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*
Department of Immunology, Imperial College School of Medicine, and
Transplantation Biology Group, Medical Research Council Clinical Sciences Centre, Hammersmith Hospital, London, United Kingdom
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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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. The
consequences of Ag presentation by these other cell types is less
clear. We have argued that costimulation-deficient Ag presentation
favors the induction of T cell unresponsiveness, and provides a
mechanism of regulating unwanted autoreactive T cells in the periphery
(4). There is a considerable body of data supporting this
concept; however, it is inconsistent with the recent suggestion that
the induction of T cell unresponsiveness requires ligation of the
second receptor for B7 molecules, namely CD152 (5, 6). In this study we have explored this issue and found that CD152 ligation can be implicated in the context of Ag presentation by costimulation-deficient or -negative cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR-transgenic DO.11.10 mice (7) were kindly provided by Drs. David Gray and Hans Reiser at the Department of Immunology (Hammersmith Hospital, London, U.K). BALB/c mice were purchased from Harlen Olac (Bicester, U.K.).
B cell lines and Chinese hamster ovary (CHO)4 transfectants
A20 (TIB-208; American Type Culture Collection (ATCC), Rockville, MD), M12, and AJ9 (both from Dr. Hans Reiser) (8) are three B lymphoma cell lines derived from BALB/c mice. A20-II is a subclone of A20. CHO-H2-Ad and CHO-H2-Ad-B7-2 transfected cells were gifts from Dr. Hans Reiser.
Peptide, mAbs, and Fab
Chicken OVA (cOVA)323–339 peptide (ISQAVHAAHAEINEAGR) was kindly provided by Dr. Sara Brett at Glaxo Wellcome (Research Triangle Park, NC). The hybridoma culture supernatants used for T cell purification were as follows: anti-H2-Ad (MKD6, TB-3; ATCC), anti-H2-Ek,d/Ab,d (M5/114, HIB-120; ATCC), anti-CD8 (53.6.7, TIB-105, and YTS 169; ATCC), and anti-CD44 (I42.5). The purified mAbs used in T cell proliferation assays included the following: anti-CD80/B7-1 (16-10A1, HB-301; ATCC), anti-CD86/B7-2 (GL1), anti-CD152/CTLA4 (UC11-4F10-11, HB-304; ATCC), anti-CD28 (37.51), and hamster IgG. Anti-clonotypic mAb, KJ1-26 (9), was kindly provided by Drs. David Gray and Hans Reiser. The references of YTS 169, I42.5, GL-1, and 37.51, the purification of mAb, and preparation of Fab have been described previously (10, 11).
Purification of naive CD4+ T cells
Spleen and pooled lymph node cells from BALB/c or DO.11.10 mice were treated with a mixture of MKD6, M5/114, 53.6.7 and YTS169, and then with a mixture of sheep anti-mouse or rat IgG Dynabeads (Dynal, Oslo, Norway). To obtain naive CD4+ T cells, the purified CD4+ T cells were further treated with I42.5 followed by sheep anti-rat IgG Dynabeads. The resulting cells were >95% CD4+CD8- H2-Ad- CD44low CD45RBhigh CD86low CD80-.
T cell proliferation assay
T cells were routinely cultured in round-bottom 96-well plates in a volume of 200 µl for 3 days, and mAbs added were usually at 10 µg/ml, unless otherwise indicated. Fixation of APC was performed by incubating A20-II cells with 1% paraformaldehyde for 15 min at room temperature. Blockade of FcR was conducted by incubating A20-II cells with anti-FcR (2.4G2, HB-197; ATCC) before fixation. CD4+ T cells were activated by immobilized Abs in some experiments. The preparation of mAb-coated plates, preparation of complete culture medium (10% FCS RPMI 1640), [3H]thymidine pulsing, cell harvest, and ß-counting have been described previously (10, 11). The results are expressed as mean cpm for duplicate cultures. SEs were routinely <10% and thus omitted.
Measurement of IL-2 production
The protocol of CTLL-2 bioassay has been described previously (10, 11).
Flow cytometric analysis
All FITC- or PE-conjugated mAbs were purchased from PharMingen (San Diego, CA), unless otherwise indicated. All flow cytometric analysis was conducted on a Becton Dickinson FACScaliber running CellQuest software (Franklin Lakes, NJ).
Generation and maintenance of T cell line
A T cell line was generated and maintained by repeatedly stimulating DO.11.10 CD4+ T cells with cOVA protein (Sigma, St. Louis, MO), irradiated BALB/c spleen cells, and recombinant human IL-2 (Boehringer Mannheim, Mannheim, Germany) according to a standard protocol (12) with some modifications.
