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*
Laboratory of Immunogenetics and Transplantation and
Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115;
Transplantation Unit, Surgical Services, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114;
Cellular and Molecular Immunology Laboratory, Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114; and
¶ Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we used mice lacking CD28 to examine the role of interactions between CTLA-4 and B7-1 vs B7-2 in regulating alloimmune responses in a model of vascularized cardiac transplantation. Our results indicate that B7-1 plays a dominant role in the down-regulatory interaction with CTLA-4 and provide evidence suggesting that B7-2 may mediate a positive costimulatory signal through a previously unrecognized third receptor that is distinct from CD28 and CTLA-4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL6J (H-2b) (B6), BALB/cJ (H-2d) and BALB/c background CD28-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B6 background B7-1-deficient, B7-2-deficient, B7-1/B7-2 double-deficient mice (19), and BALB/c background CD28/CTLA-4 double-deficient mice (20) were generated in the laboratory of Dr. A. H. Sharpe. Animals were used at 6–14 wk of age.
Heterotopic heart transplantation
Vascularized heart grafts were transplanted using microsurgical techniques essentially as described by Corry et al. (21). Briefly, the harvested donor heart was placed in 4°C saline until transplantation. The recipient mouse was anesthetized by i.p. injection of 4% chloral hydrate. The donor aorta was sutured to the recipient aorta and the donor pulmonary artery to the recipient inferior vena cava end-to-side using 10-0 suture. Transplant function was evaluated by daily abdominal palpation. Rejection was defined as complete cessation of cardiac contractility as determined by direct visualization. Loss of graft function within 48 h of transplantation was considered a technical failure (<5% on the average), and these animals were omitted from further analysis.
Reagents, Abs and in vivo T cell depletion
The anti-B7-1 (CD80) mAb 1G10 and anti-B7-2 (CD86) mAb
2D10 hybridomas were a gift from Dr. G. D. Powers (Roche Research
Laboratories, Nutley, NJ). Anti-B7-2 Fab was generated from
anti-B7-2 mAb (Bio Express, West Lebanon, NH). The fusion protein
murine CTLA4Ig (a gift from Dr. R. Peach, Bristol-Myers Squibb,
Princeton, NJ) has been described previously (18).
CTLA4IgY100F, a mutated form of CTLA4Ig that binds B7-1 but not B7-2
(22), was also a gift from Dr. R. Peach. The blocking
anti-CTLA-4 mAb hybridoma 4F10 (23) was a gift from
Dr. J. Bluestone (University of California, San Francisco, CA).
Anti-CD4- and anti-CD8-depleting mAbs were prepared from hybridomas
GK1.5 (rat anti-mouse CD4) and 2.43 (rat anti-mouse CD8),
respectively, obtained from American Type Culture Collection (Manassas,
VA). All treated mice received 0.1 ml i.p. unpurified ascites of the
appropriate Ab (roughly equivalent to 100 µg purified Ab) on -6,
-3, and -1 days before transplantation (24). This
regimen insures >95% depletion of the respective cell type in the
peripheral blood on the day of transplantation. Cell counts start
recovering by 
2 wk after the last injection with complete recovery
occurring within 10 wk.
Ab-mediated complement-dependent cytotoxicity assay
Assays were performed in two stages with minor modifications from techniques previously described (25). Sera were collected from recipients around day 14 after transplantation and were heat inactivated at 56°C for 30 min and stored at -20°C until tested. 51Cr-labeled mouse splenocytes (105) of donor type were incubated with serially diluted sera in a final volume of 50 µl in U-bottom wells for 15 min at 37°C. Cells were washed with medium, pelleted, resuspended, and incubated with rabbit complement (C-6 Diagnostic, Mequon, WI) at a final dilution of 1/12 for 30 min at 37°C. Cells were pelleted, and 100 µl of the supernatant were harvested and counted on a gamma counter (Gamma 4000; Beckman Instruments, Fullerton, CA). Percentage of lysis was calculated by comparison to the 51Cr release obtained using a rabbit anti-mouse lymphocyte serum made in our laboratory.
In vitro MLR
Untreated responder spleen cells (4 x
105) and stimulator spleen cells (4 x
105) (2000 cGy) were added in a final volume of
200 µl tissue culture medium to U-bottom wells in triplicate. The
cultures were incubated at 37°C in a humidified air containing 5%
CO2 for 3 days. [3H]TdR
(1 µCi/well; New England Nuclear, Boston, MA) was added 6–9 h before
the end of culture. The samples were harvested onto glass fiber
filters, and [3H]TdR uptake was measured by 
scintillation counting on a RackBeta model 1209 counter (Wallac,
Gaithersburg, MD).
