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T Cell Proliferation Induced by Autologous
Non-T Cells Is a Response to Apoptotic Cells
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J Immunol 2002; 169:1241-1250; ;
doi: 10.4049/jimmunol.169.3.1241
http://www.jimmunol.org/content/169/3/1241
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Copyright © 2002 by The American Association of
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References
Anna D. Chernysheva, Kyriakos A. Kirou and Mary K. Crow
The Journal of Immunology
T Cell Proliferation Induced by Autologous Non-T Cells Is a
Response to Apoptotic Cells Processed by Dendritic Cells1
Anna D. Chernysheva, Kyriakos A. Kirou, and Mary K. Crow2
T
he capacity of the immune system to respond to microbial
pathogens in preference to self-Ags is highly dependent
on the process of thymic selection, in which T cells with
high affinity for peptides derived from self-Ags are deleted. However, the persistence in the T cell repertoire of cells with some
reactivity with self-peptides, through presumably low affinity interactions between peptide-MHC class II complexes and TCRs, is
well documented (1–7). In the case of patients with autoimmune
disease, T cells specific for peptide components of the targeted
tissue have been characterized. For example, in multiple sclerosis
T cells with specificity for myelin-derived peptides contribute to
CNS inflammation, and in systemic lupus erythematosus T cells
reactive with peptides derived from nucleosomes, small nuclear
ribonucleoprotein particles, or ribosome particles circulate in peripheral blood and are associated with the presence of autoantibodies specific for components of those particles (3, 5, 7–15). Occasionally, healthy individuals have also been shown to have T
cells with low level reactivity with specific self-Ags (3–5, 16). In
contrast to these examples of T cell responses directed toward
characterized components of disease-targeted tissue, the basis of in
vitro proliferation of T cells cultured with autologous APC is not
understood (17, 18). In this autologous MLR (AMLR),3 low level
T cell proliferation can generate effector cells that either suppress
polyclonal Ab production or mediate autocytotoxic activity (19 –
23). The AMLR is MHC class II restricted (18, 23), but the antigenic peptides that are involved are not known.
A popular concept that has evolved in the context of autoreactivity involves cleaved fragments of self-Ags produced through the
process of apoptosis. Some of the Ags targeted by lupus autoantibodies are clustered in the cell membrane blebs that form in cells
undergoing apoptosis (24 –27). It has been suggested that cryptic
epitopes, revealed after enzymatic cleavage of those proteins by
caspases or granzymes, might activate persisting self-reactive T
cells and contribute to induction of autoimmune disease (26 –28).
However, no data have directly addressed the role of caspases in
the activation of autoreactive T cells. To elucidate the cellular Ags
that trigger autologous T cell proliferation in the AMLR and to
gain insight into the relevance of that interaction to immunoregulation, we studied the role of apoptosis and caspase activity in the
induction of T cell proliferation by autologous dendritic
cells (DC).
Materials and Methods
Mary Kirkland Center for Lupus Research, Hospital for Special Surgery, Weill Medical College and Graduate School of Medical Sciences of Cornell University, New
York, NY 10021
Received for publication October 12, 2001. Accepted for publication June 3, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by research grants from the National Institutes of Health
(R01AI42185 and R01AI45011 to M.K.C.), the SLE Foundation, Inc. (to M.K.C. and
K.A.K.), and the Arthritis Foundation (to M.K.C.).
T and non-T cell isolation and cell culture
PBMC were obtained from heparinized blood of healthy volunteers by
Ficoll-Hypaque (Pharmacia, Peapack, NJ) centrifugation after approval of
the research protocol by the Hospital for Special Surgery Institutional Review Board. PBMC T and non-T cell fractions were isolated using a magnetic bead separation system (Pan T Cell Isolation kit; Miltenyi Biotec,
Auburn, CA). Cells were incubated at 37°C, 5% CO2, in culture medium
containing RPMI 1640 (Life Technologies, Gaithersburg, MD), 10% heatinactivated autologous human serum, 2 mM L-glutamine, and 50 U/ml
penicillin and streptomycin (Life Technologies).
