By
From the Immunology Research Division, Department of Pathology, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
The induction of T cell anergy in vivo is thought to result from antigen recognition in the absence of co-stimulation and inflammation, and is associated with a block in T cell proliferation and Th1 differentiation. Here we have examined the role of interleukin (IL)-12, a potent inducer of Th1 responses, in regulating this process. T cell tolerance was induced by the administration of protein antigen without adjuvant in normal mice, and in recipients of adoptively transferred T cells from T cell receptor transgenic mice. The administration of IL-12 at the time of tolerance induction stimulates Th1 differentiation, but does not promote antigen-specific T cell proliferation. Conversely, inhibiting CTLA-4 engagement during anergy induction reverses the block in T cell proliferation, but does not promote full Th1 differentiation. T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti-CTLA-4 antibody are not anergized, and behave identically to T cells which have encountered immunogenic antigen. These results suggest that two processes contribute to the induction of anergy in vivo; CTLA-4 engagement, which leads to a block in the ability of T cells to proliferate to antigen, and the absence of a prototypic inflammatory cytokine, IL-12, which prevents the differentiation of T cells into Th1 effector cells. The combination of IL-12 and anti-CTLA-4 antibody is sufficient to convert a normally tolerogenic stimulus to an immunogenic one.
Clonal anergy is an important mechanism of peripheral
T cell tolerance (1, 2). The development of anergy
was first analyzed in cloned lines of mouse Th1 cells. These
studies have led to the widely accepted view that anergy is
induced when antigen is recognized in the absence of second signals (3), the best defined of which are costimulators of the B7 family (6). Thus classical studies, showing
that antigens administered with strong adjuvants elicit T
cell responses and antigens without adjuvants induce tolerance, are now interpreted to suggest that an important
function of adjuvants is to enhance the expression of the
costimulators that determine whether antigen recognition
will lead to activation or anergy (3). Based on these ideas,
many attempts have been made to prevent immune reactions, e.g., against organ allografts, and to induce tolerance
by blocking B7 molecules in vivo (7). Conversely, the
ectopic expression of B7 proteins in tissues results in an increased predisposition to tissue-specific autoimmune injury, presumably by breaking T cell tolerance to self antigens in the tissues (11).
Two sets of findings suggest that costimulators alone may
not dictate the choice between T cell activation and tolerance. First, expression of B7-1 as a transgene in tissues does
not, by itself, lead to autoimmune disease. In most of these
studies, an additional local stimulus, such as the proinflammatory cytokine TNF- It is well known that adjuvants induce the production of
numerous cytokines from APCs. One of these cytokines,
IL-12, is a potent, and obligatory, inducer of Th1 differentiation (19). Since T cell anergy preferentially affects Th1
cells (3, 20), we postulated that a deficiency of local IL-12 production may play an important role in the induction
of anergy. We have tested this hypothesis in two experimental models of T cell anergy induced in vivo, i.e., normal mice and recipients of T cells from TCR transgenic
mice treated with high doses of antigen in the absence of
adjuvant. We show that in both models of anergy, IL-12
by itself promotes Th1 differentiation, but not T cell proliferation or expansion, in response to tolerogenic antigen.
Blocking CTLA-4 during anergy induction overcomes the
block in T cell expansion, but does not lead to full Th1 differentiation. Exposure to tolerogenic antigen in the presence of both IL-12 and an anti-CTLA-4 antibody prevents
the induction of anergy, and induces a full T cell response. Thus, anergy induction in vivo involves both a block in
Th1 differentiation and in T cell proliferation. These different aspects of T cell anergy are regulated by IL-12 and
CTLA-4, respectively. Local inflammation also prevents
the induction of T cell anergy, presumably by stimulating
IL-12 secretion and changing the expression and recognition of costimulators. Our results support the potential importance of IL-12 in breaking T cell tolerance, e.g., in autoimmune diseases, and conversely, the potential of IL-12
antagonists in inducing tolerance, e.g., to allografts.
Mice.
