anergic B cells.
A hallmark of B cell responses is the ability to vary the
constant regions of expressed IgH by class switch recombi-
nation (CSR). Ig class switching has evolved to target dif-
ferent immune effector mechanisms toward invading
pathogens. For instance, IgG but not IgM antibodies can
penetrate tissues where they activate complement in situ
and bind Fc receptors on macrophages and NK cells to in-
duce antibody-dependent cellular cytotoxicity (14). Be-
cause of their ability to activate multiple immune effector
systems, IgG autoantibodies pose a greater potential threat
to the host than do IgM autoantibodies (15–19). This
threat is exemplified by the relatively common finding of
IgM but not IgG autoantibodies in sera from healthy indi-
viduals (20). Given the greater pathogenicity but reduced
prevalence of IgG autoantibodies, self-reactive B cells may
possess controls over Ig isotype switching in addition to any
that normally act to prevent their differentiation into anti-
body-secreting cells.
Despite the insights gained from earlier Ig Tg models, it
has not been possible to study the regulation of CSR by
self-reactive B cells in detail because B cells from these
models failed to undergo CSR. This problem has been
overcome by using homologous recombination in embry-
onic stem (ES) cells to target rearranged V
H
DJ
H
genes to
the 5
end of the germline IgH gene (21). Location of the
V
H
DJ
H
segment at its physiological position in the IgH lo-
cus results in a high frequency of B cells in the adult mouse
expressing the targeted variable region (21–23). This fre-
quency is typically lower than in conventional Ig Tg mice
due to recombination of upstream V
H
or V
H
plus D seg-
ments into the targeted V
H
DJ
H
exon (22, 23). Nonetheless,
B cells expressing a targeted heavy chain variable region
can undergo CSR to all downstream isotypes in a physio-
logical manner (21–23).
To combine the advantages of the well-characterized
anti-HEL Ig Tg system with the ability to study CSR, we
have targeted the V
H
10 anti-HEL heavy chain variable re-
gion gene to the endogenous C57BL/6 IgH locus. SW
HEL
mice carrying the targeted allele and an anti-HEL light
chain transgene were shown to produce HEL-binding
(HEL
) B cells capable of switching to all classes of Ig. Ad-
ditionally, a large population of non-HEL binding (HEL
)
B cells that had undergone V
H
gene replacement was gen-
erated in these mice. These replacement events occurred in
the absence of any obvious self-reactivity and may reflect
instability of rearranged V
H
DJ
H
genes during early B cell
development. When SW
HEL
mice were crossed with ML5
soluble HEL Tg mice, the resulting self-reactive B cells be-
came anergic. However, they also displayed an immature
phenotype, reduced lifespan, and were excluded from the
splenic follicle as well as the MZ, in contrast to the original
MD4 double Tg model. Detailed analysis revealed that the
changes observed in anergic SW
HEL
B cells were not linked
to their CSR potential but instead were due to competi-
tion from endogenous HEL
B cells. Under normal cir-
cumstances, self-reactive B cells constitute a small propor-
tion of the peripheral B cell repertoire and must compete
with nonself-reactive B cells. The new SW
HEL
model reca-
pitulates this repertoire diversity and is therefore a more
physiological system in which to study the fate of anergic
self-reactive B cells and the effect of tolerance induction on
regulation of CSR. When anergic self-reactive SW
HEL
B
cells were activated with BCR-independent stimuli in
vitro, they proliferated and readily secreted isotype-
switched anti-HEL Ig. These unexpected findings demon-
strate that there is no intrinsic restriction to Ig class switch-
ing by anergic B cells and separate controls are required to
prevent the secretion of IgG autoantibodies.
Materials and Methods
Mice.
Tg mice were all maintained on a pure C57BL/6
background at the Centenary Institute Animal Facility. In MD4
Ig Tg mice the heavy (V
H
10-
) and light chain (V
10-
) trans-
genes encoding IgM and IgD with the high affinity anti-HEL
specificity of the HyHEL10 mAb are cointegrated (5). In con-
trast, MD2 Ig Tg mice (24) carry only the V
H
10-
heavy chain
transgene and LC2 Ig Tg mice (produced by DNX Inc.) carry
only the V
10-
light chain transgene. Membrane-bound HEL
was expressed as a neo self-antigen in KLK3 Tg mice under the
control of the H2-K
b
promoter (2) and soluble HEL was ex-
pressed under the control of either the metallothionein promoter
(ML5 mice) or albumin promoter (AL3 mice). The latter two
lines of mice express soluble HEL at intermediate (10–20 ng/ml)
and high (80–160 ng/ml) concentrations, respectively (5, 13).
Non-Tg C57BL/6 (NB6), C57BL/6-SJL.Ptprc
a
(CD45.1 con-
genic), and BALB/c mice were obtained from the Animal Re-
sources Centre (Canning Vale, Western Australia).
To produce anti-HEL Tg mice capable of undergoing CSR,
the rearranged V
H
10 variable region gene from the HyHEL10
hybridoma was targeted to the 5
end of the endogenous IgH lo-
cus in mouse ES cells using the general approach of Taki et al.
(see Fig. 1, A and B; reference 21). 5
homology sequences for
the targeting construct were obtained from a
phage clone iso-
lated from a C57BL/6 liver genomic DNA library (CLON-
TECH Laboratories, Inc.). 3
homology sequences were ampli-
The Journal of Experimental Medicine
847
Phan et al.
fied by PCR from C57BL/6 genomic DNA and verified by
DNA sequencing. The final targeting construct included a loxP-
flanked neomycin resistance cassette in reverse transcriptional
orientation to the IgH locus that was located immediately 5
to
the rearranged V
H
10 variable region and its associated promoter
(see Fig. 1 C). ES cell electroporation, selection of homologous
recombinant clones, and production of chimeric mice were per-
formed using a C57BL/6 ES cell line (Bruce 4) as previously de-
scribed (25). Two homologous recombinant clones were identi-
fied from 192 G418 resistant clones by PCR (see Fig. 1 D).
Chimeric mice were generated from one of these clones and
then backcrossed with C57BL/6 females. Black progeny het-
erozygous for the targeted V
H
10 variable region (V
H
10
tar
; see
Fig. 1 E) were mated with LC2 Ig Tg mice to produce SW
HEL
mice heterozygous for both the V
H
10
tar
IgH allele and the V
10-
Tg. All mice were screened for the presence of transgenes by
PCR amplification of genomic DNA prepared from peripheral
blood leukocytes.
Analysis of Heavy Chain Variable Region Expression.
Spleen
cells from SW
HEL
mice were stained with anti–B220-PE and
HEL-FITC, after which HEL- (B220
, HEL
) and non-HEL–
(B220
, HEL
) binding B cells were separated to
95% purity
by sorting on a FACStarPlus™ flow cytometer (BD Bio-
sciences). PolyA
mRNA was prepared from 5
10
5
sorted
cells using the Oligotex™ Direct mRNA Kit (QIAGEN). A
modified 5
rapid amplification of cDNA end system (GIBCO
BRL) was used to generate PCR fragments for cloning and se-
quencing. PolyA
mRNA was reverse transcribed with a primer
specific to the first constant region domain of the
heavy chain
(C
H1
) and the cDNA was then dC tailed with terminal deoxy-
nucleotidyl transferase. Primary PCR amplification was per-
formed with an unabridged anchor primer that recognized the
poly-dC tail and a nested C
H1
-specific primer 5
to the first
primer. Secondary PCR amplification was performed with a
universal primer to the first framework coding region and a
third nested C
H1
-specific primer. The final PCR product was
ligated with T4 DNA ligase (Roche Diagnostics) to the pCR2.1
plasmid (Invitrogen). Plasmid DNA was purified from individ-
ual clones and the inserts were sequenced (Australian Genome
Research Facility).
Antibodies and Reagents.
The following mAbs to murine anti-
gens were purchased from BD Biosciences: anti–CD5-biotin
(clone 53-7.3), anti–CD21/CD35-FITC (7G6), anti–CD23-PE
(B3B4), anti–CD24-PE (M1/69), anti–CD45R/B220-biotin,
-PerCP, and -APC (RA3-6B2), anti–CD45.1-PE (A20), anti–
CD69-PE (H1.2F3), anti–CD86-PE (GL1), anti–IgM
b
-PE (AF6-
78), anti–IgD
b
-PE (217-170), anti–IgD-FITC (11-26c.2a),
anti–IgG1-biotin (A85-1), anti–IgG2a-biotin (R19-15), anti–
IgG2b-biotin (R12-3), anti–IgG3-biotin (R40-82), anti–IgE-
biotin (R35-118), anti–IgA-biotin (C10-1), and anti–Ig
-biotin
(187.1). Anti–IgM-PE (1B4B1) and IgD-PE (11-26) were pur-
chased from Southern Biotechnology Associates, Inc. HyHEL5,
HyHEL9, anti-IgM (Bet-2), IgM
a
(RS-3.1), and IgD
a
(AMS-
15.1) mAbs were purified from hybridoma supernatants and ei-
ther biotinylated or conjugated to FITC as previously described
(5). Conjugation to Alexa Fluor
®
647 was performed using the
Alexa Fluor
®
647 Monoclonal Antibody Labeling Kit (Molecular
Probes) according to the manufacturer’s instructions. Streptavidin
(SA)-PE, -PerCP, and -APC were purchased from BD Bio-
sciences. HEL was purchased from Sigma-Aldrich and conju-
gated to FITC and Alexa Fluor
®
647 as described above.
ELISAs.
Anti-HEL antibody levels in sera and culture su-
pernatants were measured by direct ELISA essentially as previ-
ously described (5). In brief, 96-well polystyrene plates (Nunc)
were coated overnight at 4
C with HEL at 10
g/ml. The wells
were then blocked with 100
L 1% BSA/PBS and serial dilu-
tions of either sera or culture supernatants added together with
the appropriate standards. Biotinylated isotype-specific mAb in
0.1% BSA/1% skim milk powder/PBS was used to detect bound
antibody. SA-alkaline phosphatase (Roche Diagnostics) was then
added and visualized with the substrate
p
-nitrophenyl phosphate
(ICN Biomedicals). Absorbance at 405 nm was read and the
concentration of anti-HEL antibodies was calculated from the
standard curve.
Production of HyHEL10 Anti-HEL Ig Isotype Standards.
