By
From The Wistar Institute, Philadelphia, Pennsylvania 19104
Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti-double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.
Ig transgenic models to neo-self Ags have helped to classify two manifestations of B cell tolerance: clonal deletion and functional inactivation (anergy) (1). Recently,
the distinction between these two has come into question
as "anergized" cells have been shown to have a reduced
lifespan and may be in a state of "delayed deletion" (4).
Furthermore, the relative contribution of deletion versus
receptor editing to the elimination of autoreactive B cells is
being reevaluated (5, 6). Given that most autoimmune diseases are characterized by the presence of autoantibodies
directed toward a discrete set of autoantigens, we are interested in determining whether the mechanisms described for
the maintenance of tolerance to neo-self Ags apply to disease-associated autoantigens.
Anti-double-stranded (ds)1 DNA Abs are one of the
hallmarks of SLE and the MRL-lpr/lpr murine model for
SLE, and rising titers of these Abs correlate with disease exacerbation (7). In the serum of nonautoimmune individuals, anti-dsDNA Abs are not present, suggesting that this
specificity is regulated, yet the mechanism governing this
regulation remains unclear. To follow the fate of anti-dsDNA
B cells, we have used Ig transgenic mice. The transgene (tg) being studied encodes the VH3H9 H chain, originally
isolated from anti-dsDNA Igs in diseased MRL-lpr/lpr
mice, in combination with different L chains (8). Transfection studies have shown that this H chain can pair with a
variety of different L chains to generate both anti-single-stranded (ss)DNA and anti-dsDNA Abs (9). As a tg, VH3H9
can pair with endogenous L chains to generate anti-ssDNA,
anti-dsDNA, and non-DNA B cells, allowing us to study the regulation of anti-dsDNA B cells in the presence of B
cells with other specificities (10, 11). Tracking anti-dsDNA
B cells in a diverse repertoire is important because this
more closely mimics the conditions present in SLE. In addition, precedent exists for a differential fate of autoreactive
B cells in the context of a monoclonal versus polyclonal B
cell repertoire (12, 13).
There have been conflicting reports on the fate of anti-dsDNA B cells in nonautoimmune mice. Some state that
anti-dsDNA B cells are deleted in the bone marrow; others
report that these cells exit to the periphery, but do not secrete anti-dsDNA Ab due to endogenous Ig expression or
B cell functional inactivation (14). These discrepancies
may be a reflection of the degree to which individual Ig tgs
are capable of inhibiting endogenous Ig rearrangement,
which could rescue an otherwise autoreactive B cell.
Whether endogenous Ig expression is due to the active induction of receptor editing or is the consequence of a defect on the part of the Ig tgs to inhibit rearrangement can
be difficult to assess, particularly for L chain tgs (15). A
more interesting possibility to explain these divergent outcomes is that they reflect the different specificities of the tgs
used in these studies, which may in turn differ in their regulation (14, 20). Because anti-dsDNA Abs from SLE
patients and lupus mice are heterogeneous, and the particular specificities which are significant in disease are not known,
it will be important to understand these differences (21, 22).
The VH3H9 tg offers an opportunity to study the regulation of a range of anti-DNA B cells. Initial studies using
the VH3H9 H chain tg on the BALB/c background demonstrated that neither anti-ssDNA nor anti-dsDNA serum
Abs were elevated over tg( To address the mechanisms governing the regulation of
anti-dsDNA B cells in a diverse repertoire and to avoid the
use of L chain tgs altogether, we relied on the fact that
VH3H9 can pair with endogenous V Mice
BALB/c mice were purchased from Harlan Sprague Dawley
(Indianapolis, IN). VH3H9 tg mice have been described previously (23). The VH3H9 tg mice have been backcrossed onto the
BALB/c background for at least nine generations, and have been
bred and maintained in the animal facility at The Wistar Institute
(Philadelphia, PA). In all cases, age-matched BALB/c mice or
tg( Cell Preparations
Bone marrow, spleen, and lymph node cells were removed
from VH3H9 tg and tg( Flow Cytometry Analysis
Cells (5 × 105) were surface stained according to standard protocols (25). The following Abs were used: RA3-6B2-PE or biotin (anti-B220), R11-153-FITC (anti-V All samples were analyzed on a FACScan® flow cytometer
(Becton Dickinson, Mountain View, CA) using Cellquest software. 15,000-40,000 events were collected for each sample and
gated for live lymphocytes based on forward and side scatter.
Bromodeoxyuridine Labeling
8-d Labeling.