BALB/c mice, 6-8 wk old, were purchased from the
Jackson Laboratory (Bar Harbor, ME). Transgenic mice expressing the DO.11.10 TCR (hereafter called DO.11) specific for the
chicken OVA peptide, OVA323-339, in association with I-Ad were
obtained from Dr. D. Loh (Hoffmann-La Roche, Tokyo, Japan). The mice were bred in our pathogen- and viral antibody-free facility in accordance with the guidelines of the Committee on Animals of the Harvard Medical School (Cambridge, MA) and those
prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research
Council (Washington, DC). The mice were typed for the DO.11
by staining peripheral blood cells with antibodies against CD4
and V Adoptive Transfers and In Vivo Tolerance Induction.
To induce
tolerance in normal BALB/c mice, animals were treated with
0.3-1 mg i.p. of OVA or PBS. 7-10 d later, mice were immunized with 100 µg OVA in CFA (Difco, Detroit, MI) and s.c. Draining lymph nodes were harvested 7 d later and assayed as described below. To induce tolerance in TCR transgenic T cells,
we used the protocol described by Kearney et al. (23). Lymph
node and spleen cells pooled from DO.11 transgenic mice were
prepared as previously described (15) and injected intravenously
into unirradiated syngeneic BALB/c recipients, such that each received 5 × 106 KJ-126+ CD4+ T cells. The recipients were either not immunized (naive), or 2 d after cell transfer were treated
subcutaneously in the footpad with 50 µg of the OVA323-339 peptide emulsified in IFA (immunized) or intravenously with 300 µg
of the peptide in PBS (tolerized). The peripheral lymph nodes (submandibular, axillary, brachial, inguinal, and popliteal) were harvested 3 d after treatment in all the groups except for the immunized recipients, from which only the draining lymph nodes (inguinal and popliteal) were harvested. For in vivo challenge experiments, mice treated as described above were immunized subcutaneously in the footpad with 50 µg of the peptide emulsified
in IFA 10 d after the initial treatment, and the draining lymph
nodes (inguinal and popliteal) were harvested 3 d later. To study
the effects of cytokines and costimulation on the induction of tolerance, mice were treated either with 0.3-3 µg of murine IL-12 (gift
from Dr. S. Wolf, Genetics Institute, Andover, MA) or 100 µg of
anti-CTLA-4 antibody (reference 18; gift from Dr. J. Bluestone,
University of Chicago, Chicago, IL) on days In Vitro Proliferation and Cytokine Assays.
Harvested cells were
assayed as previously described (15). In brief, 5 × 105 lymph node
cells from each of the groups were cultured in 0.2 ml of RPMI
1640 supplemented with 1 mM L-glutamine, penicillin, streptomycin, nonessential amino acids, sodium pyruvate, Hepes (all
from Life Technologies, Grand Island, NY), 5 × 10 Intracellular Staining for IFN- T cell responses to antigen are induced by administering a foreign
antigen subcutaneously with an adjuvant. In contrast, T cells
are rendered unresponsive when high doses of antigen are given systemically in the absence of adjuvant (20). A
vigorous response to OVA can be detected in the peripheral lymph node cells of BALB/c mice immunized with
OVA in CFA subcutaneously (Fig. 1). If these mice are
pretreated with 1 mg of OVA in saline i.v., they fail to
generate Th1 responses when they are subsequently immunized with antigen in CFA (Fig. 1). Noticeably, Th2 responses to OVA are not abrogated in tolerized mice. The
observation that tolerance is associated with a selective Th1
block led us to examine whether anergy induction was a
result of antigen recognition in the absence of the major
Th1-inducing cytokine, IL-12 (19). Moreover, the production of IL-12 is enhanced by inflammatory stimuli, and
it is likely to be poorly expressed under the conditions where anergy is induced. When mice are tolerized and
given three daily injections of recombinant IL-12 (1 µg
i.p.), the T cells secrete IFN-
The limitation of studying T cell responses to immunogenic and tolerogenic antigen in normal mice is that the
fate of antigen-specific T cells can not be followed. Therefore, it is difficult to determine if the inability of T cells to
respond to antigen is due to the deletion, migration, or
functional inactivation of antigen-specific T cells. To study
the regulation of T cell anergy by IL-12 in antigen-specific
T cells, we used the method described by Kearney et al.
