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Trudeau Institute, Saranac Lake, NY 12983
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent mechanism of suppression was responsible for the
apparent unresponsiveness of the CD4 T cells. In this study, we analyze
the response of CD4 T cells isolated throughout the first 10 days of
the in vivo response to injected SEA. We show that CD4 T cells isolated
at the peak of the in vivo response undergo very little
activation-induced cell death after sterile FACS sorting or
restimulation in the presence of neutralizing Abs to IFN-. We also
show that the IFN--dependent tolerance develops soon after SEA
injection in the spleens of both normal and TCR-transgenic mice. This
suppression is dependent upon myeloid cells from the SEA-treated mice
and is optimal when inducible NO synthase activity and reactive oxygen
intermediates are both present. The data indicate that IFN-, myeloid
cells, and a combination of NO and reactive oxygen intermediates all
contribute to a common pathway of T cell death that targets activated
or responding CD4 T cells. Sorted Gr-1+ cells from
SEA-treated mice also directly suppress the response of naive CD4 T
cells in mixed cultures, indicating that this tolerance mechanism may
play a role in down-regulating other vigorous immune
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains of their TCRs
(1). Although these proteins are potent T cell mitogens,
they often cause shock and immune dysfunction in vivo, which directs
the immune system away from protective immunity (2). Injected SAg stimulate a brief period of immune hyperactivity in vivo, which lasts about 3 or 4 days (3, 4, 5). During this time, the numbers of responding T cells increase substantially in the spleens and lymph nodes (LN) of the staphylococcal enterotoxin A (SEA)-treated animals, and cytokines are released in high concentrations (3, 4, 5). However, the response peaks about 3 days after the SAg is injected and then declines abruptly, leaving diminished populations of SAg-reactive CD4 T cells in the periphery of the SAg-treated animals. In some studies, the residual SAg-reactive T cells were hyporesponsive to restimulation (6). Secondary T cell responses to injected SAg are also very transient and even more rapidly aborted than the primary response (7). Although T cell responses to injected SAg have been extensively studied, the mechanisms that are responsible for the acute loss of responding T cells in vivo and the subsequent immune unresponsiveness in vitro remain controversial.
Several mechanisms that can induce CD4 T cell death or unresponsiveness
have been identified. These mechanisms include T cell anergy due to a
lack of costimulation at the time of activation (8) and
Fas-mediated activation-induced cell death (AICD) (9).
Cytokines such as IFN- (10, 11), TNF-
(12), and IL-10 (13) can also mediate
suppression of T cells. However, the detailed mechanisms involved in
the induction and execution of these cytokine-regulated death pathways
have not been clarified.
Bacterial SAg are an accepted model to analyze peripheral T cell
tolerance. Because these proteins bind to class II MHC molecules and
stimulate T cells through their Ag receptors, it is believed that they
stimulate T cells in much the same way as conventional peptide Ags. In
a previous study (11), we showed that injected SEA induced
an active mechanism of T cell suppression to develop in the spleens of
AND TCR-transgenic (Tg) mice (14). This suppression
inhibited the proliferative responses and cytokine production of
V3+ CD4 T cells after in vitro restimulation.
Although increased numbers of dead V3+ CD4 T
cells were found in the suppressed cultures, Fas expression was not
required. The T cell death was prevented when CD4 T cells from the
SEA-treated mice were highly purified or IFN- was neutralized in the
cultures, indicating that exogenous factors were involved in the
suppression. Since IFN- was not sufficient to suppress the response
of the CD4 T cells by itself, the data suggested that other cells or
factors from the SEA-treated mice were also required. This
study demonstrated that an extrinsic mechanism of suppression was
responsible for inhibiting the responses of the SEA-reactive CD4 T
cells in vitro, but the precise mechanism of T cell death was not
further analyzed.
Other evidence suggests that IFN- may play a significant role in
peripheral T cell tolerance. This evidence comes from both in vitro
(10) and in vivo studies. Some studies suggest that
IFN- may play a protective role during the active phase of
experimental autoimmune encephalomyelitis (15) and prevent
the accumulation of activated T cells (16) by a mechanism
that may involve neutrophils (17). IFN- and NO
production also prevent activated CD4 T cells from accumulating in mice
infected with bacillus Calmette-Guérin (BCG) bacteria
(18) or lymphocytic choriomeningitis virus
(19). In other studies, reactive oxygen intermediates
(ROI) were suggested to play a role in regulating T cell survival in
SAg-treated mice (20, 21). Together, these reports suggest
that reactive molecules, which can be produced by myeloid cells, may
play an important role in down-regulating T cell responses in vivo.
However, the cells and molecules responsible for inducing T cell death
have not been completely elucidated in these studies.
In this study, we have analyzed the response of
V3+ CD4 T cells harvested throughout the first
10 days of the in vivo response to injected SEA. We show that
V3+ CD4 T cells isolated at the peak of the in
vivo response undergo very little AICD after purification by sterile
FACS sorting or restimulation in the presence of neutralizing Abs to
IFN-. We found that myeloid cells, IFN-, NO, and ROI all
contribute to a single novel pathway of T cell suppression. This
suppressive pathway is responsible for the apparent unresponsiveness of
the SAg-reactive CD4 T cells in partially purified cultures. We show
that this suppressive mechanism specifically targets activated or
responding CD4 T cells, and we present evidence that this pathway is a
physiological mechanism of T cell regulation. We discuss the potential
role of this death mechanism in down-regulating other T cell responses
in immunized animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal B10.BR mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 (B6) x SJL founder Tg AND mice (14) were backcrossed to B10.BR mice for at least 10 generations. Mice between 3 and 7 mo of age received purified SEA (25 µg for AND mice and 150 µg for B10.BR mice) in HBSS by i.v. injection in the tail.
