By
From the * Immunology Research Division, Department of Pathology, and Renal Division,
Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston,
Massachusetts 02115
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Abstract |
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The costimulatory molecules B7-1 and B7-2 regulate T lymphocyte activation by delivering
activating signals through CD28 and inhibitory signals through cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The importance of CTLA-4-mediated inhibition was demonstrated by the uncontrolled T cell activation and lymphoproliferative disease that develops in
CTLA-4-deficient (/
) mice. To examine the role of B7 signaling in the activation of
CTLA-4-deficient T cells, we bred CTLA-4
/
mice with mice lacking B7-1, B7-2, or both
B7 molecules. The CTLA-4/B7-1
/
and the CTLA-4/B7-2
/
mice develop lymphoproliferation and enhanced T cell activation. Mice lacking CTLA-4, B7-1, and B7-2 have a normal
life-span, and do not have lymphocytic infiltrates in any organs, or increased T cell activation.
Therefore, the two B7 molecules have overlapping functions, since either B7-1 or B7-2 alone
can cause the CTLA-4
/
phenotype. Elimination of both B7-1 and B7-2 from the CTLA-4- deficient mouse abrogates the lymphocyte activation and disease, and does not reveal evidence
for additional stimulatory CD28 ligands. The CTLA-4
/
phenotype can be reproduced with
anti-CD28 antibody in mice lacking CTLA-4, B7-1, and B7-2, but wild-type mice are unaffected by the same treatment. This suggests that the inhibitory function of CTLA-4 can overcome strong CD28-mediated signaling in vivo.
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Introduction |
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The importance of the B7-CD28/cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) pathway has been highlighted by studies showing that blockade of B7 costimulation can prolong allograft survival (1) and suppress autoimmunity (2). The two B7 ligands, B7-1 (CD80) and B7-2 (CD86), can both stimulate immune responses by binding to CD28 and downregulate responses by binding to CTLA-4. Because B7-2 is expressed constitutively and upregulated earlier than B7-1, it has been suggested that B7-2 participates in initiating an immune response, whereas B7-1 may be more important in sustaining or regulating ongoing responses. The upregulation of CTLA-4 on activated T cells parallels the kinetics of B7-1 expression. Together with the higher avidities of B7 ligands for CTLA-4 than CD28, this has raised the possibility that B7-1/CTLA-4 interactions predominate late to terminate immune responses. However, several reports suggest that B7-1 and B7-2 can provide similar signals (3, 4). Further understanding of the complexities in this pathway is required in order to optimize therapies that target this pathway.
To study the interactions between receptors and ligands
in the B7-CD28/CTLA-4 pathway, we have generated
and characterized mice lacking expression of one or more
molecules in the pathway. The phenotype of mice lacking
CTLA-4 (CTLA-4/
) provides crucial evidence that
CTLA-4 plays a negative regulatory role in T cell activation. These mice develop a fatal lymphoproliferative disease
with lymphocytic infiltrates in multiple organs (5, 6).
CTLA-4
/
T cells are highly activated in vivo and spontaneously proliferate in vitro without any added stimuli.
Early treatment with CTLA4Ig blocks the T cell activation
and lymphoproliferation, demonstrating that B7, presumably functioning by ligating CD28, is necessary for the development of the CTLA-4
/
phenotype (7). However,
these studies do not address whether CD28 interacts only
with B7-1 and B7-2, or if additional ligands for CD28/
CTLA-4 exist, as some have proposed (8, 9). The T cell
activation observed in CTLA-4
/
mice and its dependence on B7 molecules provide a unique system for exploring interactions in this pathway.
Here we have used the CTLA-4/
mouse as a tool to
investigate interactions between CD28 and its ligands. We
have generated and characterized three novel mouse strains,
which are CTLA-4-deficient and also lack B7-1, B7-2,
or both B7 molecules. If additional stimulatory CD28 ligands existed, then mice lacking CTLA-4, B7-1, and B7-2
would be expected to develop increased T cell activation. Alternatively, if there is not increased T cell activation in these mice, then this would suggest that in the absence of
the two known B7 molecules, CD28 is no longer engaged.
