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*
Department of Clinical Immunology, Royal Free and University College Medical School, London, United Kingdom;
Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom;
Division of Immunity and Infection, Birmingham University Medical School, United Kingdom; and
§
Department of Immunopathology, Glaxo-Wellcome Medicines Research Centre, Stevenage, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. This increases cell recovery after
stimulation, which is not due to T cell proliferation. This mechanism
for apoptosis inhibition rapidly stops protein kinase C-
translocation from the cytoplasm to the nucleus, which is an early
event in the death process. A central observation was that
CD4+ T cells that are rescued from apoptosis after T:T
presentation of peptide by IFN-ß remain profoundly anergic to
rechallenge with Ag-pulsed APCs. However, anergized cells retain the
ability to respond to IL-2, showing that they are nonresponsive but
functional. The prevention of peptide-induced apoptosis in activated T
cells by IFN-ß is a novel mechanism that may enable the survival
and maintenance of anergic T cell populations after TCR engagement.
This has important implications for the persistence of anergic T cells
with the potential for immunoregulatory function in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the relationship between apoptosis and anergy as a
consequence of TCR ligation we added specific peptide to human
CD4+ T cell clones. The resulting T:T presentation of this
peptide by T cells to each other in the context of MHC class II induces
apoptosis in a proportion of the responding cells, while the nondeleted
cells are nonresponsive to subsequent challenge with Ag-pulsed APCs
(6, 8, 9, 10). Previous studies have demonstrated that
IFN-ß secreted by fibroblasts and IFN- secreted by other cell
types can prevent cytokine deprivation-induced T cell apoptosis in both
humans (11, 12) and mice (13), achieved in
part by the up-regulation of Bcl-xL, but not
Bcl-2, expression (12, 14, 15). Fibroblast-conditioned
medium
(FCM)4 and
IFN-ß could also prevent the translocation of the signaling
molecule protein kinase C- (PKC-) from the cytoplasm to the
nucleus of IL-2-deprived T cell lines, which prevented its activation
by caspase-3 (11). The active form of PKC- can induce
apoptosis when transfected into hemopoietic cells (16, 17), and specific activation of PKC- by Bistratene A can lead
to death (16, 17, 18). These results prompted us to
investigate whether FCM and IFN-ß can also prevent apoptosis
resulting from TCR ligation during T:T presentation of Ag and whether
these CD4+ T cells are responsive to subsequent
challenge with Ag-pulsed APC. We demonstrate that fibroblast-secreted
IFN-ß inhibits the peptide-induced, CD95-mediated apoptosis of
CD4+ T cell clones and that this rescue from
death enables the persistence of an anergic T cell population.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN- and IFN-ß were obtained from PeproTech (London, U.K.)
and BioSource Europe (Nivelles, Belgium). IL-2 was obtained from R&D
Systems (Abingdon, U.K.) and Roche (Mannheim, Germany). Sheep
anti-human IFN-ß and anti-hepatocyte growth factor
(anti-HGF) Abs were obtained from Sigma (Poole, U.K.). Anti-CD95
blocking Abs were a gift from Immunex Research and Development Corp.
(Seattle, WA). Anti-CD95-inducing Ab, which triggers death, was
obtained from Upstate Biotechnology (Lake Placid, NY). The MR6 (IgG1)
Ab was used as a control. Neutralizing sheep anti-human IFN-ß Ab
and a control sheep anti-human HGF Ab were obtained from Serotec
(Oxford, U.K.). Polyclonal Abs specific for STAT-1, -3, or -5 and
affinity-purified rabbit anti-human PKC- Ab were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA),
Induction of anergy in CD4+ T cell clones
The stimulation and maintenance of three human CD4+ clones, 7P.24, 7P.41, and 7P.61, which are specific for influenza hemagglutinin (HA307–319), restricted by HLA-DRB1*0701, and derived from an HLA-DRB1*0701 DRB1*1301 individual have been described in detail previously (10). Additional human CD4+ T cell clones used in this study include NF4, which is specific for HA307–319 and restricted by DRB1*0101, and HC3, which is specific for HA100–115, restricted by DRB1*1010, and obtained from a DRB*0101, DRB1*0403 individual. Polyclonal CD4+ T cell lines were generated as described previously (19). A murine IL-2-dependent cytotoxic T cell line (CTLL) was used to determine IL-2 secretion of peptide-stimulated clones as described previously (10). To induce apoptosis and anergy, 7P.24 and 7P-41 T cell clones were cultured with 1 µg/ml of peptide in the absence of IL-2 or accessory cells for 18–24 h (10). To test for anergy, the cells that were precultured with peptide were washed and recultured with irradiated B lymphoblastoid cells (B-LCL), which were pulsed with different concentrations of peptide. Proliferation was measured by [3H]thymidine uptake after 72 h of culture (5, 10). Apoptosis was also induced in T cell clones by stimulation with anti-CD3 Abs that were immobilized on plastic in the absence of costimulation as described previously (20).