ELISA spot assay
ELISPOT plates (Polyfiltronics, Rockland, ME) were coated with
the capture Abs in sterile PBS overnight: R46A2 at 2 µg/ml for
IFN-
; 11B11 at 2 µg/ml for IL-4; and TRFK5 at 4 µg/ml for IL-5
(PharMingen, San Diego, CA). The plates were then blocked for 1.5
h with sterile PBS-1% BSA and washed three times with sterile PBS.
Spleen cells (1.2 x 106 cells/well) in 100
µl AIM-V medium were placed in each well with or without stimulator
cells (1.2 x 106 cells/well, 1:1 ratio) and
cultured for 24–48 h at 37°C in 5% CO2. After
washing, the detector Abs (IFN-: XMG1.2; IL-4: BVD4-1D11; IL-5:
TRFK4, PharMingen, San Diego, CA) diluted in PBS-0.025% Tween
containing 1% BSA were added overnight. After washing, HRP avidin D
diluted 1/2000 was added for 1.5 h at room temperature. The plates
were developed using 800 µl 3-amino-9-ethylcarbazole (Sigma, St.
Louis, MO; 10 mg dissolved in 1 ml dimethylformamide) mixed in 24 ml
0.1 M sodium acetate, pH 5.0, plus 12 µl
H2O2. The resulting spots
were counted on a computer-assisted ELISPOT image analyzer (T Spot
Image Analyzer; Cellular Technology, Cleveland, OH) (26, 27).
Statistics
Kaplan-Meier survival graphs were constructed, and the log rank comparisons of the groups were used to calculate p values. Significant differences between experimental groups in ELISPOT assay were analyzed using Student’s t test. Differences were considered to be significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cardiac grafts from C57BL/6 (B6) donors
(H-2b) were transplanted into fully allogeneic
wild-type or CD28-deficient BALB/c (H-2d)
recipients. While wild-type recipients acutely rejected their grafts
between 7 and 11 days (median survival time
(MST),3 9.0 ±
0.5 days, n = 7; Fig. 1
),
CD28-deficient recipients had significant prolongation of graft
survival as compared with wild-type controls (MST 38.5 ± 12.3
days, n = 6, p < 0.002; Fig. 1).
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CTLA-4 or B7 blockade accelerates cardiac allograft rejection in CD28-deficient recipients
To examine the roles of CTLA-4 and the B7 molecules in the absence
of CD28 costimulation, we next studied the effect of CTLA-4 or B7
blockade in CD28-deficient recipients of vascularized cardiac
allografts. Administration of CTLA4Ig (250-µg single dose on day 2
posttransplant), which binds both B7-1 and B7-2, or a blocking
anti-CTLA-4 mAb (250 µg on days -1, 0, and 1), significantly
accelerated allograft rejection in CD28-deficient recipients (Fig. 2, A and B). In
both instances, the effect was more pronounced when the frequency of
administration of either CTLA4Ig (days 0, 2, 4, and 6) or
anti-CTLA-4 mAb (days 0, 2, 4, 6, 8, and 10) was increased (Fig. 2, A and B). This is in contradistinction to their
effects in wild-type recipients, in which anti-CTLA-4 treatment
resulted in only a small degree of acceleration of allograft rejection
whereas CTLA4Ig induced indefinite allograft survival (Fig. 2C). The contrasting effects of CTLA4Ig in wild-type vs
CD28-deficient recipients highlight the importance of CTLA-4-mediated
down-regulation of alloimmune responses in vivo. In the absence of
CD28-mediated costimulation, CTLA-4 on T cells interacts with B7 on
APC, resulting in down-regulation of alloimmune responses and
prolongation of allograft survival observed in CD28-deficient animals
(see above and Fig. 1
). Administration of CTLA4Ig to CD28-deficient
animals blocks B7 interaction with CTLA-4, resulting in acceleration of
graft rejection. Similarly, administration of the anti-CTLA-4 mAb
blocks an inhibitory signal, also resulting in acceleration of graft
rejection that is more pronounced in CD28-deficient mice. These
observations confirm the CD28-independent down-regulatory function of
CTLA-4 in alloimmune responses in vivo (31).
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To study whether B7-1 and B7-2 have distinct or overlapping roles
in CTLA-4-mediated down-regulation of alloimmune responses, we next
examined the effect of selective blockade of either B7-1 or B7-2 in
CD28-deficient recipients. For B7-1 blockade, we treated the recipients
with a blocking anti-B7-1 mAb or a mutant form of CTLA4Ig,
CTLA4IgY100F, which selectively binds and blocks B7-1 (13, 22, 32). Administration of either the anti-B7-1 mAb or
CTLA4IgY100F resulted in significant acceleration of graft rejection
(Fig. 3A). In contrast, and to
our surprise, administration of a blocking anti-B7-2 mAb
significantly prolonged allograft survival (Fig. 3B).