Stan-
dards expressing the HyHEL10 specificity in association with
each heavy chain isotype were produced by transiently transfect-
ing Chinese hamster ovary cells with a V
10-
light chain ex-
pression plasmid along with a heavy chain expression plasmid en-
coding V
H
10 in association with secreted constant region
sequences of either the
,
1,
2a,
2b,
3,
, or
heavy chain
isotype. Constant region coding sequences were obtained by
PCR amplification from C57BL/6 genomic DNA (IgH
b
allo-
type) and verified by DNA sequencing. Supernatants were col-
lected 3 d after transfection and the concentration of anti-HEL
antibodies was determined by ELISA using a biotinylated anti-
Ig
mAb and HyHEL10 (IgG1
a
) purified from hybridoma super-
natant as the standard.
Flow Cytometry.
Four-color flow cytometry was performed
on a dual laser FACSCalibur™ flow cytometer (BD Biosciences)
and analyzed with CELLQuest™ v.3.3 software (BD Bio-
sciences). Single cell suspensions of spleen, bone marrow, and
peritoneal exudate were prepared and 10
6
cells were stained for
surface markers in 96-well round-bottom plates and transferred
to microtiter tubes (Bio-Rad Laboratories) for data acquisition.
To reveal HEL-binding antigen receptors, cells were incubated
initially with saturating concentrations of HEL (200 ng/ml) fol-
lowed by biotinylated or fluorochrome-labeled HyHEL5 anti-
HEL mAb (HyHEL5 and HyHEL10 mAbs bind HEL noncom-
petitively). To determine receptor occupancy, splenocytes were
separately stained with anti–HEL-FITC (HyHEL5-FITC) with
and without previous incubation with HEL. All data were gated
on live lymphocytes on the basis of forward and side light scat-
ter. For calculations of frequencies of HEL-binding B cells, the
forward scatter was expanded because the high expression of
anti-HEL receptors in MD4 and SW
HEL
mice results in a propor-
tion of these B cells forming doublets when stained with HEL
plus HyHEL5.
For intracellular staining, cultured B cells were harvested and
washed twice in 1% BSA/0.1% azide/PBS and fixed in 2%
paraformaldehyde for 30 min at room temperature before perme-
abilization at 4C overnight in 0.1% Tween20/0.1% azide/PBS.
10
6
cells were then washed and transferred to new round-bottom
FACS
®
tubes (BD Biosciences) for staining with HEL-Alexa
Fluor
®
647 and biotinylated isotype-specific mAb revealed by
SA-PE. Cell division number was determined from the 5-(and
6-)carboxyfluorescein diacetate succinimidyl ester (CFSE; Mo-
lecular Probes) peaks and gates were drawn around each peak to
allow backgating (“division slicing”) to determine the proportion
of isotype-switched cells per division (26).
Bromodeoxyuridine (BrdU) Labeling. BrdU (Sigma-Aldrich)
was included in the drinking water of the mice at a concentration
of 0.25 mg/ml with the addition of 1% glucose as previously de-
scribed (11, 27). After 3 d, splenocytes from treated and control
mice were harvested and stained for cell surface markers. Cells
were then fixed, permeabilized, and stained with anti–BrdU-
The Journal of Experimental Medicine
848 Production of IgG Autoantibodies by Anergic Self-Reactive B Cells
FITC (3D4) using the BrdU Flow Kit (BD Biosciences) accord-
ing to the manufacturer’s instructions. Proportions of BrdU
cells
were determined with reference to equivalently stained cells from
mice that were not administered BrdU.
Immunohistology. Spleens were snap frozen in liquid nitrogen
and 5-m sections were acetone fixed and air dried before stain-
ing as previously described (6). HEL binding was detected by
staining with HEL at 200 ng/ml followed by polyclonal rabbit
anti–HEL sera and revealed with sheep anti–rabbit IgG-FITC
(Silenus Labs). The marginal sinus was identified by staining with
purified anti–MadCAM-1 (MECA-367) rat mAb (BD Bio-
sciences) followed by goat anti–rat IgG Texas red (Caltag Labora-
tories). To reveal the B cell area, sections were blocked with 1%
rat serum (Jackson ImmunoResearch Laboratories) and then ex-
posed to biotinylated anti-B220 and SA-FluoroBlue (Biomeda).
Bone Marrow Chimeras. 10–16-wk-old recipient mice were
lethally irradiated (950 rads) and rescued 6–8 h later by intrave-
nous injection with 2 10
7
bone marrow cells. All mice were
analyzed 2–3 mo after reconstitution.
In Vitro Cultures. The ability of B cells to respond to BCR
engagement was measured in vitro by culturing fresh splenocytes
overnight at 37C in B cell medium (BCM) containing RPMI
1640 medium supplemented with 10% heat-inactivated FCS
(Life Technologies), 2 mM l-glutamine, 1 mM sodium pyruvate,
0.1 mM nonessential amino acids, 10 mM Hepes, 100 U/ml
penicillin, 100 g/ml streptomycin, and 5 10
5
M 2-ME (all
from Sigma-Aldrich) with and without HEL at a concentration
of 500 ng/ml. Cells were then surface stained to detect up-regu-
lation of CD86 and CD69 by flow cytometry.
B cell responses to BCR-independent stimuli were deter-
mined by activating purified small B cells in vitro with either 5
g/ml agonist anti-CD40 mAb (HM40-3; BD Biosciences) or
CD40L prepared from Sf9 insect cell line transfected with a bac-
ulovirus vector containing CD40L (provided by M.R. Kehry,
Boehringer Ingelheim, Ridgefield, CT) plus 10 ng/ml IL-4
(Sigma-Aldrich) to simulate T cell–derived signals or 2.5 g/ml
LPS (Sigma-Aldrich) to simulate T-independent signals. Small B
cells were purified from spleens as described previously (28).
RBCs were lysed with hypotonic ammonium chloride and ad-
herent cells were depleted by incubation on a plastic tissue cul-
ture dish. T cells were depleted by complement-mediated lysis
using CD4- (RL172), CD8- (31M), and Thy.1- (30-H12) spe-
cific hybridoma supernatants. B cells were further purified by
Percoll (Amersham Biosciences) density gradient and small B
cells were recovered from the 65/80% interface. The recovered
B cells were 90% pure as determined by flow cytometry. Ini-
tially, cells were washed and resuspended in 0.1% BSA/PBS at
10
7
cells/ml and labeled with CFSE at a final concentration of 5
M for 10 min at 37C. Unlabeled CFSE was quenched with
ice-cold RPMI 1640 medium containing 10% FCS (Common-
wealth Serum Laboratories) and washed twice with BCM. The
labeled cells were then cultured in BCM and appropriate stimuli
for 3–4 d.
Results
Generation of SW
HEL
Mice. To produce mice in which
B cells have the same anti-HEL specificity as the MD4 Ig
Tg line but can undergo CSR, we generated two new
lines of mice. The first of these lines, LC2 Ig Tg mice,
carried the same V
10- light chain transgene present in
MD4 mice (5) but without the accompanying V
H
10-
heavy chain transgene. For the second line, gene targeting
in ES cells was used to insert the rearranged V
H
10 exon in
place of the J
H
segments at the 5 end of the endogenous
IgH gene (Fig. 1, A and B). The LC2 Ig Tg and V
H
10
tar
gene-targeted mice were produced on an inbred C57BL/6
background identical to that of MD4. When heterozygous
V
H
10
tar
and LC2 Ig Tg mice were mated, the two loci
were inherited at the expected Mendelian ratios. Mice
heterozygous for both loci (V
H
10
tar
/
LC2) were des-
ignated SW
HEL
.
Figure 1. Targeted insertion of the anti-HEL
V
H
10 variable region gene into the C57BL/6 IgH
gene. (A) Endogenous IgH locus indicating the
location of screening primers located within the
J
H
3 segment and 3 of the IgH intronic enhancer
(E
H
). U, upper primer; L, lower primer. (B) Tar-
geting construct indicating 5 and 3 homology
arms and the inverted loxP-neo
r
-loxP cassette and
rearranged V
H
10 variable region gene replacing
DQ52 and all four J
H
segments of the endogenous
IgH gene. Square arrows indicate promoters asso-
ciated with neo
r
and V
H
10. (C) Targeted IgH lo-
cus (V
H
10
tar
) after homologous recombination.
V
H
10 utilizes J
H
3 and therefore hybridizes with
the upper primer described above. Due to dele-
tion of J
H
4, amplification of the targeted locus re-
sults in a shorter PCR product than that from the
endogenous heavy chain gene (1.8 vs. 2.3 kb).
(D) PCR screening of DNA from G418-resistant
ES cell clones obtained after transfection with the
targeting construct. A single homologous recom-
binant heterozygous for V
H
10
tar
is shown (*). (E)
PCR screening of mice derived from homolo-
gous recombinant ES cells. Heterozygous
(V
H
10
tar
) mice were mated and DNA from the
blood of the resulting litter was PCR amplified
with the upper and lower screening primers de-
scribed above.
The Journal of Experimental Medicine
849 Phan et al.
SW
HEL
Mice Contain both HEL-binding B Cells and non-
HEL–binding B Cells Generated by V
H
Gene Replacement.
Flow cytometric analysis of spleen cells from LC2,
V
H
10
tar
/
, and SW
HEL
(V
H
10
tar
/
LC2) mice con-
firmed that expression of both HyHEL10 heavy and light
chain variable regions is required to generate significant
numbers of B cells with BCRs capable of binding HEL
(Fig. 2 A). Although a high proportion (40–60%) of B cells
in SW
HEL
mice bound HEL, this was significantly lower
than the frequency of 90% routinely observed in MD4 Ig
Tg mice (Fig. 2 A). The difference was not due to ineffi-
cient expression of the V
10- transgene by SW
HEL
mice
because inheritance of the same V
10- transgene in con-
junction with the V
H
10- transgene (MD2 LC2 mice)
resulted in high frequencies of HEL
B cells similar to
those observed in MD4 (not depicted).