Dual chamber coculture of T cells and fibroblasts
T cells and fibroblasts were separated by a semipermeable 0.22-µm pore size Transwell culture insert (Costar, High Wycombe, U.K.) in 24-well plates (14, 15). The T cell clone was placed into the insert above the fibroblasts. FCM was also added directly to T cells with identical results. The T cells were preincubated with the fibroblasts for 1 h before peptide was added and were then harvested at different times for characterization of anergy or apoptosis. We also used a control fibroblast cell line derived from a patient with a self-limiting viral arthritis (NR), which is the only one we have derived from any tissue used to date that does not rescue cytokine-deprived T cells from apoptosis due to defective IFN-ß secretion (12). Student’s t test was used to analyze the results.
Enumeration of viable, apoptotic, and cycling T cells
Viable cells were distinguished by their forward angle scatter and 90o side scatter profiles and were counted using a Cytoron Absolute flow cytometer (Ortho Diagnostics, High Wycombe, U.K.) and also by trypan blue exclusion as described previously (14). Apoptosis and cell cycling were measured at a particular time point by flow cytometry using propidium iodide as the DNA-specific label (12). Apoptosis was also measured by counting cytocentrifuge slides stained with May-Grunewald-Geimsa (14). We also measured the cumulative proliferation of T cells in culture after various treatments by the sequential addition of [3H]thymidine followed by harvest of radiolabeled cells 18–24 h later.
Analysis of STAT proteins
Activated T cells were cultured in the presence of cytokines for
15 min and washed in ice-cold PBS before the extraction of nuclear
proteins (12, 21). EMSA was performed using the
32P-labeled Fc
R1 DNA probe (21).
Nuclear extracts were preincubated for 10 min at room temperature
with H2O containing poly(dI-dC) (Roche). The
FcR1 DNA probe (GTATTTCCCAGAAAAGGAC and its complementary
sequence) was added, and the sample was incubated for an additional 20
min. Loading dye was added, and the mixture was loaded onto a 6%
nondenaturing 30/1 acrylamide:bisacrylamide gel made with 0.25x TBE.
After electrophoresis, the gel was dried without fixation and exposed
at -70°C to x-ray film. To identify the specificity of STAT protein
activation, nuclear protein extracts were preincubated with polyclonal
Abs specific for STAT-1, -3, or -5 as described previously
(12).
PKC- staining
T cell clones that had been cultured under different conditions
were spun onto slides using a Cytospin centrifuge (Shandon, Pittsburgh,
PA). Slides were air-dried, fixed in acetone for 10 min at room
temperature, and stained in an indirect immunofluorescence assay using
rabbit anti-human PKC- antisera (Santa Cruz Biotechnology).