Cell lines and reagents
Murine fibroblasts transfected with I-Ek class II MHC molecules and ICAM-1 (DCEK-ICAM) (22) and expressing high levels of B7.1 (23) were treated with mitomycin C (50 µg/ml) and used as APCs for in vitro experiments. NG-monomethyl L-arginine (L-NMMA), NG-monomethyl D-arginine (D-NMMA), N-acetylcysteine (NAC), reduced glutathione (GSH), and propidium iodide (PI) were all purchased from Sigma (St. Louis, MO). RB6-8C5, XMG1.2, and the other depletion Abs (11) were purified from murine ascites or cell culture supernatants. 5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR). SEA was purchased from Toxin Technologies (Sarasota, FL).
Enrichment of CD4 T cells and in vitro restimulation
CD4 T cells were enriched with Abs and complement (C'), using
anti-HSA (J11D), anti-class II MHC (M5114 and CA4), and
anti-CD8 (3.155) Abs as previously described (11).
Cell debris was removed with Lympholyte separation gradients, spun at
1,900 x g for 20 min at room temperature. These
enriched CD4 T cell populations were cultured in RPMI 1640 medium
supplemented with 200 µg/ml penicillin, 200 µg/ml streptomycin, 4
mM L-glutamine, 50 µM 2-ME, and 10% FCS as
previously described (24). Tg CD4 T cells were cultured at
3 x 105/ml with 1 x
105 APC/ml, 5 µM pigeon cytochrome c
fragment (PCCF) peptides, and 20 U/ml rIL-2. Non-Tg T cells were
cultured at 106/ml with SEA (500 ng/ml),
mitomycin C-treated APC (105/ml), and rIL-2 (20
U/ml). Purified anti-IFN- Abs (XMG1.2) and
L-NMMA (50 µM) were added to the cultures at
the time of restimulation. NAC and GSH were made up fresh before each
experiment and added 16 h after in vitro restimulation.
Measuring CD4 T cell expansion
For proliferation studies, CD4 T cells were purified from the
spleens of HBSS- or SEA-treated mice by Abs and C' depletion and
stimulated as described above. Three days after in vitro restimulation,
the numbers of live V3+ CD4 T cells were
calculated by the counting trypan blue-excluding cells and correcting
for the percentage of V3+ CD4 T cells using
FACS analysis. PI was added to stained CD4 T cells immediately before
FACS analysis to quantify dead V3+ CD4 T
cells.
CFSE analysis to identify proliferating and anergic CD4 T cells
Enriched CD4 T cells were labeled with 1 µM CFSE in PBS at
37°C for 15 min (25). The cells were then washed with
cold PBS and restimulated in vitro as described above. After in vitro
restimulation the intensity of the CFSE dye was analyzed by three-color
FACS analysis, using CyChrome-conjugated Abs to CD4 (BD PharMingen, San
Diego, CA) and PE-conjugated Abs to V3 (BD PharMingen). Rates of
cell division were assessed by the intensity of the CFSE fluorescence,
analyzed at 491–518 mM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent mechanism of T cell suppression develops after
SEA injection
We have previously shown that an active mechanism of T cell
suppression prevented V3+ CD4 T cells from
SEA-treated Tg TCR mice from proliferating or producing cytokines in
response to antigenic restimulation in vitro (11). Here,
we have used ex vivo analyses to further investigate the mechanism of
this SEA-induced T cell suppression, using both Tg TCR and normal
B10.BR mice. In these studies, we analyze the proliferative responses
of V3+ CD4 T cells as a representative
population of SEA-reactive CD4 T cells. The cultures are restimulated
in vitro with SEA or PCCF peptides and subsequently examined for live
and dead V3+ CD4 T cells. Neutralizing Abs are
also used to confirm the role of IFN- in the suppression.
B10.BR mice were used to determine when the IFN--dependent
suppression developed in vivo. The mice were given 150 µg of SEA (or
HBSS alone) by i.v. injection. Duplicate animals were euthanized at 2,
3, 4, and 7 days after immunization, and the spleens were analyzed for
live V3+ CD4 T cells, Fig. 1
a (line graph). This study
shows the kinetics of the in vivo response to injected SEA. As in other
studies, the numbers of responding V3+ CD4 T
cells increased dramatically between 2 and 3 days after SEA injection.
However, on the fourth day the numbers of T cells began to decline
substantially, leaving relatively few live V3+
CD4 T cells in the spleens of the SEA-treated animals by day 7. In
other studies, we have used an adoptive transfer system to show that
when naive V3+ CD4 T cells from AND TCR Tg
mice (9) are introduced into B10.BR hosts, virtually all
of the transferred CD4 T cells undergo rapid cell division after
systemic SEA injection (data not shown).
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-dependent T cell suppression. For this analysis,
CD4 T cells were enriched with Abs and C' to deplete CD8 T cells and B
cells as previously described (11). The recovered cells
were between 69 and 77% CD4 T cells. The others were a mixed
population of residual B cells and some nonlymphoid cells. These
enriched cell populations were then stimulated in vitro with
I-Ek-bearing murine fibroblasts and SEA. Because
naive CD4 T cells die if cultured in the absence of added cytokines
(25, 26), we also used rIL-2 to keep the SEA-nonreactive
CD4 T cells alive during the 3-day culture period. Half the cultures
also received the neutralizing Abs to IFN- (XMG1.2). Three days
later, the cultures were analyzed for live
V3+CD4 T cells by counting the trypan
blue-excluding cells and FACS analysis (Fig. 1
a, bar graph).
The results are expressed as the fold increase in the numbers of live
V3+ CD4 T cells during the 3-day culture
period. The cultures were also analyzed for the number of dead
V3+ CD4 T cells by PI staining and FACS
analysis (Fig. 1b).
After in vitro restimulation, reduced numbers of live
V3+ CD4 T cells were detected in all the
cultures from the SEA-treated mice compared with the numbers in the
cultures from HBSS-treated control animals (Fig. 1a, bar
graph). PI analysis also revealed large numbers of dead
V3+ CD4 T cells in the suppressed cultures
(Fig. 1b). This demonstrated that the injected SEA had
induced a form of T cell suppression in all the SAg-treated animals. In
every case, the suppression was blocked when neutralizing Abs to
IFN- were used (Fig. 1a), and the numbers of dead
V3+ CD4 T cells were substantially reduced
(Fig. 1b). The control cultures (day 0) were not suppressed
after HBSS injection and were unaffected by the neutralizing Abs to
IFN-.
