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Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production upon rechallenge.
Finally, in contrast to rapamycin, full T cell activation in the
presence of hydroxyurea (which inhibits the cell cycle in early S
phase) did not result in anergy. These data suggest that it is neither
the direct effect of costimulation nor the subsequent T cell
proliferation that prevents anergy induction, but rather the
biochemical events that occur upon progression through the cell cycle
from G1 into S phase. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vis-à-vis anergy, a simple interpretation of these data is that
signal 2 directly prevents the induction of anergy. However, a number
of subsequent experimental observations did not support this theory.
DeSilva et al. showed that incubating T cell clones with live APCs plus
Ag and anti-IL-2 along with anti-IL-2R Abs resulted in the
induction of anergy 9 . Beverly et al. also demonstrated the induction
of anergy in the presence of costimulation by eliminating the APCs and
changing the medium at 16 h after initiation of the cultures.
These manipulations stopped IL-2 production and removed all the IL-2
that had been produced 10 . The interpretation of both sets of
experiments was that, despite costimulation, anergy was induced by
preventing IL-2R engagement. This led Jenkins 11 and Beverly 10 to
propose that anergy is induced when signal 1 is encountered in the
absence of proliferation. In this model, costimulation prevents anergy
induction indirectly by enhancing IL-2 production and promoting cell
division. Consistent with this idea are the findings of Boussiotis et
al., who showed that signaling through the common -chain could
prevent anergy induction in human T cells 12 , as well as the work of
Gilbert and Weigle, who proposed that anergy was the result of G1a cell
cycle blockade 13 . The model also implies that TCR engagement (in
addition to providing the positive signals necessary for IL-2
production) results in the production of negative regulatory factors.
The abilities of cyclosporin and cycloheximide to inhibit anergy
induction supports this concept as these agents prevent NF-AT-induced
transcription and protein synthesis, respectively, which might be
necessary for the production of these negative regulatory factors
5, 6 . In the absence of proliferation, these factors accumulate and
thus the cell fails to respond upon restimulation.
Despite these data, the precise mechanism(s) leading to the induction of anergy have not yet been elucidated, and several groups have focused on the direct ability of costimulation to prevent anergy. Based on studies using CD28 knockout mice, Bachmann et al. suggest that signaling through CD28 has both costimulatory (signal 2c) and tolerance prevention (signal 2t) abilities 14 . Furthermore, Becker et al. have demonstrated the up-regulation and increased binding of the negative regulatory transcription factor Nil-2a when T cells are stimulated by signal 1 alone 15 . Nil-2a has been shown to bind to the IL-2 promoter at the negative regulatory element (NRE) and inhibit expression of reporter constructs 16 . In the presence of costimulation, Nil-2a does not appear to be up-regulated 15 . Thus, this group proposed that costimulation directly inhibits anergy induction by preventing the enhancement of Nil-2a function following TCR occupancy.
In the present work we sought to better define the events responsible
for anergy induction by using the immunosuppressive agent rapamycin.
Rapamycin is a macrolide antibiotic with a similar biochemical
structure to cyclosporin A
(CSA)2 and FK506 17 .
Indeed, the intracellular target for rapamycin is FK506 binding protein
(FK506BP). However, unlike CSA and FK506, rapamycin does not inhibit
IL-2 production 18 . Rather, it inhibits the ability of lymphocytes to
proliferate in response to IL-2. It is thought that the
rapamycin-FK506BP complex does this by binding to and
inhibiting the mammalian target of rapamycin (mTOR), a serine/threonine
kinase that is believed to be a critical step in the pathway leading to
the down-regulation of the cell cycle inhibitor Kip-1 following IL-2R
engagement 17 . As a result, the cell fails to progress from G1 into S
phase and does not proliferate. Therefore. we incubated the
CD4+ Th1 clone A.E7 overnight with or without costimulation
in the presence or absence of rapamycin. Our data show that even when
given full signal 1 and signal 2, A.E7 cells are rendered anergic if
cultured in the presence of rapamycin. Furthermore, the
rapamycin-induced anergy appears to be more complete than conventional
anergy in that there is also a marked decrease in IFN- and IL-3
production upon restimulation. On the other hand, A.E7 T cells
stimulated with signal 1 plus 2 in the presence of hydroxyurea, an
agent which arrests proliferation in S phase, are not rendered anergic.