(23), in which small numbers of DO.11 transgenic T cells (specific for OVA323-339+ I-Ad) are adoptively transferred
into syngeneic BALB/c mice and exposed to antigen in
immunogenic or tolerogenic form. Approximately 1-2% of
the peripheral lymph node cells in these adoptive transfer recipients are OVA-specific DO.11 T cells, and these can
be readily followed using a clonotypic mAb (KJ-126; reference 24). DO.11 T cells can be tolerized in vivo by treating adoptive transfer recipients with high doses of aqueous
OVA323-339 peptide intraveneously (see below, and references
15 and 23).
Two important features of the adoptive transfer system
are that responses of naive T cells can be measured, and that it
is possible to detect changes in functional response patterns
within 3 d of antigen exposure. At this time, one can
readily distinguish naive, activated (immunized), and tolerized T cells (Fig. 2). Naive cells produce IL-2 with a peak
at ~day 3 of in vitro stimulation with antigen, proliferate
weakly, and do not produce IFN-
To exclude a contribution of differential T cell expansion in vivo on responses to antigen restimulation, CD4+ T
cells were purified from the lymph nodes of naive, immunized, and tolerized mice that either had been treated or
had not been treated with IL-12. By determining the percentage of KJ-126+ cells present in these purified CD4+ T
cell populations, we were able to set up restimulation assays
with the same number of DO.11 T cells from each group
of mice (4,000-5,000 KJ-126+ T cells/well for proliferation assays and 20,000-25,000 KJ-126+ T cells/well for cytokine assays), and use syngeneic spleen cells from untreated
mice as APCs. As shown in Fig. 3, IFN-
Since IL-12 is known to stimulate both T cells and NK
cells to produce IFN-
Finally, we have also examined the effects of different
doses of IL-12 on T cell expansion and functional responses. The injection of tolerogenic antigen (aqueous
peptide intravenously) results in a significant expansion of
antigen-specific T cells, although this is not as great as the
expansion seen in draining lymph nodes after priming with
peptide in adjuvant (Table 1). Administration of up to 1 µg/d of IL-12 together with tolerogenic antigen has a
small effect on T cell numbers. This effect is more pronounced at higher concentrations of IL-12 (Table 1), and is
probably due to an antigen-independent polyclonal stimulation induced by IL-12 (24a). If adoptive transfer recipients are treated with IL-12 alone, without antigen, the
numbers of lymph node cells recovered, as well as the
numbers of KJ-126+ cells, increase about two- to threefold
(Table 1). Lower doses of IL-12 do not induce this polyclonal response, but at these doses the Th1 response is also
much less. Interestingly, despite the T cell expansion seen
with tolerogenic antigen, and even more with tolerogenic
antigen + IL-12, the cells do not proliferate upon in vitro
restimulation. This suggests that IL-12 may augment the
abortive T cell response to tolerogenic antigen, but is not able to prevent the proliferative block associated with anergy.
, has to be provided (13). Second,
we have recently shown that completely blocking costimulation during exposure to antigen leads to a state of clonal
ignorance and not to anergy in vivo (15). In fact, anergy
develops when antigen-specific T cells use the inhibitory
receptor for B7 molecules, CTLA-4, to interact with costimulators at the time of antigen recognition. These results provide an explanation for the severe autoimmune disease
that develops in mice in which CTLA-4 is deleted by targeted gene disruption (16, 17). Thus, the choice between
T cell activation versus anergy may be determined partly by
whether CD28 or CTLA-4 engages B7 molecules on
APCs (18). Although the mechanisms that influence this
choice are not yet defined, the findings do suggest that simply
the absence of costimulation may not be the mechanism of T cell anergy in vivo.
8.
1, 0, and +1 of
the initial antigen exposure. To study the effects of local inflammation on the induction of tolerance, tolerized mice were given
PBS emulsified in CFA subcutaneously in the footpad on day 0. After the harvest, the lymph node cells from each group were
stained with CyC-labeled anti-CD4 mAb (PharMingen, San Diego, CA) and biotinylated KJ-126 clonotypic antibody specific
for the DO.11 followed by streptavidin-FITC for FACS® analysis. Analyses were performed on a FACScan® flow cytometer.
5 M 2-ME,
and 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO). Cells were restimulated with 0-1 µg/ml of OVA323-339.
