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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1006-1012
IMMUNOBIOLOGY
Interleukin-15 redirects the outcome of a tolerizing T-cell
stimulus from apoptosis to anergy
Hans Dooms,
Tom Van Belle,
Marjory Desmedt,
Pieter Rottiers, and
Johan Grooten
From the Department of Molecular Biology, Molecular Immunology Unit,
Flanders Interuniversity Institute for Biotechnology and Ghent
University, Ghent, Belgium.
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Abstract |
Clonal deletion and anergy are 2 mechanisms used by the immune
system to establish peripheral tolerance. In vitro, these mechanisms are induced in T lymphocytes by triggering the T-cell receptor (signal
1) in the absence of costimulation (signal 2). T-cell clones have been
shown either to become anergic or to die in response to signal 1 alone;
yet the factors that govern this choice remain unknown. This study
evaluated the influence of the cytokines interleukin (IL)-2 and IL-15
on the response of the Th1 clone hemagglutinin (T-HA) to signal 1, delivered by stimulation with immobilized anti-CD3 monoclonal antibody
(mAb). The response induced by immobilized anti-CD3 mAb was dependent
on the cytokine milieu; in the presence of IL-2, T-HA cells were
subject to apoptosis, whereas in the presence of IL-15 the cells
remained viable but showed proliferative unresponsiveness. After
release from the anti-CD3 stimulus, the IL-15-rescued T-HA cells
regained responsiveness to IL-2 and IL-15 growth factor activity.
However, they were unable to proliferate when stimulated with their
cognate antigen presented by professional antigen-presenting cells
(signal 1 plus 2) and thus had acquired an anergic phenotype. These
data assign a novel function to the previously reported antiapoptotic
activity of IL-15, namely, the capacity to redirect the T-cell response
to partial stimulation from clonal deletion to anergy. Furthermore,
they emphasize that the cytokine environment can critically influence
the outcome of a tolerizing stimulus.
(Blood. 2000;96:1006-1012)
© 2000 by The American Society of Hematology.
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Introduction |
Interleukin (IL)-15 is a cytokine that shares many
biologic activities with IL-2. IL-15 induces proliferation of activated T lymphocytes and natural killer (NK) cells and release of interferon (IFN)- , stimulates cytolytic activity of cytotoxic T lymphocytes (CTL) and NK cells, and costimulates immunoglobulin synthesis by B
cells.1-4 These redundant functions of IL-2 and IL-15 are an obvious consequence of their common usage of the IL-2/IL-15  and
c receptor subunits for binding and initiation of intracellular signaling.5 However, both cytokines use a specific
high-affinity binding  chain to compose their heterotrimeric
receptor complex in T and NK cells.6 Furthermore, IL-2 and
IL-15 also differ in their cellular sources of production and in the
way their expression and secretion are controlled. IL-2 production
occurs exclusively in activated T cells and expression is mainly
controlled by transcriptional regulation and messenger RNA (mRNA)
stabilization. IL-15 mRNA is widely distributed in many different cell
types and tissues, and its expression is tightly regulated at the level
of transcription, translation, and secretion.7 Due to these
differences, IL-15 exerts several unique functions in lymphoid and
nonlymphoid tissues, not shared by IL-2.
In T lymphocytes, IL-15 and IL-2 have different roles in the regulation
of apoptosis. Although both cytokines protect resting T cells from
growth factor deprivation-induced cell death,8 they exert
opposite activities during T-cell receptor (TCR)-induced cell death.