These data suggest that it is neither direct costimulation nor
proliferation per se that prevents anergy induction, but rather the
biochemical events that occur upon progression through the cell cycle
from G1 to S phase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A.E7 is a CD4+ Th1 clone specific for the pigeon cytochrome c (PCC) peptide 81–104. It was grown and maintained as previously described 19 . Briefly, cells were stimulated for 48 h with whole PCC and irradiated (3000 rad) B10.A splenocytes as a source of APCs. The cells were next expanded 20:1 in 10 U/ml of rIL-2. After a minimum of 12 days in culture, when the IL-2 was consumed and the cells were rested, the live cells were isolated from a Ficoll gradient and utilized. Anergy induction was achieved by overnight incubation of 20–40 x 106 A.E7 cells in a T75 tissue culture flask (Costar, Cambridge, MA) that had previously been coated with anti-TCR-ß Ab H57-597 20 at a concentration of 10 µg/ml. Some cultures were also supplemented with ascitic fluid containing the anti-CD28 mAb 37.51 21 (a kind gift from Dr. James Allison) at a final dilution of 1:5000. CSA (Calbiochem, Cambridge, MA) was added to some cultures at a final concentration of 100 or 1000 nM, while rapamycin (a generous gift of Dr. S. Sehgal, Wyeth-Ayerst, Princeton, NJ) was added to other cultures at the same concentrations. Both the cyclosporin and the rapamycin were dissolved in ethanol. Preliminary studies revealed that adding an equivalent volume of ethanol (vehicle alone) did not affect cell cultures (data not shown). Hydroxyurea (Sigma, St. Louis, MO), dissolved in water, was added to some cultures at a concentration of 2 mM. None of the drugs used caused a decreased viability as determined by trypan blue exclusion during the overnight incubation period. After the overnight incubation, the cells were removed from the flask, washed, and recultured in fresh medium for 5–14 days before rechallenge. At this time, the cells were reisolated and assayed for their ability to proliferate or produce IL-2.
Proliferation assay
Anergized or control clones were assessed for their ability to proliferate to PCC by adding 2 x 104 A.E7 cells to 50 x 104 B10.A irradiated splenocytes (3000 rad) and increasing doses of PCC in 96-well plates in triplicate. After 48 h, the cells were pulsed with [3H]thymidine and harvested 16 h later, and thymidine incorporation was determined using a betaplate reader. Controls included A.E7 cells cultured in the presence of 50 U/ml of exogenous IL-2, which were pulsed and harvested as above.
Cytokine production and measurement
Conditions were determined for optimal IL-2 production by the
clones. A total of 50 x 104 anergized or control
cells were added to 24-well plates, precoated with 10 µg/ml
anti-TCR, with a 1:5000 dilution of anti-CD28 in a total volume
of 0.5 ml. The supernatant fluids were harvested after 16 h and
frozen at -20°C until they were used. IL-2 was assessed by measuring
the proliferation of the IL-2-dependent CTLL cell line (American Type
Culture Collection, Manassas, VA) 22 as described previously 23 .
Each IL-2 determination was calculated from eight twofold serial
dilutions of supernatant fluids. In addition, IL-2, IFN-, and IL-3
were assayed by ELISA (Endogen, Woburn, MA) according to the
manufacturer’s instructions. Each value was calculated from either two
or three serial dilutions of supernatant fluid.