We found that mice lacking CTLA-4 and either B7-1 or
B7-2 develop the lymphoproliferative phenotype observed
in the CTLA-4
/
strain, but that mice deficient in
CTLA-4 and B7-1 have a shorter life-span and show
greater T cell activation in vivo than the CTLA-4/B7-2-
deficient mice. In contrast, mice lacking CTLA-4, B7-1,
and B7-2 ("triple knockout," CTLA-4/B7-1/B7-2
/
TKO)
show no evidence of T cell activation or lymphoproliferation, suggesting that there are no additional stimulatory ligands for CD28. We also show that administration
of anti-CD28 antibody to CTLA-4/B7-1/B7-2
/
TKO
mice can reproduce the phenotype of the CTLA-4
/
mouse, demonstrating the critical role of CD28 in activating CTLA-4
/
T cells.
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Materials and Methods |
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Mice.
Mouse strains lacking CTLA-4 (6), B7-1 (10), B7-2, or both B7 molecules (11) have been described previously. Because CTLA-4-deficient mice die before reaching sexual maturity, the three CTLA-4/B7-deficient mice were generated by interbreeding CTLA-4 heterozygotes with B7-1-, B7-2-, or B7-1/B7-2-deficient mice. Mice heterozygous for both CTLA-4 and the B7 mutation were then intercrossed, and CTLA-4/B7-deficient mice were obtained. Because of the lethality of the CTLA-4/B7-1- and CTLA-4/B7-2-deficient phenotypes, these two strains were maintained by interbreeding CTLA-4+/In Vivo Administration of Anti-CD28.
Wild-type and CTLA-4/B7-1/B7-2Cell Preparation and Cultures.
Single cell suspensions from spleen and lymph nodes were prepared by dissociating tissue with sterile glass slides. Red blood cells were lysed by incubation in Tris-ammonium chloride for 5 min at 37°C. To assay proliferation, 2 × 105 cells/well were cultured in flat-bottomed 96-well plates in media as described previously (6). Cells were pulsed with 1 µCi [3H]thymidine for the last 8 h of the indicated day. Anti-CD3 stimulation used high titer supernatant prepared from the 145-2C11 hybridoma, obtained from the American Type Culture Collection.Cell Surface Staining.
Spleen and lymph node cells were stained with fluorochrome-conjugated mAbs including anti- CTLA-4 (4F10), anti-B7-1 (1G10), anti-B7-2 (GL1), anti-CD62L (Mel 14), anti-CD69 (H1.2F3), anti-CD25 (7D4), and anti-CD3 (145-2C11). These antibodies were purchased from PharMingen. Stained cells were analyzed on a FACSCalibur® (Becton Dickinson).Histology.
Tissue for light microscopy was fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard techniques. ![]() |
Results |
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To assess the role of B7-1 and B7-2 in producing
lymphoproliferative disease in mice lacking CTLA-4, we
crossed the CTLA-4-deficient strain with mice lacking
B7-1, B7-2, or both B7 molecules. Mice lacking CTLA-4
and B7-1 (CTLA-4/B7-1/
) and mice lacking CTLA-4
and B7-2 (CTLA-4/B7-2
/
) develop the characteristic fatal phenotype of CTLA-4
/
mice. The survival of the
CTLA-4/B7-1
/
mice (mean = 21.9 d; n = 8) was
shorter than that of the CTLA-4/B7-2
/
mice (mean = 30.5 d; n = 8) (P = 0.02 by Wilcoxon signed rank test).
The difference in survival between the CTLA-4/B7-1
/
and the CTLA-4
/
(mean = 17.5 d; n = 11) strains did
not reach statistical significance (P > 0.05; Fig. 1). In contrast, CTLA-4/B7-1/B7-2
/
TKO mice lacking CTLA-4,
B7-1, and B7-2 remain healthy and have a life-span and reproductive capacity comparable to wild-type mice.
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Both the CTLA-4/B7-1/
and CTLA-4/B7-2
/
strains develop splenomegaly, lymphadenopathy, and multiorgan lymphocytic infiltrates with tissue damage, similar
to the CTLA-4
/
mice. The CTLA-4/B7-1
/
mice
typically develop these pathologic changes earlier than the
CTLA-4/B7-2
/
mice. In contrast, CTLA-4/B7-1/B7-2
/
TKO mice develop neither splenomegaly nor lymphadenopathy. Detailed histologic examination of CTLA-4/
B7-1/B7-2
/
TKO mice ranging in age from 2 to 8 mo reveals the complete absence of lymphocytic infiltrates (Fig.