Engagement of TCR during an antigenic response results in activation of
the T cell, progression into the cell cycle, and production of IL-2,
promoting further expansion. Re-engagement of TCR during clonal
expansion activates the Fas/Fas ligand (FasL) apoptotic pathway and
results in deletion of the activated T lymphocyte.9 This
TCR-induced cell death not only restores cellular homeostasis at the
conclusion of an immune response but also preserves peripheral tolerance. Lenardo10 originally demonstrated in vitro that
the presence of IL-2 is essential for this apoptotic response of
activated T cells to TCR re-engagement. Experiments with IL-2,
IL-2R, and IL-2R knockout mice further confirmed the essential
role of IL-2 in peripheral T-cell homeostasis and tolerance; mice
lacking the IL-2,11 IL-2R,12
or IL-2R13 gene suffered from severe lymphoproliferative disease and autoimmune disorders, explained by
impaired deletion of excessive and potentially autoreactive T
lymphocytes, respectively. We and others demonstrated that IL-15 protects against TCR-induced death signaling, in contrast to IL-2. In
vitro, CD4+ T lymphocytes treated with IL-15 are resistant
to apoptosis induced by antigen, resulting in enhanced and extended
antigen-specific proliferation.14 In vivo, Bulfone-Paus and
coworkers15 reported that cells from the liver, spleen, and
thymus of mice challenged with an anti-Fas antibody were rescued from
apoptosis by an IL-15 IgG2b fusion protein.15 However,
under certain conditions rescue from TCR-induced cell death may
represent a threat to the maintenance of peripheral tolerance in the
animal, because the rescued cells may generate autoimmune activity or
become anergic. Anergized T cells remain viable but lose their capacity
for IL-2 production and proliferation in response to an otherwise full
stimulatory signal consisting of TCR-engagement (signal 1)
and costimulation (signal 2).16 Initially, anergy was
described as the consequence of stimulating a T cell with signal 1 in
the absence of signal 2 either by activating T cells either with
antigen presented by chemically fixed antigen-presenting cells (APC),
or with immobilized anti-CD3 mAb or with ConA.17 More
recently, it has been documented that anergy can also be induced in the
presence of appropriate costimulation by T-T antigen presentation or by
inhibiting correctly stimulated T cells to divide with anti-IL2R
antibody or rapamycin.18-20 Although these different
conditions of anergy induction result in the same basic characteristics
of proliferative unresponsiveness and blocked IL-2 production,
anergized T cells show discrepancies in other features such as
responsiveness to exogenous IL-2, reversibility of the anergic
condition, and suppressive activity on other T cells. It is, however,
still unknown how the 2 main mechanisms of tolerance
induction apoptosis and anergyare related. Unclear is whether a
particular T cell will preferentially die in response to a tolerizing
stimulus, whereas others will become anergic, or whether both pathways
can be induced in the same T cell, the outcome depending on
environmental factors such as the presence or absence of certain
cytokines. Considering the opposite effects of IL-2 and IL-15 on
TCR-induced cell death, we investigated whether both cytokines
differentially affect the apoptotic viz. anergic response of a
CD4+ T cell clone to partial (signal 1) stimulation. The
results demonstrate that the antiapoptotic cytokine IL-15 skews the T
cell to anergy, whereas the proapoptotic cytokine IL-2 generates
apoptosis in the same T-cell clone after stimulation with signal 1 in
the absence of signal 2. Furthermore, to determine the possible impact
on a later immune response, we followed the functionality of the anergized T cells in terms of their responsiveness to environmental IL-2 and IL-15 in the presence or absence of a normal antigenic stimulus.
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Materials and methods |
Animals
Female C57Bl/6 (H-2b) mice were purchased from the
Broekman Instituut (Eindhoven, The Netherlands). For spleen cell
preparations, mice were used at the age of 9 to 14 weeks.
Antibodies, cytokines, and cytokine assays
Purified anti-CD3 mAb (145-2C11; kindly provided by Dr G. Leclercq,
Ghent, Belgium) was used at a concentration of 10 µg/mL in
phosphate-buffered saline (PBS) to coat flat-bottom microwells (30 µL/well) for 2 hours at 37°C. Unbound antibody was removed by 4 washing steps before adding cells. For immunofluorescence, binding of
anti-CD3 mAb was detected with a fluorescein isothiocyanate (FITC)-conjugated antihamster IgG (clone G94-56; Pharmingen, San Diego,
CA). Antimouse Fas (clone Jo2), antimouse FasL (clone Kay-10), and
anticommon chain (clone TUG2m) were also purchased from Pharmingen.
Anti-IL-2R (clone PC61) and anti-IL-2/15R (clone TM-1) mAbs
were produced in our laboratory. Human recombinant IL-15 (rIL-15)
(Peprotech, London, UK) had a specific activity of
2 × 106 U/mg. Human recombinant IL-2 (rIL-2) was
produced in our laboratory and had a specific activity of
1.3 × 107 IU/mg as determined in a CTLL-2 assay (1 IU corresponds to 77 pg). Murine IL-2 present in cell culture
supernatants was quantified with a Quantikine M mouse IL-2 Immunoassay
kit (R&D Systems, Minneapolis, MN) following the manufacturer's
instructions. IFN- was assayed by a cytopathic effect inhibition
assay on murine L929 cells, using encephalomyocarditis virus as a
challenger. Granulocyte/macrophage colony-stimulating factor (GM-CSF)
was detected by measuring growth of factor-dependent FDCp1 cells. For
quantification, murine IFN- (103 U/mL; NIAID Research
Resources Section, Bethesda, MD) and mGM-CSF (1 × 105 U/µg; National Institute for Biological
Standards and Control, Potters Bar, UK) were used as standards.