After 48 h, cultures were pulsed for 6 h with 1 µCi [3H]TdR
(New England Nuclear, Boston, MA), and incorporated radioactivity was measured in a Betaplate scintillation counter (LBK
Pharmacia, Piscataway, NJ). To determine cytokine production,
2 × 106 lymphocytes were cultured in 1 ml of medium in the
presence of 0, 0.1, or 1 µg/ml of OVA323-339. Supernatants were
collected after 24, 48, and 72 h, and levels of IL-2, IL-4, and
IFN-
were assayed by ELISA as described previously (15). In
some experiments, CD4+ T cells were purified from the lymph
nodes of experimental mice using CD4+ Dynalbeads® (Dynal
A.S., Oslo, Norway), and cultured with mitomycin C-treated BALB/c splenocytes as APCs. Typically, T cell preparations were >98% CD4+ as determined by staining and flow cytometry.
.
5 × 105 lymph node cells
were restimulated with 1 µg/ml OVA323-339 for 24 h. During the
last 6 h, 10 µg/ml brefeldin A (Sigma Chemical Co.) was added
to cultures. The lymph node cells were then washed in PBS/1%
BSA containing 10 µg/ml brefeldin A, stained with antibodies to
CD4 and the DO.11 (with the monoclonal antibody KJ-126; reference 24 ), and fixed overnight with 4% paraformaldehyde (Sigma
Chemical Co.). The fixed cells were permeabilized by incubation
in PBS with 1% BSA and 2% saponin (Sigma Chemical Co.) at
room temperature for 10 min. A phycoerythrin-conjugated anti-
IFN-
antibody (PharMingen), diluted to 20 µg/ml in PBS with
1% BSA and 2% saponin was then added, and the cells were incubated for a further 30 min. Finally, the cells were washed once
with PBS with 1% BSA and 2% saponin and once with PBS with
1% BSA, and analyzed on a FACScan® flow cytometer.
IL-12 Promotes Th1 Differentiation in Response to Tolerogenic Antigen, but Does not Rescue T Cell Proliferation.
after a challenge with antigen in adjuvant (Fig. 1). However, IL-12 is not able to prevent the proliferative block associated with anergy.
Fig. 1.
IL-12 promotes Th1 differentiation in response to tolerogenic antigen in vivo. BALB/c mice were pretreated on day 0 with saline (immunized) or 1 mg of OVA i.v. (tolerized), with or without daily treatments (days 1, 0, +1) of 1 µg of IL-12 (tolerized + IL-12). All mice
were immunized with OVA emulsified in CFA in the footpad on day 10. The popliteal and inguinal lymph nodes were harvested 7 d later, and
lymph node cells were restimulated in vitro with OVA. Proliferative responses (A) were assessed after 72 h by measuring [3H]thymidine incorporation, and cytokine secretion (B) was assayed by ELISA. Results are from
one representative experiment of three. The error bars represent standard
deviations calculated from duplicate wells.
[View Larger Version of this Image (26K GIF file)]
. T cells from immunized mice produce IL-2 rapidly, show strong proliferation, and secrete IFN-
. (The decline in supernatant IL-2 levels
with increasing duration of culture may be due to absorption of secreted IL-2 by the proliferating T cells.) Tolerized
cells do not proliferate or secrete IL-2 or IFN-
. Similar to
the results obtained with normal BALB/c mice (Fig. 1), administration of IL-12 (1 µg/d) at the time of tolerance induction markedly enhances IFN-
secretion, but has no effect on IL-2 production and proliferation in this adoptive
transfer tolerance system (Fig. 2).
Fig. 2.
IL-12 promotes early Th1 differentiation, but not T cell proliferation, in antigen-specific T cells exposed to tolerogenic antigen. BALB/c recipients of DO.11 T cells were left untreated (A); immunized with OVA323-339 peptide in IFA in the footpad on day 0 (B); tolerized with OVA323-339
peptide intravenously on day 0 (C); or tolerized and treated daily (days 1, 0, +1) with 1 µg of IL-12 (D). The submandibular, axillary, brachial,
popliteal, and inguinal lymph nodes (only popliteal and inguinal lymph nodes in B) were harvested 3 d later. Lymph node cells were restimulated in vitro
with OVA323-339 peptide. Proliferative responses were assessed after 72 h by measuring [3H]-thymidine incorporation, and are corrected for the numbers of KJ-126+ CD4+ cells per well. Cytokine secretion was assayed daily by ELISA. Results are from one representative experiment of three. The error bars
represent standard deviations calculated from duplicate wells.