Mice were injected intraperitoneally with 200 µl of 3 mg/ml 5-bromodeoxyuridine (BrdU) (Sigma Chemical
Co., St. Louis, MO) in PBS every 12 h for 8 d. BrdU staining
was performed essentially as described (26), with the exception
that the cells were not fixed in ethanol. In brief, spleen and bone
marrow cells from mice were isolated and surface stained as described above. The cells were then fixed and permeabilized with
1% paraformaldehyde containing 0.1% Tween-20. The DNA was
denatured using 10 µM HCl and 100 U/ml DNase I. The incorporated BrdU was then detected using an anti-BrdU-FITC Ab
(B44) from Becton Dickinson.
2-h Pulse.
Mice were injected intraperitoneally with 200 µl
of 3 mg/ml BrdU in PBS. The spleen and bone marrow cells were
isolated 2 h later and stained as above.
Immunohistochemistry
Spleens were suspended in OCT, frozen in 2-methyl-butane
cooled with liquid nitrogen, sectioned, and fixed with acetone.
The spleen sections were stored at Hybridoma Generation
Spleen cells from a VH3H9 tg mouse were stained for B220,
IgM, and CD44, and sorted for IgMlow CD44high cells (which includes VH3H9/V ELISA Assay.
The Ig isotype of hybridomas was determined
via an indirect solid-phase ELISA assay, using anti-IgH + L
(Southern Biotechnologies) as the primary Ab and developing
with AP-labeled anti-IgM, -IgG, -Ig Sequence Analysis.
The H and L chain variable (V) regions
were sequenced from messenger RNA according to the protocol
described (29). In brief, cytoplasmic RNA was isolated and constant region-specific primers were used to direct synthesis of
cDNA copies of the H (Cµ1) and L (C Antinuclear Antibody Assay.
The presence of antinuclear antibodies in the supernatants was detected using permeabilized
HEP-2 cells as the substrate (Antibodies Incorporated, Davis,
CA). The supernatants were used undiluted and were detected
using an anti-mouse IgM or IgH + L-FITC secondary Ab
(Southern Biotechnologies). The samples were then visualized under a fluorescent microscope.
Statistical Analysis.
Statistical significance was determined using an unpaired Student's t test and Instat Software.
In this study, we use VH3H9 H chain
only tg mice to increase the frequency of anti-dsDNA B
cells while maintaining a polyclonal repertoire. Transfection and hybridoma analysis have identified germline V Table 1.
One scenario that could account for the presence of Using flow cytometry, we assessed the developmental and activation status of anti-dsDNA B cells in the spleen. The panel of developmental
markers, shown in Fig. 2, includes CD19, CD21/35, CD22, CD23, HSA, and B220. We compared the expression levels of these markers on
As an indication of activation/antigen encounter, additional cell surface markers, whose differential expression
levels have been used to mark a B cell's activation state,
were also analyzed. CD44 and MHC class II are molecules
whose expression levels increase on activated B cells (47).
L-selectin expression decreases upon activation, but given
that it is also low on immature B cells, it cannot be used to
distinguish an immature B cell from a postactivated one
(50, 51). The To determine at what point anti-dsDNA B cells have
been arrested in development, flow cytometric analysis of
VH3H9/ VH3H9 tg mice contain, in addition to anti-DNA B
cells, a population of non-DNA B cells in the repertoire
that allow us to control for and distinguish effects which
are due to autoreactive specificity versus those that are due
to the presence of the transgene (10, 23). The presence of
non-DNA B cells in VH3H9 tg mice would predict that
not all of the B cells in these mice will exhibit the immature/activated phenotype. Indeed, comparing the surface phenotype of the In summary, the VH3H9/ The in vivo
lifespan of a B cell can be estimated by continuously labeling mice with the thymidine analogue BrdU, and then measuring the incorporation of BrdU-labeled cells into the
splenic population (4, 52). A population that is rapidly
turning over will be replaced more quickly with labeled
cells from the bone marrow. Studies of BALB/c splenic B
cell turnover rates have estimated that B cells have an average lifespan of 3-4 wk (4). To estimate the lifespan of anti-dsDNA B cells, tg(
Given that there are many In the HEL B cell tolerance model, an increased in vivo
turnover rate has been hypothesized to be a mechanism
that maintains tolerance to self Ags (4). However, it is unclear whether this is dependent upon competition with
nonautoreactive B cells or an intrinsic property of anergic
B cells (4, 13). Here we have shown that when anti-dsDNA
B cells are present in a polyclonal repertoire, they are short
lived. Importantly, in another anti-dsDNA Ig tg model
(VH3H9 H chain with a mutated Lymphocytes are organized into discrete
structures within the splenic architecture. Resting T and B
cells segregate into the inner periarteriolar lymphoid sheath
(inner PALS or T cell zone) and outer PALS (B cell follicle) within the spleen. To address where the short-lived
anti-dsDNA B cells are located within the splenic architecture, spleen sections from tg(
These data are reminiscent of those obtained from the
HEL system, where under some circumstances, anergic B
cells have been reported to be excluded from the B cell follicle and accumulate in the inner PALS where they persist
for only a few days (12, 13, 54). When using B220, both
anti-HEL and anti-dsDNA B cells appear to accumulate at
the T-B interface. However, staining with anti-CD4 to
mark the T cell zone reveals that the anti-dsDNA B cells,
contrary to anergic B cells in the HEL model, are present in the follicle, accumulating at the T zone proximal end
(Fig. 4). Whether this distinction is important is presently
not clear, nor is the relationship between follicular localization and lifespan (13, 54). Cyster et al. suggest that the follicle may provide growth factors or critical cell contacts
that B cells require for survival and that these factors are not
present or are too dilute at the edge of the T cell zone to be
effective (55). Alternatively, Fulcher et al. propose that follicular localization and lifespan are determined by the degree
of receptor engagement and availability of T cell help (54).