CD4+ T-cell clone
The influenza A/H3 HA-specific and H-2b-restricted
CD4+ murine T-cell clone T-HA was developed and maintained
as previously described.14 Briefly, T-HA cells were
cultured in vitro by biweekly restimulation with 10 ng/mL
bromelain-cleaved hemagglutinin (BHA) and 5 × 107
syngenic spleen cells from C57 BL/6 mice (3000 rad gamma irradiated). On day 2, 30 IU/mL of human IL-2 was added, after which T cells were
further expanded by renewing culture medium and IL-2 every 4 days.
Culture medium consisted of RPMI 1640 buffered with 12.5 mmol/L HEPES
(Life Technologies, Paisley, Scotland) and supplemented with 10% fetal
calf serum (FCS), 2 mmol/L GlutaMAX-1 (Life Technologies), 100 U/mL
penicillin, 100 µg/mL streptomycin, 1 mmol/L sodium pyruvate, and
5 × 10 5 mol/L 2-mercaptoethanol.
Flow cytometric analysis of apoptotic cell numbers
Apoptotic cells were measured by addition of 30 µmol/L propidium
iodide (PI) (ICN Pharmaceuticals, Costa Mesa, CA) to harvested T-HA
cells. The percentage of PI-positive cells was measured with a
FACScalibur flow-cytometer (Becton Dickinson, Sunnyvale, CA) at 610 to
630 nm. To discriminate T-HA cells from irradiated spleen cells in
coculture experiments, T-HA cells were first irreversibly labeled with
the membrane dye PKH2-GL (Sigma Chemical, St Louis, MO), as
described.14 Percentages of apoptotic T-HA cells were then
obtained by flow-cytometric analysis of PI-positive cells emitting
green fluorescence (525 nm).
Proliferation assays
The T-HA cells, cultured with the indicated concentrations of IL-2
or IL-15, were harvested and washed 3 times to remove cytokines. T-HA
cells (1 × 104) were seeded in triplicate in
flat-bottom 96-well plates for 72 or 96 hours with cytokine,
immobilized anti-CD3 or antigen/APC; proliferation was measured by
addition of [3H]thymidine (0.5 µCi/well) for the last
12 hours of incubation. Cells were harvested on glass fiber filters and
incorporated [3H]thymidine was measured by a Topcount
scintillation counter (Packard Instrument, Meriden, CT). In case of
antigen-induced proliferation, 200 ng/mL BHA was used as antigen and
2 × 105 irradiated C57BL/6 spleen cells as a source
of APC.
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Results |
Anti-CD3-induced cell death, but not proliferative unresponsiveness,
is differentially regulated by IL-2 and IL-15 in T-HA cells
We have previously compared the activities of IL-2 and IL-15 on
CD4+ T lymphocytes that were stimulated with antigen
presented by professional APC (signal 1 plus 2) or that were left
unstimulated.14 In the absence of TCR engagement, both IL-2
and IL-15 delivered survival signals to CD4+ T lymphocytes,
protecting the cells against growth factor deprivation-induced cell
death. However, although IL-2, at its optimal growth factor activity
(10 ng/mL), induced survival accompanied by proliferation, unstimulated
CD4+ T lymphocytes did not proliferate with IL-15 but
acquired a quiescent phenotype. This effect was observed using a wide
dose range of IL-15 (0.1-200 ng/mL). The conditioned T lymphocytes also
showed a differential responsiveness on stimulation with antigen/APC; IL-15-conditioned cells proliferated much stronger to antigenic stimulation than IL-2-conditioned cells.14 This difference
in response was due to the fact that IL-15 protected antigen-stimulated CD4+ T lymphocytes against TCR-induced cell death, whereas
IL-2 sensitized for this form of cell death. In the current study, we
compared the response of IL-2- versus IL-15-conditioned
CD4+ T cells on stimulation with signal 1 alone. In an
introductory experiment, we wanted to ensure that IL-2 and IL-15
exerted their activity on the CD4+ T cell clone T-HA by
usage of the same receptor components. Therefore, survival of
unstimulated T-HA cells induced by IL-2 and IL-15 was measured in the
presence of blocking antibodies against the different receptor chains.
Table 1 shows that blocking the and
c chain of the IL-2/15 receptor inhibited IL-2- and
IL-15-induced survival signals in T-HA cells. As expected, mAb against
the specific IL-2R chain only blocked IL-2-induced but not
IL-15-induced survival signals. Thus, both IL-15 and IL-2 exerted their
effects on T-HA cells via the c chains of the IL-2/15
receptor complex.