Assessment of extracellular regulatory kinase (ERK) activation
Anergized and control cells (4 x 106 in 50 µl) were stimulated or mock stimulated in anti-TCR (10 µg/ml)-coated 24-well plates floated in a 37°C water bath for 10 min. At that time, the cells were lysed in 150 µl of SDS sample buffer that was supplemented with 1 mM sodium vanadate. PAGE with a 10% gel was performed on 25 µg of each sample, and the samples were then transferred to nitrocellulose. Western blot analysis was performed using 1 µg/ml of anti-phospho-ERK (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-ERK (Upstate Biotechnology, Lake Placid, NY). Secondary Ab consisted of alkaline-phosphatase-labeled anti-mouse (Santa Cruz) (1:10,000) or anti-rabbit (Sigma) (1:5000) Abs 24 . The blots were developed using Vistra ECF substrate (American Life Sciences, Arlington Heights, IL) and the blue fluorescence mode of the STORM Phosphorimager (Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The strategy behind using rapamycin was that it would enable us to
stimulate A.E7 cells with signal 1 plus 2, allow for the production and
secretion of IL-2 as well as engagement of the IL-2R, but prevent
IL-2-induced proliferation. Previously, Dumont et al. demonstrated that
rapamycin inhibited proliferation but not IL-2 production by murine T
cells 18 . Therefore, initial experiments were performed to confirm
that rapamycin was able to inhibit IL-2-induced proliferation in the
A.E7 clone and not affect IL-2 production. In these experiments,
similar doses of CSA were used for comparison. Fig. 1
A formally demonstrates that
CSA does not inhibit proliferation of the A.E7 cells in response to
exogenous rIL-2 (50 U/ml), while rapamycin inhibits proliferation in a
dose-dependent fashion. The ID50 for rapamycin was 
4 nM.
On the other hand, we see the exact opposite effect for IL-2
production. A.E7 cells were cultured with plate-bound anti-TCR and
soluble anti-CD28 in 24-well plates overnight, and supernatant
fluids were collected and assayed by ELISA for IL-2 production. As seen
in Fig. 1B, CSA inhibited IL-2 production completely at a
concentration of 100 nm. On the other hand, for the same concentrations
of rapamycin tested, IL-2 production was similar to that of the control
cells. This overnight stimulation protocol is precisely the same as
that we used to induce anergy. Of note, when the clones were stimulated
with anti-TCR and anti-CD28 overnight and then pulsed for an
additional 12 h with [3H]thymidine, proliferation
resulted in the uptake of 24,500 cpm, while in the presence of 1,000 nM
CSA or rapamycin there was only 242 and 267 cpm, respectively. In this
case, the CSA inhibited proliferation by inhibiting IL-2 production,
while the rapamycin inhibited proliferation by endogenously produced
IL-2.
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To induce anergy, A.E7 cells are normally incubated with plate-bound anti-TCR overnight (induction phase). The cells are then harvested, washed, and rested in fresh medium. After a minimum of 5 days, the cells are rechallenged with either APCs plus Ag or anti-TCR plus anti-CD28. In the following experiments, anti-CD28 with or without CSA or rapamycin was added to the induction phase. Of note is the fact that the rapamycin and CSA are only present during the 16-h overnight incubation and not during the resting or rechallenge phase. After the overnight culture to induce anergy, the cells are washed and the drugs are removed.
A.E7 cells stimulated in the presence of signal 1 alone proliferate
less effectively upon rechallenge with APCs and Ag than cells that
received signal 1 plus 2 during the induction phase; this is
conventional anergy (Fig. 2A).
As has been previously shown, the presence of CSA during the induction
phase inhibits the development of the anergic state 5 . In other
experiments, we have determined that the enhanced proliferation of the
CSA-treated cells when compared with the signal 1 plus 2-treated cells
is actually the same level of proliferation as nonmanipulated cells
(data not shown). In contrast, the presence of rapamycin during the
induction phase with signal 1 alone does not interfere with anergy
induction, but rather results in strong anergy (Fig. 2B). In
fact, in this particular experiment, the presence of rapamycin appears
to enhance conventional anergy. We have noted this enhancement in a
number of experiments, though not all, (see Fig. 3 for example), particularly if the
signal 1-induced anergy is not optimal. Rapamycin in the absence of
anti-TCR had no effect on the cell cultures (data not shown). Most
important, though, is the observation that the presence of rapamycin
promotes anergy induction even in the presence of full costimulation.
Thus, in the presence of rapamycin, costimulation does not inhibit the
induction of anergy. Finally, a characteristic of anergic T cell clones
is that they are able to proliferate to exogenous IL-2, because they
constitutively express low levels of the high-affinity IL-2R. As seen
in Fig. 2C, the cells anergized in the presence of rapamycin
proliferated to exogenous IL-2 as well as the conventionally anergized
cells and slightly less effectively than the cells that were given
signal 1 plus 2 without rapamycin. Also, as expected, the cells that
were initially incubated with CSA proliferated to the exogenous IL-2.