The localization of anti-dsDNA B cells is particularly intriguing within the framework of the model proposed by
Rothstein et al. for B cell activation in the inner PALS
(56). This model is based on immunohistochemical studies
that identified IgG2a and rheumatoid factor B cells in the
inner PALS of lpr/lpr mice, as well as data from the Goodnow lab which suggest that anergic B cells are Fas sensitive
when given appropriate T help, regardless of Ig engagement (as opposed to conventional B cells that are Fas resistant in the presence of T help and Ig engagement) (57). The model further predicts that autoantibodies that arise in
lpr/lpr mice may be a consequence of the inability to execute Fas-mediated apoptosis of anergic cells. Importantly
for this model, we show that the anti-dsDNA B cells that
are present in nonautoimmune animals are located at or
near the PALS. Studies are underway to define the role that
Fas-mediated apoptosis plays in the elimination of anti-dsDNA B cells once they have been reactivated and to determine if they form antibody-forming cells at this site in
lpr/lpr mice.
Interestingly, when we compare the frequency of In conclusion, we have used Ig H chain tg mice to boost
the frequency of anti-dsDNA B cells and follow their fate
in the context of a polyclonal repertoire. This is important
because it more accurately reflects the conditions under
which serum autoantibodies are expressed in autoimmune
disease. To this end, we used the VH3H9 H chain tg,
which pairs with the endogenous V) BALB/c control sera (23).
When hybridoma panels were generated from the spleens
of VH3H9 tg mice, anti-ssDNA and non-DNA hybridomas were recovered, but not anti-dsDNA hybridomas (23,
24). Transfection studies clearly showed that this H chain
has the capacity to generate anti-dsDNA B cells (9). Importantly, we have also recovered this specificity in hybridoma
panels generated from VH3H9 MRL-lpr/lpr spleens (10). The
absence of anti-dsDNA hybridomas from BALB/c-derived panels suggests, therefore, that they are either deleted in the bone marrow or, if present, cannot be rescued as hybridomas.
1 L chains to generate anti-dsDNA Abs (9). Thus, we were able to track the
1-bearing anti-dsDNA B cells in the context of the diverse repertoire of the VH3H9 tg mouse. We have found
that anti-dsDNA B cells are not deleted in the bone marrow, but instead exit to populate the spleen. Their unique surface phenotype suggests that they are both developmentally arrested and antigen experienced. These cells exhibit
an increased in vivo turnover rate and are localized to the
T-B interface of the splenic white pulp.
) littermates were used as controls. The presence of the
VH3H9 tg was determined by PCR amplification of tail DNA
with primers specific for VH3H9 (23).
) mice. Single-cell suspensions were
prepared and, where necessary, erythrocytes were removed by
hypotonic lysis.
1), R26-46-FITC or
-biotin (anti-V
total), R8-140-PE (anti-Ig
), 1D3-FITC (anti-CD19), 7G6-FITC (anti-CD21), Cy34.1-FITC (anti-CD22), 3/23-FITC (anti-CD40), Mel-14-FITC (anti-CD62L, L-selectin),
2G9-PE (anti-I-Ad/I-Ed, MHC class II), M1/69-FITC (anti-
heat-stable antigen [HSA]), IM7-FITC (anti-CD44) (all from
PharMingen, San Diego, CA), LS136-biotin (anti-V
1), and
JC5.1-PE (anti-V
total) (LS136 and JC5.1, gifts from J. Kearney,
University of Alabama, Birmingham, AL; JC5-PE, gift from R. Hardy, Fox Chase Cancer Center, Philadelphia, PA), polyclonal
anti-IgM-PE and SBA-1-PE (anti-IgD) (Southern Biotechnologies, Birmingham, AL), B3B4-FITC (anti-CD23) (gift from D. Conrad, Virginia Commonwealth University, Richmond, Virginia),
and streptavidin-Red670 (GIBCO BRL, Gaithersburg, MD).