As a model to assess the response of IL-2- versus IL-15-conditioned
T-HA cells to signal 1 alone, T-HA cells were treated for 48 hours with
IL-2 (10 ng/mL) or IL-15 (1 ng/mL) and subsequently stimulated with
immobilized anti-CD3 mAb in the absence of APC, a stimulation condition
that induces unresponsiveness in T cell clones.21 As
expected, T-HA cells did not proliferate in response to immobilized
anti-CD3 (Figure 1A), although amounts of
IL-2 in the nanogram range were produced (5-15 ng/mL). Also, addition of exogenous IL-2 or IL-15 during CD3 cross-linking could not restore
proliferation (data not shown). This state of proliferative unresponsiveness was not differentially influenced by conditioning with
IL-2 or IL-15. As a control, the same cells stimulated with antigen/APC
showed the expected differential response after IL-2 or IL-15
conditioning, in agreement with our previous results.14 To
verify whether the proliferative unresponsiveness induced by anti-CD3
mAb was the consequence of cell death, apoptosis was measured after 24 and 48 hours by flow cytometric analysis of PI uptake. As shown in
Figure 1B, T-HA lymphocytes conditioned by IL-2 massively underwent
apoptosis, whereas IL-15 protected against anti-CD3-induced cell death.
Although immobilized anti-CD3 mAb thus induces proliferative
unresponsiveness in both IL-2- and IL-15-conditioned T-HA cells, the
mechanism responsible for this unresponsiveness differs; IL-2 promotes
elimination of the T cells by apoptosis, whereas IL-15 promotes
survival accompanied by a proliferative block of T cells.
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| Fig 1.
Proliferative unresponsiveness of T-HA cells induced by
immobilized anti-CD3 is only after IL-2 but not IL-15 conditioning the
consequence of cell death.
(A) Immobilized anti-CD3 does not induce proliferation of T-HA cells.
T-HA cells were treated for 48 hours with IL-2 (10 ng/mL, solid bars)
or IL-15 (1 ng/mL, open bars), harvested and stimulated with antigen in
the presence of irradiated splenocytes (APC) or immobilized anti-CD3
mAb (10 µg/mL). Medium and APC alone were added as controls.
Proliferation was measured by [3H]thymidine incorporation
after 72 hours in culture. Data shown are the mean cpm of triplicate
cultures ± SD. (B) IL-15 protects against anti-CD3-induced cell
death. Viable T-HA cells (4 × 104), treated for 48 hours with IL-2 (solid bars) or IL-15 (open bars), were incubated on
96-well plates coated with anti-CD3 mAb (10 µg/mL). Anti-CD3-induced
death was measured after 24 and 48 hours by PI uptake and flow
cytometry. Cultures containing cytokine but no anti-CD3 mAb were used
as controls. Results shown represent 3 pooled wells and are
representative for several similar experiments. (C) Anti-CD3-induced
death is partially inhibited by anti-Fas plus anti-FasL. T-HA cells
treated with IL-2 (10 ng/mL) were seeded on plates coated with anti-CD3
in the absence or presence of anti-Fas (5 µg/mL) and anti-FasL (2.5 µg/mL) mAb. Cell death was determined after 24 hours as described in
panel B. (D) IL-2 and IL-15 do not differentially influence CD3
expression. IL-2-conditioned (black lines) or IL-15-conditioned (gray
lines) T-HA cells were labeled with anti-CD3 mAb and FITC-conjugated
antihamster IgG (thick lines) or with the detecting antibody alone
(thin lines). Fluorescence was measured by flow cytometry. Imm
indicates immobilized.