In addition to their ability to respond to exogenous IL-2, the
rapamycin-induced anergic cells were able to up-regulate CD25 and CD69
in response to TCR stimulation as well as normal T cell clones and
their conventionally anergized counterparts (data not shown). The TCR
level was also comparable (data not shown).
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As seen in Fig. 2
, CSA inhibits anergy induction while the
presence of rapamycin results in anergy induction even in the presence
of signal 2. Thus, the question arises as to what happens when the two
drugs are both present during the induction phase. Fig. 3 shows once
again that the presence of rapamycin during the induction phase
promotes anergy even in the presence of costimulation. On the other
hand, when CSA is also added to this culture, anergy induction is
abrogated. CSA’s ability to inhibit anergy dominates over the ability
of rapamycin to induce anergy. This is presumably because CSA blocks
anergy induction proximally by inhibiting the production of negative
regulatory factors. The results are consistent with the idea that
rapamycin works distally, possibly by inhibiting proliferation and/or
the inactivation of the negative regulatory factors.
Hierarchy of anergy induction
Cells that were stimulated during the induction phase with both
signal 1 and signal 2 appeared to be less responsive than
nonmanipulated resting cells. These cells appeared to be partially
anergized. To test this directly, we compared the proliferative
response of cells that had been stimulated overnight with signal 1 plus
2, rested, and rechallenged, to cells that were "mock" stimulated,
rested, and rechallenged. As seen in Fig. 4A, cells that were stimulated
with signal 1 plus 2 during the induction phase proliferated less
effectively upon rechallenge than cells that were not stimulated at all
(mock stimulation). This observation is consistent with the results of
Beverly et al., who showed that anergy could be induced in the presence
of signal 1 plus 2 if APCs and the medium were removed and the cells
washed free of IL-2 10 . In our system, after 16 h of signal 1
plus 2, the stimulating Abs and medium are also removed. It might be
that despite the high levels of IL-2 produced during the 16 h with
signal 1 plus 2, there is not sufficient time to induce the cells to
proliferate or completely inactivate the negative regulatory factors.
In addition, under such culture conditions, the cells are continuously
being stimulated by signal 1 and thus presumably continuously
replenishing negative factors. By adding rapamycin to this culture, the
anergy induced is more complete and rivals that of the cells anergized
conventionally with signal 1 alone. This is consistent with the idea
that rapamycin completely blocks the ability of IL-2 to prevent anergy
during the initial stimulation phase. As is the case for conventional
anergy, the partial anergy induced by signal 1 plus 2 and the profound
anergy induced by signal 1 plus 2 plus rapamycin is not secondary to
down-regulation of the TCR as determined by flow cytometry (data not
shown).
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B, there was a >220-fold
decrease in the IL-2 produced by the conventionally anergized cells.
Likewise, the cells anergized in the presence of signal 1 plus 2 plus
rapamycin demonstrated a >370-fold reduction in IL-2 production. In
addition, consistent with their decreased proliferative ability upon
rechallenge, the cells that were stimulated with signal 1 plus 2 during
the induction phase also showed a decrease in IL-2 production when
compared with the nonmanipulated cells (19-fold), though their response
was still >20-fold that of the fully anergized cells. IL-2 rescues cells from rapamycin-induced anergy
The anergic state of T cell clones is reversed by culturing the
cells in exogenous IL-2 and allowing them to proliferate. We wanted to
determine whether the anergic state induced in the presence of
rapamycin was also reversible. Anergy was induced in the presence and
absence of costimulation and the presence and absence of rapamycin. The
cells were harvested and split into either fresh medium or fresh medium
containing 50 U/ml of IL-2. The cells were cultured for 12–14 days and
then harvested. As seen in Fig. 5A, induction with signal 1 or
signal 1 plus 2 plus rapamycin results in hypoproliferation in response
to rechallenge with APCs and Ag when compared with the cells that were
induced with signal 1 plus 2. However, culturing these cells in IL-2
during the rest period reverses the anergy. In addition, we see that
culturing cells in IL-2 that were initially stimulated with signal 1
plus 2 rescues them from their state of partial anergy. As shown in
Fig. 5B, the clones were tested for their ability to produce
IL-2 after being rested in the presence or absence of exogenous IL-2.