2). These results indicate that either B7-1 or B7-2 can provide the necessary activating signal for the lymphoproliferative phenotype in CTLA-4
/
mice, but that the absence
of both B7 molecules abrogates the phenotype.
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To assess the degree of T cell activation in these mouse strains in vivo,
freshly isolated splenocytes were stained for activation markers and analyzed by flow cytometry. T cells from
CTLA-4/
, CTLA-4/B7-1
/
, and CTLA-4/B7-2
/
mice show significant increases in the percentage that are
positive for the activation marker CD69 (Fig. 3) and
CD62L-low (data not shown) compared with wild-type
mice. In four animals of each strain analyzed at 2 wk, there
was a mean of 51% CD69+ T cells in CTLA-4
/
, 48% in
CTLA-4/B7-1
/
, and 44% in CTLA-4/B7-2
/
mice.
The same trend was observed when staining for CD62L,
but the differences did not reach statistical significance. In
contrast, <10% of CTLA-4/B7-1/B7-2
/
TKO cells are
activated, as assessed by staining for CD69 (Fig. 3) and
CD62L (data not shown). None of the CTLA-4/B7-1/
B7-2
/
TKO mice analyzed show significant T cell activation. Both the CTLA-4/B7-1/B7-2
/
TKO and B7-1/
B7-2
/
strains have consistently fewer activated T cells
than wild-type mice.
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We have previously reported that unfractionated splenocytes from CTLA-4/
mice proliferate spontaneously in
vitro in the absence of exogenous stimulation (7). This assay serves as another measure of unopposed T cell activation in CTLA-4
/
mice. Proliferation in both CTLA-4/
B7-1
/
and CTLA-4/B7-2
/
strains was significantly
higher than in wild-type mice (Fig. 4 A). As might be expected from other measures of T cell activation in the two
strains, CTLA-4/B7-1
/
cells proliferate more than those
from age-matched CTLA-4/B7-2
/
cells. In contrast,
CTLA-4/B7-1/B7-2
/
TKO splenocytes do not proliferate spontaneously, suggesting that no CD28 signaling occurs in the absence of B7-1 and B7-2.
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We also assessed the in vitro proliferative response of
CTLA-4/B7-1/B7-2/
TKO T cells to anti-CD3 antibody stimulation. The response of CTLA-4/B7-1/B7-2
/
TKO splenocytes is markedly reduced compared with
wild-type mice (Fig. 4 B). This deficit is indistinguishable
from that observed with the B7-1/B7-2
/
mice. Splenocytes from CTLA-4
/
, CTLA-4/B7-1
/
, and CTLA-4/
B7-2
/
mice which are already activated in vivo showed a
proliferative response to anti-CD3 stimulation that was
highly variable between experiments, presumably because
of varying numbers of in vivo-activated T cells, which undergo activation-induced cell death upon restimulation in vitro.
The results showing that T cell activation in CTLA-4/
mice requires B7-1 or B7-2 suggest
that B7-CD28 interactions are critical for such activation.
If CD28 signaling is sufficient to cause T cell activation in
the absence of CTLA-4, then ligating CD28 artificially using
anti-CD28 antibody should induce disease in CTLA-4/
B7-1/B7-2
/
TKO mice. To test this, we administered
anti-CD28 mAb (PV-1) to adult mice for 2 wk. Mice were
analyzed 14-19 d after initial treatment with anti-CD28
because they exhibited features typical of the CTLA-4
/
lymphoproliferative disease. At this time, they developed
splenomegaly and lymphadenopathy, with greater than sixfold increases in cell numbers compared with anti-CD28-
treated wild-type mice (data not shown). CTLA-4/B7-1/
B7-2
/
TKO mice treated with anti-CD28 also developed
lymphocytic infiltrates and necrosis, in an organ distribution and severity comparable to the CTLA-4
/
mice (data
not shown). No lymphoid or other organ involvement was
observed in CTLA-4/B7-1/B7-2
/
TKO mice receiving
control IgG, or wild-type mice receiving anti-CD28 or
control IgG. Anti-CD28 administration also markedly increased the fraction of activated T cells in the CTLA-4/B7-1/
B7-2
/
TKO mice compared with wild-type, as determined by CD69 (Fig. 5) and CD62L expression (data not
shown). Thus, signaling through CD28 in the CTLA-4/
B7-1/B7-2
/
TKO strain can reproduce the lymphoproliferative disease typical of CTLA-4
/
mice. The lack of effect of anti-CD28 antibody in wild-type mice in vivo is also
noteworthy, demonstrating that engagement of CTLA-4 by endogenous levels of B7 can inhibit the proliferation of
T cells receiving a strong positive signal through CD28.