In this experiment, the numbers of V3+ CD4 T
cells increased 4- to 5-fold in the SEA-treated mice and then returned
to numbers approximating the starting population by day 7. However,
when CD4 T cells were enriched and restimulated in vitro, all of the
V3+ CD4 T cells exhibited similar
proliferative capacity throughout the response. Even the cells that
were harvested at the peak of the in vivo response (day 3) underwent
very little AICD in the presence of the neutralizing Abs. This suggests
that the mechanism that regulates normal T cell deletion in vivo was
blocked when the V3+ CD4 T cells were removed
from the animals or by neutralizing Abs to IFN- and further suggests
that fraternal Fas-Fas ligand interactions were not sufficient to
induce a majority of the T cell death. The enriched CD4 T cells from
SEA-treated B10.BR mice were also able to suppress the responses of
fresh naive CD4 T cells from untreated animals (data not shown),
indicating that the mechanism of suppression was similar to that
originally described in TCR Tg mice (11).
A role for NO in SAg-induced in vitro suppression
NO has been shown to inhibit T cell responses in several different
models (27). Since inducible NO synthase (iNOS) activity
is regulated by IFN- (28), we used a chemical inhibitor
of iNOS (L-NMMA) (29) to investigate
whether reactive nitrogen intermediates (RNI) played a role in the
IFN--dependent T cell suppression (Fig. 1c).
Enriched CD4 T cells from the same SEA-treated mice that are shown in
Fig. 1, a and b, were restimulated in vitro with
SEA and APC as before. In this case, replicate cultures received the
iNOS inhibitor L-NMMA (50 µM) or its inactive
enantomer D-NMMA (50 µM). Three days later, the
cultures were analyzed for live (Fig. 1c) and dead (data not
shown) V3+ CD4 T cells as previously
described.
The suppression was unaffected by the inactive enantomer
D-NMMA. However, L-NMMA significantly increased
the numbers of live V3+CD4 T cells in many of
the cultures from the SEA-treated mice (Fig. 1c) and reduced
the numbers of dead cells (data not shown). This effect was
particularly pronounced in the cultures that were isolated on days 4
and 7 after SEA treatment, indicating that NO was contributing to the
suppression. However, L-NMMA had relatively
little effect in cultures isolated on days 2 and 3. We have
consistently found that CD4 T cells from SEA-treated mice do not
proliferate to the same extent in the presence of
L-NMMA (Fig. 1c) as identical cells
cultured in the presence of neutralizing Abs to IFN- (Fig. 1a). This disparity was particularly pronounced in the
cultures that were harvested on days 2 and 3 after SEA injection. Other
experiments, in which T cells were restimulated at lower
concentrations, have given similar results (data not shown), indicating
that the activity of the iNOS inhibitor was not saturated. Since we
have not found 50 µM L-NMMA to be toxic to
proliferating T cells, we considered the possibility that additional
factors were contributing to the IFN--dependent suppression.
Gr-1+ cells are required for the SEA-induced in vitro suppression
Our previous study indicated that nonlymphoid cells from
SEA-treated mice were contributing to the SEA-induced suppression
(11). To identify these nonlymphoid cells, we used a panel
of mAbs to deplete different subsets of cells from the restimulated
cultures. In separate experiments, Abs were used to deplete NK cells
(DX5), 
T cells (GL3), and Gr-1+ cells
(RB6-8C5) from the enriched cultures. The efficiencies of the
depletions were confirmed by FACS staining. In these studies, only the
anti-Gr-1 Abs reversed the suppression (Fig. 2), and the other Abs had no effect (data
not shown).
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, 3) 50 µM L-NMMA, or 4)
50 µM D-NMMA. Three days later, the cultures were
analyzed for live (Fig. 2) and dead (data not shown)
V3+ CD4 T cells as before.
When only CD8 T cells and B cells were depleted from the cultures, the
SEA-induced T cell suppression was detected as before. The numbers of
live V3+ CD4 T cells in the SEA-treated
cultures were also substantially increased when neutralizing Abs to
IFN- were used (Fig. 3b)
and were partially restored in the cultures that received
L-NMMA (Fig. 3c). Strikingly, the
suppression was completely eliminated when RB6-8C5 Abs were used to
deplete Gr-1+ cells from the cultures before
restimulation, and there was no added benefit from adding Abs to
IFN- or iNOS inhibitors to the cultures (Fig. 2, b and
c). As before D-NMMA had no effect on
the suppression in any of the cultures (Fig. 3d). The
control CD4 T cells that were purified from HBSS-treated mice by the
same protocols were not suppressed after enrichment and were largely
unaffected by the presence of neutralizing Abs to IFN-,
L-NMMA, or the RB6-8C5 purification protocol
(Fig. 2, left).
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3+ CD4 T cells in the suppressed
cultures and relatively few dead cells when neutralizing Abs to IFN-
or Gr-1+ cells were used. The dead cells
accounted for only 5–10% of the recovered
V3+ CD4 T cells in these cultures (data not
shown). These data indicated that IFN- and
Gr-1+ cells were part of a common pathway of T
cell suppression. Other experiments with non-Tg mice and CD4 T cells
isolated up to 10 days after SEA injection have given similar results
(data not shown).
In this experiment, the Gr-1+ cells were
10–12% of the restimulated cell populations from SEA-treated mice
and were <3–4% of the control cultures. Because the CD4 T cells and
Gr-1+ cells were purified together, they were
present in the restimulated cultures at similar ratios to the numbers
present in the original SEA-treated spleens. These numbers were
sufficient to completely suppress the response of the CD4 T cells from
all SEA-treated mice. As before, L-NMMA was less effective
in reducing the rate of T cell death in the cultures from SEA-treated
mice than were neutralizing Abs to IFN-, further suggesting that
there may be another component to the suppression.