Biotinylated goat anti-rabbit IgG antisera (Southern Biotechnology,
Birmingham AL) was used as the secondary Ab, which was developed with
FITC-conjugated streptavidin (Life Technologies, Grand Island, NY). The
slides were then counterstained with 5 µg/ml propidium iodide in PBS
and analyzed by laser scanning confocal microscopy using a MRC 500
confocal microscope (Bio-Rad, Hercules, CA). All experiments included
slides stained with species- and isotype-matched irrelevant antisera as
negative controls, which showed no or very faint staining as described
previously (11).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We incubated 7P.24 CD4+ T cells either in
medium alone or in the presence of fibroblasts in dual chamber cultures
(Fig. 1
A). The addition of
specific peptide to the medium in the absence of fibroblasts led to a
significant reduction in viable cell recovery compared with the
controls over the 3-day culture period (p <
0.001). This peptide-induced death was CD95 mediated, because the
addition of a blocking Ab, but not an isotype-specific control Ab,
could prevent this apoptosis (Table I)
(10). When the T cells were cocultured with fibroblasts
and peptide, peptide-induced death was significantly reduced after 1
(p < 0.005), 2 (p <
0.001), and 3 (p < 0.0 01) days of culture,
respectively (Fig. 1A). We obtained identical results if we
used 50% FCM to rescue the 7P.24 cells from peptide-induced death (not
shown). We were also able to prevent peptide-induced apoptosis in the
7P.41 clone by coculturing these cells with fibroblasts (Fig. 1B; p < 0.01) or in the 7P.61 clone by
culture with 50% FCM (Fig. 1C; p < 0.01).
We could also protect three different CD4+ T cell
clones from activation-induced cell death (AICD) with FCM upon
stimulation with anti-CD3 Ab in the absence of costimulation (Table II; p < 0.01 in all
cases). In addition, FCM, but not IL-2, could inhibit
anti-CD3-induced death in a polyclonal CD4+ T
cell line that was tested at the same time (Table II; p
< 0.05).
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To identify the anti-apoptotic mediators in FCM, cultures were
treated with anti-IFN-ß Ab (Fig. 2A). There was a significant
reduction in the ability of FCM to prevent peptide-induced apoptosis at
the two concentrations of Ab used (p < 0.01;
Fig. 2A). The control HGF Ab had no effect. Although
anti-IFN-ß treatment abrogated most of the survival-promoting
activity of FCM, a small amount of activity always remained regardless
of the amount of Ab used (Fig. 2A). This suggests that
additional fibroblast-derived factors may have a role in preventing
peptide-induced death.
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B) (12, 22). The preincubation of
FCM with Abs to IFN-ß, but not IFN- or HGF, substantially
decreased its ability to induce STAT-1 (Fig. 2C). This
confirms previous studies showing that the fibroblasts do not secrete
IFN- (12). A unique synovial fibroblast cell line
derived from a patient with self-limiting arthritis is the only one we
have tested that does not prevent cytokine-deprived apoptosis of
activated T cells but otherwise grows with normal fibroblastic
characteristics (12). FCM derived from this line
(NR-FCM) does not induce STAT-1 induction in T cells (Fig. 2B), indicating that it has a defective capacity to
synthesize biologically active IFN-ß. We used FCM derived from
this line to investigate whether fibroblast-secreted mediators other
than IFN-ß may be involved in the rescue of T cell clones from
peptide-induced death. The coculture of 7P.24 T cells with FCM derived
from fibroblasts defective in IFN-ß secretion (12)
showed markedly diminished rescue of these cells from apoptosis (Fig. 2D). These observations suggest that IFN-ß is the main
mediator secreted by fibroblasts that rescues T cell clones from
peptide death. It is possible, however, that a minor contribution to
survival is made by other as yet uncharacterized fibroblast-derived
mediators.
We next treated the 7P.24 T cells with increasing concentrations of
peptide in the presence or the absence of FCM, IFN-, IFN-ß, or
IL-2 (Fig. 3A). Preincubation
of the cells with FCM, IFN-, or IFN-ß for 1 h significantly
increased the viable cell recovery after peptide activation at all
concentrations tested (p < 0.005 for FCM and
p < 0.05 for IFN-ß, respectively). Preincubation
of the cells with IL-2 did not rescue from peptide-induced death as
previously described (23). IFN-ß and FCM also blocked
cell death and increased cell recovery of 7P.24 cells induced by the
direct addition of an agonistic CD95-Ab rather than by peptide (Fig. 3B). Collectively, these results indicate that IFN-ß is
the principal mediator secreted by fibroblasts that prevents
peptide/CD95-induced death in specific T cells after T:T presentation
of Ag.