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Discussion |
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The dual specificities of B7-1 and B7-2 for CD28 and
CTLA-4 have made it challenging to elucidate physiological interactions in the B7-CD28/CTLA-4 pathway. The
uncontrolled T cell activation seen in the CTLA-4/
mice provides a valuable experimental system for dissecting
in vivo functions of B7-1 and B7-2, and for searching for
additional members of this family of costimulators. To do
this, we have generated mouse strains lacking CTLA-4 and
either B7-1, B7-2, or both B7 molecules, and analyzed
them for evidence of T cell activation in vivo and in vitro.
The phenotypes of the CTLA-4/B7-1
/
and CTLA-4/
B7-2
/
strains demonstrate overlapping roles for B7-1
and B7-2 in CD28-mediated signaling, as the presence of
either B7-1 or B7-2 is sufficient to produce the CTLA-4
/
phenotype. However, the CTLA-4/B7-1
/
mice have
shorter survival and greater T cell activation than age-matched CTLA-4/B7-2
/
mice. These differences may
reflect earlier expression and higher cell surface levels of
B7-2 compared with B7-1. Since either B7 molecule is capable of producing lymphoproliferation and T cell activation
in the CTLA-4
/
mice, it seems unlikely that B7-1 and
B7-2 produce fundamentally different signals through CD28.
The striking absence of T cell activation in the CTLA-4/
B7-1/B7-2/
TKO strain demonstrates that B7-1 or B7-2
is required to produce the CTLA-4
/
lymphoproliferative
disease. The absence of CD28-mediated costimulation in
the CTLA-4/B7-1/B7-2
/
TKO strain is demonstrated
by the (a) lack of lymphoproliferation in vivo, as evidenced
by the normal life-span and absence of splenomegaly, lymphadenopathy, and lymphocytic infiltrates; (b) naive phenotype of T cells in vivo; (c) lack of spontaneous proliferation
of splenocytes in vitro; and (d) impaired proliferative response to CD3 stimulation in vitro. Similar results have
been obtained by treating CTLA-4
/
mice with CTLA4Ig.
Our results extend these studies and show that B7-1 or
B7-2 is capable of activating CTLA-4
/
T cells.
This requirement for B7-1 or B7-2 in activating CTLA-4/
T cells is most consistent with a role of unopposed
signaling by CD28, the only known activating T cell receptor for B7 molecules. We have formally established this by
showing that anti-CD28 antibody can reproduce disease in
CTLA-4/B7-1/B7-2
/
TKO mice. Interestingly, the
same anti-CD28 antibody has no detectable effect on T cell
activation in wild-type mice in vivo. These findings demonstrate the critical balance between CD28-mediated T
cell activation and CTLA-4-mediated downregulation, and
suggest that the inhibitory function of CTLA-4 can overcome strong CD28-mediated signals in vivo. These results
may explain why many studies show no or inconsistent in
vivo effects of anti-CD28 antibodies, which are known to
be strong agonists when added to T cells in the absence of
APCs in vitro.
We have not found evidence for additional stimulatory
CD28 ligands in the CTLA-4/B7-1/B7-2/
TKO mice.
This strain provides an exquisitely sensitive means for examining whether additional stimulatory CD28 counterreceptors exist, since in the absence of CTLA-4, even the
effects of weak CD28-mediated signaling would be apparent. The profound deficits observed in the CTLA-4/B7-1/
B7-2
/
TKO mice would suggest that if additional B7
ligands exist, they either do not stimulate T cell activation
through CD28, or they are expressed in a restricted microenvironment. Such microenvironments might include
organs such as the brain or kidney, which exhibit no inflammatory changes in CTLA-4
/
mice.
The CTLA-4/B7-1/B7-2/
TKO strain has a phenotype that appears to be comparable to the B7-1/B7-2
/
strain both in vivo and in vitro. Therefore, in the absence
of B7-1 and B7-2, the presence of CTLA-4 has no detectable downregulatory effect. Because T cells from the
CTLA-4/B7-1/B7-2
/
TKO strain are mostly naive, unlike cells from CTLA-4
/
mice, CTLA-4/B7-1/B7-2
/
TKO mice also can serve as a useful tool to study the function of CTLA-4 during the initial activation of T cells.