70°C and then stained according to the protocol described (27). In brief, the sections were blocked using PBS/5% BSA/0.1% Tween 20, and then stained
with GK1.5-biotin (anti-CD4), 53-6.7-biotin (anti-CD8), RA3-6B2-biotin (anti-B220) (grown as supernatants), and/or anti-Ig
-alkaline-phosphatase (AP; Southern Biotechnologies). Streptavidin-horseradish-peroxidase (HRP; Southern Biotechnologies)
was used as a secondary antibody with the biotinylated reagents.
HRP and AP were developed using the substrates 3-amino-9-ethyl-carbazole and Fast-Blue BB base (Sigma Chemical Co., St. Louis,
MO), respectively.
1 anti-dsDNA B cells) by flow cytometry. The IgMlow CD44high cells were then cultured overnight in media
(DMEM/10% FCS) containing CD40 ligand-CD8 fusion protein (a gift of P. Lane, Basel Institute for Immunology, Basel,
Switzerland; reference 28; 1:2 dilution of culture supernatant) and
rIL-4 (2 ng/ml; Genzyme Diagnostics, Cambridge, MA). The
cells were then fused to the Ig(
) myeloma Sp2/0. Cells were
plated at limiting dilution and wells bearing single colonies were
expanded for analysis.
, or -Ig
Abs (Southern
Biotechnologies). Binding to dsDNA was detected in a similar
manner. In this case, the plates were coated with Avidin-DX
(Vector, Burlingame, CA); DNA-biotin was used in place of the
primary antibodies. DNA-biotin was prepared as described (9).
1) chain V regions. The
cDNA was then amplified using the constant region primers in
conjunction with VH5
1 or
1L primers that hybridize to the 5
ends of H and L chain V region genes, respectively (29). Amplification products were sequenced by automated analysis (Wistar
Institute Nucleic Acid Facility). Sequence translation and comparison was carried out using the Sequencher program and by
searching EMBL/GenBank/DDBJ databases.
Anti-dsDNA B Cells Are Present in the Periphery of Nonautoimmune Mice.
1
as an L chain that pairs with the VH3H9 H chain to generate an anti-dsDNA Ab (see J558LT and MRL1-45 in Table 1; references 9, 10). Because the VH3H9 H chain tg
has been shown to be a good excluder of endogenous H
chain rearrangement on the BALB/c background (Table 1;
references 23, 30), we can follow the fate of anti-dsDNA B
cells in VH3H9 tg mice using anti-
specific reagents. Several different reagents were used to track
+ and
1+ B
cells (LS136, R11-153, JC5, and R26-46). Using these reagents and flow cytometry, we have shown that the majority of
+ B cells in VH3H9 tg mice are
1 (79 ± 19%).
Therefore, for the remainder of our studies, we have used
anti-pan
reagents to detect anti-dsDNA B cells. As is
shown in Fig. 1,
+ B cells are present in the bone marrow,
spleen, and lymph node. It is also apparent that the levels of
Ig on these cells are lower in the periphery compared to
+ B cells from tg(
) mice (spleen: mean fluorescence intensity [MFI] 52 versus 181; LN: MFI 52 versus 216). Interestingly, Ig levels are also decreased on the
+ B cells
from the bone marrow (MFI 47 versus 197). There is precedent for antigen encounter leading to a decrease in Ig
density in other tolerance model systems; for example,
anti-hen egg lysozyme (HEL) B cells have a reduced level
of Ig when in the presence of HEL (3), but when these B
cells are removed from Ag, either by in vivo parking or in
vitro cultures, their surface Ig levels increase (31, 32). The
decreased Ig density on the anti-dsDNA B cells suggests
that these cells are encountering their Ag and that this encounter initially occurred in the bone marrow.
-expressing Hybridomas: Specificity and Ig Usage
L chains
H chains
Specificity
Hybridoma
1
VH3H9
endog.Ig
dsDNA§
ANA
J558LT
+
+
+
+
+
MRL1-45¶
+
+
+
+
+
BALB1-72¶
+
+
+
+
L1
+
+
+
+
+
L9
+
+
+
+
+
L12
+
+
+
+
+
L17
+
+
+
+
+
ANA, antinuclear antibody; endog, endogenous.
*
The presence of and
protein was determined by ELISA performed
on hybridoma supernatants. An OD of 5× background was considered positive.
L and H chain genes were sequenced as described in Materials and
Methods.
§
Binding to dsDNA was assessed by ELISA on hybridoma supernatants.
An OD of 4× background was considered positive.
J558LT is the previously published VH3H9/
1 transfectant and is
shown here as a positive control (9).
¶
MRL1-45 and BALB1-72 were described previously and are shown
here for comparison (10).
Fig. 1.
VH3H9/ anti-dsDNA B cells are present with a reduced Ig
density. Bone marrow (left), spleen (middle), and lymph node (right) cells
from Tg(
) (top) and VH3H9 tg (bottom) mice were stained with anti-B220-biotin/streptavidin-Red670 and anti-
-FITC. MFI is given for the
+ cells in the boxed region. These are representative plots of n = 19 mice of each genotype.
[View Larger Version of this Image (29K GIF file)]
+
B cells in the periphery of VH3H9 tg mice is coexpression
of a kappa chain or an endogenous H chain to generate a
non-dsDNA-binding Ab (10, 15, 33, 34). Indeed, our previous analysis of hybridomas generated from either unmanipulated or LPS-activated B cells from VH3H9 BALB/c
mice detected a single
+ hybrid (BALB1-72) that also coexpressed a kappa protein (Table 1). The addition of the
second L chain (kappa) disrupted DNA binding, which we
suggested most likely spared the B cell from deletion (10).
A similar scenario has been described for VH3H9 tg mice
bred to V
4 tg mice (15). VH3H9/V
4 encodes an anti-dsDNA Ig, but no peripheral B cells with this specificity
were identified in the tg mice. The fact that all the B cells
isolated as hybridomas coexpressed an endogenous L chain
was interpreted as evidence for receptor editing (15). In
contrast, the
+ B cells we detect show no evidence of L
chain coexpression by flow cytometry; the B cells all express either
or
(data not shown). Because low levels of
surface Ig are hard to detect using flow cytometry, we took
a second approach; B cells were isolated by flow cytometry
based on Ig density, cultured in a cocktail mimicking T
help (CD40 ligand and rIL-4), and then used to generate hybridoma panels. Anti-dsDNA hybridomas were recovered that only expressed a
L chain as detected by ELISA.
Importantly, messenger RNA H + L sequencing analysis
showed that these hybridomas exclusively use the VH3H9
tg and V
1 (Table 1). The ability to rescue anti-dsDNA B
cells with T help-derived factors is intriguing in light of the
requirement for T cells in murine SLE (35). In addition, this may explain why we, and others, did not previously recover anti-dsDNA B cells from LPS-derived hybridomas (10, 14, 23, 24, 34).
+ B cells from VH3H9 tg
mice with those on the tg(
)
+ B cells, as well as the total
B cell population, from tg(
) mice (Fig. 2). B220 (CD45R)
increases with maturity and, in conjunction with HSA, has
been used to define the immature to mature stages of B cell
development (25). HSA is expressed at high levels on immature (newly emerging) B cells and at a lower level on
mature B cells in the spleen (26). As is shown in Fig. 2 B,
the VH3H9/
B cells express a slightly reduced level of
B220 and a level of HSA intermediate between the HSAhigh
and HSAlow cells in the tg(
) spleen. CD22 is expressed at
a low level on immature B cells and increases with maturity, whereas CD21/35 and CD23 become surface positive
at the mature B cell stage (38). The VH3H9/
B cells
have a dramatically reduced level of CD21/35, as well as
lower levels of CD22 and CD23 on their surface (Fig. 2 B).
Because CD21/35 (complement receptors 1 and 2) and
CD22 play a role in modulating the response through the
Ig receptor (42), the low expression levels of these
coreceptors on VH3H9/
B cells may alter the signaling
threshold of these cells when stimulated through membrane Ig. CD19 is a B cell-specific marker that is expressed
on all B cells starting at the pro-B cell stage (46). As is
shown in Fig. 2 B, CD19 expression on the surface of
+ B
cells in VH3H9 tg mice is higher than on B cells from
tg(
) mice. CD40 was also examined and has a similar expression level to tg(
) B cells (data not shown). In contrast
to VH3H9 tg mice, the
+ B cells in tg(
) mice have
equivalent levels of all surface markers tested (Fig. 2), suggesting that there is nothing inherently different about B
cells with
L chains. Taken together, these data suggest that the VH3H9/
B cells are phenotypically immature.
Fig. 2.
Phenotypic analysis of VH3H9/ B cells. Spleen (A-C) and bone marrow (D) B cells were stained with anti-B220-biotin/streptavidin-Red670, anti-
-PE or -FITC, and either anti-HSA, CD19, CD21/35, CD22, CD23, CD44, CD62L-FITC, or anti-class II-PE. (A) Dot plots showing B220 versus
staining in the spleen. (B) Developmental markers and (C) activation markers on the total splenic B cell population (gating on B220+ cells)
in tg(
) mice (thin lines in B and C) and B220+
+ B cells in tg(
) mice (bold line, top), B220+
+ B cells in VH3H9 mice (bold line, middle), or B220+
+
B cells in VH3H9 tg mice (bold line, bottom). The underlayed histograms (thin lines) were scaled down to allow for the comparison to the
+ B cells
(which are present at ~0.1 the frequency of total B cells) in the upper and middle panels. (D) Histograms showing developmental and activation markers
on the
+ B cells in the spleen (thin line) and bone marrow (bold line) in tg(
) (top panels) and VH3H9 tg mice (bottom panels). These are representative plots from n = 4 mice of each genotype.
[View Larger Versions of these Images (30 + 27 + 25K GIF file)]
+ B cells in VH3H9 tg mice express increased levels of CD44 and MHC class II and decreased
levels of L-selectin (Fig. 2 C). Together, these data as well as
the increase in cell size (Fig. 2 C), suggest that the anti-dsDNA
B cells have encountered antigen.
+ bone marrow cells was performed. The majority of
+ B cells in the bone marrow of VH3H9 tg mice
express similar levels of the markers B220, HSA, CD21,
CD22, CD23, CD44, and L-selectin as the tg(
) bone
marrow B cells (compare the bold lines in Fig. 2 D). Additionally, in comparison to the bone marrow, the VH3H9/
B cells in the spleen have altered their expression level of B220, HSA, CD22, CD23, and L-selectin, consistent with
their continued maturation (compare the thin line to the
bold line in Fig. 2 D). These data suggest that B cell development proceeds in the bone marrow for the VH3H9/
+
cells in a similar fashion to
+ cells from tg(
) mice, and
furthermore, that the
+ anti-dsDNA B cells in the spleen
have matured more than the majority of bone marrow B cells.
+ cells in the VH3H9 tg mice with B
cells from tg(
) mice shows that the majority of the
VH3H9
+ B cells express cell surface densities equivalent
to their tg(
) counterparts for both the developmental
markers (CD21/35, CD22, CD23, B220, and HSA; Fig. 2
B) as well as activation markers (CD44, L-selectin, and cell
size; Fig. 2 C). In contrast, CD19 and MHC class II are
slightly elevated on all of the VH3H9 cells (Fig. 2, B and
C), implying that their alteration may be due to the presence of VH3H9 tg per se, and not to their autoreactive
specificity. Furthermore, as transfection studies and L chain
repertoire analysis of hybridoma panels from VH3H9 tg
MRL-lpr/lpr mice have revealed, there are
L chains in
addition to
1, which can pair with the VH3H9 H chain
to generate anti-dsDNA B cells (8). We predicted that
these B cells would also be regulated and exhibit an altered surface phenotype. Consistent with this, there is a small
population of VH3H9
+ B cells that exhibit a phenotype
similar to the VH3H9/
+ B cells: CD21/35low, CD22low,
CD23low, CD44high, and L-selectinlow (note the shoulders in
Fig. 2, B and C).
1 anti-dsDNA B cells have a
unique cell surface phenotype, as does a subpopulation of
VH3H9/
B cells. They show evidence of activation
(CD44high, L-selectinlow, and increased cell size), suggesting
that they have been exposed to Ag. In addition, they appear developmentally arrested in that they express reduced
levels of CD21/35, CD22, CD23, L-selectin, and B220,
and intermediate levels of HSA. These data suggest that anti-dsDNA B cells have encountered their Ag while still at
an immature stage, resulting in arrested development with
respect to some markers and a concurrent change in the
expression of activation markers. In support of this, there is
a reduced level of surface Ig in the bone marrow (Fig. 1). A
similar immature phenotype (low levels of CD21/35, CD22,
CD23, and L-selectin) was seen in the anti-HEL/membraneHEL mice in the context of a bcl-2 tg, whereas in the
absence of the bcl-2 tg, the anti-HEL B cells were deleted in the bone marrow (2, 32). Thus, the bcl-2 tg protects B cells from deletion, but does not protect them from maturational arrest. The anti-dsDNA B cells are unique in that
they are present in the periphery with an immature phenotype in the absence of a bcl-2 tg.
) and VH3H9 tg mice were labeled
with BrdU for 8 d and then the incorporation of BrdU was
measured using flow cytometry. We chose to analyze the
8-d time point since this is when relatively few B cells will be labeled, and cells with an increased turnover rate will be readily apparent (4, 26). To assess the lifespan of the anti-dsDNA population of cells, we examined BrdU incorporation in the
+ subset. Fig. 3 A demonstrates that among the
+ cells, the frequency of BrdU-labeled cells is significantly
higher in VH3H9 tg mice than in tg(
) mice (63.1 ± 5.6%
versus 9.5 ± 0.4%), suggesting that the anti-dsDNA B cells
have a decreased in vivo lifespan. Alternatively, the increased BrdU uptake in this population could be due to the
active proliferation of the
+ B cells in vivo. To address
this, we pulsed VH3H9 tg and tg(
) mice with BrdU for
2 h and then measured the uptake of BrdU in the splenic B
cell populations. This is a time point when actively proliferating cells will take up BrdU, but is not a long enough period for replacement of cells from the bone marrow pool
(52). We were unable to detect BrdU label in the
+ B
cells in VH3H9 tg mice spleens. Labeling was analyzed and
detected in the rapidly dividing populations of the bone
marrow, thus ensuring that the mice did receive BrdU
(data not shown). Together, these data suggest that the increased BrdU labeling of the VH3H9/
B cells is due to
their decreased in vivo lifespan.
Fig. 3.
VH3H9/ B cells have an increased in vivo turnover rate.
Tg(
) (top) and VH3H9 (bottom) mice were continuously labeled with BrdU for 8 d. Spleen cells were then stained with anti-
-biotin/streptavidin-Red670 and anti-B220-PE or anti-IgM + IgD-PE (Ig), fixed, permeabilized, and then incorporated BrdU was detected with anti-BrdU
Ab. (A) Dot plots show B220 versus
(left) and histograms show BrdU
label within the B220+
+ gate (right). Percentages are given for the
BrdU+ cells. (B) Dot plots show B220 versus Ig (left) and histograms show
BrdU label for the indicated B220+Ighigh and B220+Iglow gates (right).
These are representative plots for n = 4 tg(
) and n = 3 VH3H9 tg mice.
[View Larger Versions of these Images (26 + 31K GIF file)]
L chains in addition to
1
that can pair with the VH3H9 H chain to generate anti-dsDNA B cells, we predicted that they too may exhibit a
decreased lifespan. Since we do not have L chain-specific
reagents to detect these B cells (as we do for
1), we used
the phenotype of Iglow to distinguish these cells. The
+
cells are included within the Iglow cells, comprising 20-30%
of this subset. Iglow cells in tg(
) and VH3H9 tg mice were
defined by gating on cells stained with either IgM and IgD
(Fig. 3 B) or with only IgM (data not shown); both approaches yielded similar results. As shown in Fig. 3 B, the
majority of the B cells in VH3H9 tg mice have approximately the same amount of BrdU incorporation as the
tg(
) B cells. However, when we gate on the Iglow subset
of cells, 57.7 ± 5.1% of the tg B cells are labeled. These data are consistent with the idea that there are Iglow
+ cells
in VH3H9 tg mice that are dsDNA reactive, and these also have a rapid turnover rate. Interestingly, when we look in
tg(
) mice, the Iglow B cells are present, although at a
much reduced frequency (<5% of the B cells). The Iglow
cells that we do detect have a slight increase in BrdU labeling (16%); however, this is much lower than that seen in
the VH3H9 Iglow B cells (62%). It is possible that the frequency of autoreactive B cells in tg(
) mice is too low to
detect by these means, underscoring the advantage of using
the VH3H9 tg to track these cells.
2 L chain), where the repertoire is monoclonal by virtue of the Rag-2
/
background, we have shown that anti-dsDNA B cells still exhibit a decreased lifespan (53). Therefore, we favor the interpretation that anti-dsDNA B cells are dying rapidly due
to their exposure to antigen without receiving appropriate
T cell help, rather than competition from nonautoreactive
B cells.
) and VH3H9 tg mice were
stained with anti-
, anti-B220, and anti-CD4 to mark B
and T zones, respectively (Fig. 4). In tg(
) mice,
+ B cells
are scattered throughout the B cell area in the follicle as
well as in the red pulp, but are not detected within the T zone. In contrast, the
+ anti-dsDNA B cells in the
VH3H9 tg mouse are found clustered at the T-B interface
in the B cell follicle, as well as in the T cell zone (Fig. 4).
Fig. 4.
VH3H9/ B cells accumulate at the T-B
interface. Spleen sections from Tg(
) (left) and
VH3H9 (right) mice were stained with anti-
-AP and
either anti-B220-biotin (top) or anti-CD4-biotin (bottom). A similar staining pattern was observed when
anti-CD8-biotin was included with anti-CD4-biotin
(data not shown). Avidin-HRP was used as a secondary step for the biotinylated reagents. HRP and AP
were developed using the substrates, 3-amino-9-ethyl-carbazole (red) and Fast-Blue BB base (blue), respectively. These are representative sections from n = 15 Tg(
) and n = 19 VH3H9 mice. Original magnification: 100.
[View Larger Version of this Image (124K GIF file)]
+ B Cells in VH3H9 tg Mice.
+ B
cells are not only present in the periphery of VH3H9 tg
mice, but they are present at a twofold higher frequency
than in the tg(
) controls, despite the fact that VH3H9/
1
B cells are autoreactive and have an increased turnover rate
(Fig. 5). What could account for this seemingly surprising
result? Pulsing mice with BrdU showed that the increased
number of VH3H9/
B cells is not simply due to their
proliferation in the periphery (data not shown). Another
possibility is that the VH3H9/
B cells are positively selected (on some unidentified ligand) in the bone marrow.
In support of this, there is a twofold increase in
+ B cells
in the VH3H9 tg bone marrow over tg(
) bone marrow
(Fig. 5). Alternatively, the increased
frequency may be
the consequence of receptor editing where
s represent the
end result of multiple L chain rearrangement attempts (19,
33). The scenario we favor for an autoreactive Ig in
VH3H9 tg mice is that after rearrangement at the
loci is
exhausted,
rearrangement occurs, completing receptor
editing. The
1+ B cells that are generated are not deleted;
rather, they persist in a compromised state.
Fig. 5.
Increased frequency
of + B cells in VH3H9 tg mice.
Bone marrow (BM; left), spleen
(middle), and lymph node (right)
cells from Tg(
) (open bars) and
VH3H9 (shaded bars) mice were
stained with anti-B220-biotin/
streptavidin-Red670 and anti-
-FITC or -PE. The percentage of
B220+
+ cells of total B cells for
spleen and lymph node and percentage of B220+
+ cells out of total B220+Ig+ cells in the bone marrow
was determined by flow cytometry. The graph shows the mean percentage of B220+
+ B cells. There is an increased frequency of
+ B cells in
the VH3H9 spleen (10.1 ± 5.5% versus 5.0 ± 0.8%; P = 0.0011). There
is also a greater frequency of
+ B cells in the VH3H9 bone marrow
(16.8 ± 7.0% versus 8.3 ± 3.1%; P = 0.0010), but not in the lymph
node (5.4 ± 2.4% versus 4.4 ± 0.7%; P = 0.1139). Interestingly, when
we compare the frequency of
-expressing B cells within a mouse, the
frequency is highest in the bone marrow and then decreases in the spleen
(VH3H9: 16.8 ± 7.0% versus 10.1 ± 5.5%; P = 0.0087; tg(
): 8.3 ± 3.1% versus 5.0 ± 0.8%; P = 0.0017). The frequency of
s changes only
slightly between the spleen and lymph node in tg(
) mice (5.0 ± 0.8%
versus 4.4 ± 0.7%; P = 0.0251); however, it decreases drastically from the VH3H9 spleen to the lymph node (10.1 ± 5.5% versus 5.4 ± 2.4%; P = 0.0034). n = 14 tg(
) mice and n = 13 VH3H9 tg mice.
[View Larger Version of this Image (33K GIF file)]
-expressing B cells in VH3H9 and tg(
) mice, the frequency is highest in the bone marrow and then decreases in the spleen.
This is indicative of the loss of B cells from the bone marrow and recruitment into the long-lived splenic B cell pool
(60). The frequency of
s changes only slightly between
the spleen and lymph node in tg(
) mice; however, it decreases by half from the VH3H9 spleen to the lymph node
(Fig. 5). The loss of
+ B cells between the spleen and
lymph node in VH3H9 tg mice may be a consequence of
their reduced half life, resulting from autoreactivity.
1 L chain, to generate
an anti-dsDNA Ab, thus enabling us to follow anti-dsDNA B cells using
-specific reagents. We report that these anti-dsDNA B cells are not deleted in the bone marrow, but instead are present in the periphery where they exhibit features of immaturity and activation, an increased in vivo
turnover rate, and altered splenic localization. The lack of
detectable anti-dsDNA serum titers (23) suggests that anti-dsDNA B cells are actively regulated, which we show is
manifested by their unique phenotype. What accounts for
the presence of anti-dsDNA Abs in autoimmunity is unknown. Studies are underway to determine if the addition
of cognate T cell help will rescue anti-dsDNA B cells and
lead to the expression of secreted autoantibodies.
Address correspondence to Dr. Jan Erikson, The Wistar Institute, Rm 273, 3601 Spruce St., Philadelphia, PA 19104. Phone: 215-898-3823; FAX: 215-573-9053; E-mail: jan{at}wista.wistar.upenn.edu
Received for publication 17 June 1997 and in revised form 14 August 1997.
Services provided by the Wistar Institute staff were supported by the Core grant No. CA10815 and by grants from the National Institutes of Health (5R01 AI32137-06), the Arthritis Foundation, and the Pew Charitable Trust to J. Erikson. L. Mandik-Nayak is supported by the Wistar Training grant CA-09171. H. Noorchashm and A. Eaton are supported by the National Cancer Institute Training grant 2T32CA09140.We thank Dr. Andrew Caton and Eden Haverfield for critical reading of the manuscript, Dr. Andrew Caton for sequencing analysis, Dr. Garnett Kelsoe for help with setting up the histology staining protocol, Dr. Clayton Buck for use of his cryostat and microscope, Sudhir Nayak for help with the graphics, and Deepa Kurian for genotyping the mice. In addition, we acknowledge Drs. Randy Hardy and John Kearney for valuable reagents and discussion.
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