ROI play a role in the SAg-induced in vitro suppression
Blocking iNOS activity only partially prevented IFN--dependent
suppression in the cultures from SEA-treated mice (Fig. 1c).
This incomplete blocking was particularly apparent in the cultures that
were isolated on days 2 and 3 after SEA treatment, suggesting that
other factors were contributing to the suppression of CD4 T cells.
Murine neutrophils and related bone marrow precursors express Gr-1 at
high levels. Lower levels of Gr-1 can also be found on some monocytes
and macrophages (30). All of these cells produce a
combination of RNI (NO) and ROI (superoxide and
H2O2) in response to
IFN- (31, 32). Since RNI and ROI are often produced
together and have both been implicated in causing the death of CD4 T
cells in other models (21, 33, 34), we investigated
whether the response of the SEA-treated CD4 T cells was further
restored when ROI-scavenging antioxidants (NAC and GSH) were used in
combination with L-NMMA (Fig. 4). For these experiments, CD4 T cells
were again harvested at the peak of the in vivo response (3 days after
SEA injection).
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(Fig. 3a). Some
of the cultures received 50 µM L-NMMA at the
time of restimulation. Sixteen hours later, varying concentrations of
NAC or GSH were also titrated into the cultures. The live
V3+ CD4 T cells were then counted 3 days after
in vitro restimulation, and the results are expressed as the fold
increase in the number of live V3+ CD4 T cells
during the 3-day culture period, as before (Fig. 3a).
As in previous experiments, after 3 days of in vitro restimulation very
reduced numbers of live V3+ CD4 T cells were
recovered from the cultures from SEA-treated mice compared with the
control cultures. However, these numbers increased 5-fold when the
cells were restimulated in the presence of 50 µM L-NMMA.
Significant numbers of live V3+ CD4 T cells
were also detected in the cultures that were treated with NAC or GSH,
resulting in a 6-fold expansion of the V3+ CD4
T cell population at 5 mM GSH or NAC. The expansion was further
enhanced when 50 µM L-NMMA was used in combination with
one of the antioxidants, resulting in a maximum 9-fold expansion of the
V3+ CD4 T cell population at 5 mM antioxidant.
Both antioxidants appeared to show toxicity at the 10-mM concentration
for 3 days. These results demonstrated significant additive protection
when L-NMMA was used in combination with NAC or GSH,
suggesting that RNI and ROI were both contributing to the SEA-induced T
cell suppression.
In a second set of experiments, CD4 T cells were harvested from SEA-
and HBSS-treated AND mice. The CD4 T cells were enriched with Abs and
C' and restimulated in vitro with a specific peptide Ag (PCCF), APC,
and rIL-2, as described in Fig. 2. At 16 h after restimulation,
varying concentrations of NAC were added to some of the cultures. In
this experiment, PI analysis was used to quantify the percentages of
live and dead CD4 T cells in the cultures at 40 h after in vitro
restimulation (Fig. 3b). The results show the percentages of
PI+ CD4 T cells averaged from three replicate
animals.
The PI analysis revealed large numbers of dead
(PI+) CD4 T cells in the cultures from
SEA-treated mice, when no antioxidants were added. In these cultures
the dead cells were 65% of the recovered CD4 T cells after 40
h of in vitro restimulation. However, when NAC was added to the
cultures, the percentages of PI+ CD4 T cells were
substantially reduced (down to 22%). The numbers of
PI+ CD4 T cells decreased as the dose of NAC
increased. This confirmed the role of ROI in SEA-induced T cell death
in cultures isolated 3 days after SAg injection. NAC had only a slight
enhancing effect on the percentage of live CD4 T cells in the control
cultures and showed no evidence of toxicity during the 40-h incubation
period.
These experiments showed additive effects from blocking NO and ROI
production, suggesting that a combination of RNI and ROI was
responsible for mediating the death of CD4 T cells in the suppressed
cultures. We have found some experimental variability in the relative
contributions of NO and ROI to the IFN--dependent suppression
between individual animals.
Purified Thy-1-Gr-1+ cells from
SEA-treated mice kill responding V3+ CD4 T cells in
reconstituted cultures
To definitively establish the role of the
Gr-1+ cells in SEA-induced T cell suppression
(Fig. 2), we have used sterile FACS sorting to isolate
Thy-1-Gr-1+ cells from
SEA-treated AND mice. The purified
Thy-1-Gr-1+ cells were
then mixed with either highly purified CD4 T cells from SEA-treated
mice or fresh naive CD4 T cells from untreated Tg TCR mice.
Neutralizing Abs to IFN- were also added to some of the cultures to
measure suppression. These experiments showed that the
Thy-1-Gr-1+ cells from
SEA-treated mice were able to suppress the proliferative response of
the V3+ CD4 T cells in reconstituted cultures
when IFN- was present (Fig. 4).
As before, CD4 T cells were enriched from the spleens of SEA-treated
AND mice at 3 days after injection. In addition, highly purified
populations of Thy-1-Gr-1+
cells and V3+ CD4 T cells were isolated from
the same mouse spleens by sterile FACS sorting. Abs to Thy-1 were used
to exclude cross-reactive Ly6C+ T cells from the
sorted cell populations, since they do not contribute to the
suppression (data not shown). A third population of naive CD4 T cells
was isolated from untreated control AND mice with Abs and C'. Each of
the purified CD4 T cell populations was restimulated in vitro with APC,
rIL-2, and PCCF. The purified
Thy-1-Gr-1+ cells from
SEA-treated mice were then titrated into some of the cultures. Finally,
neutralizing Abs to IFN- were used to measure suppression. After 3
days of in vitro restimulation the live (Fig. 4a) and dead
(Fig. 4b) CD4 T cells were analyzed as before.