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Although most authors support the view that anti-CD3, in combination
with IL-2, activates mainly the Fas/FasL cell death
pathway,22 other effector molecules such as tumor necrosis
factor or reactive oxygen species can be used to induce apoptosis after
T-cell activation.23,24 To assess the contribution of the
Fas death pathway to apoptosis induced by anti-CD3 plus IL-2,
antagonistic Abs against Fas and FasL were applied. The combined
addition of both mAbs reduced the apoptotic response of the
IL-2-treated T-HA cells against immobilized anti-CD3 by approximately
50% (Figure 1C). This suggests that Fas/FasL interaction is an
important but not the sole death effector mechanism induced by anti-CD3
plus IL-2 in the T-HA clone. Moreover, it can be concluded that IL-15
protects against both these Fas-dependent and Fas-independent apoptotic
pathways. Finally, the possibility existed that a differential
expression of CD3 by the T-HA cells after culture in IL-2 or IL-15
caused the quantitatively different apoptotic signal induced by
anti-CD3 cross-linking. Although unlikely considering the strong
proliferative response of IL-15-treated T-HA cells against antigen/APC
and the similar up-regulation of IL-2R induced by immobilized CD3,
we nevertheless analyzed CD3 expression on IL-2- versus
IL-15-conditioned T-HA cells. As shown in Figure 1D, no difference in
CD3 levels was observed, thus ruling out this possibility. Therefore,
we conclude that the antiapoptotic activity of IL-15, previously
reported to act on appropriately stimulated CD4+ cells,
also is effective on partially, signal 1-stimulated T cells, allowing
the cells to remain viable but unresponsive under these tolerizing
stimulation conditions.
T-HA cells stimulated with signal 1 alone have prolonged sensitivity
to the mitogenic activity of IL-15
During stimulation with antigen/APC, T-HA cells are responsive to
the mitogenic activity of IL-15. However, in the absence of TCR
engagement, T-HA cells become quiescent in response to IL-15 although
they continue to proliferate with IL-2.14 To determine
whether T-HA cells activated by signal 1 alone also became quiescent
after removal of the stimulus, IL-15-conditioned cells were activated
by immobilized anti-CD3 for 48 hours, harvested, and cultured with
IL-15 or IL-2 for an additional 96 hours. In contrast to T-HA cells
stimulated with signal 1 plus 2, the cells that received signal 1 alone
proliferated not only strongly with IL-2 but also with IL-15 (Figure
2A,B). This result was surprising because
T-HA cells are unable to proliferate as long as their CD3 molecules are
cross-linked by immobilized anti-CD3 Ab (Figure 1A), although they
produce a mitogenic amount of IL-2 (14 ng/mL) under this condition.
After release from the stimulus, however, they transgress to an
activated, IL-2- and IL-15-responsive state. The persistence of this
semiactivated state was evaluated by continued culture of T-HA cells
with a mitogenic dose (10 ng/mL) of IL-2 or with IL-15 (1 ng/mL) for 12 days and subsequently measuring proliferation in response to the
respective cytokines. Figure 2, panels C and D, show that the partially
stimulated T-HA cells remained responsive to IL-2 but finally lost
their responsiveness to the mitogenic activity of IL-15, thus behaving
in a similar manner as appropriately stimulated T-HA cells. In
conclusion, IL-15-treated T-HA cells stimulated with immobilized
anti-CD3 remain viable, incapable of autocrine growth, but can still
respond to the exogenous growth factors IL-2 and IL-15 for some days
after removal of the TCR stimulus. Then they gradually lose the
capacity to proliferate in response to IL-15, whereas IL-2
responsiveness remains unaffected.
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| Fig 2.
T-HA cells rescued from anti-CD3-induced death by IL-15
transgress from a semiactivated to a quiescent state.
T-HA cells were treated for 48 hours with IL-15 (1 ng/mL). On day 0, the cells were harvested, washed, and stimulated for 48 hours with
immobilized anti-CD3 (solid lines) or with antigen/APC (dotted lines).
On day 2, viable cells were recovered and 1 × 104
cells were incubated with the indicated doses of IL-2 (A) or IL-15 (B)
for 96 hours. Proliferation was analyzed by measuring
[3H]thymidine incorporation on day 6. In parallel,
long-term cultures in IL-2 (10 ng/mL) or IL-15 (1 ng/mL) were set up
with the T-HA cells recovered on day 2. On day 14, these cultures were
harvested and 1 × 104 cells were seeded and tested
for proliferative responsiveness to IL-2 (C) or IL-15 (D) by pulsing
with [3H]thymidine for the last 12 hours of the 72-hour
assay period (day 17). All results are expressed as mean cpm from
triplicate cultures ± SD.
