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Effects of anti-CD154 treatment on B cells in murine systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 2, February 2003, pp 495–506
DOI 10.1002/art.10929
© 2003, American College of Rheumatology
Effects of Anti-CD154 Treatment on B Cells in
Murine Systemic Lupus Erythematosus
Xiaobo Wang,1 Weiqing Huang,1 Lena E. Schiffer,1 Masahiko Mihara,2 Alla Akkerman,1
Kenji Hiromatsu,1 and Anne Davidson1
attenuated IgM response to the hapten oxazolone and
produced no IgG antioxazolone antibodies.
Conclusion. Anti-CD154 is a B cell–depleting
therapy that affects multiple B cell subsets. Activation of
both B and T cells is prevented during therapy. After
treatment cessation, autoreactive B cells progress
through a series of activation steps before they become
fully competent antibody-producing cells. The general
immunosuppression induced during treatment will need
to be taken into account when using B cell–depleting
regimens in humans.
Objective. To determine the immunologic effects
of anti-CD154 (CD40L) therapy in the (NZB ⴛ NZW)F1
mouse model of systemic lupus erythematosus.
Methods. Twenty-week-old and 26-week-old
(NZB ⴛ NZW)F1 mice were treated with continuous
anti-CD154 therapy. Mice were followed up clinically,
and their spleens were studied at intervals for B and T
cell numbers and subsets and frequency of anti–doublestranded DNA (anti-dsDNA)–producing B cells. T cell–
dependent immunity was assessed by studying the humoral response to the hapten oxazolone.
Results. IgG anti-dsDNA antibodies decreased
during therapy and disease onset was delayed, but
immune tolerance did not occur. During treatment,
there was marked depletion of CD19ⴙ cells in the
spleen; however, autoreactive IgM-producing B cells
could still be detected by enzyme-linked immunospot
assay. In contrast, few IgG- and IgG anti-dsDNA–
secreting B cells were detected. Eight weeks after treatment cessation, the frequency of B cells producing IgG
anti-dsDNA antibodies was still decreased in 50% of the
mice, and activation and transition of T cells from the
naive to the memory compartment were blocked. AntiCD154 treatment blocked both class switching and
somatic mutation and induced a variable period of
relative unresponsiveness of IgG anti-dsDNA–producing B
cells, as shown by decreased expression of the CD69
marker and failure to generate spontaneous IgG antidsDNA–producing hybridomas. Treated mice mounted an
The interaction of CD40 on B cells with its ligand
CD154 (CD40L) on activated T cells provides a B cell
costimulatory signal that induces B cell proliferation and
formation of germinal centers (1–3). Further cell–cell
interactions within the germinal center, also involving
CD40/CD154, lead to immunoglobulin isotype switching, somatic mutation, clonal expansion of high-affinity
B cells, and terminal differentiation to memory cells or
antibody-forming cells (1,4,5). In addition, activation of
antigen-presenting cells by CD40 ligation is required for
optimal priming and clonal expansion of CD4⫹ T cells
(2). CD40–CD154 interactions are also important in the
effector arm of the inflammatory response (6,7). In
particular, CD40 engagement plays a role in activation
of endothelial cells, including up-regulation of adhesion
molecules and chemokines that help direct cell traffic to
sites of inflammation (7). The multiple functions of
CD40/CD154 in the immune and inflammatory response
have made it an attractive target for therapeutic intervention in autoimmune disease.
Disease expression in 3 independent murine
models of systemic lupus erythematosus (SLE) has been
shown to depend on T cell help mediated by CD40–
CD154 interactions. In the MRL/lpr mouse, the absence
of CD40 in T cell receptor–intact mice resulted in a
decrease in IgG levels but not IgM levels, markedly
Supported by the Sklarow Trust and by NIH grant AI-47291.
1
Xiaobo Wang, MD, Weiqing Huang, MD, Lena E. Schiffer,
MD, Alla Akkerman, BSc, Kenji Hiromatsu, PhD, Anne Davidson,
MBBS: Albert Einstein College of Medicine, Bronx, New York;
2
Masahiko Mihara, PhD: Chugai Pharmaceuticals, Shizuoka, Japan,
and Albert Einstein College of Medicine, Bronx, New York.
Address correspondence and reprint requests to Anne Davidson, MBBS, Albert Einstein College of Medicine, 1300 Morris Park
Avenue, U505, Bronx, NY 10461. E-mail: davidson@aecom.yu.edu.
Submitted for publication August 16, 2002; accepted in
revised form November 13, 2002.
495
496
WANG ET AL
lower titers of IgG autoantibodies, and decreased expression of clinical renal disease (8). In the (SWR ⫻
NZB)F1 model, 3 doses of anti-CD154 antibody given
early in life delayed disease onset by an average of 3
months (9). In these mice, a high-dose continuous
treatment regimen was able to stabilize or reverse early
nephritis, although it had much less effect on mice that
had already developed proteinuria of ⬎300 mg/dl
(10,11). In (NZB ⫻ NZW)F1 mice, long-term administration of anti-CD154 antibody prevented development
of high-titer autoantibodies and disease onset (12).
Finally, a 2-week combination protocol of anti-CD154
and CTLA4-Ig was highly synergistic in the (NZB ⫻
NZW)F1 model, resulting in prolonged survival of the
mice (13,14).
The goal of this study was to determine the
effects, and to further evaluate the mechanism of action,
of continuous anti-CD154 treatment in the (NZB ⫻
NZW)F1 model with respect to its effect on autoreactive
B cells. We confirmed that disease onset is prevented in
mice treated 20 weeks before the development of anti–
double-stranded DNA (anti-dsDNA) antibodies. In 26week-old prenephritic mice, prevention of disease onset
necessitated higher doses of anti-CD154.
There was a substantial decrease in the frequency
of B cells in the spleens of mice during treatment,
showing that anti-CD154 is a B cell–depleting agent.
Secretion of autoantibodies by IgM anti-dsDNA
antibody–producing B cells was relatively unaffected by
treatment, but class switching and somatic mutation
were suppressed during therapy, as was the IgG humoral
response to a foreign antigen. Activation of T cells was
also suppressed, and there was a delay in the abnormal
accumulation of T cells bearing the activated/memory
phenotype. After cessation of therapy, there was a
variable period during which autoreactive B cells were
not fully activated, and there was a consequent delay in
disease onset for an average of 4 months. This maintenance of an immunosuppressive effect suggests that
CD154 blockade might be useful for intermittent
therapy.