As expected, the enriched CD4 T cells from SEA-treated mice did not
proliferate unless neutralizing Abs to IFN- were used (Fig. 4a). However, when the CD4 T cells were isolated by sterile
FACS sorting, they responded vigorously to antigenic restimulation in
vitro (Fig. 4a) and were not affected by the neutralizing
Abs to IFN-. The suppression reappeared when sorted
Gr-1+ cells were titrated back into the cultures
and was inhibited by the neutralizing Abs to IFN-. Naive CD4 T cells
from treated mice were also suppressed in the presence of the sorted
Gr-1+ cells.
These experiments showed that a ratio of 1:8
Gr-1+ cells from the SEA-treated mice was
sufficient to completely inhibit the proliferative response of the
sorted CD4 T cells from SEA-treated mice. Slightly larger numbers of
Gr-1+ cells were required to suppress the
response of the naive CD4 T cells, suggesting that recently activated
CD4 T may be slightly less sensitive to suppression. This may be
because naive CD4 T cells make very little IFN- in response to a
primary stimulation. Small numbers of
Thy-1-Gr-1+ cells were
also purified from the spleens of the HBSS-treated control mice.
However, titration experiments showed that much larger numbers of these
cells (a 1:2 ratio) were required to suppress the proliferative
response of the V3+ CD4 T cells in mixed
cultures (data not shown).
Again, PI analysis revealed a reciprocal pattern of results. In
general, large populations of dead V3+ CD4 T
cells were recovered from all of the suppressed cultures from the
SEA-treated mice (Fig. 4b). The numbers of dead cells were
substantially reduced when the CD4 T cells were highly purified by
sterile FACS sorting or when neutralizing Abs to IFN- were used. In
these rescued cultures, only 5–10% of the
V3+ CD4 T cells underwent AICD after in vitro
restimulation.
The Thy-1-Gr-1+ cells were
further analyzed by three-color FACS analysis. This analysis showed two
populations of Thy-1-Gr-1+
cells in the spleens of the SEA-treated mice (Fig. 5a), which expressed high and
low levels of Gr-1. Both populations of Gr-1+
cells also expressed high levels of Mac-1 and LFA-1 (Fig. 5b). Hematoxylin- and eosin-stained cytospins were used to
further analyze the sorted Gr-1+ cells by
standard differential analysis. This analysis showed a mixed population
of mature neutrophils with highly segmented nuclei (70–80%) and
smaller numbers of macrophage/monocytes (20–30%). Although a very
small number of contaminating lymphocytes and some other unidentified
cells were also found (<5%), no eosinophils or basophils were
detected. The Gr-1+ cells from control mice
included some Mac-1-negative cells (Fig. 5b) and some
immature neutrophils with banded nuclei (data not shown). This suggests
that some of the Gr-1+ cells from control animals
may have a less activated phenotype than the
Gr-1+ cells from SEA-treated animals.
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-dependent suppression develops in the spleens, but not the
peripheral LN, of SEA-treated animals
Two groups of four AND mice were given 25 µg of SEA or HBSS
alone by i.v. injection. Five days later, the spleens from each group
of four animals were pooled and enriched for CD4 T cells with Abs and
C' as described in Fig. 2. The peripheral LN from each group of four
animals were also pooled and enriched for CD4 T cells by the same
protocol. After purification the CD4 T cells were restimulated in vitro
with 5 µM PCCF, APC, and IL-2 as before. Neutralizing Ab to IFN-
were also added to half the cultures. Four days later, the cultures
were analyzed for live CD4 T cells by counting and FACS analysis (Fig. 6). This analysis showed the
IFN--dependent suppression only in the spleen cultures from the
SEA-treated mice. The LN cultures and cultures from control animals
were not suppressed.
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We further investigated the mechanism of SEA-induced in vitro
suppression by analyzing the kinetics of the proliferative response of
the SEA-reactive V3+ CD4 T cells to in vitro
restimulation. In this case, the live CD4 T cells were counted on
consecutive days after restimulation to measure the size of the
proliferating T cell population (Fig. 7a). CFSE was used to follow
the kinetics of T cell division in the cultures (Fig. 7, b–e). B10.BR mice were used for this analysis to compare
the response of the SEA-reactive V3+ CD4 T
cells with the response of CD4 T cells that express SEA-nonreactive
V-chains in the same cultures. Recombinant IL-2 was used to keep the
nonresponding T cells alive throughout the 4-day culture period.
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were added to half the cultures to
produce suppressed and nonsuppressed cultures. On consecutive days
after in vitro restimulation the cultures were analyzed for live
V3+ CD4 T cells (Fig. 7a). The CD4
T cells were also analyzed for the intensity of their CFSE
fluorescence, which was reduced by half with each cell division. The
overlaid histograms show gated populations of
V3+ CD4 T cells from cultures stimulated in
the presence (heavy lined open histograms) and absence (shaded
histograms) of neutralizing Abs to IFN- (Fig. 7, b and
c).
When Gr-1+ cells and IFN- were both present in
the cultures, the V3+ CD4 T cell populations
from SEA-treated mice did not expand (Fig. 7a). However,
many more live V3+ CD4 T cells were recovered
when Gr-1+ cells or IFN- were removed. In
these cultures T cell response peaked between 2 and 4 days after
restimulation, and there were no appreciable additive effects of
combining the Gr-1 depletion protocol with blocking Abs to IFN-. The
cultures from the control mice did not begin to expand until 3 days
after stimulation (Fig. 7a) and were not significantly
affected by the presence of Gr-1 cells or IFN-. Since the
V3+CD4 T cells from SEA-treated mice expanded
with slightly accelerated kinetics in the rescued cultures compared
with the cultures from control animals, these data suggest that some
cells had been preactivated by their in vivo exposure to the
injected SEA.
The CFSE analysis showed dividing V3+ CD4 T
cells in the control cultures on days 2, 3, and 4 after stimulation
(Fig. 7, b and c). This rate of cell division was
not influenced by the Abs to IFN- or treatment to remove
Gr-1+ cells. In contrast, very few dividing
V3+ CD4 T cells were recovered from the
cultures from SEA-treated mice unless neutralizing Abs to IFN- were
used (Fig. 7d) or Gr-1+ cells were
removed (Fig. 7e). The V3+ CD4 T
cells proliferated extensively in these cultures and were not further
enhanced when Abs to IFN- and Gr-1+ cell
depletion were used together (Fig. 7e).