[View Larger Version of this Image (34K GIF file)]
is detected in
cultures of purified T cells from tolerized mice given IL-12, demonstrating that they are able to secrete this cytokine
when restimulated with antigen in vitro. These T cells do
not produce IL-2 or proliferate when restimulated with
OVA323-339 peptide, confirming that IL-12 does not prevent the block in T cell proliferation associated with anergy
induction. The results obtained in this set of experiments,
using equivalent numbers of purified antigen-specific T
cells from different experimental groups of mice, were
identical to those obtained with bulk lymph node cultures
(Fig. 2). We therefore used unfractionated lymph node
cells in all subsequent experiments.
Fig. 3.
Purified, anergic antigen-specific T cells treated with IL-12 in vivo produce IFN-, but do not proliferate. BALB/c recipients of DO.11 T
cells were treated as in Fig. 2. CD4+ T cells were purified from lymph node cells. An aliquot of the purified CD4+ T cells was stained with antibodies to
CD4 and the DO.11 (KJ-126) to determine the percentage of KJ-126+ T cells present. Equal numbers of antigen-specific T cells were restimulated in
vitro with OVA323-339 peptide and syngeneic spleen cells from BALB/c mice as APCs. Proliferative responses of 5,000 DO.11 T cells/well, and cytokine secretion from 25,000 DO.11 T cells/well, were assayed as in Fig. 2. Results are from one representative experiment of two. The error bars represent
standard deviations calculated from duplicate wells.
[View Larger Version of this Image (31K GIF file)]
, an important question is, what cells
produce IFN-
in mice given tolerogenic antigen + IL-12.
When lymph node cells from tolerized mice treated with
IL-12 are restimulated in vitro, the secretion of IFN-
is
antigen dose-dependent, suggesting that OVA323-339-specific DO.11 T cells are the source of this cytokine. The
production of IFN-
can be directly visualized by intracellular staining and flow cytometry. As shown in Fig. 4, antigen-specific T cells exposed in vivo to immunogenic antigen or tolerogenic antigen and IL-12 synthesize detectable
levels of IFN-
when restimulated with antigen in culture.
In contrast, naive or tolerized T cells do not make IFN-
,
and under these conditions KJ-126
cells also do not synthesize IFN-
. Therefore, in lymph node cells stimulated
with antigen in vitro, the major source of IFN-
is the antigen-specific T cells.
Fig. 4.
Intracellular staining for IFN- in immunized and
tolerized DO.11 T cells. BALB/c
recipients of DO.11 T cells were
treated as in Fig. 2. CD4+ T cells
were purified from lymph node
cells. Lymph node cells were restimulated in vitro with 1 µg/ml
OVA323-339 peptide for 24 h,
stained for expression of CD4, the
DO.11, and intracellular IFN-
as described in Materials and
Methods, and analyzed by flow
cytometry. The dot plots show
expression of KJ-126 and IFN-
in CD4+ T cells, and the histograms show the expression of
IFN-
in KJ-126+ CD4+ T cells
(continuous black lines). Control
stains are represented by the discontinuous grey lines.
[View Larger Version of this Image (37K GIF file)]
We have recently
shown that tolerance induction in vivo is due to an active
inhibition that requires the recognition of B7 molecules by
the inhibitory T cell receptor for B7, CTLA-4 (15). This
raises the possibility that blocking CTLA-4 may enable T cells to proliferate in response to tolerogenic antigen and,
together with IL-12, may convert a normally tolerogenic
stimulus to an immunogenic one. To test this, DO.11
transfer recipients were tolerized by injection of aqueous
peptide antigen, and treated with anti-CTLA-4 mAb, IL-12, or both. As shown in Fig. 5, in mice given tolerogenic
antigen, anti-CTLA-4 mAb significantly enhances IL-2
production and antigen-specific T cell proliferation, IL-12 alone promotes IFN- production, and the two together
stimulate responses to tolerogenic antigen that are comparable to those induced in draining lymph node cells after
immunization with antigen in adjuvant. Furthermore,
blocking CTLA-4 enables T cells to expand in vivo after
administration of tolerogenic antigen (Table 2).