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T-HA cells rescued by IL-15 from immobilized
anti-CD3-induced cell death are anergic
Hargreaves and colleagues25 showed that T-cell clones
stimulated by T-T antigen presentation died from Fas/FasL interaction. Rescue from cell death by an anti-Fas mAb rendered the cells anergic to
appropriate restimulation with antigen/APC. Because IL-15 similarly rescued the cells from immobilized anti-CD3-induced cell death, we
questioned whether T cells could still respond when appropriately restimulated with signal 1 plus 2. Therefore, IL-15-conditioned T-HA
cells were stimulated for 48 hours on anti-CD3-coated plates, detached,
and further cultured with IL-2 (10 ng/mL) or IL-15 (1 ng/mL) for 12 days. In parallel, control cultures of T cells stimulated with
antigen/APC were maintained under the same conditions. On day 12, the
cultures were harvested, labeled with a green fluorescent dye, and
stimulated with antigen/APC. The proliferative response, IL-2
production, and occurrence of cell death were measured. As shown in
Figure 3A, no proliferative response could
be induced by antigen/APC in T-HA cells that had previously been
stimulated with immobilized anti-CD3 mAb, irrespective of the cytokine
(IL-2 or IL-15) added during the intermittent culture period. As
opposed to these partially stimulated T-HA cells, T-HA cells that
received signal 1 plus 2 as first stimulus proliferated in response to a rechallenge with antigen/APC, although proliferation after culture in
10 ng/mL IL-2 was limited for the reasons mentioned above. In addition,
partially stimulated T-HA cells were unable to produce IL-2 after
restimulation with antigen/APC (Figure 3A, inset). Thus, although IL-15
protects T-HA cells against immobilized anti-CD3-induced cell death,
the surviving cells have become unresponsive to restimulation with
antigen/APC. It should also be noted here that addition of a dose of
IL-2 (10 ng/mL) that clearly induces cell division during the
intermittent culture period (Figure 2A,C) apparently does not break the
state of unresponsiveness in our T-cell clone. To resolve the question
whether unresponsiveness was the consequence of cell death or anergy,
that is, a state of proliferative unresponsiveness to antigen coupled
to viability, we followed the emergence of dead T-HA cells during the
antigenic challenge. Figure 3B shows that cell death occurred in the
absence of any stimulus or growth factor but was prevented by signals
derived from the APC. The presence of antigen did not further enhance
nor reduce the number of living cells, in agreement with the anergic
state of the cells. Hence the unresponsiveness to antigen/APC of T-HA
cells rescued from immobilized anti-CD3-induced cell death by IL-15 is
the result of anergy and not of TCR-induced cell death.
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| Fig 3.
T-HA cells rescued from anti-CD3-induced death have
become anergic.
IL-15-conditioned T-HA cells were stimulated for 48 hours with
immobilized anti-CD3 mAb or antigen/APC (first stimulus), recovered,
and further cultured for 12 days with IL-2 (10 ng/mL) or IL-15 (1 ng/mL) (intermittent culture). On day 14 after the initial stimulation,
viable cells were harvested, labeled with the green fluorescent
membrane marker PKH2-GL and 1 × 104 stained cells
were stimulated with medium (open bars), APC (hatched bars), or
antigen/APC (solid bars). (A) Proliferation was measured by addition of
[3H]thymidine for the last 12 hours of the 72-hour assay
period. Results are expressed as mean cpm from triplicate cultures ± SD. (A, inset) Supernatants were taken from the cultures and IL-2
content was determined. (B) The percentage of apoptotic cells of the
stained cell population was determined at the indicated time points by
flow cytometry and PI uptake. The background percentage of dead cells
present in the population at the time the stimulation was started is
represented by the gray bars. Imm indicates immobilized.
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Anergic T-HA cells stimulated with antigen/APC
proliferate in response to IL-15 and IL-2 and produce cytokines
To verify whether anergic T cells may still contribute to immune
responses despite their proliferative block, we investigated whether
TCR signaling in anergic cells induced responsiveness to exogenous
growth factors and production of endogenous cytokines. T-HA cells
rendered anergic by IL-15 treatment, followed by 12 days of culture in
IL-2 or IL-15, were restimulated with antigen/APC in the absence or
presence of a suboptimal, nonmitogenic concentration of exogenous IL-2
(1 ng/mL) or a standard, likewise nonmitogenic, concentration of IL-15.
Figure 4 shows that antigen/APC
activation of anergic cells makes them responsive to the growth factor
activity of exogenous IL-15, and especially of low-dose IL-2. This
result suggests that availability of even low concentrations
of bystander growth factors such as IL-2 and IL-15 in the
microenvironment of antigen-activated, anergic cells may lead to the
further expansion of these cells. Furthermore, in culture supernatants
from antigen-activated, anergic cells we detected a significant amount
of IFN- and marginal GM-CSF production as compared to appropriately
stimulated T-HA cells (Table 2).
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| Fig 4.
Antigenic stimulation makes anergic T-HA cells responsive
to nonmitogenic concentrations of growth factors.