MATERIALS AND METHODS
Mice. Female (NZB ⫻ NZW)F1 mice were purchased
from The Jackson Laboratory (Bar Harbor, ME) and maintained in a conventional animal housing facility. Mice were
treated at the age of 20 weeks or 26 weeks with 250 ␮g of
hamster anti-CD154 (MR1; a kind gift of Dr. Susan Kalled,
Biogen, Cambridge, MA) given intraperitoneally biweekly for
6 months (groups A20 and A26, respectively; n ⫽ 10 mice per
group) until age 46 weeks. Because this protocol was ineffec-
tive in 26-week-old mice, a group of 26-week-old mice (group
B; n ⫽ 10 mice) was treated for 10 weeks with a higher dose
protocol of 500 ␮g/week for 3 weeks followed by biweekly
treatment for a further 4 doses (total of 7 doses given from
ages 26–36 weeks). This protocol was designed to replicate the
dosing schedule of a simultaneous human clinical trial of an
anti-CD154 antibody based on the half-lives of the two antibodies (15). Control mice received either hamster IgG (group
C; n ⫽ 14 mice) or no treatment (group D; n ⫽ 17 mice). Prior
to treatment, mice were randomized into treatment groups
depending on the titer of anti-dsDNA antibodies. Mice were
bled every 2–4 weeks. Urine was tested for proteinuria by
dipstick (Multistick; Fisher, Pittsburgh, PA) every 2 weeks.
Survival splenectomies were performed at intervals following
treatment. Mice were followed up until death.
Anti-dsDNA antibodies. Enzyme-linked immunosorbent assay (ELISA) plates (Falcon, Springfield, NJ) were
coated with 100 ␮l of 100 ␮g/ml salmon sperm DNA made
double stranded by passage through a 45␮ filter (USA Scientific, Ocala, FL). After drying, the plates were blocked with 5%
fetal calf serum (FCS)/3% bovine serum albumin (BSA) in
phosphate buffered saline (PBS) and then incubated sequentially for 1 hour at 37°C with serial dilutions of serum followed
by peroxidase-conjugated F(ab⬘)2 goat anti-mouse immunoglobulin (1:8,000) or anti-IgG2a (1:5,000) in PBS/1% BSA
(Accurate, Westbury, NY) and then by ABTS substrate
(Kirkegaard & Perry, Gaithersburg, MD).
Anti-hamster antibodies. Anti-CD154 antibody (BD
PharMingen, San Diego, CA) was plated at 2 ␮g/well onto
ELISA plates (Falcon) overnight at 4°C. Plates were blocked
with 5% FCS/3% BSA in PBS for 90 minutes at 37°C and then
incubated with a 1:500 dilution of serum taken 6 weeks after
the first injection of anti-CD154 antibody or hamster Ig. This
was followed by peroxidase-conjugated anti-mouse IgG2a (1:
5,000; Accurate) or anti-mouse total Ig (1:5,000; Accurate) for
1 hour at 37°C and then by ABTS peroxidase substrate. A
high-titer serum was plated in serial dilutions on each plate for
quantification.
Enzyme-linked immunospot (ELISpot) assay. Spleens
were harvested by survival splenectomy either 2 or 8 weeks
after cessation of treatment. Splenectomy has been shown not
to affect the course of disease in (NZB ⫻ NZW)F1 mice (16),
and it allowed us to correlate the immunologic phenotype with
the eventual clinical outcome. Threefold serial dilutions of
spleen cells in Dulbecco’s modified Eagle’s medium (DMEM)/
10% FCS were plated in quadruplicate on DNA-coated plates
starting at 1 ⫻ 106/well. The plates were spun and incubated
for 2 hours at 37°C, after which 50 ␮l of biotin-conjugated
anti-IgM or anti-IgG (Southern Biotechnology, Birmingham,
AL) diluted 1:700 in DMEM/10% FCS was gently added to
each of two wells, and the plates were allowed to incubate
overnight. The cells were then washed off, and the plates were
incubated with streptavidin–alkaline phosphatase (Southern
Biotechnology) diluted 1:1,000 in PBS/1% BSA for 45 minutes.
Plates were then developed with 1 mg/ml of BCIP (Sigma, St.
Louis, MO) in AMP buffer (0.75 mM MgCl2, 0.01% Triton
X-405, 9.58% 2-amino-2-methyl-1-propanol, pH 10.25). Spots
were counted using a dissecting microscope. Total numbers of
immunoglobulin-secreting cells were measured the same way
using anti-mouse immunoglobulins (Cappel, West Chester,
PA) to coat the plates.
ANTI-CD154 IN MURINE SLE
Generation of hybridomas. Highly activated B cells
from (NZB ⫻ NZW)F1 mice will spontaneously generate
hybridomas without the need for immunization (14). Hybridomas were therefore generated from spleen cells by standard
techniques using NSO cells as fusion partners. In some cases,
spleen cells were also stimulated with 20 ␮g/ml lipopolysaccharide (LPS; Sigma) for 48 hours prior to fusion. Hybridomas
were screened for anti-dsDNA activity by ELISA as above.
Positive hybridomas were then isotyped using specific
peroxidase-conjugated secondary antibodies for IgM and IgG
(Cappel).
Analysis of class switching. Evidence of active class
switching was sought by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) analysis of I-C
transcripts from the spleens of 4–6 treated mice from groups
A20 and B, and from the spleens of 5 nephritic mice from
group C. Complementary DNA (cDNA) was generated using
random primers, normalized for cDNA content using actin
primers, and subjected to PCR using primers for the I␥2b and
C␥2b exons as previously described (17).
Analysis of the VHBW-16 gene. To determine the effect
of treatment on somatic mutation, we performed a detailed
analysis of the VHBW-16 gene as we have previously described
(17). Briefly, IgG cDNA libraries were constructed by RTPCR from the 8 week–posttreatment spleens of 6 group A20
mice, 6 group B mice, and 8 age-matched controls from groups
C and D. Between 200 and 400 colonies from each library were
hybridized at 54°C with two VHBW-16–specific oligomers
(for the first complementarity-determining region [CDR1],
5⬘-CTGCTGCAAGGCTTCTGGTT-3⬘ and for CDR2, 5⬘GGAATTAATCCTTACTATGGT-3⬘). Positive colonies were
selected, and inserts from purified plasmids were sequenced in
the Albert Einstein College of Medicine sequencing facility.
Sequences were compared with the germline
VHBW-16 sequence using a BLAST search (online at http://
www.ncbi.nlm.nih.gov/blast/). Mutations were analyzed in the
V region only. Replacement mutation to silent mutation ratios
were calculated, and the frequency of mutations at any position
in the RGYW hot spot on either strand was determined (18).
Mutations shared by more than one clonally related sequence
were analyzed only once.
Flow cytometry. Spleen cells were analyzed for B and T
cell markers using antibodies to CD4 (Caltag, Burlingame,
CA), CD8 (Caltag), and CD19 (BD PharMingen). The presence of activated CD4 cells was determined by double staining
with fluorescein isothiocyanate (FITC)–anti-CD4 and phycoerythrin (PE)–anti-CD69 (PharMingen). The presence of naive and activated/memory CD4 cells was determined by triple
staining with FITC–anti-CD4, Cy-Chrome–anti-CD44
(PharMingen), and PE–anti-CD62L (PharMingen). The presence of activated B cells was determined by double staining
with FITC–anti-CD19 and PE–anti-CD69. Transitional, marginal zone, and follicular B cells were stained as previously
described (19) using anti-CD19 allophycocyanin, PE–anti–
heat-stable antigen (PE–anti-HSA), FITC–anti-CD21, and
biotin–anti-CD23, followed by streptavidin–peridin chlorophyll protein. CD5⫹ cells were identified as B220intermediate
and CD5⫹ using PE–anti-B220 and Cy-Chrome–anti-CD5. All
antibodies to B cell markers were obtained from PharMingen.
497
Oxazolone immunization. To determine whether therapy had affected the T cell–dependent immune response to
foreign antigen, 5 mice from group B and 5 age-matched
controls from group C were immunized with 750 ␮g of the
hapten oxazolone (Sigma) by skin paint in olive oil/acetone on
the shaved abdomen 3 days after the fourth dose of treatment
(age 30 weeks). Mice were bled at weekly intervals thereafter
for 3 weeks. Antioxazolone antibodies were measured by
ELISA as previously described (20). Briefly, plates were coated
overnight with 100 ␮g/ml of oxazolone coupled to BSA in PBS,
blocked, and then incubated sequentially with serum at 1:500
and 1:2,000 dilutions in PBS/1% BSA at 37°C for 1 hour,
peroxidase-conjugated anti-mouse IgM, IgG1, IgG2a, or IgG3
(Accurate) at a 1:5,000 dilution in PBS/1% BSA at 37°C for 1
hour, and ABTS substrate.
Statistical analysis. Proteinuria and survival data
shown in Figure 1 were analyzed using Kaplan-Meier curves
and the log rank test. Comparisons shown in Figures 3 and 8
were performed using the Wilcoxon rank sum test. Comparisons shown in Figures 4 and 6 and in Table 2 were performed
using chi-square analysis or Fisher’s exact test. Only significant
P values are shown.
RESULTS
Clinical outcomes. Mice treated with 250 ␮g of
anti-CD154 biweekly from age 20 weeks until age 46
weeks (group A20) and mice treated with 7 doses of 500
␮g of anti-CD154 from age 26 weeks until age 36 weeks
(group B) had a markedly delayed onset of proteinuria
(Figure 1). Proteinuria onset was delayed for an average
of 19 weeks after cessation of therapy in both groups.
Similarly, death was delayed by an average of 27–28
weeks after treatment cessation in both groups (Figure
1). Once proteinuria became manifest, the course of
disease was similar to that in controls. There was no
difference in disease course or survival between mice in
the two control groups. The 26-week-old mice treated
with 250 ␮g of anti-CD154 biweekly (group A26) did not
respond to therapy and were not studied further. This
response failure was associated with the prompt development of anti-hamster antibodies in this group, but not
in the other groups (data not shown), indicating that
higher doses of therapy are needed to suppress the
immune response as the degree of B cell activation
increases with aging (12).
Anti-dsDNA antibody findings. IgG anti-dsDNA
antibodies and anti-dsDNA antibodies of the IgG2a
isotype, the predominant isotype deposited in the kidneys of (NZB ⫻ NZW)F1 mice (21), were measured
biweekly. Development of anti-dsDNA antibodies was
delayed by an average of 20 weeks in group A20 (data
not shown), a time equivalent to the treatment window.
Six of 10 group B mice had high titers of anti-dsDNA
498
WANG ET AL
Figure 1. Percentages of mice with fixed proteinuria ⬎300 mg/dl (left) and of mice surviving (right) after
each treatment regimen (see Materials and Methods for a description of the treatment groups).
Comparisons for proteinuria yielded the following P values: P ⫽ 0.008 for hamster IgG–treated control
group (n ⫽ 14) versus group A20 (treatment commenced at 20 weeks); P ⫽ 0.024 for hamster IgG–treated
control group versus group B (treatment commenced at 26 weeks); P ⫽ 0.0016 for untreated controls (n ⫽
17) versus group A20; P ⫽ 0.001 for untreated controls versus group B. Comparisons for survival yielded
the following P values: P ⫽ 0.0037 for hamster IgG–treated control group versus group A20; P ⫽ 0.043 for
hamster IgG–treated control group versus group B; P ⫽ 0.0042 for untreated controls versus group A20;
P ⬍ 0.02 for untreated controls versus group B. Group A26 mice did not respond to therapy and were not
studied further.
antibodies at the beginning or within the first 2–3 weeks
of treatment initiation. In all these mice, anti-dsDNA
antibody titers stabilized or diminished during treatment
and rose again after the treatment course was completed
(Figure 2).
ELISpot assays. We have previously shown that
neither long-term CTLA4-Ig nor short-term combination CTLA4-Ig/anti-CD154 treatment alters either the
polyclonal expansion of IgM antibodies or the serum
Figure 2. Measurement of IgG2a anti–double-stranded DNA (antidsDNA) antibodies (see Materials and Methods for description of the
treatment groups). A, Data for 6 individual mice from group B that had
serum IgG anti-DNA antibodies prior to treatment. Anti-dsDNA titers
stabilized and, in most instances, decreased between 8 and 16 weeks
after the start of therapy, reaching a low point at 16 weeks. Levels of
autoantibodies rose again, peaked, and then fell concomitant with the
onset of proteinuria. Mice were followed up until death. B, Representative data from 4 control mice. Anti-DNA antibody titers rose
between 28 and 32 weeks and fell at the time of onset of proteinuria.
All 4 mice were dead by age 40 weeks. C, Twofold serial dilutions of a
high-titer serum. OD ⫽ optical density.
levels of IgG antibodies in (NZB ⫻ NZW)F1 mice
(14,17). Similarly, total serum IgM and IgG levels were
unaffected by anti-CD154 treatment throughout the
treatment course (data not shown).
B cells of (NZB ⫻ NZW)F1 mice spontaneously
secrete anti-dsDNA antibodies in vitro. To enumerate
the frequency of these cells, ELISpot assays were performed on spleen samples from (NZB ⫻ NZW)F1 mice
after cessation of anti-CD154 treatment. Frequencies of
antibody-producing cells were compared with those in
nephritic controls from groups C and D. Two mice in
group B chosen at random from those whose antidsDNA antibody titers had declined after treatment
were examined 2 weeks after cessation of treatment.
These two mice displayed a marked suppression of
IgG-secreting B cells, indicating a substantial suppression of splenic B cell maturation and class switching
during anti-CD154 treatment. No effect was seen in
these mice on the frequency of IgM anti-dsDNA
antibody–secreting B cells, suggesting that CD40 ligation
is not required for the selection of autoreactive B cells
into the naive B cell compartment (Figure 3).
The remaining mice in groups A20 and B were
studied 8 weeks after cessation of treatment, at which
time the anti-CD154 antibody had cleared from the
serum. Eight mice from each group were examined.
There was no difference in the frequency of IgMsecreting or IgM anti-dsDNA–secreting B cells between
any of the treated mice and age-matched controls. In
contrast, the effect of treatment on the frequency of IgG
ANTI-CD154 IN MURINE SLE
Figure 3. A, Frequencies of total IgM- and IgG-producing cells per
103 spleen cells and of IgM and IgG anti–double-stranded DNA
(anti-dsDNA)–producing B cells per 105 spleen cells measured by
enzyme-linked immunospot assay 2 weeks (n ⫽ 2 mice) and 8 weeks
(n ⫽ 16 mice) after the final treatment (see Materials and Methods).
Bars show the mean and SD. 22–25w ⫽ 22–25-week-old predisease
control mice; 34–38w ⫽ 34–38-week-old nephritic control mice from
groups C and D (see Materials and Methods for a description of the
treatment groups). B, Dot-plot analysis of 16 anti-CD154–treated
mice. Eight weeks after cessation of treatment, 8 mice from the treated
groups had frequencies of IgG anti-dsDNA–producing B cells comparable with those in untreated controls, and 8 mice had fewer than 5
detectable IgG anti-dsDNA–secreting B cells per 105 spleen cells.
anti-dsDNA antibody–secreting B cells was variable.
(NZB ⫻ NZW)F1 mice age 22–25 weeks had low
frequencies of IgG anti-dsDNA antibody–producing
cells by ELISpot, and the frequencies increased ⬃20fold by the time of nephritis onset (Figure 3A). Eight of
the 16 treated mice examined by ELISpot had a frequency of IgG anti-dsDNA–secreting cells that was not
different from that in group C and D controls. However,
these 8 mice had 4–10-fold lower titers of IgG antidsDNA antibodies in their serum than did age-matched
controls (not shown) and had not yet developed renal
disease. The other 8 treated mice had fewer than 5 IgG
anti-dsDNA–secreting B cells per 105 spleen cells, a
frequency comparable to that in pretreatment controls
(Figure 3B).
The degree of suppression of the anti-dsDNA
response 8 weeks after cessation of treatment did not
correlate with the treatment dose or the age at which
treatment was instituted. The suppression of IgG antidsDNA–producing B cells was transient, however, and
nearly all mice eventually developed high-titer IgG
anti-dsDNA antibodies in the serum. Nevertheless, all 3
long-term survivors (⬎18 months) were in the group of
8 mice that demonstrated suppression of IgG antidsDNA antibody–secreting cells 8 weeks after treatment
cessation, with a trend toward longer survival in this
group (P ⫽ 0.09).
499
Analysis of hybridomas. Spleen cell fusions were
performed to determine the activation state of antidsDNA antibody–producing B cells. Hybridomas were
generated with the standard NSO fusion partner immediately after splenectomy and following a 2-day stimulation with LPS. In 8 control mice from group C, an
average of 3.7% of the hybridomas bound dsDNA, and
the ratio of IgG:IgM DNA-binding hybridomas was
3.5:1. No increase in hybridoma recovery or change in
this ratio was observed after 2 days of LPS stimulation.
In contrast, 10-fold fewer dsDNA-binding hybridomas
were recovered from 9 anti-CD154–treated mice from
either group A20 or group B 8 weeks after cessation of
treatment (P ⬍ 0.0001), and the ratio of IgG to IgM
DNA-binding hybridomas was 2:3 (P ⬍ 0.03), indicating
that the IgG-producing B cells detected by ELISpot
were not activated enough to produce hybridomas. After
LPS stimulation, an increased number of hybridomas of
the IgM isotype were recovered from 4 mice that had
anti-dsDNA–producing cells detected by ELISpot (Figure 4). No increase in the frequency of either IgM or
IgG anti-dsDNA–producing hybridomas was found after
LPS stimulation in 5 treated mice that had a low
frequency of IgG anti-dsDNA–producing B cells by
ELISpot.
Flow cytometry findings. Spleen cells from 6
nephritic controls from groups C and D (age 34–38
weeks), from 6 anti-CD154–treated mice from group
Figure 4. Analysis of hybridomas. Hybridomas were generated from 9
mice 8 weeks after cessation of therapy (anti-CD154 treated) and from
8 control mice ages 34–38-week. Spleen cells from each mouse were
divided in two, and fusions were performed immediately after harvest
and after 2 days of stimulation with lipopolysaccharide (LPS). Data are
shown as the mean and SD percentage of hybridomas of each isotype
that were anti–double-stranded DNA (anti-dsDNA) positive. A total
of 1,273 hybridomas from controls and 2,375 from treated mice were
screened. The frequency of IgM or IgG anti-dsDNA–producing hybridomas from 2 mice that were examined 2 weeks after cessation of
treatment was 2 of 437, with no increase after LPS stimulation (data
not shown). See Materials and Methods for a description of the
treatment groups.
500
WANG ET AL
Table 1. B and T cell subsets in the spleens of control mice and mice examined 8 weeks after cessation of anti-CD154 treatment*
Mice†
No. of spleen cells (⫻ 107)
CD19⫹‡
CD19⫹,CD69⫹§
CD19⫹,HSA⫹§
CD4⫹‡
CD4:CD8 ratio
CD4⫹,CD69⫹‡
CD4⫹,CD44⫺,CD62L⫹¶
CD4⫹,CD44⫹,CD62L⫺¶
CD8⫹‡
Age 14–16 weeks
(n ⫽ 4)
Age 22–25 weeks
(n ⫽ 3)
Age 34–38 weeks
(n ⫽ 6)
Group A20
(n ⫽ 6)
Group B
(n ⫽ 4)
ND
ND
2.2 ⫾ 0.8
44.2 ⫾ 7.3
ND
ND
ND
ND
ND
ND
9 ⫾ 1.0
42 ⫾ 13.6
ND
ND
29.5 ⫾ 8.4
1.9 ⫾ 0.4
7.4 ⫾ 3.3
44 ⫾ 11.5
35.9 ⫾ 6.9
15.7 ⫾ 4.5
13.3 ⫾ 5.4
46 ⫾ 6.2
13 ⫾ 4.1
19.1 ⫾ 3.6
34.4 ⫾ 8.9
6.6 ⫾ 2.4
15 ⫾ 6.2
9.2 ⫾ 4.0
74.4 ⫾ 6.6
6.1 ⫾ 3.1
7.5 ⫾ 2.5
38.6 ⫾ 3.8
5.5 ⫾ 1.0
24.2 ⫾ 6.9
25.5 ⫾ 4.0
2.1 ⫾ 0.8
4.1 ⫾ 1.3
36.6 ⫾ 5.8
43.9 ⫾ 5.4
12.8 ⫾ 2.9
5.3 ⫾ 1.3
39.4 ⫾ 15.8
3.2 ⫾ 2.0
29 ⫾ 5.7
30.6 ⫾ 6.0
1.7 ⫾ 0.1
2.5 ⫾ 1.1
44.7 ⫾ 8.4
45.2 ⫾ 8.2
17.5 ⫾ 2.7
* Except where indicated otherwise, values are the mean ⫾ SD percentage of the given cell type. ND ⫽ not determined; HSA ⫽ heat-stable antigen
(see Materials and Methods for detailed description of treatment groups).
† Negative controls were ages 14–16 weeks and ages 22–25 weeks. Nephritic controls (from groups C and D) were ages 34–38 weeks. Group A20
mice were treated starting at age 20 weeks. Group B mice were treated starting at age 26 weeks with a higher dose protocol.
‡ Percentage of live cells.
§ Percentage of CD19⫹ gated cells.
¶ Percentage of CD4⫹ gated cells.
34–38-week-old controls) and 50 ⫾ 4.4% in 4 mice in
which these cells were detected, compared with 42.9 ⫾
4.2% in 34–38-week-old controls (P not significant).
Flow cytometry was also performed to determine
whether particular B and T cell subsets were preferentially depleted by anti-CD154 therapy. Treated mice had
fewer CD19⫹,CD69⫹ B cells than 34–38-week-old controls (Tables 1 and 2). There was no change in the
frequency of B220intermediate,CD5⫹ cells between 22–25week-old and 34–38-week-old control mice or between
treated and control groups (data not shown). Mice ages
14–16 weeks had more immature (HSA⫹) cells than did
controls ages 34–38 weeks, and the treated mice had a
phenotype intermediate between these 2 groups (Table
1). There was a trend toward an increase in marginal
zone B cells and follicular B cells with age, but there was
no statistically significant difference between treated
mice and either 14–16-week-old or 34–38-week-old controls (data not shown). In both treatment groups, anti-
A20, and from 4 anti-CD154–treated mice from group B
were analyzed by flow cytometry for expression of B and
T cell markers 8 weeks after cessation of treatment. Four
14–16-week-old and three 22–25-week-old mice were
examined as negative controls. The total number of
spleen cells was decreased in the treated mice compared
with either the 22–25-week-old or the 34–38-week-old
control mice (Table 1). There was also a decrease in B
cell frequency during treatment compared with 34–38week-old controls; this decrease persisted in some mice
after cessation of treatment and correlated with a decrease in the frequency of Ig-secreting cells and with the
presence or absence of anti-dsDNA antibody–producing
cells by ELISpot. The mean ⫾ SD frequency of CD19⫹
B cells was 21.8 ⫾ 6.6% in the 2 mice whose spleens
were harvested immediately after high-dose treatment
ended. The frequency of B cells was 30.6 ⫾ 9% in 4 mice
from the group with no detectable anti-dsDNA
antibody–producing cells by ELISpot (P ⬍ 0.03 versus
Table 2. Significant P values for the flow cytometry data shown in Table 1*
Comparison
No. of spleen cells
(⫻ 107)
CD19⫹,
CD69⫹†
CD19⫹,
HSA⫹†
CD4:CD8
ratio
CD4⫹,
CD69⫹‡
CD4⫹,CD44⫺,
CD62L⫹§
CD4⫹,CD44⫹,
CD62L⫺§
34–38-week-old group vs. group A20
34–38-week-old group vs. group B
34–38-week-old group vs. 22–25-week-old group
22–25-week-old group vs. group B
0.03
0.004
NS
0.04
0.003
0.024
0.001
NS
NS
NS
0.03
NS
0.002
0.001
0.024
NS
0.003
0.01
0.03
NS
0.0001
0.0001
0.0001
NS
0.0001
0.0001
0.0001
NS
* HSA ⫽ heat-stable antigen; NS ⫽ not significant (see Table 1 and Materials and Methods for detailed description of treatment groups).
† Percentage of CD19⫹ gated cells.
‡ Percentage of live cells.
§ Percentage of CD4⫹ gated cells.
ANTI-CD154 IN MURINE SLE
Figure 5. Effect of treatment on class switching. Shown are the results
of reverse transcription–polymerase chain reaction of spleen mRNA
for IgG2b sterile class-switch transcripts. The upper band in each panel
is the IgG2b sterile transcript. The lower band is actin. Three 5-fold
serial dilutions of cDNA are shown for each mouse. Panels 1–6, Mice
treated at age 26 weeks with high-dose therapy (group B). Panels 1 and
2, Treated mice whose spleens were harvested 2 weeks after cessation
of treatment (IgG2b undetectable). Panels 3–6, Mice whose spleens
were harvested 8 weeks after cessation of treatment (mean ⫾ SD
IgG2b:actin ratio 0.08 ⫾ 0.10, n ⫽ 5 mice). Panel 7, A representative
untreated control (IgG2b:actin ratio 0.98 ⫾ 0.43, n ⫽ 5 mice). Panels
8–12, Mice treated at age 20 weeks with low-dose therapy (group A20)
whose spleens were harvested 8 weeks after cessation of treatment
(IgG2b:actin ratio 0.52 ⫾ 0.32). Single lanes at left show the 2.1-kb
marker. P not significant for group A20 versus control mice; P ⫽ 0.008
for group B versus control mice. See Materials and Methods for a
description of the treatment groups.
CD154 treatment prevented the loss of CD8⫹ T cells
that occurs in these mice with age and inhibited the
accumulation of both activated and memory CD4⫹ T
cells as assessed by the markers CD69, CD44, and
CD62L (Tables 1 and 2).
Class switching. To determine the effect of treatment on class switching, semiquantitative RT-PCR was
performed on spleen messenger RNA for sterile I␥2b–
C␥2b class-switch transcripts. No transcripts were detected using a single round of PCR in the 2 mice whose
spleens were harvested 2 weeks after cessation of treatment, indicating that anti-CD154 suppresses class
switching. In the mice examined 8 weeks after treatment,
class-switch transcripts were detected in all those in
group A20, and results were variable in group B. This
points to a dose-dependent effect of treatment on the
length of time needed to recover the ability to class
switch to IgG (Figure 5). Class-switch transcripts were
absent only in mice that had a low frequency of IgG
anti-dsDNA antibody–producing B cells detectable by
ELISpot.
Somatic mutation. The VHBW-16 gene, a member of the J558 gene family, has been shown to be
strongly associated with pathogenic anti-dsDNA antibodies in (NZB ⫻ NZW)F1 mice (22). VHBW-16 is not
expressed in normal mice, even by naive cells, unless
tolerance to DNA is broken by immunization with DNA
complexed to a DNA-binding protein. In these cases, the
501
autoantibodies are either all of the IgM isotype or of low
affinity (23). These findings indicate that antibodies
using this gene are regulated in the peripheral B cell
compartment of normal mice. High-affinity anti-dsDNA
antibody activity in (NZB ⫻ NZW)F1 mice is associated
with class switching from IgM to IgG and with the
presence of basic amino acids in the CDRs (17,23). We
have previously shown that somatic mutations accumulate in the CDR2 of this gene with age (17). Thus, the
VHBW-16 gene is an excellent marker to examine the
effect of costimulatory blockade, both on the selection
of naive autoreactive B cells and on activated B cells
undergoing somatic mutation.
Analysis of the VHBW-16 sequences from antiCD154–treated mice revealed findings similar to those
we have previously reported with other forms of costimulatory blockade (14,17). In 1 of the 2 mice from
group B harvested 2 weeks after cessation of treatment,
we could not produce any IgG VHBW-16 product by
PCR. In the other mouse, we recovered a few sequences
that had 0–1 mutations, consistent with the ELISpot
data. VHBW-16 IgG was detected in all 4 group A20
mice examined and in 6 of the 8 group B mice. There
was no difference in the number of sequences containing
arginines in the CDR3 compared with group C or D
controls, indicating that regulation of naive bone marrow emigrants did not appear to be affected by therapy
(data not shown). However, the frequency of somatic
mutation was decreased in both the low- and highdose–treated mice compared with controls. Sixty percent
of sequences had 0–1 mutations in mice from group
A20, but ⬍30% had 0–1 mutations in mice from group
B, suggesting that the effect of treatment on somatic
mutation decreased with increasing disease activity.
Nevertheless, ⬎90% of the sequences had 0–4 mutations in group B mice compared with ⬍60% in the
controls, suggesting that accumulation of mutations
could be blocked even in older mice (Figure 6).
As we have previously observed with costimulatory blockade, there were also differences in the nature
of the mutations that accumulated in the CDR2 of the
treated mice. In nephritic mice, a mutation to arginine
was a frequently observed change in positions 55, 56, and
58, where 4 RGYW hot spots overlap. In contrast,
mutation to arginine was infrequent in the treated mice,
being present in 9 of 44 control sequences, but in only 1
of 54 sequences from the treatment group (P ⬍ 0.005)
(Figure 7). Analysis of RGYW hot-spot mutations was
performed in the early treatment group in which mutations were occurring during therapy. The analysis was
performed only for silent mutations that are not subject
502
WANG ET AL
Figure 6. Effect of treatment on the frequency of somatic mutation of
the VHBW-16 gene. In the early treatment group (group A20), 60% of
the sequences had 0–1 mutations, compared with only 20% of the
sequences in 34–38-week-old controls (P ⫽ 0.0023). In the late
treatment group (group B), the frequency of somatic mutation was still
significantly lower than that in 34–38-week-old controls (P ⫽ 0.023).
See Materials and Methods for a description of the treatment groups.
to selection bias. Despite what appeared to be a normal
targeting of replacement mutations to the hot spots in
the CDR2, there was a significant decrease in the
frequency of silent hot-spot mutations, but not in that of
silent non–hot-spot mutations, in the treated mice versus
the hamster IgG–treated or untreated controls (P ⬍
0.05) (Table 3).
Immunization with oxazolone. In order to determine whether anti-CD154 treatment resulted in
immunosuppression of humoral immune responses, we
immunized mice in group B with the hapten oxazolone
by skin painting without adjuvant 3 days after the fourth
anti-CD154 treatment. Control age-matched mice from
group C mounted a strong IgG antioxazolone response.
In contrast, mice treated with anti-CD154 were unable
to mount a response of either the IgG2a or the IgG1
isotype to oxazolone. Anti-CD154–treated mice developed an IgM antioxazolone response at week 1 postimmunization, but this had stabilized or diminished by
week 3 postimmunization, whereas it continued to increase in controls (Figure 8). These studies show that
treated mice are immunosuppressed during treatment,
with failure to maintain or expand a mature classswitched humoral response after initial priming.
DISCUSSION
The SLE-like disease that develops in (NZB ⫻
NZW)F1 mice is dependent on the spontaneous activation of both T and B cells. The CD40/CD154 receptor/
ligand pair has been an attractive target for therapeutic
intervention in SLE because it is involved both in
costimulation of antigen-presenting cells and B cells by
T cells (2) and in the effector arm of the immune
response (7,24). Up-regulation of CD154 expression on
T and B cells has been found very early in the life of
lupus-prone mice (9), and similar findings have been
reported in humans with SLE (25,26). CD40/CD154
blockade has been successful in preventing or stabilizing
SLE nephritis in spontaneous murine models, even at
relatively late stages of disease (10).
In both murine and human SLE, there is spontaneous overproduction of polyclonal IgM antibodies,
including dsDNA-binding antibodies. It is possible that
aberrant expression of CD154 on T cells early in the
course of murine SLE (9) might rescue autoreactive B
cells from apoptosis during early B cell activation. If this
were the case, treatment with anti-CD154 might induce
deletion of naive autoreactive B cells. In contrast to
observations of mice made deficient in CD40 or CD40L
from birth (27,28), we found that spleens were smaller in
treated mice than in young controls, and the percentage
of spleen B cells was decreased by 50% during CD154
blockade. This effect was not specific for autoreactive B
Figure 7. Pooled mutation analysis of the second complementaritydetermining region (CDR2) of VHBW-16 genes from the IgG cDNA
libraries compared with the germline sequence. The middle of the
diagram shows the germline sequence of the CDR2 (boldface); pooled
data from treated (54 sequences) and control untreated (44 sequences)
mice are shown below and above the germline sequence, respectively.
Positions 57–60 (underlined) of the germline sequence contain an area
of 4 overlapping RGYW hot spots. In this area, there are 9 mutations
to arginine in the sequences from control untreated mice and only 1
such mutation in the sequences from treated mice.
ANTI-CD154 IN MURINE SLE
503
Table 3. Mutation frequencies and patterns of the VHBW-16 gene*
Group
No. of
sequences
analyzed
No. of
mutations
analyzed
No. of
mutations
per sequence†
Ages 34–38 weeks
Anti-CD154 treated
Ages 22–25 weeks
44
54
24
211
132
30
4.8
2.4
1.3
Replacement mutation:
silent mutation ratio
Framework
CDR
No. of silent
mutations in
hot spots‡
1.8
2.7
2.4
3.3
3.6
3.3
19
7
–
No. of silent
mutations in
non–hot spots
18
12
–
* CDR ⫽ complementarity-determining region (see Materials and Methods for a description of the treatment groups).
† P ⬍ 0.0001 for 34–38-week-old control mice versus pooled anti-CD154–treated mice; P ⫽ 0.001 for 22–25-week-old control mice versus
anti-CD154–treated mice.
‡ Thirty control sequences and 28 early treatment sequences were included in this analysis. There are 86 RGYW hot spots and 202 non–hot spots
in the VHBW-16 gene. Hot spot targeting was seen in the control sequences (ratio 2.47:1), but not in the treatment sequences (ratio 0.58:1) (P ⬍
0.05).
cells, since there was no change in the relative frequency
of naive IgM anti-dsDNA–producing B cells enumerated by ELISpot. In addition, there was no apparent
effect of anti-CD154 on selection of B cells into the B1
or marginal zone subsets that are a significant source of
IgM anti-dsDNA antibodies (29,30). Finally, it appears
that CD40 ligation is not required for priming of an
initial IgM response to the hapten oxazolone in this
autoimmune strain.
Anti-CD154 treatment resulted in a block in the
expansion and activation of autoreactive B cells, and
class switching from IgM to IgG did not occur during the
treatment period. Furthermore, as indicated by the
studies using the marker gene VHBW-16, the frequency
of somatic mutation was decreased and its pattern was
altered. We observed a decrease in the frequency of
RGYW hot-spot targeting in unselected mutations in
our early treatment group, supporting the contention
Figure 8. IgM and IgG isotype–specific antioxazolone antibodies.
Antibodies were measured by enzyme-linked immunosorbent assay at
weeks 1 and 3 after immunization with oxazolone in treated and
age-matched control (NZB ⫻ NZW)F1 mice. Data for the 1:500
dilution of serum are shown for IgM and for the 1:2,000 dilution for
IgG. Bars show the mean and SD. There was an increase in IgM
antioxazolone titer between weeks 1 and 3 in all the control mice (P ⫽
0.0039), but not in the treated mice. Treated mice did not mount an
IgG antioxazolone response. Naive mice had optical density (OD)
values ⬍ 0.05.
that CD40 signaling has an effect on hot-spot targeting
(31), but these data need to be confirmed with a larger
number of sequences. Anti-CD154–treated mice had
fewer mutations to arginine in the CDR2 than did
untreated (NZB ⫻ NZW)F1 controls, a finding that we
have previously observed following treatment with
CTLA4-Ig (17) and short-term combination CTLA4-Ig/
anti-CD154 (14).
We speculate that costimulatory blockade,
through its effects on T cells or dendritic cells (32), alters
B cell signaling thresholds, leading to regulation of
high-affinity autoreactive B cells by deletion. The absence of high-affinity autoantibody-producing B cells
will in turn result in the formation of less pathogenic
immune complexes. Since activated autoreactive B cells
have additional functions, including antigen presentation to T cells and secretion of cytokines (33,34), antiCD154 treatment should result in a global decrease in
immune activation and consequent beneficial effects on
disease.
It is of potential clinical relevance that treatment
with high-dose anti-CD154 therapy was successful even
in mice that had already developed IgG anti-dsDNA
antibodies. The fall in anti-dsDNA titers in these mice
was associated with a decrease in total IgG-secreting B
cells and with the disappearance of anti-dsDNA–
secreting B cells from the spleen. These findings indicate
that most of the anti-dsDNA response in early disease
derives from B cells that are CD40 dependent.
It appears from the results of ELISpot assays and
hybridoma analysis that B cell function resumed in a
stepwise manner after cessation of anti-CD154 treatment. During treatment, B cell numbers were substantially diminished. In the early stage of recovery, total B
cell frequency increased, but the frequency of B cells
producing IgG or IgG anti-dsDNA antibodies remained
504
low. In the next stage of recovery, high frequencies of
IgG anti-dsDNA–producing B cells were readily detectable by ELISpot assay and were similar to those found in
older (NZB ⫻ NZW)F1 mice with renal disease. However, serum titers of IgG anti-dsDNA antibodies were
still 4–10-fold lower than in untreated age-matched
controls, and no spontaneous IgG anti-dsDNA–
producing hybridomas could be recovered from these
mice even after LPS stimulation. The lack of response to
LPS did not appear to be due to anergy, since spontaneous secretion of anti-dsDNA antibodies could be
detected by ELISpot. Nevertheless, at this stage, the B
cells of treated mice remained functionally different
from those in the diseased controls. Flow cytometry
analysis revealed that the frequency of CD69⫹ B cells in
all mice examined 8 weeks after cessation of therapy was
similar to that in young controls, even when anti-dsDNA
antibody–producing cells were detected by ELISpot,
showing that excessive B cell activation is a late step in
the process of development of a high-titer serum antidsDNA response.
A possible explanation for the inability of the IgG
anti-dsDNA–producing B cells detected by ELISpot to
secrete high titers of antibodies in vivo could be a lack of
sufficient T cell help in the treated mice, since T cell
function was also affected by treatment with antiCD154. We observed a striking difference in the effect
of long-term administration of anti-CD154 on T cell
activation compared with long-term administration of
CTLA4-Ig. In mice treated with CTLA4-Ig, the abnormal accumulation of memory T cells that is associated
with disease in older mice was not blocked despite the
absence of the early activation marker CD69 on these T
cells. This may be because CTLA4-Ig, in addition to
blocking B7–CD28 interactions, blocks negative regulatory signals mediated via CTLA4 (17,35,36), or because
activation of naive T cells by resting or anergic B cells
results in only partial T cell activation in the presence of
CTLA4-Ig (37). In contrast, anti-CD154 blocked memory T cell accumulation even after anti-dsDNA titers
were beginning to rise, perhaps reflecting the need for
CD40 ligation on antigen-presenting cells and/or memory B cells to generate or maintain an activated T cell
response (38).
Permanent tolerance was not achieved in the
majority of treated mice. After a variable period of time
following cessation of treatment, the T cell phenotype of
the treated mice became indistinguishable from that of
aged controls (data not shown), and the B cells appeared
to attain a fully activated phenotype, in that pathogenic
IgG anti-dsDNA antibodies began to appear in high titer
WANG ET AL
in the serum, and the mice developed proteinuria and
progressive renal disease.
Recently, two clinical trials of anti-CD154 antibody treatment in humans have been performed using
humanized mouse anti-CD154 antibodies different from
the antibody used in the present study. In one placebocontrolled trial, no efficacy of treatment was observed
(39). It is possible that the dosage of drug used in that
study was too low to achieve efficacy in most of the
patients who already had active disease, analogous to the
low-dose, 26-week-old group in our study. A second
phase II study that used a dose of 10 mg/kg in a regimen
analogous to the high-dose, 26-week-old group we describe here was stopped prematurely because of thrombotic events in 2 patients. Nevertheless, some efficacy
was shown in the 18 patients who received at least 4
doses of the drug (15). In that study, only a 40–50%
decline in anti-dsDNA titers was observed following
anti-CD154 treatment despite the rapid disappearance
of plasmablasts from the peripheral blood (40,41).
As discussed above, most autoreactive B cells in
the (NZB ⫻ NZW)F1 SLE model are CD40 dependent
early in the disease course. However, after renal disease
has developed, anti-CD154 treatment does not alter
anti-dsDNA titers in (NZB ⫻ NZW)F1 mice (42). These
antibodies are probably derived from long-lived plasma
cells that are thought to be responsible for maintaining
long-term antibody synthesis (43–45). These cells do not
appear to require either T cells or the continuing
presence of antigen to survive or secrete antibody (44),
and they are therefore resistant to costimulatory blockade. Thus, patients with early disease (who have not yet
developed a large autoreactive plasma cell population)
may be more likely than patients with late disease that
has proven refractory to other treatments to benefit
from anti-CD154 treatment and other B cell–depleting
treatments that spare plasma cells.
Although the monoclonal antibody approach to
CD40/CD154 blockade has not yet been successful in
human clinical trials in SLE, the sequence of events that
we have described here may be relevant to the application of other B cell–depleting therapies. Treatment with
such agents may be useful in early disease or when given
intermittently in combination with T cell blockade. With
the appropriate combination of drugs and timing of
treatment, it may be possible to achieve long-lasting
effects on autoreactive cells with less global immunosuppression (14), pointing the way to safer and more
effective therapy of human autoimmune disease.
ANTI-CD154 IN MURINE SLE
505
ACKNOWLEDGMENTS
We thank Drs. B. Diamond and H. Keiser for critical
reading of the manuscript.
18.
19.
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lupus, effect, treatment, murine, cd154, systemic, erythematosus, anti, cells
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