Induction of T cell clonal anergy in vitro
Ten to 12 days after their last stimulation, the DO.11.10 T cell line was harvested. To remove any remaining splenic accessory cells, T cells were treated with a mixture of MKD6 and M5/114 followed by a mixture of sheep anti-mouse or rat IgG Dynabeads. Flow cytometric analysis showed that the resulting cells were >98% CD4+KJ1-26+. These T cells were incubated with peptide plus fixed CHO-H2-Ad cells in the absence or presence of 10 µg/ml anti-CD80 or anti-CD152 overnight in 24-well plates (Costar, Cambridge, MA). APCs were then removed using biotinylated M5/114 followed by streptavidin-Dynabeads, and T cells were allowed to rest for 2–5 days. After resting, viable T cells were restimulated with peptide plus fixed CHO-B7-2-H2-Ad cells or irradiated BALB/c spleen cells. At day 2 of restimulation culture, 50 µl cell-free supernatants were transferred into a new set of plates, and IL-2 activity was measured by CTLL-2 bioassay.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies with naive CD8+
TCR-transgenic T cells had raised the possibility that CD80 expressed
by responder T cells had a negative regulatory role (13).
This was further examined using APC that expressed no (A20-II), or very
low (M12) levels of CD80, and low amounts of CD86. To exclude any
change in the phenotype of the B cells during the cultures with T
cells, the APC were paraformaldehyde-fixed before culture. The results
of staining the B cell lines used in these experiments for B7 and H2-A
expression are shown in Fig. 1
. AJ9 cells
were used as a positive control, and expressed high levels of both B7
isoforms.
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A). The T cells did not
proliferate to Con A alone, confirming their purity. Addition of A20-II
cells induced low-level proliferation, which was almost completely
abolished by the presence of anti-CD86 mAb. In marked contrast,
addition of anti-CD80 mAb led to a substantial increase in
proliferation and IL-2 production (Fig. 2, A and
B). Identical results were obtained when M12 cells were used
as accessory cells (data not shown).
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C, anti-CD80 and
anti-CD86 had opposing effects. Anti-CD86 suppressed, whereas
anti-CD80 augmented proliferation, especially at suboptimal
concentrations of peptide. Furthermore, in the absence of
anti-CD80, proliferation induced by 1 µg/ml of peptide peaked at
day 3 and fell rapidly at day 4; however, in the presence of
anti-CD80, proliferation was maintained at a high level at day 4
(Fig. 2D), suggesting that anti-CD80 not only increased
the level of proliferation but also prolonged its duration. The results
were most striking when a suboptimal dose of peptide (i.e., 0.1
µg/ml) was used in a kinetic study (Fig. 2E). The effects
of anti-CD80 were further examined in an APC-free system where
naive CD4+ T cells were stimulated with
coimmobilized anti-CD3 and anti-CD28. As shown in Fig. 2F, the proliferation induced by coimmobilized anti-CD3
and anti-CD28 was markedly enhanced by the addition of soluble
anti-CD80 mAb.
One explanation for the enhancing effects of anti-CD80 Ab was that
it prevented ligation of CD152, thereby releasing T cells from negative
regulation. Consistent with this suggestion, anti-CD152 mAb caused
comparable enhancement of proliferation to that seen with anti-CD80
in response to CD80-negative B cells (Fig. 2G). Blocking
CD152 using soluble anti-CD152 mAb leads to an enhancement of T
cell response was also observed by Alegre et al. (14).
Given that the B cell APC were CD80 negative, we investigated the
expression of B7 and CD152 by the T cells after antigenic stimulation
by the fixed A20-II cells. Naive DO.11.10 T cells are CD80 and CD152
negative, but express a low level of CD86 (Fig. 2, H and
I). After 3 days culture with Ag and B cells, the pattern of
expression of CD86 and CD80 was sharply reversed; CD80 was markedly
up-regulated, whereas CD86 was down-regulated. Cell surface expression
of CD152 was also detectable (Fig. 2, J and K).
Thus, it is possible that CD152-CD80 interactions may occur following
activation.
Anti-CD80 does not signal T cells directly
An alternative explanation for the anti-CD80-mediated
amplification of the T cell response was that the Ab was delivering a
positive signal directly to the T cells, mimicking costimulation. This
possibility was addressed in three ways; first, the ability of the
anti-CD80 to costimulate the T cell response to immobilized
anti-CD3 mAb was examined. As presented in Fig. 3A, coimmobilized
anti-CD28 provided very efficient costimulation. In contrast,
anti-CD80 used at the same concentration had no effect. Second, as
shown in Fig. 3B, a Fab of anti-CD80 caused comparable
augmentation of proliferation by the transgenic T cells to that induced
by intact Ab. Third, the effect of anti-CD80 Ab was not diminished
by pretreating the B cells with anti-FcR mAb, preventing Ab
immobilization (Fig. 3C).
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The effects of anti-CD80 and anti-CD152 Abs are not additive, suggesting that they interrupt the same molecular interaction
A second alternative explanation for the effects of anti-CD80
Ab was that T cell-expressed CD80 was sequestering CD28 at the
interface between two T cells and inhibiting its migration into the
"synapse" between the T cell and the APC. Given that costimulation
is most efficient when in cis rather than in
trans, interruption of the CD80:CD28 interaction would
release CD28 to cocluster with the TCR/CD3 complex. If this were the
case, the enhancement caused by anti-CD152 would result from the
interruption of a separate interaction, namely the ligation of CD152 by
the low levels of CD86 expressed by the B cells. Under these
circumstances, it would be predicted that the effects of anti-CD80
and anti-CD152 would be additive. This possibility was tested in
the context of the DO.11.10 T cell response to peptide presented by A20
cells. As shown in Fig. 3D, maximal enhancement was achieved
by Ab alone, and no additive effects were seen, further suggesting an
inhibitory interaction between CD80 and CD152 at the surface of T
cells.
Anti-CD80 partially, and anti-CD152 totally, prevent the induction of anergy induced by TCR engagement in the absence of costimulation
Two apparently contradictory observations have been made
concerning peripheral T cell tolerance. The first is that
costimulation-deficient Ag presentation leads to T cell
unresponsiveness (15). The second is that CD152 ligation
on the T cell is required for unresponsiveness to be induced (5, 6). If the APC express no B7 family molecules, how can CD152,
expressed by the T cell, be ligated? The contribution of T
cell-expressed CD80 to the induction of nonresponsiveness was tested
using DO.11.10 T cells. It has been reported previously that only
activated DO.11.10 T cells are sensitive to anergy induction in vitro
whereas naive DO.11.10 T cells are not (16). A T cell line
was therefore generated using DO.11.10 T cells. This T cell line
displayed Ag-specific proliferation which usually peaked at 0.01
µg/ml peptide (Fig. 4A).
Immediately before Ag stimulation (10 days after the last
restimulation) the cells expressed an intermediate level of CD86, a low
level of CD80, and a trace level of CTLA4 (data not shown).
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B, culture with the CHO
transfectants that lacked B7 molecule expression induced profound
unresponsiveness. The maximum effect was observed when 10 µg/ml
peptide was used. Addition of anti-CD80 mAb to the anergy induction
culture led to partial protection, as judged by proliferation (Fig. 4C) and by IL-2 secretion (data not shown). However,
addition of anti-CD152 caused almost complete protection from the
induction of unresponsiveness, revealed by measurement of proliferation
(Fig. 4, D and E) and IL-2 production (Fig. 4F), indicating that CD152 ligation by the CD80 expressed on
the T cells was responsible for the inhibitory effects of
costimulation-deficient Ag presentation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The function of B7 molecule expression by T cells has remained obscure, particularly for murine CD4+ T cells which cannot present Ag to other CD4+ T cells due to lack of MHC class II expression. Regulatory effects have been suggested based on results obtained using an APC-free system involving immobilized anti-CD3 and anti-CD28 mAbs (17). However, no effects of anti-CD80 Ab were seen in the absence of anti-CD28 (equivalent to costimulation-deficient Ag presentation), and no data concerning the induction of unresponsiveness have been described previously.
The amplifying effects of anti-CD80 Ab described in this paper were only seen when APC-derived costimulation was limiting or absent; if CHO-H2-Ad+CD86high cells were used as APC the anti-CD80 mAb had no detectable effect on T cell reactivity (data not shown). Previous reports have suggested that CD152 is preferentially ligated when APC B7 molecule expression is limiting (13). The findings described in this paper strongly suggest that CD152 ligation, under these circumstances, is due to CD80 expressed by the T cells. It is difficult to rule out the possibility that CD80+ splenocytes contaminated the cultures; however, the T cells had been cultured for 2 wk since the last addition of feeder cells, and were treated with anti-MHC class II Ab followed by Dynalbeads to remove any residual APC before use as responder cells, making it highly unlikely that the CD80 was provided by another cell type. Anti-CD152 was consistently more effective than anti-CD80 at preventing the induction of anergy. The most likely explanation for this is that CD86 also contributes to CTLA-4 ligation. The higher affinity/slower off rate of CD80 makes this the dominant ligand for CD152, but CD86 may also contribute. Although no gross differences in reactivity have been observed with T cells from CD80/CD86 knockout (KO) mice (18), the results obtained in this experiment suggest that differences would only be expected if costimulation-incompetent APC were used (19).
Several reports have suggested that CTLA4-transduced signals modify
CD3-mediated signaling. Indeed, one study described a physical
association between CTLA4 and the 
-chain of the CD3 complex
(20). These events are likely to be most efficient when
they occur in cis (i.e., at the same pole of the T cell as
the TCR:CD3 complex). It is possible that the CD80:CD152 interaction
implied by these results is occurring between neighboring T cells that
are interacting with the same APC (21), or that it
involves CD80 and CD152 expressed on the same T cell surface
(22). In the former case the interaction would be in
trans, occurring away from the TCR:CD3 cluster. In the
latter case, the interaction could be physically proximal to the
cognate-recognition complex. What is not clear is why CD80 on T cells
should preferentially signal through CD152, rather than CD28, as
implied by these results. It has been suggested that the B7 molecules
expressed by T cells fail to ligate CD28, due to altered glycosylation,
but retain the ability to ligate CD152. Using CTLA4-Ig and CD28-Ig
fusion proteins, we found that T cells bound both equally well
suggesting that differential binding of the two ligands is unlikely to
provide an explanation for the dominant effect of CD152 in these
experiments (13). Two alternative explanations for the
dominance of CD152-mediated effects are first, the unusual ratio of B7
molecules expressed by T cells. The 5-fold excess of CD80 has not been
described for any other B7+ cell population.
Given the slower off rate of CD152 from CD80 than CD86, this pattern of
expression may favor preferential ligation of CD152 (23).
A second possibility is that CD152-transduced signals can be delivered
in trans more efficiently than those transduced by CD28.
This explanation is consistent with the general observation that B7
expression by T cells fails to provide transcostimulation, and
overcomes the effects of costimulation-deficient Ag presentation
(24), as would be predicted if CD28 signaling occurred
with any appreciable efficiency in trans.
Our present study may provide an additional explanation as to why CTLA-4 KO (1, 2) and CD80 KO (25) mice display a different phenotype. Our data suggest that CTLA-4 is influential in the induction of T cell unresponsiveness. It follows that self-tolerance in CTLA-4-deficient mice cannot be induced due to lack of this anergy-inducing molecule; therefore, these mice develop fatal lymphoproliferative disease. In contrast, in CD80 KO mice, CD152 can be ligated by CD86, thereby promoting the induction of anergy in response to costimulation-deficient Ag presentation. The situation in CD80/CD86 double-KO mice is more complex due to alterations in delivery of costimulation.
A recent study from Frauwirth et al. (26) indicated that CD152-deficient TCR-transgenic T cells were as susceptible to anti-CD3-induced anergy as normal cells from the same transgenic strain. The most likely explanation for this discrepancy is that the two T cell populations that they studied were CD8+, while the T cells in our study were CD4+. The Frauwirth results are in keeping with those from Bachmann et al. (27) who reported, while using a different CD8+ TCR-transgenic mouse that CD152 played little, if any, role in regulating CD8+ T cell responses. This contrasts with the in vivo observations for CD4+ T cells (5, 6), and with the fact that the predominant cell type that accumulates in the CD152 KO mouse is CD4+ (28).
Incorporating these findings into the physiology of T cell responses in vivo, we would propose that this mechanism of T cell regulation contributes to the effects of Ag presentation by costimulation-deficient cells in two ways. First, it may be one of a number of mechanisms that limit clonal expansion during the evolution of an immune response. Second, it may lead to the silencing of autoreactive T cells that enter inflamed tissues and encounter MHC class II-expressing parenchymal cells presenting tissue-specific autoantigens. Testing of these hypotheses will require additional in vivo experiments.
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Acknowledgments |
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Footnotes |
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2 J.-G.C. and S.V. contributed equally to this manuscript.
3 Address correspondence and reprint requests to Dr. Robert Lechler, Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K.
4 Abbreviations used in this paper: CHO, Chinese hamster overy; ATCC, American Type Culture Collection; cOVA, chicken OVA; KO, knockout.
Received for publication March 8, 2000. Accepted for publication June 28, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
)
or Con A plus A20-II cells (2 x 104/well) in the
absence (•) or presence of anti-CD80 (
), hamster IgG (
), or
anti-CD86 (
) for 3 days. B, BALB/c naive
CD4+ T cells (4 x 104/well) were
stimulated with Con A alone (
) or presence of 10
µg/ml soluble anti-CD80 (
) or 10 µg/ml hamster IgG (
).
G, DO.11.10 naive CD4+ T cells (4 x
104/well) were stimulated with 0.1 µg/ml peptide alone
(