Note the hierarchy of IL-2 production in the control groups in this
experiment: the nonmanipulated cells produced the greatest amount of
IL-2, followed by the cells stimulated with signal 1 plus 2 and then
the cells stimulated with signal 1 alone or 1 plus 2 plus rapamycin.
Thus, 14 days after their exposure to signal 1 plus 2 and rapamycin the
clones remain anergic. In contrast, when these cells were stimulated
with IL-2, following removal of rapamycin, the anergic state was
reversed. In terms of IL-2 production, the reversal was not as complete
as that seen for the reversal of proliferation (Fig. 5A). In
this experiment, there was a 5.4-fold increase in IL-2 production after
culture of anergic cells in IL-2, while in three other experiments we
have seen between a 5- and 12-fold increase. The failure to completely
reverse anergy and achieve maximum IL-2 production upon rechallenge
might relate to the deeper state of anergy induced with rapamycin (Fig. 2B and Fig. 7 to be discussed below).
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Anergic cells have a defect in their
12–0-tetradeconoylphorbol-13-acetate responsive element (TRE)-mediated
transcription upon rechallenge 25, 26 . Recently, it was shown that
there is a block in the MAP kinase pathway in anergic cells 27, 28 ,
which could be responsible for the decrease in TRE-mediated
transcription. To determine whether the induction of anergy in the
presence of costimulation and rapamycin resulted in a block in the MAP
kinase pathway, Western blot analysis was performed on extracts derived
from cells anergized under various conditions. As shown in Fig. 6, mock-anergized, conventionally
anergized, partially anergized (signal 1 plus 2), and
rapamycin-anergized cells were either left unstimulated or stimulated
with plate-bound anti-TCR for 10 min. For all conditions, there was
relatively little phosphorylated ERK found in the extracts from the
unstimulated cells. The extracts from the mock stimulated cells showed
a marked increase in phosphorylated ERK following TCR signaling. In
comparison, there was less phoshorylated ERK in the extracts derived
from the conventionally anergized, the rapamycin-anergized, as well as
the partially anergized cells. In this particular experiment, the
relative decrease in ERK phosphorylation follows the hierarchy of
anergy induction (rapamycin-anergized < conventionally
anergized < signal 1 plus 2). However, in other experiments we
have seen a less profound decrease in phosphorylated ERK in the
rapamycin-anergized extracts, even though such cells were profoundly
anergic. The range of inhibition relative to the phosphorylated
ERK induced in the mock cells was 60–95%. Thus, our data
show that cells anergized in the presence of costimulation and
rapamycin display a similar biochemical block in the MAP kinase pathway
to that seen in cells anergized by signal 1 alone.
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production
Despite the profound block in IL-2 production by anergic cells,
they still produce other cytokines upon rechallenge, albeit in
decreased amounts. Typically, the anergic clones in our laboratory
display a 50% decrease in IFN- production and a 10-fold decrease in
IL-3 production under optimal activation conditions 10 . To examine
the production of these two cytokines by the rapamycin-induced anergic
cells, supernatant fluids from 16-h stimulated cells were assayed by
ELISA for IFN- and IL-3 levels. These are the same supernatant
fluids from Fig. 5B that demonstrated 240 U/ml IL-2 for the
stimulated cells vs 8 U/ml and 7 U/ml for the signal 1 and
rapamycin-induced anergic cells, respectively. As can be seen in Fig. 7A, there is a 50%
decrease in IFN production as measured in the signal 1-induced anergic
supernatants, when compared with the nonanergic supernatant fluid. On
the other hand, there is a >10-fold reduction in IFN production in the
supernatant fluid derived from the rapamycin-induced anergic cells.
Likewise, as seen in Fig. 7B, there is a typical sixfold
reduction in IL-3 production between the supernatant fluids derived
from the conventionally anergized cells and the nonanergic cells, while
the supernatant fluids derived from the rapamycin-induced anergic cells
showed nearly a 25-fold difference. Thus, it appears as if the cells
anergized in the presence of costimulation and rapamycin have a more
profound block in the production of these other cytokines than the
decrease seen in cells that are anergized by stimulation with signal 1
alone. This observation was also made for cells treated with signal 1
and rapamycin in the absence of costimulation, suggesting that
costimulation is not necessary for the deeper state of
rapamycin-induced anergy (data not shown). Furthermore, as is the case
for signal 1-induced anergy, the hierarchy of inhibition (IL-2 >
IL-3 > IFN-) is also maintained in the rapamycin-induced
anergy. Interestingly, this hierarchy of inhibition is also observed
for the signal 1 plus 2-induced partial anergy. As seen in Fig. 7, A and B, there is essentially no inhibition of
IFN- in the supernatant fluid from the cells incubated initially
with signal 1 plus 2, while there is a 2.5-fold decrease in the
production of IL-3 and a 5-fold decrease in the production of IL-2
(Fig. 5B).
Cell cycle progression from G1 to S phase prevents the induction of anergy
In as much as we have been able to demonstrate the induction of T
cell clonal anergy for cells cultured in the presence of signal 1 plus
2 and rapamycin, the data thus far support the Jenkins and Beverly
hypothesis that TCR engagement in the absence of proliferation leads to
anergy induction. Because rapamycin blocks IL-2-induced proliferation
at the level of mTOR, it was possible that it was not proliferation and
subsequent dilution of negative regulatory factors that prevented
anergy induction, but rather the biochemical events that are the result
of progression through the cell cycle. If indeed such were the case,
then we would predict that by incubating the clones with signal 1 plus
2 and hydroxyurea, which blocks proliferation in early S phase, anergy
would not be induced. Fig. 8A
demonstrates that increasing concentrations of hydroxyurea inhibit
IL-2-induced T cell proliferation as potently as rapamycin (see Fig. 1A). In addition, the clones still produce IL-2 in the
presence of hydroxyurea, although there is a slight attentuation of
IL-2 production at the highest concentration of the drug (Fig. 8A). Cell cycle analysis by propidium iodide staining
confirmed that for the A.E7 cells rapamycin blocks IL-2-induced
proliferation in late G1, while hydroxyurea blocks cell cycle
progression in S phase (data not shown). As seen in Fig. 8B,
like rapamycin, hydroxyurea does not inhibit anergy when the clones are
induced with anti-TCR alone. However, in contrast to rapamycin,
when the clones are stimulated with signal 1 plus 2 in the presence of
hydroxyurea they do not become anergic. These findings are consistent
with the data of Gilbert and Weigle, who also found that hydroxyurea
did not cause anergy in negative controls for their butyrate-induced
anergy model 13 . Thus, inhibition of proliferation in early S phase
does not result in anergy if the cells are stimulated through the TCR
in the presence of costimulation. Furthermore, in sharp contrast to CSA
(Fig. 3), hydroxyurea does not inhibit rapamycin-induced anergy (Fig. 8B). This is consistent with the idea that rapamycin acts
proximally to hydroxyurea and that the mechanism responsible for
preventing anergy lies between G1 and S phase. Overall, these data are
consistent with a model in which CSA blocks the up-regulation of
"anergic factors" and rapamycin blocks their
degradation/inactivation, while hydroxyurea, despite its ability to
block proliferation, neither blocks the up-regulation nor
down-regulation of the factors that promote anergy.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clearly, we have not formally ruled out the possibility that rapamycin
prevents the signaling of an as-yet unidentified CD28
"anti-anergy" or signal 2t pathway 14 . In fact, it has been
shown that rapamycin can inhibit CD28-mediated down-regulation of
I
B and up-regulation of CTLA4 29, 30 . However, at the very
least, as shown in Fig. 1B, in the presence of rapamycin,
the CD28-mediated signaling pathway responsible for the up-regulation
of IL-2 production appears to be intact. Furthermore, our findings are
consistent with the observations of Gilbert and Weigle, who used the
histone deacetylase inhibitor n-butyrate to induce anergy
13 . They proposed that anergy is the result of TCR stimulation and
G1a sequestration. However, unlike rapamycin, histone deacetylase
inhibitors also inhibit IL-2 production 13, 31 , and, thus, the
ability of n-butyrate to induce anergy could also be
consistent with the model of Jenkins and Beverly 10, 11 . Finally, in
terms of whether or not regulation of the transcription factor Nil-2a
contributes to the antagonism of the anergic state, using
electrophoretic mobility shift assays we could not demonstrate the
down-regulation of Nil-2a binding to the NRE in A.E7 cells stimulated
with anti-TCR and anti-CD28 Abs (data not shown). Thus, at this
time, the role, if any, that Nil-2a plays in anergy induction in the
A.E7 clone is not clear.
The anergic state induced in the presence of rapamycin shares many
similarities with conventionally anergized cells. They both display a
block in the MAP kinase pathway as determined by decreased ERK
phosphorylation 27, 28 and they both were reversed by adding
exogenous IL-2. On the other hand, unlike conventionally anergized
cells, the rapamycin-induced anergic cells appeared to have a more
profound block in the production of the cytokines IL-3 and IFN-. It
is not clear whether this is due to more complete anergy, as a result
of the ability of rapamycin to fully prevent the G1 to S phase
transition, or the ability of rapamycin to affect an additional
pathway. It is also of note that both IL-3 and IFN- contain AP-1
sites in their promoters 32, 33 . Because it has been shown that there
is a decrease in TRE-mediated transcription in anergic cells, it might
be that the differences seen in the production of these cytokines in
anergy is related to the relative contribution of these sites to
TCR-induced activation. The consistency of the hierarchy of
inhibition between IL-2, IL-3, and IFN- is striking and is
observed under conditions of partial anergy (induced by 16 h of
signal 1 plus 2 and removal of IL-2), conventional anergy (signal 1
alone), and rapamycin-induced anergy. Interestingly, this same pattern
is observed when examining the effect of costimulation on cytokine
production (Ref. 34 and our unpublished observations). That is, the
production of IL-2 by A.E7 is costimulation dependent, while IL-3 and,
to a greater degree, IFN- are produced in response to signal 1 alone
34 . This hierarchy may ultimately provide insight into the precise
molecular mechanisms of cytokine induction and inhibition in Th1 cells.
Fig. 9 depicts a general model for anergy
induction. We have adapted concepts from the kinetic proofreading model
of TCR signal transduction to incorporate a number of observations
concerning the anergy induction process 35, 36 . TCR engagement leads
to a series of reactions (A 
B C) that ultimately lead to full
signal 1-induced changes. At reaction B, the negative regulatory
pathway leading to anergy is initiated. It has been shown that certain
variant peptides with lower affinities for a particular TCR can induce
anergy even in the presence of costimulation, a so-called partial
agonist anergy 2, 37, 38 . A partial agonist may have an affinity for
the TCR that only permits signaling up to B, never leading to full
signal 1. Thus, in this case, the negative regulatory factors are
produced but there is insufficient signaling to produce IL-2 and the
subsequent G1 to S phase transition. This is consistent with the data
of Madrenas et al., who showed that anergy induction for partial
agonists could be overcome by the addition of exogenous IL-2 39 . In
the case of conventional anergy, TCR engagement results in both the
initiation of the negative regulatory pathway as well as full signal 1.
However, in the absence of costimulation, little or no IL-2 is
produced, and, as a result, there is no entry into S phase. In
rapamycin-induced anergy, there is production of the negative
regulatory factors as well as full signal 1 and signal 2. This leads to
IL-2 production, release, and IL-2R engagement. However, the cell is
blocked from proceeding through the cell cycle by rapamycin. As a
result, there remains a build up of negative regulatory factors, and
the cell is hyporesponsive upon rechallenge. In this model, hydroxyurea
blocks proliferation distal to the events responsible for the
abrogation of anergy, and, as a result, even though cells incubated in
the presence of signal 1 plus 2 and hydroxyurea do not proliferate,
signaling through the IL-2R leads to the prevention of anergy. Finally,
signal 1 plus 2 followed by the removal of IL-2 results in a small
anergic effect. We postulate that this is due to the fact that the
cells are continuously being stimulated through the TCR and thus the
IL-2 produced during the 16-h culture period is unable to accomplish
the complete dissipation of the negative regulatory factors.
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and IL-2 production secondary to
increases in cAMP is the result of the up-regulation of the negative
transcription factor inducible cAMP early repressor (ICER)
42 . ICER is an isoform of cAMP-response-element modulator
(CREM) that has the ability to bind to DNA, but lacks a
transactivating domain 43 . It remains to be seen if this protein
contributes to the decrease in IL-2 and IFN- production seen in
anergy.
Because rapamycin does not appear to inhibit IL-2-induced
p21ras activation or the induction of
c-myc mRNA, presumably these biochemical consequences of
IL-2R engagement do not play a role in inhibiting anergy 44 . A role
for signaling via the common -chain of the IL-2R is supported by
findings in human T lymphocytes that activating Abs against this
chain can antagonize anergy induction 12 . The prevention of anergy
induction by IL-2 must involve mTOR, the serine/threonine kinase
blocked by rapamycin 17, 44 . At this time, it is unclear if mTOR is
directly or indirectly involved in the degradation or inactivation of
negative factors up-regulated by TCR engagement. In this regard, mTOR
is an obligate participant in the activation of
p70s6k, which is proposed to be essential
for G1 cyclin-cdk activation and progression of the cell from G1 into S
phase 44 . Although the precise mechanism whereby
p70s6k promotes this transition is not
known, it is believed that progression through the cell cycle is
mediated in part by the kinase’s ability to enhance translational
initiation 44 . This is mediated in part by its ability to
phosphorylate and inactivate translational repressors. Likewise,
p70s6k might serve the same function in
terms of inactivating the negative regulatory factors that maintain
anergy. In addition, p70s6k has been shown
to phosphorylate and increase the transcriptional activity of CREM
45 . It remains to be seen if this kinase has additional effects on
other CREB family members, for example those that might be involved in
the cis-dominant repression of IL-2 transcription.
TCR engagement results in the transition of the cells into the late G1 phase of the cell cycle 17 . Further progression is blocked by the accumulation of the inhibitor Kip-1 44 . Signaling through the IL-2R results in the degradation of Kip-1 through the ubiquitin-proteasome pathway and the subsequent assembly of G1 cyclin-cdk complexes 46 . This in turn leads to progression into S phase and cellular proliferation. The presence of rapamycin inhibits IL-2-mediated degradation of Kip-1. Similarly, we propose that rapamycin promotes anergy by blocking cell cycle progression and promoting the build up of negative regulatory factors. As is the case for the inhibitor Kip-1, it might be that progression through the cyclin-dependent kinase pathway leads to the degradation/inactivation of the negative regulatory factors that mediate anergy. In this regard, it has been shown that the transcriptional repressor ICER is degraded by the ubiquitin-proteasome pathway 47 . If such is the case, then Kip-1 itself may be central to the maintenance of the anergic state by its ability to inhibit the cyclin-dependent kinase cascade. Thus, anergy might be viewed as part of the mechanism of check point cell cycle growth arrest adopted by the lymphocyte for an immunological purpose. Current studies are focused on determining the level at which anergy is prevented in the cyclin-dependent kinase cascade between G1 (rapamycin) and S phase (hydroxyurea).
Finally, the data described herein have potential clinical
implications. As seen in Fig. 2A and as has been
demonstrated previously, CSA inhibits anergy induction 5, 6 . Indeed,
in a mouse allograft model, it has been shown that long-term graft
acceptance in the absence of long-term immunosuppression can be induced
by the infusion of CTLA4 Ig and anti-CD40 ligand Abs in the
peritransplant period 48 . However, if CSA is added to this regimen,
the grafts are rejected. These observations suggest the potential
importance of signal 1 in the induction of graft tolerance in this
model. By contrast, in a swine transplant model, it has been shown that
peritransplant transfusion of donor-derived, dendritic cell-depleted
PBMC under the cover of rapamycin results in long-term graft survival
without long-term pharmacologic immunosuppression 49 . This finding is
consistent with our in vitro data that T cell activation in the
presence of rapamycin can lead to tolerance. Ultimately, it might be
possible to design immunosuppressive regimens that do not inhibit
TCR-mediated signaling and thus facilitate graft tolerance in the
absence of long-term immunosuppression.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CSA, cyclosporin A; mTOR, mamalian target of rapamycin; MAP, mitogen-activated protein; TRE, 12–0-tetradeconoylphorbol-13-acetate-responsive element; FK506BP, FK506 binding protein; PCC, pigeon cytochrome c; ERK, extracellular regulatory kinase.
Received for publication September 4, 1998. Accepted for publication December 3, 1998.
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