The few live CD4 T cells that were recovered from the suppressed
cultures appeared to initiate cell division on day 2, but no further
divisions were detected on the subsequent days of the analysis (Fig. 7d). These data strongly suggest that responding
V3+ CD4 T cells were deleted from suppressed
cultures as they started dividing on day 2. Very few live
V3+ CD4 T cells remained in the suppressed
cultures by day 4. Some residual populations of naive CD4 T cells that
did not express V3 also remained in all the cultures throughout the
3-day restimulation period. The size of this unstimulated cell
population remained relatively unchanged throughout the analysis in
both the SEA-treated and control cultures (data not shown). Together
these data suggest that naive or resting CD4 T cells are resistant to
the deletion and that only activated or responding CD4 T cells are
targeted by the IFN--dependent death mechanism. Additional
experiments were used to further address this possibility.
Activated or responding CD4 T cells are the targets of the
IFN--dependent suppression
To determine when CD4 T cells became susceptible to the
IFN--dependent death mechanism, we analyzed the surviving CD4 T
cells from the suppressed and rescued cultures for the expression of T
cell activation Ags, using three-color FACS analysis. B10.BR mice were
used for this analysis to compare the response of SEA-reactive
V3+ CD4 T cells with the response of CD4 T
cells that express SEA-nonreactive V-chains in the same cultures.
Total populations of live CD4 T cells are shown (Figs. 8, a–d). The live
V3+ CD4 T cells from the cultures that were
rescued with neutralizing Abs to IFN- are also shown as a separate
analysis (Fig. 8, e and f). There were
insufficient numbers of live V3+ CD4 T cells
in the suppressed cultures for extensive phenotypic analysis.
|
, indicating the presence of enlarged CD4 T
cell blasts (Fig. 8a). In contrast, relatively few live CD4
T cells were recovered from the suppressed cultures, and residual cells
were low in forward scatter, indicating that they were primarily
unstimulated CD4 T cells. Further analysis showed that the enlarged
cells in the rescued cultures were also primarily
V3+ CD4 T cells (Fig. 8b), which
expressed elevated levels of CD44 (Fig. 8c) and reduced
levels of CD62L (Fig. 8d), indicating that they were
Ag-experienced T cells. The V3+ T cells were
virtually all activated CD4 T cells with high levels of CD44 (99%) and
reduced CD62L expression (81%), suggesting that they were also
activated T cells (Fig. 8, e and f). The residual
CD4 T cells in the suppressed cultures did not express V3,
indicating that they were primarily SEA-nonreactive T cells. These
cells were naive in phenotype, with low CD44 and high CD62L expression.
Small populations of nonlymphoid cells were also recovered from all the
cultures, which are not shown in this analysis.
These data suggest that only CD4 T cells that did not express
SEA-reactive TCRs, and were thus unable to interact with the SAg,
survived in the suppressed cultures when IFN- was present, whereas
the responding CD4 T cells, including the V3+
cells, were deleted. In light of these data, we conclude that
responding or activated CD4 T cells were specifically targeted by the
death/deletion mechanism in the suppressed cultures.
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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the myeloid cells suppress the proliferative
response of the SEA-reactive CD4 T cells by triggering cell death as
the CD4 T cells initiate cell division. Purified
Gr-1+ cells from the SEA-treated animals are also
able to suppress the proliferative response of fresh naive CD4 T cells
from untreated animals when stimulated with PCCF peptides. This
suggests that once the conditions for the IFN--dependent suppression
have been established in vivo, SAg may not be directly required to
induce T cell death in vitro. We therefore suggest that this
mechanism of suppression may contribute to the down-regulation of T
cell responses to other immune stimuli.
We have used cell purification and Ab-mediated depletion protocols to
identify the cells responsible for the IFN--dependent suppression.
We show that as an alternative to removing IFN-, the SEA-induced
suppression can be prevented by depleting Gr-1+
myeloid cells from the cultures before restimulation (Figs. 3 and 5).
In contrast, depleting NK cells or T cells did not prevent the
suppression (data not shown). Since Abs to IFN- did not further
enhance the survival of the CD4 T cells in the restimulated cultures
once the Gr-1+ cells had been removed, our data
suggest that IFN- induces suppression by regulating the activity of
the Gr-1+ cells.
In most experiments, Gr-1+ cells accounted for
about 10–15% of the spleen cells from SEA-treated mice and <3–4%
in control animals. In several experiments, the suppressive cells and
SEA-reactive V3+ CD4 T cells were purified
together (
Figs. 1–3). Since all of the cultures were profoundly
suppressed after restimulation, these data demonstrate that there were
sufficient numbers of Gr-1+ cells in the spleens
of the SEA-treated mice to completely suppress the proliferative
response of V3+ CD4 T cells at physiological
ratios. In other experiments,
Thy-1-Gr-1+ cells were
isolated from the spleens of SEA-treated mice by sterile FACS sorting
and titrated into mixed cultures of CD4 T cells. These experiments
confirmed that as little as 5–10% Gr-1+ cells
(i.e., a ratio of 1 Gr-1+ cell to 10 T cells)
were sufficient to suppress the proliferative response of fresh naive
CD4 T cells from untreated animals as well as sorted
V3+ CD4 T cells isolated from SEA-treated mice
(Fig. 4). Although small numbers of Gr-1+ cells
were also isolated from untreated control animals, much higher ratios
of these cells (i.e., 1:2) were required to induce equivalent
suppression in the mixed cultures (data not shown).
Cytospin analysis shows that 70–80% of the sorted
Gr-1+ cells from SEA-treated mice are mature
neutrophils, with highly segmented nuclei (Fig. 5a) and
about 20–30% are monocyte/macrophages. In other
experiments, we have found that purified neutrophils (98% pure) are
able to suppress T cell responses in the presence of IFN- by a
mechanism that can be inhibited with L-NMMA (L.S.
Cauley and S L. Swain, unpublished observations). As in other studies,
we also find that purified macrophages are able to suppress T cell
responses; however, in the SAg model, subfractionation experiments
indicate that neutrophils and macrophages both contribute to the
suppression (data not shown). Both groups of
Gr-1+ cells from the SEA-treated mice also
expressed high levels of Mac-1 and LFA-1, which is characteristic of
highly activated granulocytes (30). In contrast, the
Gr-1+ cells recovered from control animals
included some immature neutrophils with banded nuclei (data not shown)
and some Mac-1-negative cells, suggesting that the granulocytes from
the control animals were less activated.
Our studies indicate that LN cultures are not affected by the
IFN--dependent mechanism of T cell suppression after SEA injection.
Others have shown that neutrophils rapidly down-regulate L-selectin
(CD62L) upon activation (35), which may explain why much
larger numbers of these cells are found in the spleens of the
SEA-treated mice than in their peripheral LNs (data not shown). We also
usually find slightly larger numbers of neutrophils in the spleens of
the Tg AND mice than in the spleens of normal animals (data not shown),
which may explain why smaller doses of SEA are required to induced the
IFN--dependent suppression in AND mice than in normal animals.
Although neutrophils are short lived cells that turn over very rapidly
in vivo, their half-life can be significantly prolonged by several
cytokines, including IFN- released from T cells stimulated with SAg
(36). Activated neutrophils also express a variety of
chemokines that are known to be chemotactic to activated T cells,
including the IFN- (MIG), IFN-inducible T cell
chemoattractant (I-TAC), and IFN--inducible protein-10 (IP-10)
(37), which are likely to bring the neutrophils into
close proximity with activated T cells within the SAg-treated
animals.
Neutrophils and monocytes release a variety of metabolic products
during the oxidative burst that is stimulated by IFN-, including
superoxide, H2O2, and NO
(31, 32). We find that the iNOS inhibitor
(L-NMMA) and the two ROI-scavenging antioxidants (NAC and
GSH) substantially reduce the IFN--dependent suppression (
Figs. 1–3) and show substantial additive effects in many of the cultures.
These data indicate that NO and ROI both contribute to the SEA-induced
suppression. However, the antioxidants were most effective in cultures
isolated at the peak of the in vivo response (Fig. 3). Others have
shown that superoxide is produced only transiently after exposure to
receptor-dependent agents, such as IFN- (32), whereas
iNOS will continue to produce NO until the substrate has been consumed
or the enzyme itself is degraded. The different reaction kinetics of
these two enzymes may explain why NO appears to play a greater role in
SEA-induced T cell suppression in cultures isolated >4 days after SEA
injection (Fig. 2).
Neither NO nor superoxide behaves as a strong oxidant toward most types of organic compounds. However, they react rapidly with one another to generate a variety of more potent oxidants, including peroxynitrite (ONOO), hydroxyl anions (·OH), and, in a reaction catalyzed by myeloperoxidase, hypochlorous acid (HOCl) (reviewed in Refs. 38 and 39). These potent oxidants can cause tissue damage through a variety of mechanisms, including interference with cellular respiration and direct DNA damage. DNA damage, in turn, stimulates repair activity by poly(ADP-ribose) synthase and depletes cellular energy supplies (40, 41). Peroxynitrite can also modify tyrosine residues, interfering with cell functions such as cell cycle progression (42) and inactivating enzymes such as superoxide dismutase (SOD) (43) and promoting apoptosis in activated T lymphocytes (34). NAC reacts slowly with superoxide and H2O2, but is an excellent scavenger of hydroxyl radicals and hypochlorous acid (44). Since we found significant additive effects of inhibiting both NO and ROI, it is possible that peroxynitrite contributes to the SEA-induced suppression.
Many previous studies have shown a dramatic decline in the numbers of
responding T cells during the in vivo response to injected SAg (Fig. 1). Some studies suggested that Fas-mediated AICD may be largely
responsible for the loss of responding T cells in mice primed with
staphylococcal enterotoxin B (SEB) (45). However, others
have found that the Fas mutation in lpr mice is not
sufficient to prevent T cell deletion in SEB-treated mice
(46, 47, 48) (L. S. Cauley and S. L. Swain,
unpublished observations). One study went on to show that Tg Bcl-2 also
had little effect on the T cell response to SEB in vivo. However, when
the Tg Bcl-2 mice were cross-bred with lpr mice, significant
accumulation of V8+ CD4 T cells was detected 7
days after SEB injection (48). Although it is likely that
injected SAg stimulates some Fas-mediated AICD in normal animals
(45), recent evidence suggests that Fas-mediated AICD in
lymphocytes is not sensitive to inhibition by Bcl-2 (49).
Together, these data suggest that two or more independently
regulated mechanisms with some redundant function may be responsible
for removing activated CD4 T cells from the circulation after in vivo
SAg treatment. It is likely that at least one of these mechanisms may
be sensitive to inhibition by Bcl-2 (50). Some evidence
suggests that NO-induced apoptosis in T cells may be sensitive to
inhibition by Bcl-2 (51). Bcl-2 has also been shown to
influence the levels of intracellular antioxidants, including SOD and
GSH, and thus inhibit apoptosis in cells exposed to
H2O2 (52, 53).
Further studies are needed to determine whether the IFN--dependent
mechanism of T cell suppression described in this study contributes to
the deletion of responding CD4 T cells during the in vivo response to
injected SAg. However, circumstantial evidence suggests that related
mechanisms may be involved. In particular, CD4 T cells harvested from
the SEA-treated animals at the peak of the in vivo response (day 3)
undergo very little AICD in the presence of neutralizing Abs to IFN-
(Figs. 1 and 4) or after sterile FACS sorting (Fig. 4). This indicates
that the mechanism that would normally have deleted many of these
SEA-reactive CD4 T cells in vivo had they remained in the SAg-treated
animals was blocked because the cells were removed from the animal or
by the Abs to IFN-. Either alternative suggests that there is an
active component to the mechanism that regulates T cell deletion in
vivo. The suppressed cultures also contain large numbers of dead
V3+ CD4 T cells, indicating that T cell death
is the cause of the in vitro suppression.
Other studies also suggest that cytokine-regulated mechanisms may
contribute to the loss of responding T cells in SAg-treated mice. In
particular, LPS treatment has been shown to inhibit the loss of
responding CD4 T cells in mice given relatively low doses of injected
SEA (54). The mechanism of this LPS-mediated rescue is not
yet clear; however, some evidence suggests that TNF- may play a role
(55). More recently, a mimetic of SOD was used to show
that low doses of injected SEA can induce a superoxide-mediated
mechanism of cell death in T cells from SAg-treated mice
(21). This study did not show in vivo data; however, an
earlier study showed that NAC slightly enhanced T cell survival in mice
stimulated with a mouse mammary tumor virus-derived SAg
(20). The origin of the ROI was not investigated in either
of these studies. A potential link between the ROI-mediated cell death
and the enhancing effects of LPS on T cell survival in SAg-treated mice
may lie in the ability of TNF- to up-regulate expression of the SOD
enzyme (56). Alternatively, TNF- may induce apoptosis
in neutrophils (57), preventing them from releasing ROI
and NO in response to IFN-. Priming with LPS has also been shown to
inhibit IFN- production in SEB-treated mice (58), which
could reduce NO and ROI production by both macrophages and
neutrophils.
Superantigens are produced by a wide variety of bacteria and viruses
(59). In many respects, T cell responses to SAg are
similar to the response to conventional Ags, and it is likely that both
responses are regulated by similar mechanisms. IFN-,
Gr-1+ cells, and RNI have all been suggested to a
play role in the effector phase of an experimental autoimmune
encephalomyelitis model (15, 16, 17). Large numbers of
activated CD4 T cells have also been shown to accumulate in IFN-
knockout mice after BCG infection, and CD4 T cells from BCG-infected
wild-type mice underwent apoptosis by a mechanism that required IFN-
and NO (18). Another study has reported T cell
hyperproliferation in IFN- knockout mice infected with lymphocytic
choriomeningitis virus and partial resistance to cell death
(19). These studies support the suggestion that
IFN- may play an important role in controlling T cell responses in
vivo through mechanisms similar to those described here and suggest
that these mechanisms may play a role in preventing autoimmunity.
A model that may explain our results is that in addition to activating
large numbers T cells in vivo and stimulating the release of large
quantities of cytokines (1, 2, 3, 4, 5, 6), injected SEA may also
promote activated neutrophils and macrophages to colocalize with the
responding T cells in an inflammatory site in the spleens of the
SAg-treated animals. We postulate that once the conditions for
suppression have been established in vivo, subsequent antigenic
restimulation in vitro promotes further IFN- production, stimulating
small numbers of Gr-1+ cells in the enriched T
cell cultures to produce a combination of RNI and ROI. Our data suggest
that RNI and ROI directly cause activated or responding CD4 T cells to
undergo apoptosis, as has been suggested in other models (20, 21, 33, 34). This possibility will be further investigated in future
studies. IFN- is produced by a variety of cells in vivo, including
CD8 T cells and NK cells. It is likely that macrophages and/or
responding CD4 T cells produce IFN- in the restimulated
cultures.
In conclusion, we find that the CD4 T cells that persist in vivo after
systemic SEA injection are not intrinsically unresponsive to antigenic
stimulation, but become targets of an active mechanism of suppression
upon in vitro restimulation. This suppression is IFN- dependent,
requires a mixed population of Gr-1+ myeloid
cells, and targets activated or responding CD4 T cells for death in the
presence of NO and ROI. We suggest that the mechanism described here
may constitute a general form of peripheral down-regulation of intense
immune responses that would otherwise lead to massive CD4
expansion.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Susan L. Swain, Trudeau Institute, P.O. Box 59, 100 Algonquin Avenue, Saranac Lake, NY 12983.
3 Abbreviations used in this paper: SAg, superantigen; AICD, activation-induced cell death; BCG, bacillus Calmette-Guérin; CFSE, 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester; iNOS, inducible NO synthase; LN, lymph node; NAC, N-acetylcysteine; L-NMMA, NG-monomethyl L-arginine; D-NMMA, NG-monomethyl D-arginine; PCCF, pigeon cytochrome c fragment; PI, propidium iodide; RNI, reactive nitrogen intermediate; ROI, reactive oxygen intermediate; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; SOD, superoxide dismutase; Tg, transgenic.
Received for publication June 27, 2000. Accepted for publication September 1, 2000.
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L. I. Terrazas, K. L. Walsh, D. Piskorska, E. McGuire, and D. A. Harn Jr. The Schistosome Oligosaccharide Lacto-N-neotetraose Expands Gr1+ Cells That Secrete Anti-inflammatory Cytokines and Inhibit Proliferation of Naive CD4+ Cells: A Potential Mechanism for Immune Polarization in Helminth Infections J. Immunol., November 1, 2001; 167(9): 5294 - 5303. [Abstract] [Full Text] [PDF]
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O. Atochina, T. Daly-Engel, D. Piskorska, E. McGuire, and D. A. Harn A Schistosome-Expressed Immunomodulatory Glycoconjugate Expands Peritoneal Gr1+ Macrophages That Suppress Naive CD4+ T Cell Proliferation Via an IFN-{gamma} and Nitric Oxide-Dependent Mechanism J. Immunol., October 15, 2001; 167(8): 4293 - 4302. [Abstract] [Full Text] [PDF]
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S. L. Swain Interleukin 18: Tipping the Balance towards a T Helper Cell 1 Response J. Exp. Med., August 6, 2001; 194(3): f11 - f14. [Full Text] [PDF]
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) or neutralizing Abs to
IFN-
). a, The numbers of live
V
) or 50 µM
D-NMMA (
). Three days later the cultures were
analyzed for live V