|
The expression of IL-12 and costimulatory molecules by
tissue APCs is enhanced by adjuvants and various microbial
products (6, 19). We therefore asked if local inflammation
provided by a strong adjuvant would also prevent the induction of T cell tolerance. To do this, DO.11 transfer recipients were given tolerogenic antigen (aqueous peptide
intravenously) and treated with CFA, a potent adjuvant,
subcutaneously. The expansion and functional responses of
T cells were assayed in peripheral lymph nodes which did
or did not drain the site of inflammation. As shown in Fig. 6, anergy is induced in lymph nodes away from the site of
CFA injection. However, T cells in lymph nodes which
are near the sites of inflammation are not anergized, and respond as if they had encountered immunogenic antigen.
Furthermore, the expansion of T cells in response to
tolerogenic antigen is significantly enhanced in the lymph
nodes near the source of inflammation. In two experiments, the numbers of KJ-126+ cells (× 104) in lymph
nodes of different mice were: naive, 2.3 ± 0.6; immunized (draining nodes only), 27.1 ± 4.8; tolerized, 17.1 ± 5.9;
tolerized + local CFA (draining nodes only), 45.6 ± 12.3;
and tolerized + distant CFA (nondraining nodes), 15.8 ± 11.1. Thus, a potent inflammatory stimulus leads to local T
cell expansion and Th1 differentiation even in response to
the systemic administration of normally tolerogenic antigen.
The studies described in this paper were aimed at analyzing the mechanisms of peripheral T cell tolerance in vivo. Specifically, we examined the roles of a prototypic inflammatory cytokine, IL-12, and costimulatory molecules in this process. The experimental models we have chosen involve treating normal mice, or mice into which TCR transgenic T cells are transferred, with high doses of antigen without adjuvants. In the TCR transgenic system, it is possible to formally demonstrate that antigen-specific T cells are not killed when they are exposed to tolerogenic antigen, but are rendered functionally unresponsive, in that they become incapable of producing IL-2, proliferating, and differentiating into Th1 effectors (Figs. 2 and 3, and references 15 and 23). By these criteria, the form of tolerance induced by aqueous peptide antigen appears to be an in vivo counterpart of the phenomenon of clonal anergy first described in T cell clones (3, 4).
Our recent results have demonstrated that the role of
B7-mediated costimulation in T cell anergy in vivo is complex, and somewhat unexpected. We have found that a B7
antagonist does not promote tolerance, as might have been
expected based on the results of in vitro studies (3, 4), but
rather it prevents the induction of anergy and keeps T cells
in a naive but functionally competent state. The induction
of tolerance depends on the engagement of B7 molecules,
presumably on APCs, by the CTLA-4 counter receptor on
antigen-responsive T cells (15). In striking contrast, T cell
priming, as measured by proliferation and differentiation into effector cells, is dependent on B7 recognition by the
activating counter-receptor, CD28 (15, 25). These results
have led to the conclusion that a key determinant of T cell
activation versus tolerance is which receptor, CD28 or
CTLA-4, is used to engage B7 costimulators during T cell
antigen recognition (15). During these studies, we realized
that the block in Th1 differentiation associated with tolerance induction in vivo is unlikely to be solely due to
CTLA-4-B7 interactions, because giving anti-CTLA-4 antibody at the time of tolerance induction led to variable
amounts of IFN- produced by antigen-specific T cells
(15). Moreover, in the earlier studies, tolerance was induced by intraperitoneal injection of peptide in IFA, and in
subsequent experiments, when we induced tolerance with
aqueous peptide without any adjuvant, anti-CTLA-4 alone
was ineffective at allowing Th1 differentiation (Fig. 5).
This is not surprising, because the development of Th1 effector cells requires IL-12, which is produced by macrophages and dendritic cells usually in response to microbes
and other inflammatory stimuli (25). Tolerogenic antigens,
such as aqueous peptides administered intraveneously, are
unlikely to stimulate IL-12 production, since such antigens
do not contain macrophage-activating substances. We
therefore reasoned that to completely prevent T cell anergy
and allow effector cell development, it might be necessary both to change the pattern of costimulator recognition and
to provide IL-12. Our experimental results support this notion. A combination of anti-CTLA-4 antibody and recombinant IL-12 is sufficient to convert a tolerogenic form of
peptide antigen to a fully immunogenic form, whereas neither alone is sufficient (Fig. 5). These conclusions are most
convincingly demonstrated in the TCR transgenic adoptive transfer system, where the fates of antigen-specific
TCR transgenic T cells can be followed quantitatively.
However, it is likely that the same is true of tolerance induced in normal T cell populations, since IL-12 promotes
Th1 differentiation but does not fully restore T cell expansion in normal mice (Fig. 1). Furthermore, a strong adjuvant, which induces local inflammation, has the same functional effects on tolerance induction as the combination of
anti-CTLA-4 antibody and IL-12 (Fig. 6). Although this
does not prove that the effects of the adjuvant are due to a
change in costimulation and IL-12 production, the findings
are consistent with this idea. Importantly, IL-12 alone stimulates production of the signature Th1 cytokine, IFN-
,
but does not prevent the proliferative block associated with
tolerance induction (Figs. 2 and 3). Therefore, the inflammatory cytokine by itself is insufficient for preventing T cell
tolerance.
The mechanisms by which anti-CTLA-4 antibody and
IL-12 cooperate to convert a tolerogenic stimulus to an
immunogenic one are not precisely defined. It is likely that
tolerogenic antigens administered without adjuvants induce
low levels of B7 costimulator expression on APCs. Such
low levels may preferentially engage the high-affinity receptor for B7 molecules, which is CTLA-4, triggering inhibitory biochemical signals which block IL-2 production and T cell proliferation (26, 27). Antigen recognition by T cells in the absence of IL-2 and proliferation is known to
lead to functional anergy (3, 5). Immunogenic forms of antigen, such as peptides administered with strong adjuvants,
may induce higher levels and more prolonged expression of
B7 molecules, resulting in the engagement of CD28 and T
cell activation. Thus, changing B7 recognition from
CTLA-4 to CD28 may be sufficient to release the block in
IL-2 production and T cell proliferation. It appears unlikely
that this alone would induce T cell differentiation into effectors, because differentiation requires another signal(s), which, for the Th1 pathway, is provided by IL-12. Thus,
adjuvants may trigger two distinct reactions that contribute
to preventing T cell tolerance and eliciting immune responsesthey stimulate IL-12 production, and they alter
costimulator expression in a way that promotes engagement by CD28.
The ability of IL-12 to function in preventing tolerance
induction is consistent with the finding that IL-12 is produced locally in lesions associated with autoimmune diseases (28, 29). However, in these situations it is not possible
to establish if IL-12 production is a cause of the autoimmune reactions or the consequence of local T cell activation and IFN- secretion. Moreover, it is not known if
blocking IL-12 will promote tolerance induction. Clearly,
this may not be an adequate treatment for inducing tolerance by itself, because if mice are immunized and treated
with IL-12 antagonists, the result is a defect in Th1 differentiation, and not anergy (30). The same result is seen in
mice lacking the p40 chain of IL-12, and in mice in which
the major IL-12-triggered transcription factor, Stat4, is deleted (31). However, IL-12 antagonists administered with agents that favor CTLA-4-B7 interactions, or with
forms of antigens that preferentially trigger such interactions, may enhance the induction of anergy. It remains to
be determined if this will be useful for the thaerapeutic induction of tolerance to prevent allograft rejection, or to
treat Th1-dependent autoimmune diseases.
Address correspondence to Dr. Abul K. Abbas, Brigham & Women's Hospital and Harvard Medical School, LMRC-521, 221 Longwood Ave., Boston, MA 02115. Phone: 617-732-6523; Fax: 617-732-5795. Present address for V.L. Perez is Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02108.
Received for publication 15 April 1997 and in revised form 16 June 1997.
1 Abbreviations used in this paper: DO.11, DO.11.10 TCR.We thank Dr. Jeff Bluestone for generous gifts of anti-CTLA-4 antibody and for his critical comments on the manuscript, and Dr. Stan Wolf for gift of recombinant IL-12. Supported by National Institutes of Health grants AI-35297 and AI-25022 (A.K. Abbas), T32 HL-07267 (V.L. Perez), and K01 RR-00121 (C.A. London), and a National Cancer Institute training grant (R.G. Maki).
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