IL-15-conditioned T-HA cells were stimulated for 48 hours with
immobilized anti-CD3 mAb (first stimulus), recovered, and further
cultured for 12 days with IL-2 (10 ng/mL) or IL-15 (1 ng/mL)
(intermittent culture). On day 14, 1 × 104 of these
anergic T-HA cells were restimulated with medium (open bars), APC
(middle bars), or antigen/APC (solid bars) in the absence
or presence of IL-2 (1 ng/mL) or IL-15 (1 ng/mL) (restimulation).
Proliferation was measured by addition of [3H]thymidine
for the last 12 hours of the 72-hour assay period. Data shown are mean
cpm from triplicate cultures ± SD. This experiment was performed 3 times with comparable results.
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The observation that anergized T cells still possess the capacity to
produce cytokines in response to antigenic stimulation emphasizes the
potential of anergic cells to play a regulatory role during immune reactions.
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Discussion |
Apoptosis and anergy have both been proposed as important mechanisms
for the establishment of tolerance in the periphery. It is, however,
still unknown how both mechanisms are related. In the past, various
T-cell clones have been examined for the nature of their response to a
tolerizing stimulus. Studies using immobilized anti-CD3 mAb or other
stimuli to tolerize clearly showed that some T-cell clones responded by
undergoing apoptosis, whereas others became anergic.26-30
Furthermore, in T cells that undergo apoptosis induced by T-T antigen
presentation, blockade of apoptosis with an anti-Fas mAb resulted in
anergy induction, thus showing that both pathways can be activated
simultaneously.25 So far, however, no physiologic factors
have been identified that can discriminate between anergy and apoptosis
induction in a single T cell clone. So it remains an open question
which inactivating mechanism is chosen by a particular, potentially
autoreactive T-cell clone in response to a tolerizing stimulus and
which factors govern this choice. In the present study, we used
immobilized anti-CD3 mAb to induce unresponsiveness in the
CD4+ T-cell clone T-HA and compared the influence of the
cytokines IL-2 and IL-15 on the type of unresponsiveness induced in
this clone. These cytokines were chosen because they exert opposing activities with respect to cell death initiated by TCR engagement; IL-2
promotes and IL-15 inhibits TCR-induced cell death.10,14,15 In our T-HA clone, immobilized anti-CD3 mAb induced proliferative unresponsiveness. However, the cytokine added during the culture period
preceding anti-CD3 treatment critically determined the nature of this
unresponsiveness. Thus, T-HA cells that had been cultured with IL-2
underwent robust apoptosis after CD3 cross-linking, explaining the
absence of proliferation. IL-15-treated cells, although also incapable
of division, remained on the contrary viable. These surviving cells no
longer responded by proliferation to a normally full stimulatory
condition, namely, antigen presented by professional APC, demonstrating
that they had acquired an anergic phenotype. These results indicate
that cytokines can govern the mechanism by which a particular T-cell
clone is tolerized. Delivery of signal 1 alone in the presence of IL-2
results in rapid apoptosis, whereas in the presence of IL-15 anergy is
favored. Because anti-CD3-induced cell death of IL-2 treated T-HA cells
is at least partially mediated by Fas/FasL interaction, the rescue from
apoptosis by IL-15 correlates with the described protective activity of
IL-15 against Fas-induced cell death. Apparently, IL-15 skews a signal
1 response from apoptosis to anergy by blocking the Fas death signaling
pathway. Clearly, Fas/FasL-induced apoptosis is extremely important for
maintenance of tolerance in the secondary lymphoid
organs,31 and sensitization for this Fas death pathway by
IL-2 is accordingly a key regulatory event to avoid autoimmunity.
Inhibition of this tolerizing death pathway by IL-15 might result in
the establishment of a less absolute form of tolerance, namely, anergy
instead of depletion. Recently, also IL-12 has been attributed
protective capacity against Fas-mediated cell death of antigen-specific
T cells.32 Addition of an anti-IL-12 neutralizing antibody
to mice tolerized for OVA enhanced peripheral tolerance, possibly by
allowing apoptosis to prevail. Thus, whereas IL-2 clearly promotes a
tolerizing response to signal 1, other cytokines such as IL-12 and, as
we show here, IL-15 may abate such a response by preventing apoptosis
but instead generating anergy.