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Footnotes |
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Address correspondence to Didier A. Mandelbrot, 221 Longwood Ave., LMRC Rm. 512, Boston, MA 02115. Phone: 617-732-6316; Fax: 617-732-5795; E-mail: damandelbr{at}bics.bwh.harvard.edu
Received for publication 23 October 1998.
We thank Abul Abbas for careful review of the manuscript, Vijay K. Kuchroo for the gift of anti-CD28 antibody, Frank Borriello and Elizabeth A. Tivol for initiating mouse breeding, and Sumi Scott for technical assistance.
This work was supported by National Institutes of Health grants K11 AI01212 to D.A. Mandelbrot, AI09709 to A.J. McAdam, and RO1 AI38310, RO1 AI40614, and PO1 AI35297 to A.H. Sharpe.
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References |
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1. | Turka, L.A., P.S. Linsley, H. Lin, W. Brady, J.M. Leiden, R.Q. Wei, M.L. Gibson, X.G. Zheng, S. Myrdal, D. Gordon, et al . 1992. T-cell activation by the CD28 ligand B7 is required for cardiac allograft rejection in vivo. Proc. Natl. Acad. Sci. USA. 89: 11102-11105 [Abstract]. |
2. | Finck, B.K., P.S. Linsley, and D. Wofsy. 1994. Treatment of murine lupus with CTLA4Ig. Science. 265: 1225-1227 [Medline]. |
3. |
Lanier, L.L.,
S. O'Fallon,
C. Somoza,
J.H. Phillips,
P.S. Linsley,
K. Okumura,
D. Ito, and
M. Azuma.
1995.
CD80 (B7)
and CD86 (B70) provide similar costimulatory signals for T
cell proliferation, cytokine production, and generation of
CTL.
J. Immunol.
154:
97-105
|
4. | Schweitzer, A.N., F. Borriello, R.C. Wong, A.K. Abbas, and A.H. Sharpe. 1997. Role of costimulators in T cell differentiation: studies using antigen-presenting cells lacking expression of CD80 or CD86. J. Immunol. 158: 2713-2722 [Abstract]. |
5. | Waterhouse, P., J.M. Penninger, E. Timms, A. Wakeham, A. Shahinian, K.P. Lee, C.B. Thompson, H. Griesser, and T.W. Mak. 1995. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science. 270: 985-988 [Abstract]. |
6. | Tivol, E.A., F. Borriello, A.N. Schweitzer, W.P. Lynch, J.A. Bluestone, and A.H. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity. 3: 541-547 [Medline]. |
7. | Tivol, E.A., S.D. Boyd, S. McKeon, F. Borriello, P. Nickerson, T.B. Strom, and A.H. Sharpe. 1997. CTLA4Ig prevents lymphoproliferation and fatal multiorgan tissue destruction in CTLA-4-deficient mice. J. Immunol. 158: 5091-5094 [Abstract]. |
8. | Boussiotis, V.A., G.J. Freeman, J.G. Gribben, J. Daley, G. Gray, and L.M. Nadler. 1993. Activated human B lymphocytes express three CTLA-4 counterreceptors that costimulate T-cell activation. Proc. Natl. Acad. Sci. USA. 90: 11059-11063 [Abstract]. |
9. |
Murakami, M.,
Y. Takahashi,
Y. Isashi,
S. Kon,
W.Y. Jia,
M. Inobe,
R. Abe, and
T. Uede.
1996.
Identification and characterization of an alternative cytotoxic T lymphocyte-associated protein 4 binding molecule on B cells.
Proc. Natl. Acad.
Sci. USA.
93:
7838-7842
|
10. | Freeman, G.J., F. Borriello, R.J. Hodes, H. Reiser, K.S. Hathcock, G. Laszlo, A.J. McKnight, J. Kim, L. Du, D.B. Lombard, et al . 1993. Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice. Science. 262: 907-909 [Medline]. |
11. | Borriello, F., M.P. Sethna, S.D. Boyd, A.N. Schweitzer, E.A. Tivol, D. Jacoby, T.B. Strom, E.M. Simpson, G.J. Freeman, and A.H. Sharpe. 1997. B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity. 6: 303-313 [Medline]. |