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We found that the greater cell recovery induced by either FCM or
IFN-ß upon culture of peptide-stimulated T cells was not due to
induction of proliferation. We first investigated the ability of T
cells that were rescued from peptide-induced death after T:T
presentation to incorporate [3H]thymidine over
a 3-day period compared with that of control cells. In these
experiments [3H]thymidine was added to parallel
cultures 18–24 h before harvest, and the uptake of this label at 1, 2,
and 3 days reflects the cumulative proliferation during this period
(Fig. 4). While cells that were cultured
with peptide-pulsed APCs or IL-2 showed increasing proliferation after
3 days, peptide-cultured cells that had been rescued from death by FCM
or cells cultured in peptide alone did not proliferate (Fig. 4).
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ß worked
in part by the ability of these cytokines to prevent the translocation
of PKC- from the cytoplasm to the nucleus of these cells
(11). These studies showed that the PKC- translocation
could be quantitated by either Western blot analysis or confocal
microscopy (11). We now show, in agreement with our
previous observations, that IFN-ß also prevents PKC-
translocation to the nucleus during rescue of 7P.24 T cells from
peptide-induced death as assessed by confocal microscopy (Fig. 5). This suggests that this
translocation may be a common process that occurs when apoptosis is
induced by different routes, and that part of the broad range of
anti-apoptotic activity mediated by IFN-ß is related to their
ability to block this translocation regardless of the apoptosis
induction process.
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ß-rescued T cells remain anergic to
peptide-pulsed APCs
We next tested the ability of T cells that had been protected from
peptide-induced death by FCM to respond to challenge with Ag-pulsed APC
(Fig. 6). Cells cultured with FCM alone,
without peptide, responded to Ag-pulsed APCs when tested after 1, 2, or
5 days after culture (Fig. 6A). Cells exposed to 1 µg/ml
of peptide alone or peptide together with FCM in the first culture were
completely anergic and unresponsive to further challenge (Fig. 6A). This nonresponsiveness was observed over a wide
concentration range of peptide on APCs (Fig. 6B). There was
significantly greater cell recovery of peptide-stimulated T cells when
cultured in the presence of FCM (see above). In an additional set of
experiments we showed that CD4+ T cells that were
rescued from peptide-mediated death upon direct addition of IFN- or
IFN-ß instead of FCM were also anergic to challenge with Ag-pulsed
APC (Fig. 6C). The presence of IFN-ß in the control T
cell cultures, which were incubated without peptide, did not affect
proliferation or IL-2 secretion of the CD4+ T
cells in response to Ag-APC in the second culture (Fig. 6C).
These results were completely reproducible in the 7P.41 T cell clone
tested (see Fig. 7) and also in the NF-4
clone (not shown). Collectively, these results suggest that IFN-ß
secreted by fibroblasts can maintain the survival of anergic
CD4+ T cells after Ag-specific activation.
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We next investigated whether T cell clones rescued from
peptide-induced apoptosis were an end-stage nonfunctional population or
retained some functional capacity (5, 24). We incubated
two different CD4+ T cell clones with peptide in
the presence or the absence of FCM (Fig. 7, A and
B). The peptide-induced apoptosis was significantly
diminished in the presence of FCM in both clones tested (see Fig. 1).
However, the rescued T cells were anergic to rechallenge with
peptide-pulsed APCs as assessed by their IL-2 secretion. We next
investigated the ability of the anergized cells to respond to IL-2. We
cultured 7P.41 (Fig. 7C) and 7P.24 (Fig. 7D) T
cells in medium alone, with peptide, or with FCM or FCM together with
peptide for 24 h as in Fig. 7, A and B. The
cells were then washed and cultured with exogenous IL-2 for a further
48 h before assessment of [3H]thymidine
incorporation. In clone 7P.41, the addition of IL-2 to the
peptide-anergized T cells, regardless of whether they had been rescued
with FCM, significantly enhanced their ability to proliferate to IL-2
compared with control cells cultured in medium or FCM alone (Fig. 7C). This shows that the rescued anergic cells are
nonresponsive to peptide-pulsed APCs but remain functional. In clone
7P.24, the cells that were anergized in the absence of FCM showed an
apparently diminished capacity to respond to IL-2 (Fig. 7D).