The failure of HEL
B cells from SW
HEL
mice to express
the targeted allele raised the possibility that this was due to
V
H
gene replacement of the V
H
10 exon. V
H
gene replace-
ment has been reported in other mice carrying targeted
IgH alleles whereby the V
H
segment of the targeted V
H
DJ
H
exon is replaced by upstream V
H
or V
H
plus D elements
during early B cell development (22, 23). To confirm this,
HEL
and HEL
B cells from SW
HEL
mice were isolated to
95% purity by FACS
®
sorting. PCR analysis of purified
genomic DNA showed that the neo
r
selection marker lo-
cated 5 of the targeted V
H
10 exon (Fig. 1 C) was deleted
from HEL
but not HEL
B cells (not depicted), consistent
with recombination of upstream V
H
/V
H
plus D elements
specifically in the HEL
B cells. RT-PCR cloning and se-
quencing of heavy chain variable region genes present in
mature heavy chain mRNAs detected only unaltered
V
H
10 variable regions in HEL
B cells (Table I). In con-
trast, about half (9 out of 21) of the cDNA clones derived
from HEL
B cells expressed hybrid variable region exons
consisting of the D and J
H
segments of the targeted V
H
10
exon recombined in frame with upstream V
H
plus D se-
quences whereas 11 out of 21 clones expressed V
H
DJ
H
ex-
ons derived from the nontargeted IgH allele (the single un-
altered V
H
10 clone from the sorted HEL
B cell population
was presumably derived from one of the 5% contaminat-
ing HEL
B cells; Table I). Flow cytometric analysis of
spleen cells from SW
HEL
mice on a C57BL/6 BALB/c
(IgH
b
IgH
a
) F1 genetic background confirmed that
HEL
B cells expressed only the targeted IgH
b
allele (Table
I). Consistent with the sequencing results, approximately
equal proportions of the HEL
B cells expressed either the
targeted IgH
b
or the nontargeted IgH
a
allele (Table I). The
fact that heavy chain allelic exclusion was maintained in the
HEL
B cell population indicated that rearrangement and
expression of the nontargeted allele occurred only when a
nonproductive V
H
replacement event had taken place at the
targeted IgH allele.
HEL
SW
HEL
B Cells Belong to the B2 Lineage and Mature
Normally. Analysis of naive splenic B cells by flow cytom-
etry revealed that the HEL
B cells from SW
HEL
mice have
an IgM
hi
IgD
lo-hi
antigen receptor phenotype identical to
that of MD4 Ig Tg B cells (Fig. 2 B). By contrast, HEL
SW
HEL
B cells have the same BCR phenotype as wild-type
B cells, including the IgM
lo
IgD
hi
subset lacking in the
HEL
B cell populations from SW
HEL
and MD4 mice (Fig.
2 B). Despite the differences in BCR phenotype between
HEL
and HEL
B cells, expression of non-Ig maturation
markers and localization to anatomical compartments
Figure 2. P
h
enotypic c
h
aracterization o
f
B ce
ll
s
from SW
HEL
mice. (A) Spleen cells from 10–14-wk-
old NB6, LC2, V
H
10
tar
, SW
HEL
(V
H
10
tar
LC2), and MD4 Ig Tg mice were harvested and
stained with anti–B220-APC, HEL plus anti–HEL-
FITC, anti–IgD-PE, and anti–IgM-biotin plus
SA-PerCP. Cells were analyzed by flow cytometry
and the frequency of HEL
(top box) and HEL
(bottom box) B cells in each density plot was indi-
cated. Values from each mouse are representative of
more than five independent experiments. (B) Spleen
cells were stained as for A. IgM and IgD expression
data are displayed for all B220
B cells (NB6 and
MD4, top) or specifically for HEL
and HEL
B
cells (SW
HEL
, bottom) using the gates shown in A.
Numbers indicate the proportions of gated cells
within the indicated quadrants. Data are representa-
tive of more than five independent experiments. (C)
Cells were isolated by peritoneal lavage from the in-
dicated mice and stained with anti–B220-APC,
HEL plus anti–HEL-FITC, anti–IgM-PE, and anti–
CD5-biotin plus SA-PerCP. Cells were analyzed by
flow cytometry and gated on live cells that were
B220
and/or IgM
to include both the B1 and B2
cell populations. For SW
HEL
mice the peritoneal
cavity B cells were further gated into HEL
and
HEL
subpopulations. Numbers indicate the propor-
tion of gated cells in the B220
hi
CD5
window. Data
are representative of three independent experiments.
The Journal of Experimental Medicine
850 Production of IgG Autoantibodies by Anergic Self-Reactive B Cells
within the spleens of SW
HEL
mice were similar. Thus,
HEL
and HEL
B cells both contained normal propor-
tions of cells with immature, follicular, and MZ pheno-
types as defined by expression of CD21/CD35 and CD23
(see Fig. 6 A; not depicted) and both populations localized
to the follicle and MZ of the spleen (see Fig. 5 D).
Analysis of cells obtained by peritoneal cavity lavage in-
dicated that HEL
SW
HEL
B cells mirror the behavior of
MD4 B cells in their failure to differentiate along the B1
cell lineage. Accordingly, HEL
B cells belong to the con-
ventional B2 cell lineage, with B1 lineage (B220
lo
CD5
/
)
cells in SW
HEL
mice residing within the HEL
B cell pool
(Fig. 2 C).
HEL
SW
HEL
B Cells Can Switch to all Ig Isotypes. Sera
from nonimmunized MD4 Ig Tg mice contain 10 g/ml
anti-HEL IgM antibody (Fig. 3 A; reference 5). When
tested for the presence of class-switched antibodies, MD4
mice were also found to have detectable, albeit low, levels
of anti-HEL IgG2b, IgG3, and IgA in their sera (Fig. 3 A),
presumably due to rare interchromosomal CSR events that
have been reported in other Ig Tg models (29). By con-
trast, sera from nonimmunized SW
HEL
mice contained anti-
HEL antibodies of all isotypes (Fig. 3 A) with the exception
of IgE (not depicted). Because IgE is typically present in
mouse serum at 1,000-fold lower levels than other isotypes,
we tested for IgE production by stimulating SW
HEL
B cells
in vitro with CD40L plus IL-4. Stimulation with LPS was
used as a control. Not only were SW
HEL
B cells shown to
secrete anti-HEL IgE in response to CD40L plus IL-4, but
the different classes of anti-HEL antibody produced by
these cells (Fig. 3 B) corresponded to those typically se-
creted by non-Tg B cells under the same conditions (30,
31). Thus, HEL
SW
HEL
B cells produced IgG1 and IgE
but not IgG2b nor IgG3 in response to CD40L plus IL-4
and vice versa for LPS (Fig. 3 B). HEL
MD4 B cells, on
the other hand, produced negligible quantities of class-
switched antibodies under both conditions.
Exposure to Soluble HEL As a Neo Self-Antigen Induces
Anergy in HEL
SW
HEL
B Cells. Having established that
HEL
SW
HEL
B cells undergo CSR normally, we sought to
determine the effects of self-reactivity on this process. In the
MD4 nonswitching model, HEL
B cells produced in mice
expressing membrane-bound HEL fail to enter peripheral
lymphoid tissues and are deleted in the bone marrow (2).
Similarly, self-reactive HEL
SW
HEL
B cells were deleted in
the bone marrow of irradiated membrane HEL Tg mice re-
constituted with SW
HEL
bone marrow (not depicted). Thus,
the ability to undergo CSR does not affect the deletion of
high avidity self-reactive B cells in the bone marrow.
Table I. IgH Expression by HEL
and HEL
Splenic B Cells
from SW
HEL
Mice
HEL
B cells HEL
B cells
IgH V-region expression
a
Intact V
H
10 10/10 1/21
Disrupted V
H
10 0/10 9/21
Endogenous IgH allele 0/10 11/21
IgH allotype expression
b
Targeted allotype (IgH
b
) 99.6 0.1% 52.5 1.3%
Endogenous allotype (IgH
a
) 0.4 0.1% 47.5 1.3%
a
To analyze IgH V-region expression, SW
HEL
spleen cells were stained
with anti–B220-PE and HEL-FITC, and HEL
and HEL
splenic B
cells (refer to Fig. 2 A) were separated to 95% purity by FACS
®
sorting. RT-PCR, cloning, and sequencing of heavy chain cDNA
clones was performed and the number of clones expressing the intact
targeted V
H
10 allele, disrupted V
H
10 allele that had undergone V
H
region
replacement, and endogenous IgH allele was determined. The
denominator indicates the total number of clones isolated for each group.
b
SW
HEL
(IgH
b
) mice were crossed with non-Tg BALB/c (IgH
a
) mice
and spleen cells from SW
HEL
F1 progeny stained with anti–B220-APC,
HEL plus anti–HEL-FITC, anti–IgM
a
-PE, and anti–IgM
b
-biotin plus
SA-PerCP. The proportions of HEL
and HEL
B cells that stained for
IgM
a
or IgM
b
were calculated separately for each mouse. Percentages
represent mean SD for four mice.
Figure 3. Ig class switching by SW
HEL
HEL
B
cells. (A) Constitutive production of anti-HEL Ig
by NB6, MD4, and SW
HEL
mice. Serum were ob-
tained from 8–12-wk-old mice and the concentra-
tion of anti-HEL Ig isotypes was quantitated by
ELISA. (B) In vitro production of anti-HEL Ig by
NB6, MD4, and SW
HEL
splenic B cells. Small, rest-
ing splenic B cells were purified from 8–12-wk-old
NB6, MD4, and SW
HEL
mice and stimulated in
vitro with either CD40L plus IL-4 or LPS. Culture
supernatants were harvested after 4 d and analyzed
for the presence of anti-HEL Ig using isotype-spe-
cific secondary antibodies. Precise antibody con-
centrations were not calculated and the data are ex-
pressed as the absorbance readings obtained from
neat culture supernatants. Data are representative of
two independent experiments.
The Journal of Experimental Medicine
851 Phan et al.
Of more relevance to the regulation of isotype-switched
autoantibody production is the scenario where anergic self-
reactive B cells with the capacity to undergo CSR colonize
peripheral lymphoid tissues. To study this, the develop-
ment and responsiveness of self-reactive SW
HEL
and MD4
B cells were compared in double Tg mice expressing inter-
mediate (ML5) or high (AL3) levels of soluble HEL. As
previously demonstrated, HEL
B cells are not deleted in
MD4 ML5 mice (Fig. 4 A) but become anergic. This is
manifested by down-regulation of surface IgM (not de-
picted), greatly reduced secretion of anti-HEL IgM (Fig. 4
C), and the inability to up-regulate CD86, CD69, or in-
crease cell size in response to stimulation with HEL in vitro
(Fig. 4 D and not depicted). A very similar picture was ob-
served in MD4 AL3 mice that expressed higher levels of
soluble HEL (Fig. 4 and not depicted). When SW
HEL
mice
were crossed with ML5 mice, the frequency of self-reactive
HEL
B cells was reduced compared with that in SW
HEL
mice (Fig. 4 A). Although such a decrease was not observed
in either MD4 ML5 nor MD4 AL3 mice, the induc-
tion of anergy in self-reactive SW
HEL
ML5 B cells other-
wise appeared to be similar to that in the two MD4 double
Tg combinations. Thus, self-reactive SW
HEL
B cells exhib-
ited the IgM
lo
phenotype characteristic of anergic B cells
(Fig. 4 B), secreted little anti-HEL Ig of any isotype in vivo
(Fig. 4 C), and were unable to up-regulate CD86, CD69,
or increase in size after antigenic stimulation in vitro (Fig. 4
D and not depicted). Therefore, the ability to undergo
CSR does not appear to affect the induction of anergy to
soluble self-antigen in SW
HEL
B cells.
Self-reactive SW
HEL
B Cells Display an Immature Phenotype
and Impaired Migration to the Splenic Follicle and MZ. Anal-
ysis of surface expression of the CD21/CD35 and CD23
molecules allowed immature (CD21/CD35
lo
CD23
lo
), ma-
ture follicular (CD21/CD35
int
CD23
hi
), and MZ (CD21/
CD35
hi
CD23
lo
) B cell subpopulations to be resolved
within the spleen (32–34). In the absence of self-antigen,
the proportions of MD4 and SW
HEL
HEL
B cells exhibit-
ing the three phenotypes were comparable to those seen in
non-Tg splenic B cells (Fig. 6 A). Correspondingly, HEL
B cells were located in both the follicle and MZ of spleens
from MD4 (6) and SW
HEL
mice (Fig. 5, A and D). In MD4
double Tg mice, very few HEL
B cells displayed the MZ
phenotype (Fig. 6 A), consistent with previous histological
analyses showing that the MZ is poorly developed in these
mice and consists almost exclusively of rare HEL
B cells
(Fig. 5, B and C; reference 6). In contrast to the MZ, the
immature and follicular splenic B cell subpopulations varied
little between MD4 single and double Tg mice when ana-
lyzed by immunohistology (Fig. 5, A, B, and C) and flow
cytometry (Fig. 6 A).
Histological analysis of SW
HEL
ML5 mice not only
confirmed the reduction in numbers of splenic HEL
B
cells compared with SW
HEL
mice (Fig. 5, D and E), but also
illustrated differences in their migratory properties. Thus,
HEL
B cells in SW
HEL
ML5 mice do not efficiently col-
Figure 4. Induction of anergy in SW
HEL
double
Tg mice. (A) Reduced frequency of HEL
B cells
in SW
HEL
ML5 but not MD4 ML5 nor MD4
AL3 mice. Spleens from 8–14-wk-old mice of
the indicated genotypes were harvested and stained
for B220 and HEL binding as in Fig. 2 A. HEL
B
cells were quantitated as a proportion of total sple-
nocytes. Columns indicate the mean of the individ-
ual data points. Dashes on the x axis indicate data
from MD4 or SW
HEL
mice not expressing HEL. (B)
SW
HEL
ML5 HEL
B cells down-regulate surface
IgM expression. Spleen cells from SW
HEL
and
SW
HEL
ML5 littermates were stained for B220,
HEL binding, and IgM as described in Fig. 2.
Overlayed histograms indicate IgM fluorescence of
HEL
B cells. The data are representative of four
pairs of similarly analyzed littermates. (C) Reduced
production of anti-HEL antibodies in SW
HEL
ML5 mice. Sera from 8–14-wk-old mice of the in-
dicated genotypes were analyzed by ELISA for
anti-HEL IgM and total anti-HEL Ig using anti-
IgM and anti-Ig secondary antibodies, respec-
tively. In each case the data were quantitated
against an HyHEL10 IgM standard. Dashes on the
x axis indicate data from MD4 or SW
HEL
mice not
expressing HEL. (D) SW
HEL
ML5 HEL
B cells
have inactive BCR signaling. Spleen cells from
8–10-wk-old mice of the indicated genotypes were
cultured for 24 h in the presence or absence of 500
ng/ml HEL and then stained with anti–B220-
PerCP, HEL plus anti–HEL-FITC, and anti–
CD86-PE. Relative CD86 levels were obtained by gating either on total B220
B cells (NB6) or HEL
B cells (all other mice) and calculating the mean
fluorescence intensity (m.f.i.) of CD86 staining. Similar results were obtained in an independent experiment.
The Journal of Experimental Medicine
852 Production of IgG Autoantibodies by Anergic Self-Reactive B Cells
onize either the splenic MZ nor follicle, but instead localize
near the boundary between the PALS and the follicle (Fig.
5 E). Flow cytometric analysis revealed that few HEL
B
cells from SW
HEL
double Tg mice display the CD21/
CD35
hi
CD23
lo
MZ phenotype. Moreover, the frequency
of CD21/CD35
int
CD23
hi
follicular B cells is reduced com-
pared with SW
HEL
mice (Fig. 6 A). Conversely, the propor-
tion of anergic HEL
SW
HEL
ML5 B cells with an imma-
ture CD21/CD35
lo
CD23
lo
phenotype is greatly increased
(Fig. 6, A and C), which clearly distinguishes them from
MD4 double Tg B cells. Identification of immature B cells
on the basis of CD24 (HSA) and CD21/CD35 expression
(CD21/CD35
lo
CD24
hi
) gave similar results to those ob-
tained using CD23 and CD21/CD35 (not depicted).
Failure of Anergic SW
HEL
Double Tg B Cells to Mature and
Localize to the Follicle Is Not Due to Increased BCR Occupancy
by Self-Antigen. One interpretation of the results de-
scribed above is that the ability to undergo CSR may cause
self-reactive B cells to be more readily purged from the
mature B cell repertoire. However, interaction with in-
creased levels of self-antigen as well as competition with
nonself-reactive B cells have previously been claimed to
influence the fate of anergic B cells (11–13), raising the
possibility that variability in one or both of these parame-
ters could explain the different maturation and migration
patterns of self-reactive B cells in SW
HEL
versus MD4 dou-
ble Tg mice. To distinguish between these possibilities, we
initially compared the levels of BCR occupancy by self-
antigen between the HEL
B cell populations from the
various double Tg mice. As expected, the proportion of
BCRs occupied by HEL on B cells from SW
HEL
ML5
mice (70–80%) was greater than MD4 ML5 mice (40–
50%) due to the presence of fewer B cells capable of
binding available HEL (Fig. 6 B). MD4 AL3 B cells,
however, exhibited significantly higher levels of receptor
occupancy (90–100%; Fig. 6 B). Therefore, simultaneous
analysis of receptor occupancy and B cell maturity clearly
showed that the failure of self-reactive SW
HEL
ML5 B
cells to mature could not be explained by greater interac-
tion with self-antigen (Fig. 6 C).
The alternative possibility that self-reactive SW
HEL
ML5 B cells exhibited impaired migration and maturation
due to competition from nonself-reactive B cells was con-
sistent with the data obtained. Thus, HEL
B cells were
more prevalent in SW
HEL
double Tg mice (60–80% of B
cells) than in MD4 double Tg mice (10% of B cells; Fig.
6 B). Nevertheless, a possible role for intrinsic differences
between anergic SW
HEL
and MD4 B cells, including the
ability to Ig class switch, was yet to be excluded.
Failure of Anergic SW
HEL
Double Tg B Cells to Mature and
Localize to the Follicle Is Not Due to Their CSR Potential but to
Competition from Nonself-reactive B Cells. To determine
whether CSR potential plays any role in the impaired mat-
uration and migration of anergic SW
HEL
double Tg B cells,
mixed radiation chimeras were produced by reconstituting
ML5, AL3, and non-Tg recipients with a mixture of bone
marrow cells from SW
HEL
, MD4, and non-Tg mice. This
strategy allowed the development of SW
HEL
and MD4
HEL
B cells to be compared in an identical environment
where they were both exposed to the same level of self-
antigen and the same amount of competition from nonself-
reactive B cells. In non-Tg recipients, both SW
HEL
and
MD4 HEL
B cells (resolved by the CD45.1 congenic
marker on SW
HEL
B cells) matured normally and contained
only low frequencies of immature B cells (Fig. 6 D). In sol-
uble HEL Tg recipients, both SW
HEL
and MD4 HEL
B
cells displayed a phenotype comparable to that seen in
SW
HEL
double Tg mice, with very few MZ cells being de-
tectable (not depicted) and 40–50% of the remaining B
cells expressing the immature CD21/CD35
lo
CD23
lo
phe-
notype (Fig. 6 D). In other words, anergic MD4 B cells
also failed to mature in the presence of competitor non-Tg
Figure 5. Anatomica
l
l
oca
l
ization o
f
nontolerant and anergic B cells in the
spleens of MD4 and SW
HEL
mice. Im-
munofluorescent staining was performed
on spleen sections from the following
9–12-wk-old mice: (A) MD4, (B) MD4
ML5, (C) MD4 AL3, (D) SW
HEL
, (E)
SW
HEL
ML5, and (F) NB6. B cells
were stained purple (anti–B220-biotin
plus SA-FluoroBlue) and HEL binding
BCRs were stained green (HEL plus
rabbit polyclonal anti–HEL plus anti–
rabbit IgG-FITC). HEL
B cells overlay
purple and green and therefore appear
cyan whereas HEL
B cells are purple.
Immature HEL
B cells express low lev-
els of B220 and therefore appear greener
than mature B cells. The marginal sinus
was stained red (anti–MadCAM-1 plus
anti–rat IgG-Texas red) to delineate the MZ (bar) and the inner follicle (FO). The T cell–rich PALS is also shown. Note the thin MZ in MD4 ML5
and MD4 AL3 mice made up predominantly of HEL
B cells (B and C) and the concentration of immature HEL
B cells in the border between the
follicle and the PALS in SW
HEL
ML5 mice (E). The section from the non-Tg NB6 mouse (F) is distal to the central arteriole and therefore does not
show the PALS. Data from flow cytometric analysis of the same mice are shown in Fig. 6, A and B. Similar data were obtained from independently ana-
lyzed mice of the same genotypes.
The Journal of Experimental Medicine
853 Phan et al.
B cells. Thus, SW
HEL
B cells do not possess any intrinsic
properties such as the ability to Ig class switch that could
explain why they undergo a more severe developmental
arrest than MD4 B cells on exposure to soluble self-anti-
gen. Rather, the impairment in both maturation and follic-
ular localization of self-reactive HEL
B cells in SW
HEL
double Tg as opposed to MD4 double Tg mice is due
solely to competition with the greater numbers of HEL
B
cells present in SW
HEL
ML5 mice.
Anergic SW
HEL
Double Tg B Cells Have a Half-Life of
3 d. Anergic MD4 B cells have been shown to have a
short lifespan (half-life of 3 d) in the presence of compet-
ing HEL
B cells (11, 27). Therefore, BrdU labeling in vivo
was used to determine the relative turnover rates of HEL
B cells in SW
HEL
double Tg versus SW
HEL
mice (11, 27,
35). After 3 d of BrdU administration, 60% of self-reac-
tive B cells from the spleens of SW
HEL
ML5 mice were
labeled with BrdU. In contrast, only 20–30% of SW
HEL
HEL
B cells were BrdU
and this labeling was confined
almost exclusively to the CD24
hi
(HSA
hi
) immature B cell
pool (Fig. 6 E and not depicted). Thus, the half-life of an-
ergic HEL
B cells from SW
HEL
ML5 mice was 3 d in
the spleen, which is similar to that of follicularly excluded
MD4 B cells (11) and immature non-Tg B cells (36). The
rate of BrdU incorporation into anergic B cells from the
spleen of MD4 AL3 mice was similar to that in naive B
cells from MD4 and SW
HEL
mice (Fig. 6 E), reflecting the
unchanged follicular localization and maturation of the self-
Figure 6. Anergic SW
HEL
ML5 HEL
B cells have an immature phenotype and re-
duced lifespan due to competition with
nonanergic HEL
B cells. (A) Splenic B cell
subsets in anergic and nontolerant B cell
populations. Splenocytes from 9–12-wk-old
mice of the indicated genotypes were
stained with anti–B220-PerCP, HEL plus
anti–HEL-biotin plus SA-APC, anti–
CD21/CD35-FITC, and anti–CD23-PE.
Data represent either total (NB6) or HEL
B cells (all others). Windows indicate fol-
licular (CD21/CD35
int
CD23
hi
) and MZ
(CD21/CD35
hi
CD23
lo
) phenotype B cells
and numbers indicate the percentage of the
displayed cells within these windows. (B)
BCR occupancy. Splenocytes from the
same mice analyzed in A were stained with
anti–B220-PerCP and anti–HEL-FITC
(HyHEL5) with (open histogram) and with-
out (filled histogram) previous incubation
with 200 ng/ml soluble HEL. Histograms
represent anti–HEL-FITC fluorescence pro-
files of total (B220
) B cells. To calculate
receptor occupancy, HEL
B cells were
gated (refer to Fig. 2 A) and the anti–HEL-
FITC m.f.i. obtained without added HEL
divided by the m.f.i. obtained with added
HEL. Also indicated are the proportions of
HEL
cells within the splenic B220
popu-
lation. The low level of HEL staining on the
HEL
B cells from SW
HEL
mice is not due
to the expression of anti-HEL Ig (refer to
Table I) but probably represents “cytophilic” antibody derived from the HEL
B cells (reference 6). For A and B, the data are representative of at least
four different mice of each genotype. (C) Relationship between receptor occupancy by HEL and B cell maturation. An independent set of mice of the
same genotypes and age as those described in A and B were analyzed as above. For each mouse, a receptor occupancy was calculated as was the proportion
of splenic HEL
B cells (excluding MZ B cells) with an immature (CD21/CD35
lo
CD23
lo
) phenotype. Individual data points from SW
HEL
(), SW
HEL
ML5 (), MD4 (), MD4 ML5 (), and MD4 AL3 () are shown, with lines connecting the midpoint of duplicate data points for SW
HEL
-
and
MD4-derived mice. Connecting lines serve only to indicate the trend within the SW
HEL
- and MD4-derived mice and do not imply any linear relationship
between maturation and receptor occupancy. (D) Maturation of SW
HEL
and MD4 HEL
B cells exposed to the same level of HEL and B cell competition.
NB6 (n 3), ML5 (n 2), and AL3 (n 1) mice were lethally irradiated and reconstituted with a mixture of bone marrow cells from SW
HEL
.CD45.1
(75%), MD4 (23%), and NB6 (2%) donors. After 8 wk, spleen cells from recipient mice were stained with anti–B220-PerCP, HEL plus anti–HEL-biotin
plus SA-APC, anti–CD45.1-FITC, and anti–CD23-PE. After gating on small lymphocytes to exclude data from MZ B cells (confirmed by separate anti-
CD21/CD35 stain), CD45.1
(SW
HEL
) and CD45.1
(MD4) HEL
B cells were gated separately and the proportion of gated cells with an immature
CD23
lo
phenotype were measured. Data show the readings obtained from SW
HEL
() and MD4 () HEL
B cells in recipients of the indicated genotypes.
(E) Relative lifespan of SW
HEL
ML5 versus MD4 AL3 anergic B cells. Mice 9–10 wk of age of the indicated genotypes were fed BrdU via their
drinking water for 72 h. Spleen cells were then stained with anti–B220-PerCP, HEL plus anti–HEL-Alexa Fluor
®
647, anti–CD24-PE, and anti–BrdU-
FITC. The proportion of HEL
B cells that stained BrdU
was calculated with reference to identically stained and analyzed spleen cells from a mouse that
had not been fed BrdU. BrdU
cells were predominantly localized to the immature CD24
hi
(HSA
hi
) compartment in all mice (not depicted), which com-
prised 20–30% of HEL
B cells in the MD4, MD4 AL3, and SW
HEL
mice and 70–80% in the SW
HEL
ML5 mice. Receptor occupancy was calculated
as for B and was 95% in the three MD4 AL3 mice used in this experiment and 72 and 75% in the two SW
HEL
ML5 mice.
The Journal of Experimental Medicine
854 Production of IgG Autoantibodies by Anergic Self-Reactive B Cells
reactive B cells in these double Tg mice (Figs. 5 C and 6 A)
and the relative prolongation of lifespan of anergic B cells
observed in the absence of competition from nonself-reac-
tive B cells (11).
Immature Anergic B Cells Proliferate and Secrete IgM Autoan-
tibodies upon BCR-independent Activation. Previous experi-
ments have shown that anergic B cells from MD4 ML5
mice can efficiently proliferate and secrete IgM autoanti-
bodies when activated by T cell–derived stimuli such as
CD40L plus IL-4 (7, 8) or T cell–independent stimuli such
as LPS (37). It is not known, however, whether immature
anergic B cells excluded from the follicle can also respond
to these stimuli. To test this, small resting splenic B cells
were purified from SW
HEL
and SW
HEL
ML5 mice, la-
beled with the division-tracking dye CFSE, and cultured in
vitro with the BCR-independent stimuli anti-CD40 mAb
plus IL-4 (Figs. 7 and 9) and LPS (Figs. 8 and 10).
Proliferation of the various B cell populations was visual-
ized by monitoring the serial twofold reductions in CFSE
fluorescence intensity that accompany cell division (26).
When responses to anti-CD40 mAb plus IL-4 were exam-
ined, nontolerant HEL
SW
HEL
B cells proliferated less than
both HEL
SW
HEL
B cells and non-Tg B cells (Fig. 7, A
and B). Thus, a greater proportion of the HEL
SW
HEL
B
cells either failed to divide or underwent only a few cell di-
visions after 4 d in culture. Proliferation by MD4 B cells
was similar, indicating that small resting Tg B cells express-
ing the HyHEL10 specificity respond less efficiently to
anti-CD40 mAb plus IL-4 than non-Tg B cells. On the
other hand, anergic B cells from both SW
HEL
ML5 (Fig.
7, A and B) and MD4 ML5 mice (not depicted) re-
sponded better than the corresponding nontolerant B cells,
with proliferation being comparable to that of non-Tg B
cells. Because the anergic B cells had matured in the pres-
ence of HEL, this finding underscored the role of the BCR
in modulating responsiveness to this T-dependent stimulus.
By contrast, proliferative responses to the T-independent
stimulus LPS did not vary greatly between the different B
cell populations and were identical for HEL
SW
HEL
single
and double Tg B cells (Fig. 8, A and B). Both anti-CD40
mAb plus IL-4 as well as LPS elicited comparable amounts
of anti-HEL IgM antibodies from anergic and nontolerant
HEL
SW
HEL
B cells (Figs. 7 C and 8 C). Thus, prolifera-
tion and production of IgM autoantibodies in response to
these BCR-independent stimuli are not reduced in imma-
ture, follicularly excluded anergic B cells.
Immature Anergic B Cells Undergo CSR and Secrete IgG
Autoantibodies upon BCR-independent Activation. To test
whether anergic B cells are intrinsically compromised in
their ability to undergo CSR, naive and anergic SW
HEL
B
cells were cultured in parallel with C57BL/6 and MD4 B
cells and then analyzed for production of IgG1 and IgG3
in response to anti-CD40 mAb plus IL-4 (Fig. 9) and LPS
(Fig. 10) respectively. Flow cytometric analysis of surface
expression of IgG revealed that both SW
HEL
and SW
HEL
ML5 HEL
B cells switched efficiently to IgG1 and IgG3
whereas minimal switching occurred in MD4 B cells
(Figs. 9, A and B, and 10, A and B). When the propor-
tions of IgG1
B cells in each cell division were calculated,
SW
HEL
and SW
HEL
ML5 HEL
B cells were found to
have identical cell division–dependent patterns of IgG1
switching (Fig. 9 B). The same was true for switching to
IgG3 in response to LPS (Fig. 10 B). ELISA of culture su-
pernatant for secretion of anti-HEL IgG1 and IgG3 anti-
bodies mirrored the degree of switching observed by flow
cytometry. That is, the anergic SW
HEL
B cells in each case
Figure 7. Anergic SW
HEL
B cells readily proliferate and
secrete IgM autoantibodies in response to T cell–derived
signals. CFSE-labeled small resting B cells purified from
8–12-wk-old NB6, MD4, SW
HEL
, and SW
HEL
ML5 mice
were stimulated in vitro with anti-CD40 mAb plus IL-4
for 4 d. Cells were then fixed, permeabilized, and stained
with HEL plus anti–HEL-Alexa Flour
®
647. (A) Density
plot of HEL binding versus CFSE cell division profile for
mice of the indicated genotypes. (B) Overlays of CFSE cell
division profiles demonstrating the lower proliferative re-
sponses of MD4 and HEL
SW
HEL
B cells compared with
NB6 B cells and HEL
SW
HEL
ML5 B cells. HEL
B
cells from SW
HEL
and SW
HEL
ML5 mice exhibited
CFSE profiles similar to that of NB6 B cells (A and not de-
picted). The number of divisions undergone by cells in
specific CFSE peaks is indicated. Note that peaks do not
exactly correspond between samples due to minor differ-
ences in CFSE labeling efficiency. (C) Secretion of IgM
anti-HEL antibodies by NB6, MD4, SW
HEL
, and SW
HEL
ML5 B cells. Duplicate cultures of purified B cells were
stimulated in vitro with anti-CD40 mAb plus IL-4 and
anti-HEL IgM detected by ELISA of culture supernatants.
Data are representative of four independent experiments.
The Journal of Experimental Medicine
855 Phan et al.
produced at least as much anti-HEL IgG antibody as the
nontolerant SW
HEL
B cells (Figs. 9 C and 10 C). There-
fore, anergic B cells have no intrinsic block to CSR nor to
subsequent secretion of potentially pathogenic IgG1 or
IgG3 autoantibodies.
Discussion
The MD4 Ig Tg model has been used for more than a
decade to study the regulation of B cell responses, both in
tolerance and immunity. With the development of SW
HEL
mice, it is now possible to extend the observations made in
the original model to encompass the full range of B cell re-
sponses. This is due primarily to the ability of HEL
B cells
from SW
HEL
but not MD4 mice to undergo CSR to all Ig
isotypes (Fig. 3, A and B). Although SW
HEL
mice will be
valuable for future studies of B cell memory, plasma cell
development and other aspects of late B cell differentiation,
we have used them initially to investigate the effect of the
anergic state on CSR. The experiments described here
show for the first time that CSR and secretion of IgG au-
toantibodies are not intrinsically blocked after induction of
tolerance in B cells.
Comparison of SW
HEL
with MD4 mice revealed that the
phenotypes of naive HEL
B cells from the two lines are
very similar. Both populations of B cells are restricted to
the B2 lineage (Fig. 2 C), migrate to the splenic follicle and
MZ (Fig. 5, A and D), and share the same differential ex-
pression of B cell surface markers such as CD21/CD35 and
CD23 (Fig. 6 A). In addition, SW
HEL
and MD4 HEL
B
cells express uniformly high levels of surface IgM (Fig. 2 B)
regardless of their CD21/CD35 and CD23 surface pheno-
type or anatomical localization in the spleen. Although MZ
B cells are also IgM
hi
in non-Tg mice, the long-lived recir-
culating and follicular B cell pool normally exhibits a range
from low to high expression of surface IgM (38). The ab-
sence of IgM
lo
follicular HEL
B cells cannot be ascribed to
trivial factors such as transgene copy number or integration
site because it occurs in both SW
HEL
and MD4 mice.
Rather, the established role of BCR specificity in shaping
the differentiation of maturing B cells (39) suggests instead
that the uniformly IgM
hi
phenotype of HEL
follicular B
cells reflects the unique interactions of the HyHEL10 BCR
specificity with the endogenous antigenic milieu.
Apart from the ability to undergo CSR, SW
HEL
mice are
also distinguished from MD4 Ig Tg mice by the presence
of significant numbers of HEL
B cells (Fig. 2 A). This dif-
ference is due to the targeted insertion of the V
H
10 heavy
chain gene into the IgH locus in the SW
HEL
mice that al-
lows replacement of the rearranged V
H
segment by up-
stream V
H
or V
H
plus D elements during early B cell devel-
opment (Table I and not depicted). Consistent with results
from other mice carrying targeted V
H
DJ
H
genes (22, 23),
recombination events in SW
HEL
mice are mediated by the
embedded RSS heptamer located at the 3 end of the rear-
ranged V
H
segment of V
H
10 (not depicted). In mice carry-
ing a targeted insertion of an anti-DNA V
H
DJ
H
gene, this
phenomenon may represent a receptor-editing mechanism
activated in response to the binding of self-antigen (22).
Such a scenario is unlikely in SW
HEL
mice however, be-
cause anti-HEL B cells show no evidence of self-reactivity
in the absence of transgenically expressed HEL and un-
dergo profound changes when HEL is expressed as a solu-
ble- or membrane-bound self-antigen (2, 5–11, and this
study). Therefore, it is more likely that the V
H
replacement
events in SW
HEL
mice reflect a general instability of rear-
Figure 8. Anergic SW
HEL
B cells readily proliferate and
secrete IgM autoantibodies in response to LPS. CFSE-
labeled small resting B cells purified from 8–12-wk-old
NB6, MD4, SW
HEL
, and SW
HEL
ML5 mice were stim-
ulated in vitro with LPS for 3 d. Cells were then fixed,
permeabilized, and stained with HEL plus anti–HEL-
Alexa Flour
®
647. (A) Density plot of HEL binding versus
CFSE cell division profile for mice of the indicated geno-
types. (B) Overlays of CFSE cell division profiles demon-
strating the slightly lower proliferative responses of MD4,
SW
HEL
HEL
, and SW
HEL
ML5 HEL
B cells com-
pared with NB6 B cells. HEL
B cells from SW
HEL
and
SW
HEL
ML5 mice exhibited CFSE profiles identical to
that of NB6 B cells (A and not depicted). The number of
divisions undergone by cells in specific CFSE peaks are
indicated. Note that peaks do not exactly correspond be-
tween samples due to minor differences in CFSE labeling
efficiency. (C) Secretion of IgM anti-HEL antibodies by
NB6, MD4, SW
HEL
, and SW
HEL
ML5 B cells. Dupli-
cate cultures of purified B cells were stimulated in vitro
with LPS and anti-HEL IgM detected by ELISA of cul-
ture supernatants. Data are representative of four inde-
pendent experiments.
The Journal of Experimental Medicine
856 Production of IgG Autoantibodies by Anergic Self-Reactive B Cells
ranged V
H
DJ
H
genes during early B cell development be-
fore the down-regulation of the RAG recombinases.
The presence of HEL
B cells in SW
HEL
but not MD4 Ig
Tg mice became of particular relevance when the two lines
of mice were crossed with mice expressing soluble HEL.
The resulting populations of self-reactive B cells were aner-
gic as indicated by surface IgM down-regulation (Fig. 4 B
and not depicted), low levels of antibody secretion in vivo
Figure 9. Anergic SW
HEL
B cells readily class
switch and secrete IgG1 autoantibodies in response
to T cell–derived signals. CFSE-labeled small rest-
ing B cells were prepared, stimulated, and stained as
in Fig. 7 with an additional stain of anti–IgG1-
biotin plus SA-PE. Data were gated on HEL
and
HEL
B cells using the regions shown in Fig. 7 A.
(A) Density plot of IgG1 expression versus CFSE
cell division profile for HEL
(top) and HEL
B
cells (bottom) from mice of the indicated geno-
types. (B) Analysis of switching per cell division.
Gates were drawn around each CFSE peak as
shown in Fig. 7 B to determine the cell division
number and the percentage of IgG1
cells in each
division calculated by backgating using the region
shown in A. NB6 (), HEL
MD4 (), HEL
SW
HEL
(), and HEL
SW
HEL
ML5 B cells ()
are shown. HEL
B cells from SW
HEL
and SW
HEL
ML5 mice had comparable profiles to NB6 (A and
not depicted). (C) Secretion of IgG1 anti-HEL an-
tibodies by NB6, MD4, SW
HEL
, and SW
HEL
ML5 B cells. Duplicate cultures of purified B cells
were stimulated in vitro with anti-CD40 mAb plus
IL-4 and anti-HEL IgG1 detected by ELISA of cul-
ture supernatants. Data are representative of four
independent experiments.
Figure 10. Anergic SW
HEL
B cells readily class
switch and secrete IgG3 autoantibodies in response
to LPS. CFSE-labeled small resting B cells were
prepared, stimulated, and stained as in Fig. 8 with
an additional stain of anti–IgG3-biotin plus SA-PE.
Data were gated on HEL
and HEL
B cells using
the regions shown in Fig. 8 A. (A) Density plot of
IgG3 expression versus CFSE cell division profile
for HEL
(top) and HEL
B cells (bottom) from
mice of the indicated genotypes. (B) Analysis of
switching per cell division. Gates were drawn
around each CFSE peak as shown in Fig. 8 B to de-
termine the cell division number and the percent-
age of IgG3
cells in each division calculated by
backgating using the region shown in A. NB6 (),
HEL
MD4 (), HEL
SW
HEL
(), and HEL
SW
HEL
ML5 B cells () are shown. HEL
B
cells from SW
HEL
and SW
HEL
ML5 mice had
comparable profiles to NB6 (A and not depicted).
(C) Secretion of IgG3 anti-HEL antibodies by
NB6, MD4, SW
HEL
, and SW
HEL
ML5 B cells.
Duplicate cultures of purified B cells were stimu-
lated in vitro with LPS and anti-HEL IgG3 de-
tected by ELISA of culture supernatants. Data are
representative of four independent experiments.
The Journal of Experimental Medicine
857 Phan et al.
(Fig. 4 C), and loss of BCR signaling capabilities (Fig. 4 D).
However, in contrast to the anergic B cells from MD4
double Tg mice, self-reactive SW
HEL
double Tg B cells
were present at reduced frequencies (Fig. 4 A), arrested in
their migration at the outer PALS (Fig. 5 E), failed to ma-
ture (Fig. 6, A and C), and exhibited a shortened life-span
(Fig. 6 E). The demonstration that the differences in phe-
notype between the two types of anergic B cells are due
solely to the presence of competitor B cells in the SW
HEL
mice has finally resolved a long-standing point of contro-
versy surrounding the fate of anergic B cells.
This controversy has stemmed from independent studies
based on the original anti-HEL double Tg model that have
given rise to two differing theories as to why follicular ex-
clusion and reduced survival of anergic B cells occur in the
presence of nonself-reactive B cells. On the one hand it has
been argued that anergic B cells compete poorly with non-
self-reactive B cells for essential migratory and survival sig-
nals (11, 12). Alternatively, the relative increase in available
self-antigen accompanying reduced frequencies of HEL
B
cells, as observed in SW
HEL
ML5 versus MD4 ML5
mice (Fig. 6 B), may provide anergic B cells with a stronger
BCR signal leading in turn to outer PALS arrest and short-
ened lifespan (13). In support of this interpretation is the
previous demonstration of outer PALS arrest in some MD4
AL3 mice and in MD4 ML5 mice induced to secrete
higher levels of HEL (13).
Simultaneous measurement of competitor (HEL
) B cell
frequency and receptor occupancy (Fig. 6 B) was com-
bined with analysis of mixed bone marrow chimeras (Fig. 6
D) to clearly demonstrate that the impaired migration,
maturation, and survival of anergic SW
HEL
B cells is due to
competition with HEL
B cells. Critical to this conclusion
was that the MD4 AL3 mice examined in the current
experiments contained 10% HEL
B cells and possessed
anergic HEL
B cells that had 90–100% saturation of their
BCRs with self-antigen (Fig. 6, B and C). Nonetheless,
these anergic MD4 AL3 B cells survived at normal fre-
quencies (Fig. 4 A), colonized the follicle (Fig. 5 C), ma-
tured normally to the follicular stage (Fig. 6 A), and had a
normal lifespan (Fig. 6 E). The discrepancy between our
results and those obtained by Cook et al. (13) almost cer-
tainly relates to the older age of the mice used in their ex-
periments compared with ours (4–9 mo vs. 8–12 wk) be-
cause HEL
B cells accumulate with age (27) and are
clearly present at high frequencies in the mice used in their
experiments (13). Therefore, maximal signaling by soluble
HEL is insufficient in itself to exclude anergic B cells from
the follicle and competition from nonself-reactive B cells is
necessary for this to occur.
Although the induction of anergy and follicular exclu-
sion can be achieved independently by manipulating the
amount of competition from nonself-reactive B cells (11),
anergy and follicular exclusion are in fact likely to be coin-
cident in the normal B cell repertoire where competition is
always present. Therefore, many if not all anergic B cells
generated in the normal repertoire might be immature and
behave in a similar way to those in the new SW
HEL
double
Tg model rather than the original MD4 double Tg model.
Thus, as is observed in SW
HEL
double Tg mice, self-reac-
tive B cells that lack sufficient avidity to be deleted in the
bone marrow are likely to be eliminated from the long-
lived peripheral B cell pool by competition from nonself-
reactive B cells (12). What then is the basis for competitive
exclusion of anergic B cells from the long-lived peripheral
B cell pool? If survival of immature B cells is contingent on
their migration into the follicle, one possibility is that aner-
gic B cells fail to compete efficiently for the follicular
chemokine CXCL13 (BLC; reference 40). In support of
this notion, anergic B cells with anti-DNA specificity that
localize to the outer PALS express low levels of the
CXCL13 receptor CXCR5 (BLR1; reference 41). Alter-
natively, anergic B cells may not compete effectively for
signals mediated by the TNF family ligand BAFF because B
cells from BAFF
mice have an immature phenotype re-
sembling that of SW
HEL
double Tg B cells (42).
Because anergic B cells present in the normal repertoire
not only have the potential to undergo CSR but are likely
to be immature, the availability of the SW
HEL
double Tg
mice with a similar immature phenotype makes it timely to
reevaluate the physiological relevance of the findings ob-
tained in the original model. For instance, the immature
phenotype could be associated with poor responsiveness to
BCR-independent stimuli (43) compared to the behavior
of MD4 double Tg B cells (7, 8, 37). Nevertheless, this
proved not to be the case because the responsiveness of
SW
HEL
double Tg B cells to anti-CD40 mAb plus IL-4
(Fig. 7) and LPS (Fig. 8) clearly showed that immature an-
ergic B cells retain the ability to proliferate and secrete anti-
HEL IgM autoantibodies when activated by BCR-inde-
pendent stimuli. Moreover, the switching capability of
SW
HEL
B cells enabled us to study for the first time whether
anergic B can undergo CSR. Given that IgG autoantibod-
ies are more pathogenic (15–19) and less prevalent (20)
than IgM autoantibodies, we anticipated that intrinsic con-
trols over Ig class switching would be required to reinforce
peripheral B cell self-tolerance and prevent autoimmunity.
Surprisingly, anergic SW
HEL
B cells were shown to undergo
efficient isotype switching and secrete IgG1 and IgG3 au-
toantibodies when they were activated independently of
the BCR with both T cell–derived (Fig. 9) and T-inde-
pendent (Fig. 10) stimuli.
The lack of intrinsic controls over CSR and secretion of
IgG by anergic B cells highlights the need to impose addi-
tional controls on these cells to prevent their activation by
BCR-independent stimuli. In the case of T cell–dependent
signals, the requirement for BCR-mediated CD86 up-reg-
ulation to facilitate delivery of productive T cell help (44,
45), and the abrogation of this in anergic B cells (Fig. 4 D),
means that T cell–derived signals such as CD40L plus IL-4
are unlikely to induce IgG production by anergic B cells
under normal circumstances. In contrast, type I T-indepen-
dent stimuli such as LPS activate B cells in the absence of
BCR engagement, meaning that access to such stimuli by
self-reactive B cells must be restricted to prevent IgG au-
toantibody production. Two ways in which this appears to
The Journal of Experimental Medicine
858 Production of IgG Autoantibodies by Anergic Self-Reactive B Cells
be achieved were shown here for SW
HEL
double Tg B cells.
The first involved limiting the survival of anergic B cells in
the periphery (Fig. 6 E). Second, the restriction of their
migration into the splenic follicle and MZ reduces the pos-
sibility of contact with type I T-independent stimuli. In
this regard, the purging of self-reactive B cells from the
MZ is likely to be particularly important because blood-
borne pathogens (such as Gram-negative bacteria bearing
LPS) are directly filtered through the MZ via the marginal
sinus (46). Significantly, although the inhibition of matura-
tion, survival, and follicular homing by anergic B cells de-
pends on the presence of competitor B cells, the purging of
anergic B cells from the MZ does not (Fig. 5), suggesting
that this aspect of anergic B cell regulation is fundamental
to B cell self-tolerance.
Therefore, how are pathogenic IgG autoantibodies pro-
duced? Based on the data presented here, any perturbation
that increases the chances of self-reactive B cells coming
into contact with type I T-independent stimuli may facili-
tate the production of IgG autoantibodies. This would in-
clude mutations that either increase the lifespan of anergic
B cells or their entry into microenvironments from which
they are normally excluded. As discussed above, the accu-
mulation of self-reactive B cells in the MZ would be
particularly dangerous. Interestingly, many models of anti-
body-mediated autoimmune diseases, including estrogen-
induced lupus (47), the NZB/NZW F1 lupus model (48),
and the BAFF-Tg model of Sjögren’s syndrome (49), have
established correlations between the onset of disease and an
expanded MZ compartment. Our data also suggest that
acute exposure to LPS may trigger IgG production by an-
ergic B cells despite their restricted lifespan and migration.
This is consistent with previous studies showing that LPS
administration results in the secretion of pathogenic IgG
autoantibodies, induction of nephritis in normal mice, and
exacerbation of nephritis in lupus-prone mice (50–52). A
similar mechanism may underlie the long-recognized asso-
ciation between systemic infection and clinical relapses of
lupus (53). Exposure of anergic B cells to LPS may not
only induce production of IgG autoantibodies directly.
Through its ability to up-regulate CD86 expression on B
cells (54), LPS may circumvent the BCR signaling defi-
ciency of anergic B cells and facilitate their T cell–depen-
dent activation (44, 45). As demonstrated here, T cell–
dependent activation by CD40L plus IL-4 delivered in this
scenario would indeed trigger subsequent production of
IgG autoantibodies (Fig. 9).
We thank Jenny Kingham, Chris Brownlee, and the staff of the
Centenary Institute Animal Facility for animal husbandry, Tyani
Chan for mouse screening, and Joseph Webster and Tara Mac-
Donald for FACS
®
sorting. We also thank Dr. Sigrid Ruuls for
generously providing the loxP-neo
r
-loxP cassette and Drs. Stephen
Adelstein, Barbara Fazekas de St. Groth, and Stuart Tangye for their
critical discussions and review of the manuscript.
This work was supported by a program grant from the National
Health and Medical Research Council of Australia. T.G. Phan re-
ceived a National Health and Medical Research Council postgrad-
uate research scholarship.
Submitted: 16 December 2002
Revised: 24 January 2003
Accepted: 5 February 2003
References
1. Nemazee, D., and K. Buerki. 1989. Clonal deletion of au-
toreactive B lymphocytes in bone marrow chimeras. Proc.
Natl. Acad. Sci. USA. 86:8039–8043.
2. Hartley, S.B., J. Crosbie, R. Brink, A.B. Kantor, A. Basten,
and C.C. Goodnow. 1991. Elimination from peripheral lym-
phoid tissues of self-reactive B lymphocytes recognizing
membrane-bound antigens. Nature. 353:765–769.
3. Radic, M.Z., J. Erikson, S. Litwin, and M. Weigert. 1993. B
lymphocytes may escape tolerance by revising their antigen
receptors. J. Exp. Med. 177:1165–1173.
4. Tiegs, S.L., D.M. Russell, and D. Nemazee. 1993. Receptor
editing in self-reactive bone marrow B cells. J. Exp. Med.
177:1009–1020.
5. Goodnow, C.C., J. Crosbie, S. Adelstein, T.B. Lavoie, S.J.
Smith-Gill, R.A. Brink, H. Pritchard-Briscoe, J.S. Wother-
spoon, R.H. Loblay, K. Raphael, et al. 1988. Altered immu-
noglobulin expression and functional silencing of self-reactive
B lymphocytes in transgenic mice. Nature. 334:676–682.
6. Mason, D.Y., M. Jones, and C.C. Goodnow. 1992. Devel-
opment and follicular localization of tolerant B lymphocytes
in lysozyme/anti-lysozyme IgM/IgD transgenic mice. Int.
Immunol. 4:163–175.
7. Eris, J.M., A. Basten, R. Brink, K. Doherty, M.R. Kehry,
and P.D. Hodgkin. 1994. Anergic self-reactive B cells present
self antigen and respond normally to CD40-dependent T-cell
signals but are defective in antigen-receptor-mediated func-
tions. Proc. Natl. Acad. Sci. USA. 91:4392–4396.
8. Cooke, M.P., A.W. Heath, K.M. Shokat, Y. Zeng, F.D.
Finkelman, P.S. Linsley, M. Howard, and C.C. Goodnow.
1994. Immunoglobulin signal transduction guides the speci-
ficity of B cell–T cell interactions and is blocked in tolerant
self-reactive B cells. J. Exp. Med. 179:425–438.
9. Healy, J.I., R.E. Dolmetsch, L.A. Timmerman, J.G. Cyster,
M.L. Thomas, G.R. Crabtree, R.S. Lewis, and C.C. Good-
now. 1997. Different nuclear signals are activated by the B
cell receptor during positive versus negative signaling. Immu-
nity. 6:419–428.
10. Glynne, R., G. Ghandour, J. Rayner, D.H. Mack, and C.C.
Goodnow. 2000. B-lymphocyte quiescence, tolerance and
activation as viewed by global gene expression profiling on
microarrays. Immunol. Rev. 176:216–246.
11. Cyster, J.G., and C.C. Goodnow. 1995. Antigen-induced
exclusion from follicles and anergy are separate and comple-
mentary processes that influence peripheral B cell fate. Immu-
nity. 3:691–701.
12. Cyster, J.G., S.B. Hartley, and C.C. Goodnow. 1994. Com-
petition for follicular niches excludes self-reactive cells from
the recirculating B-cell repertoire. Nature. 371:389–395.
13. Cook, M.C., A. Basten, and B. Fazekas De St Groth. 1997.
Outer periarteriolar lymphoid sheath arrest and subsequent
differentiation of both naive and tolerant immunoglobulin
transgenic B cells is determined by B cell receptor occu-
pancy. J. Exp. Med. 186:631–643.
14. Goldsby, R.A., T.J. Kindt, and B.A. Osborne. 2000. Immu-
noglobulins: structure and function. In Kuby Immunology.
R.A. Goldsby, T.J. Kindt, and B.A. Osborne, editors. W.H.
Freeman and Company, New York. 95–96.
The Journal of Experimental Medicine
859 Phan et al.
15. Steward, M.W., and F.C. Hay. 1976. Changes in immuno-
globulin class and subclass of anti-DNA antibodies with in-
creasing age in N/ZBW F1 hybrid mice. Clin. Exp. Immunol.
26:363–370.
16. Papoian, R., R. Pillarisetty, and N. Talal. 1977. Immunolog-
ical regulation of spontaneous antibodies to DNA and RNA.
II. Sequential switch from IgM to IgG in NZB/NZW F1
mice. Immunology. 32:75–79.
17. Panosian-Sahakian, N., J.L. Klotz, F. Ebling, M. Kronen-
berg, and B. Hahn. 1989. Diversity of Ig V gene segments
found in anti-DNA autoantibodies from a single (NZB
NZW)F1 mouse. J. Immunol. 142:4500–4506.
18. Tillman, D.M., N.T. Jou, R.J. Hill, and T.N. Marion. 1992.
Both IgM And IgG anti-DNA antibodies are the products of
clonally selective B cell stimulation in (NZB NZW)F1
mice. J. Exp. Med. 176:761–779.
19. Peng, S.L., S.J. Szabo, and L.H. Glimcher. 2002. T-Bet reg-
ulates IgG class switching and pathogenic autoantibody pro-
duction. Proc. Natl. Acad. Sci. USA. 99:5545–5550.
20. George, J., and Y. Shoenfeld. 1996. Natural autoantibodies.
In Autoantibodies. J.B. Peter and Y. Shoenfeld, editors. Else-
vier, Amsterdam. 534–539.
21. Taki, S., M. Meiering, and K. Rajewsky. 1993. Targeted in-
sertion of a variable region gene into the immunoglobulin
heavy chain locus. Science. 262:1268–1271.
22. Chen, C., Z. Nagy, E.L. Prak, and M. Weigert. 1995. Im-
munoglobulin heavy chain gene replacement: a mechanism
of receptor editing. Immunity. 3:747–755.
23. Cascalho, M., A. Ma, S. Lee, L. Masat, and M. Wabl. 1996.
A quasi-monoclonal mouse. Science. 272:1649–1652.
24. Hartley, S.B., and C.C. Goodnow. 1994. Censoring of self-
reactive B cells with a range of receptor affinities in trans-
genic mice expressing heavy chains for a lysozyme-specific
antibody. Int. Immunol. 6:1417–1425.
25. Lemckert, F.A., J.D. Sedgwick, and H. Korner. 1997. Gene
targeting in C57BL/6 ES cells. Successful germ line transmis-
sion using recipient Balb/C blastocysts developmentally ma-
tured in vitro. Nucleic Acids Res. 25:917–918.
26. Lyons, A.B., and C.R. Parish. 1994. Determination of lym-
phocyte division by flow cytometry. J. Immunol. Methods.
171:131–137.
27. Fulcher, D.A., and A. Basten. 1994. Reduced life span of an-
ergic self-reactive B cells in a double-transgenic model. J.
Exp. Med. 179:125–134.
28. Hodgkin, P.D., and M.R. Kehry. 1996. Methods for poly-
clonal B lymphocyte activation to proliferation and Ig secre-
tion in vitro. In Weir’s Handbook of Experimental Immunol-
ogy. L.A. Herzenberg, D.M. Weir, and C. Blackwell,
editors. Blackwell Science, Cambridge. 89.1–13.
29. Gerstein, R.M., W.N. Frankel, C.L. Hsieh, J.M. Durdik, S.
Rath, J.M. Coffin, A. Nisonoff, and E. Selsing. 1990. Isotype
switching of an immunoglobulin heavy chain transgene oc-
curs by DNA recombination between different chromo-
somes. Cell. 63:537–548.
30. Deenick, E.K., J. Hasbold, and P.D. Hodgkin. 1999. Switch-
ing to IgG3, IgG2b, and IgA is division linked and indepen-
dent, revealing a stochastic framework for describing differ-
entiation. J. Immunol. 163:4707–4714.
31. Hasbold, J., J.S. Hong, M.R. Kehry, and P.D. Hodgkin.
1999. Integrating signals from IFN-gamma and IL-4 by B
cells: positive and negative effects on CD40 ligand-induced
proliferation, survival, and division-linked isotype switching
to IgG1, IgE, and IgG2a. J. Immunol. 163:4175–4181.
32. Waldschmidt, T.J., D.H. Conrad, and R.G. Lynch. 1988.
The expression of B cell surface receptors. I. The ontogeny
and distribution of the murine B cell IgE Fc receptor. J. Im-
munol. 140:2148–2154.
33. Takahashi, K., Y. Kozono, T.J. Waldschmidt, D. Berthi-
aume, R.J. Quigg, A. Baron, and V.M. Holers. 1997. Mouse
complement receptors type 1 (Cr1;CD35) and type 2 (Cr2;
CD21): expression on normal B cell subpopulations and de-
creased levels during the development of autoimmunity in
MRL/Lpr mice. J. Immunol. 159:1557–1569.
34. Oliver, A.M., F. Martin, G.L. Gartland, R.H. Carter, and
J.F. Kearney. 1997. Marginal zone B cells exhibit unique
activation, proliferative and immunoglobulin secretory re-
sponses. Eur. J. Immunol. 27:2366–2374.
35. Forster, I., and K. Rajewsky. 1990. The bulk of the periph-
eral B-cell pool in mice is stable and not rapidly renewed
from the bone marrow. Proc. Natl. Acad. Sci. USA. 87:4781–
4784.
36. Fulcher, D.A., and A. Basten. 1997. Influences on the
lifespan of B cell subpopulations defined by different pheno-
types. Eur. J. Immunol. 27:1188–1199.
37. Goodnow, C.C., R. Brink, and E. Adams. 1991. Breakdown
of self-tolerance in anergic B lymphocytes. Nature. 352:532–
536.
38. Hao, Z., and K. Rajewsky. 2001. Homeostasis of peripheral
B cells in the absence of B cell influx from the bone marrow.
J. Exp. Med. 194:1151–1164.
39. Hardy, R.R., and K. Hayakawa. 2001. B cell development
pathways. Annu. Rev. Immunol. 19:595–621.
40. Gunn, M.D., V.N. Ngo, K.M. Ansel, E.H. Ekland, J.G.
Cyster, and L.T. Williams. 1998. A B-cell-homing chemo-
kine made in lymphoid follicles activates Burkitt’s lymphoma
receptor-1. Nature. 391:799–803.
41. Seo, S.J., M.L. Fields, J.L. Buckler, A.J. Reed, L. Mandik-
Nayak, S.A. Nish, R.J. Noelle, L.A. Turka, F.D. Finkelman,
A.J. Caton, et al. 2002. The impact of T helper and T regula-
tory cells on the regulation of anti-double-stranded DNA B
cells. Immunity. 16:535–546.
42. Schiemann, B., J.L. Gommerman, K. Vora, T.G. Cachero, S.
Shulga-Morskaya, M. Dobles, E. Frew, and M.L. Scott.
2001. An essential role for BAFF in the normal development
of B cells through a BCMA-independent pathway. Science.
293:2111–2114.
43. Rolink, A.G., J. Andersson, and F. Melchers. 1998. Charac-
terization of immature B cells by a novel monoclonal anti-
body, by turnover and by mitogen reactivity. Eur. J. Immu-
nol. 28:3738–3748.
44. Rathmell, J.C., S.E. Townsend, J.C. Xu, R.A. Flavell, and
C.C. Goodnow. 1996. Expansion or elimination of B cells in
vivo: dual roles for CD40- and Fas (CD95)-ligands modu-
lated by the B cell antigen receptor. Cell. 87:319–329.
45. Rathmell, J.C., S. Fournier, B.C. Weintraub, J.P. Allison,
and C.C. Goodnow. 1998. Repression of B7.2 on self-reac-
tive B cells is essential to prevent proliferation and allow Fas-
mediated deletion by CD4
T cells. J. Exp. Med. 188:651–
659.
46. Cyster, J.G. 2000. B cells on the front line. Nat. Immunol.
1:9–10.
47. Grimaldi, C.M., D.J. Michael, and B. Diamond. 2001. Cut-
ting edge: expansion and activation of a population of autore-
active marginal zone B cells in a model of estrogen-induced
lupus. J. Immunol. 167:1886–1890.
48. Wellmann, U., A. Werner, and T.H. Winkler. 2001. Altered
The Journal of Experimental Medicine
860 Production of IgG Autoantibodies by Anergic Self-Reactive B Cells
selection processes of B lymphocytes in autoimmune NZB/W
mice, despite intact central tolerance against DNA. Eur. J.
Immunol. 31:2800–2810.
49. Groom, J., S.L. Kalled, A.H. Cutler, C. Olson, S.A. Wood-
cock, P. Schneider, J. Tschopp, T.G. Cachero, M. Batten, J.
Wheway, et al. 2002. Association of BAFF/BLys overexpres-
sion and altered B cell differentiation with Sjögren’s syn-
drome. J. Clin. Invest. 109:59–68.
50. Hang, L., J.H. Slack, C. Amundson, S. Izui, A.N. Theo-
filopoulos, and F.J. Dixon. 1983. Induction of murine au-
toimmune disease by chronic polyclonal B cell activation. J.
Exp. Med. 157:874–883.
51. Cavallo, T., and N.A. Granholm. 1990. Lipopolysaccharide
from gram-negative bacteria enhances polyclonal B cell acti-
vation and exacerbates nephritis in MRL/Lpr mice. Clin.
Exp. Immunol. 82:515–521.
52. Granholm, N.A., and T. Cavallo. 1992. Autoimmunity,
polyclonal B-cell activation and infection. Lupus. 1:63–74.
53. Wallace, D.J., and E.L. Dubois. 1987. Prognostic subsets,
natural course, and causes of death in systemic lupus erythe-
matosus. In Dubois’ Lupus Erythematosus. D.J. Wallace and
E.L. Dubois, editors. Lea & Febiger, Philadelphia. 583–584.
54. Hathcock, K.S., G. Laszlo, C. Pucillo, P. Linsley, and R.J.
Hodes. 1994. Comparative analysis of B7-1 and B7-2 costim-
ulatory ligands: expression and function. J. Exp. Med. 180:
631–640.