Indeed, administration of a single dose of anti-B7-2 mAb resulted
in 67% indefinite allograft survival, and administration of multiple
injections (days 0, 2, 4, and 6) resulted in 83% indefinite allograft
survival (Fig. 3B). However, blockade of B7-1 had no effect,
and B7-2 was only marginally effective in prolonging allograft survival
in wild-type recipients (Fig. 3C). These results indicate
that B7-1 is the dominant ligand for CTLA-4 in mediating an inhibitory
signal to T cells during alloimmune responses in vivo. Moreover, they
show that even in the absence of CD28 costimulation, B7-2 can deliver a
positive signal that promotes graft rejection.
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); control
CD28-deficient recipients produced very low level of anti-donor
alloantibodies. Anti-B7-1 mAb, CTLA4Ig, and anti-CTLA-4 mAb
treatment of CD28-deficient recipients resulted in restoration of
alloantibody production up to a level comparable to that of wild-type
recipients with no treatment. Anti-B7-2 had no effect on the already
low alloantibody levels detected in sera of CD28-deficient
recipients.
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Differential effect of B7-1 vs B7-2 blockade in CD28-deficient recipients in vitro
To determine the role of B7-1 and B7-2 molecules in the
costimulation of T cells from CD28-deficient mice in vitro, we
performed primary MLR with wild-type BALB/c or CD28-deficient
responders. It has previously been reported that the alloantigen- and
mitogen-induced proliferation of CD28-deficient T cells is reduced
compared with that of wild-type responders (4). As shown
in Fig. 5B, selective blockade
of B7-1 with either anti-B7-1 mAb or CTLA4IgY100F augmented the
proliferative response of CD28-deficient responders, whereas
anti-B7-2 mAb had no effect. These results are consistent with our
in vivo finding that B7-1 is the dominant ligand for CTLA-4-mediated
inhibition of T cells.
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C,
selective blockade of B7-1 or CTLA-4 augmented the frequency of
IFN--producing cells, whereas selective B7-2 blockade resulted in a
slight decrease. We also measured the frequency of IL-4- and
IL-5-producing cells, but there was no significant difference between
selective B7-1 blockade and B7-2 blockade (data not shown) as compared
with untreated controls. These results suggest that B7-1 blockade
augments IFN- production in CD28-deficient mice after
allostimulation, whereas B7-2 blockade inhibits its production. These
data support our in vivo data indicating a differential role for B7-1
vs B7-2 in down-regulating alloimmune responses in the absence of CD28
costimulation. Evidence for a B7-2-mediated positive costimulatory signal provided through a third receptor on T cells
The finding that B7-2 blockade prolongs allograft survival in
CD28-deficient recipients could be interpreted in one of three ways.
First, it is possible that B7-2 may be delivering a positive signal
through CTLA-4 (Fig. 6A). In
that case, concomitant blockade of CTLA-4 plus B7-2 should not affect
the acceleration of allograft rejection mediated by CTLA-4 blockade.
Second, B7-2 may be delivering a positive signal through a yet
unidentified third receptor distinct from CD28 and CTLA-4 (Fig. 6B). Third, the anti-B7-2 mAb may be binding B7-2
molecules expressed on T cells, thus delivering a negative regulatory
signal. The ability of T cells to express B7 molecules has been
previously published (33). Indeed, flow cytometry analysis
of T cells from CD28-deficient animals also shows that they can express
B7-2 (62% of naive wild-type CD3+ T cells and
59% of naive CD28-deficient CD3+ T cells express
B7-2; data not shown). The possibility that the anti-B7-2 mAb is
signaling B7-2 on T cells leading to an inhibitory effect is highly
unlikely, because administration of anti-B7-2 mAb had only a
marginal effect in prolonging graft survival in wild-type recipients
(Fig. 3C). However, to definitely rule out this possibility
in CD28-deficient recipients, we administered a similar protocol of the
Fab of the anti-B7-2 mAb, thus eliminating any possibility for
signaling, to CD28-deficient recipients of cardiac allografts. The Fab
resulted in prolongation of graft survival (MST 78.6 ± 13.2 days,
n = 5) that was not significantly different from the
whole Ab (MST 88.0 ± 9.5 days, n = 6, NS)
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), we treated CD28-deficient recipients with anti-B7-2
mAb plus anti-CTLA-4 mAb. As shown in Fig. 7, B7-2 blockade overcomes the effects of
CTLA-4 blockade in accelerating graft rejection. In contrast, grafts
treated with a combination of CTLA4IgY100F to block B7-1 plus
anti-CTLA-4 mAb rejected their grafts within the same tempo as
animals treated with each reagent alone (Fig. 7). These data suggested