This reflects the fact that the 7P.24 cells are extremely sensitive to
apoptosis induced by peptide compared with the 7P.41 clone
(10). Most of the recovered 7P.24 cells that are used in
the IL-2-responsive assay are therefore already dead or are in the
early stages of apoptosis. Nevertheless, the observation that induction
of anergy in the presence of FCM, which prevents this death,
significantly increases the responsiveness of 7P.24 cells to IL-2
compared with that of cells cultured with peptide alone further
strengthens the idea that FCM rescue enables the persistence of
nonresponsive, but functional, T cells (Fig. 7D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell clones both phenotypically and functionally resemble highly differentiated CD4+ T cells that are found in the blood and other tissues of normal individuals, because they are CD45ROhigh, CD45RBlow and have reduced capacity to secrete IL-2 (25, 26). In addition, clones that have been maintained in culture have experienced more rounds of proliferation and have shorter telomeres than recently established T cell lines, further indicating that they are an older and more differentiated population (27). The decreased capacity of CD4+ T cell clones to secrete IL-2 (25) may make them more sensitive to the survival-promoting effects of type 1 IFN. Hence, the impact of type 1 rescue of T cells from peptide-induced death may have greater bearing on the function of highly differentiated CD4+ T cells, with important consequences for immune function in older individuals.
In previous studies we have shown that a wide range of fibroblasts can
prevent the apoptosis of optimally stimulated T cells
(15), and in this situation the prevention of activated T
cell death may enable the persistence of responding cells and
contribute to the maintenance of immune memory (28). We
now show that when T cells are stimulated to specifically induce
anergy, IFN-ß and IFN- can prevent the CD95-mediated apoptosis of
these cells (5, 6, 7). These results suggest that IFN-ß
may have a general role in regulating the survival of T cells after TCR
ligation. However, the context of T cell activation, depending on
whether there is complete or partial TCR signaling (10) or
whether costimulatory signals are present or absent (1),
may determine whether the rescued cells retain responsiveness or are
anergic.
It has been shown that IFN-1 may have anti-proliferative activity for activated T cells (29) and also that these cytokines may induce, rather than prevent, apoptosis under certain conditions (30). Both these studies were performed on T cells that were activated from an initially resting state, while we investigated the effects of IFN-1 on T cells that were already activated. This indicates that apart from age and/or state of differentiation, the activation status of the T cell may also determine the type of effect that IFN-1 can induce. This suggests that the timing of IFN-1 secretion during an immune response is a crucial factor in regulating the extent of T cell death and proliferation (31). While anti-proliferative effects of these cytokines may manifest themselves during the induction of stimulation, the anti-apoptotic effects may have a role once T cell activation has taken place (31).
It is unclear whether the signaling pathways for the
anti-proliferative and anti-apoptotic actions of IFN-1 are
interlinked. IFN-1 has been shown to induce
Bcl-xL expression (12, 15), inhibit
caspase-3 activation, and prevent PKC- translocation and activation
(11) and can therefore interfere with the induction,
commitment, and execution phases of apoptosis (31). The
present study suggests that the ability of IFN-1 to prevent the death
of anergic cells, which can have inhibitory activity on some immune
responses, may be another way in which the immune system may return to
a quiescent state after activation. It would be important to clarify
whether the efficacy of IFN-1 treatment in various diseases is related
to the anti-viral, anti-proliferative, or anti-apoptotic
activity of these cytokines (31).
Many recent studies support the possibility that T:T presentation of Ag occurs in vivo. Inflammatory processes may generate degraded peptides from the Ag by enzymatic cleavage (9). These peptides could then bind to the HLA-DR of the activated T cells, in analogy to the addition of peptide to specific T cells in vitro (6, 8, 9). In addition, activated T cells can actively acquire MHC/peptide complexes from APCs, which can then be presented to other specific T cells (32, 33), and this may be associated with the induction of tolerance (33). Furthermore, T cells themselves may process and present Ag under certain conditions (34). Alternatively, activated T cells can internalize their TCR and process and present fragments of these molecules in the context of class II on their surfaces to other T cells, leading to the induction of anergic immunoregulatory cells (35). In support of this latter possibility, TCR peptide-specific T cells that recognize processed TCR components exist in vivo and are able to play a regulatory role in autoimmunity (36).
The results presented here have several important implications. The
ability of IFN-ß to promote the survival of anergic T cells may
not only be restricted to situations of T:T presentation of Ag. Anergy
can also be induced by presentation of Ag to T cells by endothelial and
also epithelial cells in the context of MHC class II, which is induced
on these cells during inflammatory responses (37). The
secretion of IFN-ß by these cells themselves or by neighboring
cells may also promote anergic T cell survival in the tissues in these
situations. Furthermore, during viral infections the secretion of
IFN-ß can be up-regulated considerably, especially by certain
specialized cells, such as type 2 dendritic cells and their precursors
(38, 39). This may promote the survival of both responsive
and anergic T cells after viral infection, and the balance between
these functionally different populations may determine the subsequent
responsiveness to the virus. It is also possible that IFN-ß
therapy in various diseases may affect the balance between anergy and
apoptosis in responding T cells. The most intriguing possibility
however is that the induction of anergic T cell survival induced by
IFN-ß may promote the persistence of immunoregulatory populations
in vivo and contribute to maintenance of the tolerant state to self and
other Ags by active regulation rather than deletion of responsive cells
(24, 40, 41, 42, 43).
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Footnotes |
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2 G.L. and P.J.D. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Arne N. Akbar, Department of Clinical Immunology, Royal Free and University College Medical School, Pond Street, Hampstead, London, United Kingdom NW3 2QG.
4 Abbreviations used in this paper: FCM, fibroblast-conditioned medium; PKC-, protein kinase C-; HGF, hepatocyte growth factor; HA, hemagglutinin; B-LCL, B lymphoblastoid cells; AICD, activation-induced cell death.
Received for publication May 3, 2000. Accepted for publication July 13, 2000.
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References |
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by an ICE-like protease in apoptotic cells. EMBO J. 14:6145.
by an ICE/CED-3-like protease induces characteristics of apoptosis. J. Exp. Med. 184:2399.
and its association with nuclear lamins. J. Biol. Chem.
275:S19B.
-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression. Eur. J. Immunol. 26:294.[Medline]
of STAT factors following T cell activation. Eur. J. Immunol. 26:704.[Medline]
inhibits proliferation in human T lymphocytes by abrogation of interleukin 2-induced changes in cell cycle-regulatory proteins. Cell Growth Differ. 10:575. augments activation-induced cell death by upregulation of Fas (CD95/APO-1) and Fas-ligand expression. Cytokine 11:736.[Medline]
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) or a 1/200 (
) dilution of neutralizing
anti-IFN-ß Ab in the pretitrated optimal range for 1 h
(A). The treated or untreated FCM was then added to
IL-2-deprived 7P.24 T cells for an additional 1 h before the
addition of 1 µg/ml of specific peptide. Viable cell recovery was
determined after 24 h. Anti-HGF Ab was used as a control.
Identical results were obtained in a second separate experiment.
B, A CD4+ T cell line was deprived of IL-2
and cultured for 15 min in the presence of 10 ng/ml of IFN-ß, FCM
(50%), or FCM from the only fibroblast line tested to date that does
not rescue these cells from cytokine deprivation-induced death
(NR-FCM). Nuclear extracts were analyzed for the presence of STAT-1,
-3, and -5 by adding polyclonal Abs to these proteins in supershift
assays (B). C, Treatment of FCM with
polyclonal Abs to IFN-ß for 60 min inhibits its ability to induce
STAT proteins. The Ab-treated FCM was added to a CD4+ T
cell line for 15 min, and nuclear extracts were then tested for the
presence of STAT proteins. Lane a, Control, where FCM
was not added to the cells. FCM was added to the cells in lanes
b—f: lane b, no Ab; lane c, with
Abs to both IFN-