Whereas our data clearly demonstrate a decisive, albeit opposite role
of the c-signaling cytokines IL-2 and IL-15 in induction of anergy, other data seem to contradict such a role. Thus, Boussiotis and associates33 clearly demonstrated that signaling via
the c chain, leading to Jak3 tyrosine-phosphorylation,
prevented induction of unresponsiveness to a tolerizing stimulus in
human T cell clones. However, these authors used antigen presented by APC with blocked B7 function to stimulate the T lymphocytes. Thus, alternative costimulatory molecules expressed by the APC could influence the response of the T cells to signal 1 and the
c-signaling cytokine. In our model, using anti-CD3 mAb
as signal 1, this problem is avoided. Also, a weaker aggregation of
TCRs by antigen presented via APC, as opposed to immobilized anti-CD3
mAb, could facilitate escape from unresponsiveness provided additional
cytokine signaling is available.
To further characterize the state of (un)responsiveness of anergized
T-HA cells, we analyzed the activity of IL-2 and IL-15 on anergized
cells maintained for prolonged periods. T-HA cells released from
immobilized anti-CD3 regained proliferative responsiveness to exogenous
IL-2 as well as IL-15. IL-2 induced proliferation for indefinite
periods of time (up to 15 days), whereas IL-15 only exerted growth
factor activity for a few days, after which the cells proceeded to a
resting phase. The phenomenon that T cells are not responsive to
endogenous IL-2 when bound on immobilized CD3 mAb, but regain
responsiveness after release from the stimulus, has been described
before for cloned human T cells.34 Our data demonstrate
that this regulation also applies to the growth factor activity of
IL-15. Also the previously reported promotion by IL-15 of a quiescent
state on disappearance of antigen still applies to the anergized cells
with, however, the deviation that the cells once released from
immobilized anti-CD3 remained sensitive to IL-15 growth factor activity
longer than those cleared from antigen. Because residual anti-CD3 mAb
stayed bound to the cell surface for some days (data not shown), the
cells were kept in an activated condition for an additional period.
Possibly, this explains the prolonged responsiveness of the cells to
IL-15 growth factor activity. Eventually, however, these cells acquire
a resting state when further cultured in IL-15, as concluded from the
absence of proliferation and down-regulation of IL-2R expression on
day 17 (data not shown). However, independently of their proliferating
viz. resting state, T-HA cells maintained in IL-2 or IL-15,
respectively, retained their anergic condition. This result is similar
to that of other investigators who could not break anergy by the
induction of proliferation with IL-2 in anergized
cells35,36 and argues against the idea that anergy is
maintained by factors in the cytosol that dilute after a few cell
cycles, resulting in reversal of anergy.19,37,38
Although blocked in their proliferative response to signal 1 plus 2, T
cells nevertheless still exhibited responsiveness, apparent from their
production of IFN- and GM-CSF, but not IL-2, and their increased
sensitivity to the growth-factor activity of IL-2 and IL-15. The
observation that delivery of signal 1 plus 2 to anergized cells induced
sensitivity to IL-15 growth factor activity can have important
consequences for certain in vivo situations. Thus, anergized cells
stimulated by signal 1 plus 2, although failing to produce autocrine
IL-2, nevertheless may start to divide due to the availability of IL-15
in the microenvironment. This IL-15 could even be produced by the
dendritic cells presenting the cognate antigen to the anergic cells,
for example when TRANCE/TRANCE-R interactions would occur under these
circumstances.39 This activation condition could possibly
represent a breach in the defense line against self-reactive lymphocytes.
In summary, the results described in this paper demonstrate that the
outcome of a partial T-cell stimulation may be reoriented in a single
T-cell clone from apoptosis to anergy, depending on the cytokine
available to the cell. This result directly implicates that cell death
can be considered the dominating response of a T cell to signal 1 and
its inhibition by whatever means, IL-15 in our case or anti-Fas as
reported by Hargreaves and coworkers, automatically leads to emergence
of the back-up anergic pathway. Furthermore, our results emphasize the
critical influence of the cytokine environment on the final outcome of
a tolerizing stimulus. Availability of IL-2 will favor apoptosis to
occur, whereas IL-15 avoids deletion of the cell while preserving
anergy induction. Thus, in models of T-cell stimulation with signal 1 in the absence of signal 2, the cytokine could be considered as the
"signal 2," determining what tolerizing response is induced.
| |
Acknowledgments |
We are grateful to D. Ginneberge and W. Burm for technical assistance.
T.V.B. is a fellow with the Vlaams Instituut voor de Bevordering van
het Wetenschappelijk-technologisch Onderzoek in de Industrie.
| |
Footnotes |
Submitted October 18, 1999; accepted March 21, 2000.
Supported by the Interuniversitaire Attractiepolen.
Reprints: J. Grooten, Department of Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium; e-mail:
johang{at}dmb.rug.ac.be.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction