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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1324-1329
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGYAU#0
Prevention and treatment of factor VIII inhibitors in murine
hemophilia A
Jiahua Qian,
Mary Collins,
Arlene H. Sharpe, and
Leon W. Hoyer
From the Holland Laboratory, American Red Cross, Rockville, MD; the
Genetics Institute, Cambridge, MA; Brigham and Women's Hospital,
Harvard Medical School, Harvard University, Boston, MA; and the
Department of Medicine, George Washington University School of Medicine
and Health Sciences, Washington, DC.
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Abstract |
Inhibitory antibody formation is a major complication of factor VIII
replacement therapy in patients with hemophilia A. To better understand
the pathogenesis of this immunologic reaction, we evaluated the role of
T-cell costimulatory signals for antifactor VIII antibody formation in
a murine model of hemophilia A. Repeated intravenous injections of
factor VIII in these factor VIII-deficient mice induced an antifactor
VIII inhibitor antibody response. This response was shown to be T-cell
dependent by its absence in hemophilic mice also deficient for the
T-cell costimulatory ligand B7-2. In separate experiments, injection of
murine CTLA4-Ig completely blocked the primary response to factor VIII
in hemophilic mice with intact B7 function. This reagent also prevented
or diminished further increases in antifactor VIII when given to
hemophilic mice with low antifactor VIII antibody titers. These studies
suggest that strategies targeting the B7-CD28 pathway are potential
therapies to prevent and treat inhibitory antifactor VIII antibodies.
Moreover, because the development of antibodies to replaced proteins
may limit the success of many human gene therapy approaches, our
results may be broadly applicable.
(Blood. 2000;95:1324-1329)
© 2000 by The American Society of Hematology.
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Introduction |
Hemophilia A is an X-linked bleeding disorder caused by
the deficiency in plasma of the glycoprotein factor VIII.1
However, approximately one-third of patients with severe hemophilia A
develop an antifactor VIII inhibitor following factor VIII replacement therapy.2 Current strategies to blunt the antibody response in these patients have only been marginally successful. Moreover, the
development of antibodies to replaced proteins is a critical problem
that needs to be solved if gene therapy is to be successful in the
treatment of hemophilias and other deficiency diseases.3,4
We have used a mouse model of hemophilia A to evaluate new methods for
the prevention and treatment of inhibitor formation. Hemophilia A mice,
generated by targeted disruption of exon 16 (E-16) of the factor VIII
gene, have no detectable factor VIII activity in their
plasma5 and are similar in this way to patients with severe
hemophilia A. As expected, hemophilia A mice have in vivo signs of a
coagulation pathway defect with fatal bleeding if tails are cut without
use of hemostatic measures, and they develop subcutaneous and
intramuscular bleeding after handling or temporary
immobilization.6-8
Intravenous infusions of 0.2 µg human factor VIII, a dose equivalent
on a weight basis to that given to hemophilia A patients, resulted in
minimal or no antibody response in these hemophilia A mice after a
single injection, but repeat infusions led to high titer inhibitory
antifactor VIII.6 However, before antibodies were detected,
a factor VIII-specific T-cell proliferative response was detected 3 days after the first exposure to human factor VIII. This suggested that
the antibody response to factor VIII is T-cell dependent, so that
blockade of T-cell activation might prevent inhibitor antibody
formation in hemophilia A mice.
Optimal T-cell activation requires signaling through the
antigen-specific T-cell receptor (TCR) by its engagement with peptide major histocompatibility complex (MHC) complexes on antigen-presenting cells. This is completed in combination with costimulatory signals typically delivered through the T-cell surface glycoprotein,
CD28.9 CD28 interactions with B7-1 (CD80) and B7-2 (CD86),
costimulatory ligands on antigen-presenting cells, are essential for
initiating antigen-specific T-cell responses, up-regulating cytokine
expression, and promoting T-cell expansion and
differentiation.9
CTLA4 is a second high-affinity T-cell receptor for both the B7-1 and
B7-2 ligands.9 CTLA4 is a down-regulatory molecule in
T-cell activation, as demonstrated by the lymphoproliferative phenotype
of the CTLA4-deficient mouse strain.10,11
CTLA4-immunoglobulin (CTLA4-Ig), a soluble fusion protein in which the
extracellular domain of CTLA4 is fused to the heavy chain
constant regions 2 and 3 (CH2-CH3) tail of IgG1, has been shown to be
an effective reagent for blocking CD28-B7 interactions in vivo because
CTLA4-Ig binds to B7-1 and B7-2 ligands and blocks B7 interactions with both CD28 and CTLA4.12 Blockade of the CD28 signaling
pathway has been used successfully to prevent T-cell dependent
responses in many animal models of autoimmunity and
transplantation.9,13,14 Very recently, the first successful
clinical applications of CTLA4-Ig-mediated blockade of T-cell
costimulation have been reported.15,16
We report here evidence that the development of inhibitory antibodies
to factor VIII is T-cell dependent. Moreover, murine CTLA4-Ig
(mCTLA4-Ig) blockade is an effective means of preventing the induction
of an antifactor VIII response and suppressing secondary antifactor
VIII responses.
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Materials and methods |
Animals
The characteristics of the E-16 strain of hemophilic mice have been
reported.5,17 Adult male and homozygous E-16 female mice,
aged 10-20 weeks, were used for these studies. Blood samples were
obtained by orbital venous plexus bleeding, and the serum was separated
by centrifugation at 600g for 3 minutes. The serum samples were stored
at 20°C until the samples were assayed. To avoid severe bleeding
and death of animals, ear tags were not used to identify the mice in
some experiments. For this reason, Figure 1
does not indicate sequential data for individual mice.
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| Fig 1.
The role of B7-1 and B7-2 antigens in the antifactor VIII
antibody response.
Hemophilia A/B7-1 / ( ) and hemophilia
A/B7-2/ ( ) mice were injected
intravenously with 0.2 µg factor VIII at 2-week intervals. Serum
samples for antifactor VIII assay were obtained 12 days after the
second and sixth factor VIII injections.
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Generation of E-16/B7-1 and E-16/B7-2 double knockout mice was
accomplished by cross breeding of E-16 with B7-1 and B7-2 knockout mice.18 Homozygous E-16/B7-1 and E-16/B7-2 double knockout
mice were identified by genotype determination.5,18 Reduced
factor VIII activity was verified using a chromogenic bioassay
(Coatest; Chromogenix, MoIndal, Sweden).17 The
factor VIII activity was less then 1% in both
E-16/B7-1/ and E-16/B7-2/ mice.
Antigens
Recombinant human factor VIII (Baxter Healthcare, Hyland Division;
Glendale, CA) was used in the study.
Murine CTLA4-Ig
A mCTLA4-Ig complementary DNA (cDNA) expression plasmid was prepared
by ligation of the leader and extracellular domains of mCTLA4 to the
hinge, the CH2 and CH3 domains of IgHg2a that had been mutated to
remove effector functions, as described in Streurer.19 The
insert was cloned into the expression vector pED and stably transfected
into Chinese hamster ovary cells as previously
described.20 Concentrated conditioned media was loaded onto
a chromatography column (Protein A Sepharose Fast Flow; Amersham
Pharmacia Biotech, Piscataway, NJ). The column was washed with
phosphate-buffered saline (PBS, pH 7.1), and the mCTLA4-Ig was eluted
with 20 mmol/L citrate (pH 3.0). The peak pool was neutralized with 1 mol/L tris(hydroxymethyl) aminomethane (Tris, pH 8.0) to a final pH of
7.5 and formulated into PBS (pH 7.1) using an
Amicon-stirred cell with a YM30 membrane (Millipore Corp,
Bedford, MA). The mCTLA4-Ig was depyrogenated using a chromatography
column (Poros PI; Perceptive Biosystems, Framingham, MA),
and the product was eluted from the column in a linear sodium chloride
(NaCl) gradient from 0 to 1 mol/L NaCl in 25 mmol/L Tris (pH
7.5). The mCTLA4-Ig was then formulated into PBS (pH 7.1)
using an Amicon-stirred cell with a YM30 membrane.
Antibody measurements
The antifactor VIII titer was determined by enzyme-linked
immunosorbent assay (ELISA).6 The ELISA tests were carried
out using microtiter wells coated with 0.8 µg/mL recombinant human factor VIII in 0.05 mol/L carbonate-bicarbonate (pH 9). After mouse
plasma samples were incubated in the wells at 4°C overnight and
then washed, alkaline-phosphatase-conjugated goat antimouse IgG
(Southern Biotechnology Associates, Birmingham, AL) was added for 2 hours at room temperature. After washing, we added 2 mg/mL P-nitrophenyl phosphate (Sigma, St. Louis, MO) in 100 mmol/L glycine, 1 mmol/L magnesium dichloride (MgCl2), and 2 mmol/L
zinc dichloride (ZnCl2) (pH 10.4). The absorbance was read
at 410 nm using an automated micro titer plate ELISA reader. The
concentration of antifactor VIII antibody was estimated from a
standard curve obtained using a factor VIII monoclonal
antibody (mAb) that binds to the A2 domain, mAb
413.21 The titer was calculated from points that fell on
the linear portion of the assay standard curve. Antifactor VIII
inhibitor titers in Bethesda units (BU) were measured by the Bethesda
assay.22
T-cell proliferation assays
The spleen was used as the source of T cells for proliferation
assays. Spleen cells were then cultured (5 × 105
cells/well) in 96-well flat-bottom plates. Varying amounts of
recombinant factor VIII were added to the culture medium consisting of
complete RPMI 1640 (Gibco BRL, Rockville, MD) containing
0.5% hemophilic mouse serum. After 72 hours of culture at 37°C, we
added 0.074 MBq (2 µCi) 3H-thymidine/well (ICN
Pharmaceuticals, Irvine, CA). The cultures were harvested 16 hours
later (Matrix 9600; Packard, Meriden, CT). Data are expressed as the
mean for triplicate wells of the cpm incorporated into insoluble DNA.
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Results |
Primary immune response to factor VIII
The roles of B7-1 and B7-2 costimulatory ligands present on
antigen-presenting cells were evaluated to determine if the T-cell CD28
signaling pathway is essential for the development of inhibitory antibodies to factor VIII. To do this we crossed hemophilia A mice with
B7-1/ and
B7-2/knockout mice.18,23 Mice
deficient in both factor VIII and either B7-1 or B7-2 ligands were
selected by genotype analysis. The hemophilia
A/B7-1/ and hemophilia
A/B7-2/mice were then injected intravenously
with 0.2 µg human factor VIII at 2-week intervals. After 4 injections, all 9 hemophilia A/B7-1/mice had
developed antifactor VIII, with ELISA antibody titers greater than 350 µg/mL and a mean inhibitor level of 712 BU (Figure 1). These are
values similar to those for otherwise normal hemophilia A mice injected
with factor VIII.6 In contrast, none of the 8 hemophilia
A/B7-2/mice had detectable antifactor VIII.
To evaluate the T-cell response of these B7-1- and B7-2-deficient
hemophilia A mice, spleen cells were obtained 3 days after a fifth
intravenous injection of factor VIII. The T-cell proliferative activity
determined by 3H-thymidine incorporation showed a factor
VIII dose-dependent response for cells from the hemophilia
A/B7-1/mice (Figure
2). In contrast, T-cell response was not
detected at any factor VIII level for spleen cells from hemophilia
A/B7-2/mice. Thus, the B7-2 ligand is
essential for the development of an immune response to factor VIII
injected intravenously, and antifactor VIII formation is prevented if
it is missing.
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| Fig 2.
T-cell response to factor VIII for hemophilia
A/B7-1/ and hemophilia A/B7-2/
mice.
Spleen cells were obtained 3 days after the fifth intravenous injection
of human factor VIII. Pooled spleen cells from 3 mice were used to
establish the proliferation data. The open and closed squares are for
cells from untreated hemophilia A/B7-1/ and
B7-2/mice. Five intravenous
injections of factor VIII were given to hemophilia
A/B7-1/mice () and hemophilia
A/B7-2/mice (). The concentration of
factor VIII in the cultures is indicated on the horizontal axis.
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Blocked induction of an antifactor VIII response by mCTLA4-Ig
As mCTLA4-Ig has been shown to be an effective reagent for blocking
CD28-B7-2 interactions in vivo,12 we tested the
effectiveness of this blockade of the CD28 signaling pathway in
preventing antifactor VIII inhibitor antibody formation. Antifactor
VIII inhibitory antibodies were induced in control hemophilia A mice by
repeated intravenous injections of 1 µg recombinant human factor VIII
at 3-week intervals (group G-1, Figure 3).
Antifactor VIII was detected in 4 of the 5 mice 20 days after the first
injection, and all control mice developed high-titer antifactor VIII
after receiving 2 to 4 injections. The mean inhibitor level after 4 injections was 1860 BU. Antifactor VIII antibody formation was markedly
suppressed in mice injected intraperitoneally with 250 µg of murine
CTLA4-Ig on the day before and the day after the first factor VIII
injection (group G-2, Figure 3), even though there was no further
mCTLA4-Ig given with 3 subsequent factor VIII injections on days 23, 44, and 66. Antifactor VIII was not detectable in any group G-2 mice after the first or second factor VIII injection. Three weeks after the
third injection of factor VIII, a weak immune response was detected in
2 of the 6 mice in group G-2.
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| Fig 3.
The effect of mCTLA4-Ig on antifactor VIII antibody
formation.
Four groups of hemophilia A mice were injected with recombinant human
factor VIII on days 0, 23, 44, and 66 (initially 1 µg intravenously
and then 0.2 µg for the second, third, and fourth injections). Mice
in groups G-3 and G-4 were also injected intraperitoneally with 0.2 µg factor VIII on days 2-12. Blood samples for the antifactor VIII
assay were obtained on days 20, 37, 58, and 82. Control groups G-1 and
G-3 () were injected with only factor VIII. Groups G-2 and G-4 ()
were also injected intraperitoneally with 250 µg mCTLA4-Ig on the day
before and the day after the first factor VIII injection. The
antifactor VIII antibody concentration was determined by ELISA.
Antifactor VIII assay data points indicated as less than 0.16 µg/mL
were similar to those for plasma samples obtained from unimmunized
hemophilia A mice.
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To investigate if the limited duration of unresponsiveness is a result
of the short half-life of human factor VIII in these mice (4-5 hours in
murine hemophilia A8), control and mCTLA4-Ig-treated mice
(groups G-3 and G-4, Figure 3) were injected with 1 µg factor VIII
intravenously on day 0. This was followed by daily intraperitoneal injections of 1 µg factor VIII on days 2-12. On day 20, high-titer antifactor VIII was present in the control mice (group G-3), as determined by ELISA, and the mean inhibitor titer was 694 BU. In
contrast, the group G-4 mice that were injected with mCTLA4-Ig on the
day before and the day after the first exposure to factor VIII had no
detectable antifactor VIII on day 20. The delayed antifactor VIII
antibody response after 3 additional factor VIII injections was the
same in these mice as that in the group G-2 animals. Thus, the limited
persistence of factor VIII in the plasma after CTLA4-Ig injection was
not the reason for a limited duration of unresponsiveness in CTLA4-Ig-
treated mice.
Because a delayed antifactor VIII response was detected after repeated
factor VIII infusions when mCTLA4-Ig was given only at the time of the
first factor VIII exposure, we determined if mCTLA4-Ig might prevent
antifactor VIII development if given with each factor VIII infusion. In
that experiment (Figure 4), hemophilia A
mice were simultaneously infused 6 times at 3-week intervals with both
factor VIII and mCTLA4-Ig. There was no detectable antifactor VIII in
any of 10 mice treated in this manner when they were tested 4 weeks
after the sixth factor VIII injection. In contrast, high-titer antifactor VIII was present in serum from mice that had received only 1 mCTLA4-Ig injection (at the time of the first exposure to factor VIII)
followed by 5 injections of factor VIII alone.
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| Fig 4.
The effect of repeated administration of mCTLA4-Ig on
antifactor VIII antibody formation.
After the first injection, which contained both factor
VIII and mCTLA4-Ig, hemophilia A mice were injected intravenously, at
3-week intervals, with both 1 µg factor VIII and 250 µg mCTLA4-Ig
() or with factor VIII alone (). Serum samples for the antifactor
VIII assay were obtained 4 weeks after the sixth factor VIII
injection.
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These mCTLA4-Ig-treated mice were then tested to determine if they
would have an immune response after additional factor VIII injections
in the absence of mCTLA4-Ig. After 2 intravenous injections at 3-week
intervals, none of the 5 mice developed antifactor VIII, while
low-level antifactor VIII was detected in 2 of 4 control mice not
previously exposed to either factor VIII or mCTLA4-Ig (Figure
5). After 6 injections of factor VIII
alone, the mean factor VIII titer was 93 µg/mL for mice that had
previously received both factor VIII and mCTLA4-Ig, while the mean
titer was 155 µg/mL for the control mice. These data document the
limits of antigen-specific immune suppression that followed from
repeated coadministration of mCTLA4-Ig with factor VIII.
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| Fig 5.
The effect of simultaneous administration of mCTLA4-Ig
and factor VIII.
Hemophilia A mice treated as described for Figure 4, with 6 injections
of both factor VIII and mCTLA4-Ig, were subsequently given 6 intravenous injections of 0.2 µg of factor VIII at 3-week intervals
without additional mCTLA4-Ig (). Control mice with no prior factor
VIII exposure were immunized in parallel (). Serum samples for the
antifactor VIII assay were obtained 3 weeks after the second and sixth
injections.
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Secondary immune response to factor VIII suppressed by mCTLA4-Ig
To determine if mCTLA4-Ig modifies a secondary immune response to
factor VIII, we injected mCTLA4-Ig at the same time that factor VIII
was given to hemophilia A mice that had already developed antifactor
VIII. Initially, all the hemophilia A mice had been injected 3 times
with 0.2 µg factor VIII, and the level of antifactor VIII was
determined by ELISA. The control mice then received 3 additional
injections of factor VIII, while the remaining mice were given
mCTLA4-Ig at the same time as they received the first of 3 additional
factor VIII injections. While many mice died of bleeding complications
during this experiment because of the repeated injections and blood
sample collections, the results were clearly different for the 2 groups. An increase in the antifactor VIII titer was noted after the
fourth injection of factor VIII for the control mice, with the mean
titer ranging from 16 to 230 µg/mL (Figure
6A). After the fifth factor VIII injection,
all antifactor VIII titers were greater than 350 µg/mL in the 4 remaining control mice. In contrast, mice treated with mCTLA4-Ig at the
fourth factor VIII injection had minimal or no increases in antifactor
VIII (Figure 6B and C). The administration of mCTLA4-Ig
(Figure 6C) inhibited this secondary immune response to factor VIII in
mice that had already developed relatively high antifactor VIII levels (5-90 BU). This also occurred in mice with minimal antifactor VIII
after the 3 initial injections (less than 5 BU; Figure 6B).
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| Fig 6.
The effect of mCTLA4-Ig on the secondary immune response
to factor VIII.
All mice initially received 3 intravenous injections of 0.2 µg factor
VIII at 2-week intervals. (A) Control mice () were then injected
with factor VIII an additional 3 times, and blood samples were obtained
for assay. (B, C) The other mice () were injected intraperitoneally
with 250 µg mCTLA4-Ig the day before and the day after the fourth
factor VIII injection (as indicated by the arrow). This was followed by
2 more injections of only factor VIII at 3-week intervals. The number
of factor VIII injections prior to the blood sample tested for
antifactor VIII is indicated on the horizontal axis.
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Discussion |
We used a mouse model of hemophilia A to establish the T-cell
dependence of inhibitory antibody formation to factor VIII and to
evaluate the potential use of a reagent that blocks the B7/CD28/CTLA4 costimulation pathway. The murine fusion protein, mCTLA4-Ig, blocks this pathway, and it prevented the initiation of the antibody response and suppressed the development of a secondary
antibody response to factor VIII. These data gave direct evidence, for the first time, that the antibody response to factor VIII is T-cell dependent and can be modulated by costimulatory blockade.
While these studies in mice were done with recombinant human factor
VIII, we believe that they are relevant for our efforts to prevent and
treat factor VIII inhibitor antibodies in patients with hemophilia A. As a practical matter, mouse factor VIII is not available in the
quantity and purity needed to carry out these experiments. While the
gene has been cloned, the protein has not been produced in quantity by
recombinant technology. Moreover, as the hemophilic mice lack mouse
factor VIII, having no detectable factor VIII activity in their plasma
and no detectable factor VIII light chain protein by
immunoassay,17 they would be expected to respond to both
human and mouse factor VIII as completely foreign proteins. While it is
possible that there are some "mouse-specific" determinants on
murine factor VIII that are shared with other mouse proteins, this
would only make the response to human factor VIII somewhat greater than
that to mouse factor VIII. However, since we have demonstrated that the
blockade of a costimulatory pathway can prevent immunization, the
response to human factor VIII would be, if anything, more difficult to prevent.
To the extent possible, the experimental design incorporated all
concerns that may be important in the application of costimulatory blockade in patients. For this reason, the dose chosen for most studies, 0.2 µg factor VIII per intravenous injection, was comparable to that of a single therapeutic dose given to a patient with severe hemophilia A (with the goal of raising the plasma factor VIII level to
100%). For the initial experiments, the blockade was limited to a
single pair of injections of mCTLA4-Ig at the time of the first factor
VIII exposure, an approach that would be expected to have less effect
on the patient's overall immunologic status than repeated CTLA4-Ig
administration at each factor VIII injection. However, our experiments
showed that an immune response to factor VIII returned 6-9 weeks after
a single pair of CTLA4-Ig injections (Figure 1) or even after a series
of 6 paired injections of factor VIII and CTLA4-Ig at 3-week intervals
(Figure 3). The latter experiment did demonstrate, however, that
blockade of antifactor VIII antibody was maintained while CTLA4-Ig was
given at 3-week intervals over a 5- month period (Figure 2).
It is not certain why the hemophilic mice formed antifactor VIII after
they were given additional factor VIII injections in the absence of
mCTLA4-Ig. However, similar reversal of specific unresponsiveness to a
T-dependent antigen (sheep red blood cells [SRBC]) has been described
by Wallace and colleagues.24 In those studies, a single
dose of murine CTLA4-Ig prevented antibody formation immediately after
the first and second SRBC injections, but a high-titer response
followed the third SRBC injection. Their experiments also showed that
unresponsiveness to SRBC persisted while mice were successfully
immunized with a different T-dependent antigen. Finally, T and B cells
from unresponsive mice were shown to be functional when transferred to
irradiated mice subsequently immunized with SRBC. Thus, they had been
neither depleted of responsive cells nor rendered permanently
"tolerant" by CTLA4-Ig.24
In our studies, the development of antifactor VIII after repeated
antigen exposure is likely to be due to the combination of mCTLA4-Ig
clearance over time (its serum half-life  -phase being 6 days24) and emigration from the thymus of naive T cells that are capable of an antifactor VIII response.25 However, Wallace et al24 found that mice thymectomized before the
initial exposure to SRBC and CTLA4-Ig also develop anti-SRBC after the third injection. The down-regulatory role of B7-CTLA4 interactions is
also important in the induction and maintenance of T-cell
anergy.14 Thus, CTLA4-Ig blockade of the B7-CTLA4
interaction could also prevent maintenance of anergy in hemophilia A mice.
Blockade of CD28/B7 interactions using either CTLA4-Ig or anti-B7
antibodies has previously been shown to be an effective method for
blocking T-cell responses in several in vivo animal models of
transplantation and autoimmunity9 and in recent clinical studies.15,16,26,27 CD28 is expressed constitutively on T cells, and the ligands for CD28, B7-1 and B7-2, are expressed on B
cells as well as other antigen-presenting cells such as dendritic cells
and monocytes. Blockade of CD28 signaling in T cells results in a
failure in the activation of the antigen-specific T-cell subset.
In the absence of CD28 costimulation and IL-2 production, T-cell
activation does not occur efficiently. In vitro, exposure of T cells to
antigens in the absence of costimulation can render a T cell
unresponsive to reactivation, a condition termed anergy.28 Whether this mechanism is effective in vivo is unknown. Additional effects of costimulation blockade on the T-cell-B-cell interactions in
the germinal centers may be critical in preventing antibody formation.
Because the blockade of the B7-1 and B7-2 ligands individually can lead
to distinct effects in models of autoimmune disease, it has been
suggested that B7-1 and B7-2 may have unique biological roles.29,30 Alternatively, the earlier expression of B7-2
has suggested a greater role for B7-2 in initiating a T-cell response and a role for B7-1 in amplifying or regulating a T-cell response. We
have examined the roles of B7-1 and B7-2 in the initiation of the
antifactor VIII antibody response using hemophilia A mice deficient for
the expression of either B7-1 or B7-2. Although the antibody response
was not significantly altered in the B7-1 deficient mice, the B7-2
deficient mice formed no detectable antifactor VIII antibodies (Figure
1). In addition, the T-cell proliferative response to factor VIII was
greatly suppressed in the B7-2 deficient mice, although it was normal
in B7-1 deficient mice (Figure 2). This indicates that the
B7-2 ligand is critical for T-cell priming in hemophilia
A mice given factor VIII intravenously.
B7-2 is expressed in lymph node germinal centers of both humans and
mice,31,32 and the blockade of B7-2 in mice has been shown
to block antibody affinity maturation in germinal centers and to impair
the development of B-cell memory.32 Moreover, studies of the T-cell-dependent antibody response to the
trinitrophenol (TNP) hapten in B7-1- and B7-2-deficient mice
indicated that B7-2 is essential for isotype switching and for the
formation of germinal centers when antigen is given intravenously and
in the absence of an adjuvant.18 Under those conditions of
antigen administration, B7-1 did not compensate for the absence of
B7-2, while B7-1-deficient mice generated an antibody response
comparable to wild type mice.14,18 However, mice lacking
either B7-1 or B7-2 antigens had high-titer TNP-specific IgG responses
when immunized subcutaneously with TNP in complete Freund's adjuvant.
Mice lacking both B7-1 and B7-2 antigens, however, failed to class
switch or form germinal centers when immunized using these conditions.
Thus, B7-1 and B7-2 antigens can have compensating, overlapping roles
for isotype switching and germinal center formation when inflammation
leads to up-regulation of B7-1 and B7-2 expression.14,18
The formation of inhibitory antibodies to factor VIII is a critical
problem for hemophilia A patients treated with protein replacement
therapy and may be a major problem for gene therapy in hemophilic
patients. The results of our studies with the hemophilia A mouse
suggest that a costimulation blockade may be an effective therapy for
the prevention of antifactor VIII antibodies in these patients. Data
from the B7-2-deficient mice suggest that the blockade of the
CD28-B7-2 interaction is more important in the prevention of an
antibody response to factor VIII injected intravenously and that
anti-B7-2 may be as effective as CTLA4-Ig in blocking the initiation
of an antifactor VIII response. If this is the case, B7-1 function
would remain intact, and the treatment could be less generally
immunosuppressive than would be the case if CTAL4-Ig were used.
Although we have not verified that the antibody-free hemophilia A mice
treated with CTLA4-Ig have normal factor VIII recovery after an
infusion of factor VIII, published studies have addressed this
question.33 Using immunologic and
coagulation-based assays, gene therapy experiments with this mouse
model of hemophilia A have documented normal factor VIII recovery and
clearance in mice treated with a human factor VIII-encoding adenoviral
vector when their plasma was free of antifactor VIII.33
At the present time, it is not possible to identify which hemophilia A
patients will develop inhibitory antibodies after factor VIII
treatment.1,2 For this reason, the clinical application of
our studies would be primarily for patients who have already developed
detectable antifactor VIII. In evaluating this possibility, the ability
of anti-B7-2 to block antibody-affinity maturation32 suggests that blockade of the B7/CD28 pathway might be effective in
suppressing a secondary antifactor VIII antibody response, at least in
part, by preventing maturation of the B-cell response. In the single
experiment reported here (Figure 6), combined treatment of hemophilia A
mice with CTLA4-Ig and factor VIII after detection of antifactor VIII
antibody prevented a further increase in antibody titer in most mice
and a fall in titer in some. If the pattern is similar in patients,
B7-2 costimulation blockade at the time of factor VIII treatment may
stabilize the inhibitor level for patients treated early after
detection of antifactor VIII. For low-titer factor VIII inhibitor
patients, this costimulation blockade might then permit an escalation
of factor VIII doses to therapeutically effective levels without
further boosting the inhibitor titer. As antibody suppression by
costimulation blockade appears to last several weeks, intermittent
dosing may then be sufficient to prevent or modulate inhibitor
formation. In this way, therapeutic costimulation blockade would reduce
antifactor VIII while leaving the immune responses to other antigens intact.
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Acknowledgments |
We thank G. Gray for helpful discussions, Baxter Healthcare for
providing recombinant human factor VIII, Marina Borovok for technical
assistance, and Carolyn Murray and Debbie Wilder for manuscript preparation.
| |
Footnotes |
Submitted July 12, 1999; accepted October 7, 1999.
Supported in part by grants HL 36099 (L.W.H.) and AI 38310 (A.H.S.)
from the National Institutes of Health, Bethesda, MD.
Reprints: Leon W. Hoyer, Holland Laboratory, American Red
Cross, 15601 Crabbs Branch Way, Rockville, MD 20855; email: hoyer{at}usa.redcross.org.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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