that B7-2 is not delivering a positive signal to T cells through
CTLA-4. They raise the possibility that B7-2 has an alternative third
receptor that is delivering a positive signal to T cells. To further
confirm this hypothesis, we used BALB/c background CD28/CTLA-4
double-deficient mice that have recently generated in the laboratory
of Dr. A. H. Sharpe (20). CD28/CTLA-4
double-deficient recipients rejected cardiac allografts with the same
tempo as wild-type recipients (MST 10.3 ± 0.8 days,
n = 6, vs MST 9.0 ± 0.5 days, n =
7, NS). Anti-B7-2 mAb treatment resulted in significant (Fig. 8, MST 45.3 ± 18.6 days,
n = 4, p < 0.005 compared with
untreated controls) prolongation of graft survival in CD28/CTLA-4
double-deficient recipients. These results provide further evidence
that B7-2 has a costimulatory receptor other than CD28 and CTLA-4 on T
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The second conclusion from our data is that B7-2 may have an additional
receptor other than CD28 that provides a positive costimulatory signal
for T cells. Our observation that B7-2 blockade in CD28-deficient
recipients resulted in significantly prolonged allograft survival
raised two main possibilities. First, B7-2 may provide a stimulatory
signal through CTLA-4 on T cells. Indeed, it has been reported that
CTLA-4 may provide a positive costimulatory signal under some
circumstances (40). Second, B7-2 may have an additional
receptor other than CD28 which mediates T cell costimulation. To
address this question, we blocked both CTLA-4 and B7-2 in
CD28-deficient recipients at the same time after transplantation. If
B7-2 provided a positive signal through CTLA-4, the acceleration of
rejection produced by anti-CTLA-4 mAb would not be affected by
addition of anti-B7-2 mAb. If B7-2 blockade reversed the
accelerated graft rejection produced by CTLA-4 blockade in
CD28-deficient recipients, this would support the notion of an
additional receptor for B7-2. Indeed, the latter case proved true,
providing evidence for the occurrence of a third receptor for B7-2
costimulation. This is also supported by the finding that concomitant
blockade of both B7-1 and CTLA-4 resulted in rejection of the graft
(Fig. 7), presumably by B7-2-third receptor-mediated costimulation.
Interestingly, B7-2 blockade not only overcomes the effect of CTLA-4
blockade but also increases graft survival compared with untreated
recipients (Fig. 7). Therefore, CTLA-4-negative signaling is not
absolutely required for the induction of long term graft survival, in
the absence of CD28. Furthermore, our results with B7-2 blockade in
CD28/CTLA-4 double knockout animals showing increased graft survival
provide further support to the existence of a third receptor other than
CD28 or CTLA-4. This third receptor is unlikely to be inducible
costimulator (ICOS) (41), because ICOS ligand is B7RP-1
(41) and ICOS does not appear to bind B7 molecules
(42).
Finally, our in vitro cytokine studies show that in the absence of
CD28, B7-1-CTLA-4 but not B7-2-CTLA-4 interactions inhibited IFN-
production. These data parallel our in vivo results with graft survival
and alloantibody production in that B7-1-CTLA-4 interactions
down-regulate alloimmune responses in CD28-deficient animals.
We conclude that in the absence of CD28 during alloimmune responses, B7-1 provides a negative signal through CTLA-4, whereas B7-2 provides a positive signal through a third receptor. Determining the exact identity of the putative third receptor and its role in normal animals should help to further our understanding of the mechanisms of CD28-independent costimulation of T cells in transplant rejection. Preclinical studies in primates indicate that CD28-B7 blockade alone is not sufficient to induce long term allograft survival and tolerance (43, 44). Whether this is due to inefficient blockade of B7-1-B7-2 interactions with CD28 and/or the third receptor requires further investigation, particularly if the putative third receptor is proved to exist and to play an important role in normal animals and humans. Therefore, understanding the mechanisms of CD28-independent costimulation of T cells in alloimmune responses is clinically relevant and should help in developing new therapies aimed at inducing long term allograft survival and tolerance.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mohamed M. Sayegh, Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: msayegh{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: MST, median survival time; ICOS, inducible costimulator.
Received for publication January 11, 2001. Accepted for publication April 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 
, 
) or B6 (
, •, 
) mice were
cultured for 3 days with increasing doses of anti-B7-1 mAb (