2
Address correspondence and reprint requests to Dr. Mary K. Crow, Hospital for
Special Surgery, 535 East 70th Street, New York, NY 10021. E-mail address:
[email protected]
3
Abbreviations used in this paper: AMLR, autologous MLR; CI, caspase inhibitor;
DC, dendritic cell; Z-VAD.fmk, N-benzyloxycarbonyl Cbz-Val-Ala-Asp fluoromethylketone; Z-DEVD.fmk, N-benzyloxycarbonyl Cbz-Asp-Glu-Val-Asp fluoromethyl-
Copyright © 2002 by The American Association of Immunologists, Inc.
ketone; Z-IETD.fmk, N-benzyloxycarbonyl Cbz-Ileu-Glu-Thr-Asp fluoromethylketone; Ac-YVAD.cho, N-acetyl-Tyr-Val-Ala-Asp aldehyde; PI, propidium iodide;
PARP, poly(ADP-ribose) polymerase; TT, tetanus toxoid; PDB, phorbol dibutyrate.
0022-1767/02/$02.00
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Self-reactive T cells are present in the mature immune repertoire as demonstrated by T cell proliferation induced by autologous
non-T cells in the autologous mixed lymphocyte reaction. This reaction generates regulatory T cells in vitro and may reflect
immune regulatory pathways in vivo, but the antigenic peptides recognized remain uncharacterized. We revisited this issue in light
of the importance of apoptosis in immune regulation. We found that apoptosis among peripheral blood non-T stimulator cells is
associated with augmented induction of autologous T cell proliferation. Our data show that caspase activity in the non-T stimulator
population is essential for induction of autologous T cell proliferation, suggesting that cellular components in the non-T cell fraction are
enzymatically modified, most likely by effector caspases, and have a direct or indirect effect on autoreactive T cell activation. Furthermore, exposure of macrophage-derived dendritic cells to apoptotic non-T cells augments autologous T cell proliferation, and
blockade of ␣v␤5 integrin, but not ␣v␤3, inhibits the capacity of irradiated non-T cells or dendritic cells to stimulate autologous
T cell proliferation. These experiments, using an entirely autologous system, suggest the interpretation that autoreactive T cells
may recognize self-Ags modified through the actions of caspases and presented to T cells by dendritic cells. Induction of an in vivo
autologous mixed lymphocyte reaction by caspase-modified self-Ags present in apoptotic cells may represent a mechanism to
maintain peripheral immune tolerance. The Journal of Immunology, 2002, 169: 1241–1250.
1242
Caspase inhibitors (CI)
The broad spectrum CI N-benzyloxycarbonyl Cbz-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk, targeting caspase-1, -3, -4, and -7) was purchased from Calbiochem (San Diego, CA). Additional CI were used in
some experiments: N-benzyloxycarbonyl Cbz-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD.fmk, predominantly targeting caspase-3 and -7, as
well as caspase-6, -8, and -10), N-benzyloxycarbonyl Cbz-Ileu-Glu-ThrAsp fluoromethylketone (Z-IETD.fmk, predominantly targeting caspase-8
and granzyme B, as well as caspase-6 and -10), and N-acetyl-Tyr-Val-AlaAsp aldehyde (Ac-YVAD.cho, targeting caspase-1 and -4) were obtained
from Calbiochem. The designated selectivities of the CI are derived from
Calbiochem and BIOMOL (www.biomol.com) catalogs. CI were used at a
25 ␮M concentration unless otherwise indicated. All inhibitors were kept
as stock solutions of 20 mM in DMSO and diluted 1/10 (2 mM) in culture
medium before addition to cell cultures. The final concentration of DMSO
in the cultures was ⱕ0.5%.
Induction of apoptosis
used to generate DC, was incubated for 24 h untreated or after gamma
irradiation with 1000 rad and then added at a 1:1 ratio to the non-T cells
exposed to GM-CSF and IL-4 for the prior 24 h. In some experiments,
mAbs to ␣v␤3 or ␣v␤5 integrin at 10 ␮g/ml were added to DC induction
cultures, together with the nonirradiated or gamma-irradiated non-T cells,
24 h after the initiation of culture. At days 6 –7, DC were collected,
washed, and used as stimulators in AMLR at various stimulator-responder
cell ratios. T cell proliferation was assessed by [3H]thymidine incorporation at day 5.
Fluorescence flow cytometric analysis
The phenotype of cells in non-T or DC cultures was assessed on day 0, 24 h
after induction of apoptosis, or at day 6 or 7 of culture with DC-inducing
cytokines. Cells were reacted for 30 min on ice with mAb specific for
CD83 (Immunotech, Marseille, France), HLA-DR, CD14, CD19 (BD
PharMingen, San Diego, CA), or ␣v␤5 (Chemicon) and then analyzed using a FACScan flow cytometer. The initial non-T cell preparation contained 27.8, 24.3, and 2.1% CD19, CD14, and CD83⫹ cells, respectively.
Results
Induction of non-T cell apoptosis by gamma irradiation and
staurosporine
Among the well-described triggers of apoptotic cell death are ionizing radiation and inhibition of protein kinases by agents such as
Determination of apoptosis
Non-T cell apoptosis was assessed by cell cycle analysis and poly(ADPribose) polymerase (PARP) cleavage flow cytometric analysis. For cell
cycle analysis, cells were washed, alcohol fixed, stained with 50 ␮g/ml
propidium iodide (PI), and analyzed using a FACScan (BD Biosciences,
San Diego, CA) flow cytometer for DNA content, as previously described
(29). The percentage of mononuclear cells containing subdiploid DNA
reflects the proportion of apoptotic cells. In the PARP cleavage assay, cells
were fixed in 1% formaldehyde, washed, fixed in alcohol, permeabilized in
0.25% Triton, and stained with polyclonal rabbit anti-PARP p89 Ab (antiPARP-85 fragment; Promega, Madison, WI), followed by secondary fluorescein-conjugated anti-rabbit Ig Ab (DAKO, Carpinteria, CA) (30). Cells
were washed, and 20 ␮g/ml PI were added to the samples 20 min before
analysis using a FACScan flow cytometer.
T cell proliferation assays
The proliferative response of T lymphocytes cultured with autologous
non-T cells (AMLR) was evaluated by [3H]thymidine incorporation.
Non-T stimulator cells were untreated or induced to undergo apoptosis by
gamma irradiation or treatment with staurosporine and then cocultured at a
1:1 or 2:1 ratio with 1 ⫻ 105 autologous responder T cells in culture
medium containing 10% autologous heat-inactivated human serum in
round-bottom 96-well microtiter plates. [3H]Thymidine (1 ␮Ci/well) was
added to each microwell at day 5. After 16 h, cells were harvested, and
DNA-associated radioactivity was counted by liquid scintillation (MicroBeta TRILUX; Wallac, Turku, Finland) and expressed as a mean cpm ⫾
SEM of quadruplicate cultures. In some experiments, the non-T cells were
cultured with 50 ␮g/ml mAb to ␣v␤3 or ␣v␤5 integrin (Chemicon International, Temecula, CA) or with control anti-TNP mAb for 1 h before
induction of apoptosis and washed from the cell preparation before initiation of the AMLR culture.
The proliferative response of T cells to 50 ng/ml phorbol dibutyrate
(PDB; Sigma, St. Louis, MO) and 500 ng/ml ionomycin (Sigma) was assessed after 5 days of culture by addition of [3H]thymidine. The proliferative response of T cells to the soluble Ag tetanus toxoid (TT) was assessed
after 5 days by addition of [3H]thymidine to the cultures of T cells with
irradiated (1000 rad) autologous non-T cells prepulsed for 16 h with 0.5 or
10 ␮g/ml TT (Massachusetts Department of Public Health Biological Laboratories, Jamaica Plain, MA).
Generation and use of DC as stimulators of AMLR
DC were generated from the T cell-depleted fraction of PBMC by culturing
non-T cells at 0.67 ⫻ 106/ml in six-well plates for 6 –7 days in the presence
of GM-CSF (Sigma), IL-4 (Sigma), and 1% autologous plasma. GM-CSF
(30 ng/ml) and IL-4 (20 ng/ml) were added to the cultures on days 0, 2, and
4. At days 6 –7, the cells had typical DC morphology and expressed CD83,
HLA-DR, and ␣v␤5 integrin. An aliquot of non-T cells, autologous to those
FIGURE 1. Induction of non-T cell apoptosis by gamma irradiation and
staurosporine. Peripheral blood non-T cells were gamma irradiated with
250-4600 rad and assessed for apoptosis by PI cell cycle analysis after 24 h
of culture. A, Mean percentage of cells expressing subdiploid DNA, ⫾
SEM, based on data from four experiments. B, To assess apoptosis by
cleavage of PARP, non-T cells were gamma irradiated with 500, 1000, or
2000 rad; after 20 h of culture, the cells were fixed, permeabilized, and
stained with polyclonal rabbit anti-PARP p89 Ab, followed by secondary
fluorescein-conjugated anti-rabbit Ig. The mean percentage of cells expressing the p89 cleavage fragment of PARP from four experiments is
shown. C, Non-T cells were cultured for 24 h with the indicated concentrations of staurosporine and apoptosis assessed by PI cell cycle analysis.
Mean data from six experiments are shown.
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Non-T cells were gamma-irradiated with 250-4600 rad from a cesium
source (intensity, 770 rad/min) and assayed for apoptosis or used as stimulators in AMLR culture. Alternatively, non-T cells were incubated for
24 h at 37°C, 5% CO2 with staurosporine (0.5–1 ␮g/ml) for induction of
apoptosis or with staurosporine (0.5 ␮g/ml) for 16 h for use as stimulators
in AMLR. The lower concentration of staurosporine was used in AMLR
cultures because higher concentrations proved toxic to T cells in the those
cultures. In some experiments, apoptosis of Jurkat T cells was induced by
incubation with anti-Fas Ab (0.5 ␮g/ml; clone CH11; Upstate Biotechnology, Lake Placid, NY) for 3 h.
AUTOLOGOUS T CELL ACTIVATION BY APOPTOTIC CELLS
The Journal of Immunology
1243
staurosporine. To determine the experimental conditions that induce apoptosis of human peripheral blood non-T cells, including B
cells, monocytes, and DC, PBMC were negatively depleted of T
cells and exposed to varying doses of gamma irradiation. After
24 h of culture, the non-T cells were assessed using two measures
of apoptosis, quantitation of cells containing subdiploid DNA by
PI cell cycle analysis and detection of cells expressing the 89-kDa
fragment of PARP. Both assays reflect the coordinate action of a
series of caspase enzymes. As shown in Fig. 1A, gamma irradiation
increased the percentage of non-T cells containing subdiploid
DNA from a baseline level of 9.1 ⫾ 1.2% to a maximum of 30.5 ⫾
5.0% after 1000 rad. Induction of apoptosis by gamma irradiation
showed a dose-related effect to the maximum at 1000 rad, followed
by a dose-related decrease, such that 4600 rad induced no apoptosis over the baseline level, consistent with a previous report (31).
These results were confirmed by FACS analysis of the percentage
of non-T cells containing the p89 caspase cleavage fragment of
PARP, again demonstrating maximum PARP cleavage after 1000
rad of gamma irradiation, with a 5-fold increase in the percentage
of p89-positive cells observed (Figs. 1B and 2). Immunofluorescence analysis of CD14, CD19, and CD83 expression to detect
monocytes, B lymphocytes, and DC, respectively, along with PI
incorporation, 24 h after gamma irradiation showed that B cells
were preferentially affected, compared with monocytes and DC, by
1000 rad gamma irradiation, consistent with a prior report (32). Of
non-T cells expressing the B cell lineage marker CD19, 22.2%
incorporated PI after gamma irradiation, indicating cell death,
whereas only 3.3% of the untreated CD19⫹ non-T cells incorporated PI (data not shown).
Similar experiments were performed to document the effective
dose of staurosporine for induction of non-T cell apoptosis. Incubation of non-T cells for 24 h with staurosporine at a concentration
of 0.5 or 1.0 ␮g/ml increased the proportion of non-T cells containing subdiploid DNA from a baseline 5.6 ⫾ 0.6% to 13.7 ⫾
2.4% and 43.5 ⫾ 2.9%, respectively (Fig. 1C). These data were
confirmed by induction of the PARP p89 cleavage fragment (data
not shown).
Augmented T cell proliferation induced by gamma-irradiated or
staurosporine-treated autologous non-T cells
We hypothesized that induction of apoptosis among non-T cells
might modify self-proteins such that they become available for
effective presentation to autologous T cells, resulting in T cell
activation and proliferation. To investigate this possibility, T cells
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FIGURE 2. Induction of PARP cleavage by gamma
irradiation. A, To demonstrate the efficacy of the PARP
cleavage assay, Fas⫹ Jurkat T cells were preincubated
for 1 h with culture medium, 25 ␮M Z-VAD.fmk, or
25 ␮M Ac-YVAD.cho and then induced to undergo
apoptosis by incubation with 0.5 ␮g/ml anti-Fas Ab for
3 h. The cells were then fixed, permeabilized, and
stained with polyclonal rabbit anti-PARP p89 Ab, followed by secondary fluorescein-conjugated anti-rabbit
Ig. Cells were treated with 20 ␮g/ml PI and analyzed
using a FACScan flow cytometer. Abscissa, DNA content; ordinate, percent of cells expressing the p89
cleavage fragment of PARP. The percent of PARP
p89⫹ cells is indicated on each histogram. B, Peripheral blood non-T cells were preincubated for 1 h with
culture medium, 25 ␮M Z-VAD.fmk, or 25 ␮M AcYVAD.cho and then induced to undergo apoptosis by
gamma irradiation with 500-2000 rad. Expression of
the PARP p89 fragment was quantitated by indirect
immunofluorescence, as in A, after 20 h of culture. The
experiment shown is representative of four performed.
Ctrl, Control.
1244
Abrogation of gamma irradiation-induced non-T cell apoptosis
and AMLR stimulation by caspase inhibitors
Induction of apoptosis among cells of the AMLR non-T cell stimulator population might modify self-Ags or nonspecifically augment Ag presenting function of surviving cells. Among several
possible mechanisms, caspase-mediated cleavage of self-proteins
might expose cryptic epitopes on self-proteins or promote Ag processing, inducing activation and proliferation of circulating T cells
with autoantigen-specific TCRs. To investigate the possibility that
apoptosis increases induction of autologous T cell proliferation
FIGURE 3. Induction of apoptosis among non-T cells is associated with
increased AMLR stimulatory capacity. A, Peripheral blood non-T cells
were gamma irradiated with 250 to 4600 rad, and both were assessed for
the percentage of subdiploid DNA by cell cycle analysis (f) or used as
stimulators in AMLR (u). Results are expressed as a percentage of the
optimal result (observed with 800 rad in this experiment). B, Non-T cell
apoptosis was induced with 1000 rad or 0.5 ␮g/ml staurosporine, and those
cells were used as stimulators in AMLRs at a 1:1 (f) or 2:1 (u) ratio of
stimulator to responder cells (S:R). Mean cpm ⫾ SEM of 11 experiments
for gamma irradiation treatment and six experiments for staurosporine
treatment are shown.
through effects of caspase enzymes, non-T cells were incubated
with CI (25 ␮M) for 1 h before gamma irradiation and then cultured for 24 h, and the percentage of apoptotic cells was determined by PI cell cycle analysis (histograms shown in Fig. 4) or
PARP cleavage (histograms shown in Fig. 2). Z-VAD.fmk, which
inhibits a broad spectrum of caspases including caspase-3 and -7,
inhibited gamma irradiation-mediated non-T cell apoptosis by
74% (Fig. 4A). Z-IETD.fmk (relatively selective for caspase-6, -8,
and -10 and granzyme B) and Z-DEVD.fmk (relatively selective
for caspase-3 and -7) blocked 55 and 49% of gamma irradiationmediated apoptosis, respectively, while the caspase-1 (IL-1-converting enzyme-like protease) inhibitor Ac-YVAD.cho was not effective (Fig. 5A). Although the CI are only relatively specific for
the designated enzymes, the data are consistent with previous studies of gamma irradiation-induced apoptosis in other cell types and
suggest that the effector caspase-3 or -7, as well as caspase-6, -8,
or -10, mediate gamma irradiation-induced non-T cell apoptosis
under these culture conditions (35).
To investigate a role for caspases in the augmented AMLR stimulatory capacity of gamma-irradiated non-T cells, those cells were
incubated with CI for 1 h before gamma irradiation, cultured for
16 h, washed, and placed in AMLR cultures (Fig. 5B). Z-VAD.fmk
inhibited the induction of AMLR T cell proliferation by 92%, and
Z-IETD.fmk and Z-DEVD.fmk were also effective, decreasing the
magnitude of the AMLR response by 63 and 61%, respectively. In
contrast, Ac-YVAD.cho, the caspase-1 inhibitor, did not substantially affect proliferation. In separate experiments, Z-VAD.fmk did
not impair viability of monocyte-derived CD83⫹ DC, ruling outloss of effective APC as an explanation for the decreased stimulation of AMLR (data not shown).
A dose-response analysis of the effect of the CI on non-T cell
apoptosis and stimulation of the AMLR response confirmed the
efficacy of Z-VAD.fmk, but not Ac-YVAD.cho, at concentrations as low as 6.25 ␮M (Fig. 5, C and D). A modest inhibition
FIGURE 4. Abrogation of gamma irradiation-induced non-T cell apoptosis by caspase inhibitors. Cytofluorograph histograms demonstrate PI
cell cycle analysis of non-T cells treated with 25 ␮M CI before 1000 rad
gamma irradiation. The percentage of cells expressing subdiploid DNA (in
the M1 region) after 24 h of culture is indicated. The experiment is representative of seven performed.
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were isolated from PBMC using a magnetic bead separation system that depletes non-T lymphoid populations, avoiding exposure
of T cells to stimuli with activating potential, such as SRBC, activating Abs, or FCS. Non-T stimulator cells were induced to undergo apoptosis by gamma irradiation or treatment with staurosporine and cocultured with autologous T cells in autologous
serum-supplemented medium. Treatment of stimulator cells with
250-4600 rad demonstrated a dose-related increase in AMLRstimulatory capacity, concurrent with increased apoptosis, with
800-1000 rad inducing maximum apoptosis and optimal augmentation of T cell proliferation (Fig. 3A). Similarly, preculture of
non-T cells with 0.5 ␮g/ml staurosporine for 16 h induced a 4-fold
increase in proliferation of T cells cultured at a 1:2 ratio with
autologous non-T cells (Fig. 3B). Data in some systems have suggested that apoptotic cells trigger maturation of APC, as assessed
by increased expression of MHC class II or costimulatory molecules (33, 34). However, immunofluorescence analysis of cell surface MHC class II expression showed no increase in the irradiated
non-T cell population (mean channel fluorescence, 689), containing increased apoptotic cells, compared with the untreated non-T
cells (mean channel fluorescence, 675) after 24 h culture. Thus,
induction of apoptosis in the non-T cell stimulator population, by
either gamma irradiation or staurosporine, increased the capacity
of those cells to induce autologous T cell proliferation in the absence of xenogenic proteins and without apparent maturation of
potential stimulator cells among the non-T cells.
AUTOLOGOUS T CELL ACTIVATION BY APOPTOTIC CELLS
The Journal of Immunology
1245
of the T cell proliferative response, but not apoptosis, was observed after treatment of gamma-irradiated non-T cells with
high doses of Ac-YVAD.cho. Caspase-1 acts on IL-1 to generate immunostimulatory IL-1␤, and inhibition of that effect
might account for the observed decrease in AMLR at 50 and
100 ␮M Ac-YVAD.cho.
Abrogation of staurosporine-induced non-T cell apoptosis and
AMLR stimulation by CI
To assess the effect of caspase inhibition on the augmented AMLR
induced by staurosporine-treated non-T cells, similar experiments
were performed. As was noted with the gamma-irradiated non-T
cells, the broad spectrum CI Z-VAD.fmk, as well as Z-IETD.fmk
and Z-DEVD.fmk, markedly inhibited both non-T cell apoptosis
(Fig. 6A) and T cell proliferation induced by staurosporine-treated
non-T cells (Fig. 6B). Preculture of non-T cells with the caspase-1
inhibitor Ac-YVAD.cho did not reproducibly inhibit AMLR stimulatory capacity at a 1:1 stimulator-responder cell ratio. Taken
together, the data described thus far suggest that induction of apoptosis among the non-T stimulator population is accompanied by
caspase-dependent modifications in components of that population
that are recognized by cocultured autologous T cells. It is likely
that several caspases are involved in the apoptotic events that confer
non-T cell stimulatory capacity. Although the CI used are broadly
active, the data are consistent with an important role for effector
caspases, including caspase-3, -6, or -7, in AMLR stimulation.
Treatment of responder T cells with caspase inhibitors does not
significantly inhibit the AMLR induced by gamma irradiation- or
staurosporine-modified non-T cells or the T cell response to
polyclonal activators
In view of recent data indicating that caspases may be required
for optimum T cell proliferation induced by mitogens or antiCD3 Ab stimulation (36, 37), we considered the possibility that
the inhibition of AMLR activity after preincubation of non-T
cells with CI might be attributable to persistent CI in the AMLR
culture and direct inhibition of T cells. Experiments parallel to
those just described were performed in which the T cell fraction, rather than the non-T cell fraction, was preincubated with
CI for 1 h and the CI was washed from the cells before culture
in the AMLR. As demonstrated in Fig. 7A, although ZVAD.fmk had a modest inhibitory effect on T cell proliferation
stimulated by gamma-irradiated non-T cells in the AMLR (29%
inhibition with 1:1 T-non-T cell ratio and 39.2% inhibition with
a 2:1 non-T-T cell ratio), the other CI had no effect, and none
of the CI substantially modified the T cell response stimulated
by staurosporine-treated non-T cells (Fig. 7B). To investigate
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FIGURE 5. Dose-dependent abrogation of gamma
irradiation (XR)-induced non-T cell apoptosis and
AMLR stimulation by caspase inhibitors. A and B,
Peripheral blood non-T cells were cultured with medium alone or with CI for 1 h before gamma irradiation with 1000 rad; the cells were incubated for
24 h and then assessed for apoptosis by PI cell cycle
analysis (A; mean of eight experiments). Alternatively, those cells were incubated for 16 h, washed,
and assessed for AMLR stimulatory capacity by
[3H]thymidine incorporation (B; mean of nine experiments). In AMLR cultures, the non-T cells were
used as stimulators at a 1:1 (f) or 2:1 (u) stimulator
to responder cell ratio (S:R). C and D, Peripheral
blood non-T cells were preincubated with medium
or with 6.25–100 ␮M Z-VAD.fmk or Ac-YVAD
.cho before 1000 rad gamma irradiation. The cells
were then assessed for apoptosis by PI cell cycle
analysis (C) and used as stimulators in AMLRs at a
1:1 (f) or 2:1 (u) stimulator-responder cell ratio
(S:R) (D). The experiment is representative of four
performed.
1246
AUTOLOGOUS T CELL ACTIVATION BY APOPTOTIC CELLS
FIGURE 6. Abrogation of staurosporine (ST)induced non-T cell apoptosis and AMLR stimulation by caspase inhibitors. Peripheral blood non-T
cells were preincubated with 25 ␮M CI for 1 h before culture with 0.5 ␮g/ml staurosporine. After
16 h, the non-T cells were washed and assessed for
apoptosis by PI cell cycle analysis (A; mean of three
experiments) and for AMLR stimulatory capacity
by [3H]thymidine incorporation (B; mean of nine
experiments). Non-T cells were placed in culture
with autologous T cells at a 1:1 (f) or 2:1 (u) stimulator-responder cell ratio (S:R). Mean [3H]thymidine incorporation of three experiments is shown.
an effect on [3H]thymidine incorporation in this assay (Fig. 7C).
It appeared unlikely, then, that the CI were acting directly on
the responder T cells to inhibit the proliferative response to
either autologous non-T cells or polyclonal activators.
FIGURE 7. Caspase activity is not required for
the T cell response to autologous non-T stimulator
cells or mitogens, or for non-T cell presentation of
optimal concentrations of soluble Ag. A and B, T
cells were preincubated with 25 ␮M CI for 1 h,
washed, and then cultured with autologous non-T
cells gamma irradiated (XR) with 1000 rad (A) or
treated with 0.5 ␮g/ml staurosporine (ST) (B) at a
1:1 (f) or 2:1 (u) stimulator-responder cell ratio
(S:R). Mean [3H]thymidine incorporation data from
six experiments for gamma irradiation treatment and
from three experiments for staurosporine treatment
are shown. C, T cells were incubated with PDB and
ionomycin (Iono), along with 25 ␮M CI, for the duration of the culture. Incorporation of [3H]thymidine
was measured on day 5 of culture. The mean cpm ⫾
SEM of three experiments is shown. D, Non-T cells
were precultured for 1 h with 25 ␮M CI before
gamma irradiation with 1000 rad and addition of TT
at 0, 0.5, or 10 ␮g/ml for an additional 16 h. After
two washes, AMLR cocultures were established
with autologous T cells (2:1 stimulator-responder
cell ratio). T cell proliferation for each condition was
compared with the corresponding value for non-T
cells preincubated without CI pretreatment and expressed as a percentage. Mean [3H]thymidine incorporation cpm values in the samples without CI were
3,701 (AMLR), 24,689 (TT 10 ␮g/ml), and 8,370
(TT 0.5 ␮g/ml). Means and error bars (SEM) were
derived from five experiments for the AMLR and
TT 10 ␮g/ml conditions and from three experiments
for 0.5 ␮g/ml.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
whether CI have an inhibitory effect on T cell proliferation
triggered by polyclonal activators, T cells were incubated with
Z-VAD.fmk or Ac-YVAD.cho and stimulated with PDB and
ionomycin. At the concentration (25 ␮M) used, neither CI had
The Journal of Immunology
Caspase inhibitors do not abrogate the capacity of non-T cells
to present optimal concentrations of soluble Ag to T cells
In the next set of experiments, the capacity of gamma-irradiated
non-T cells to stimulate Ag-specific T cell proliferation was studied using TT. None of the CI used, including Z-DEVD.fmk, ZIETD.fmk, and Ac-YVAD.cho, substantially modified the capacity
of gamma-irradiated non-T cells to present an optimal concentration (10 ␮g/ml) of TT to autologous T cells (Fig. 7D), whereas in
the same cultures, the CI abrogated non-T cell induction of autologous T cell proliferation in the absence of TT. Moreover, gamma
irradiation was not required for effective presentation of TT by
non-T cells (data not shown). When TT was used at 0.5 ␮g/ml,
generating T cell proliferation with a stimulation index of 2 compared with the AMLR, that low level response was abrogated by
the CI. These results suggest that caspase enzymes and apoptosis
may play important roles in the induction of the AMLR, and perhaps in responses to low concentrations of Ag, although not being
required for Ag-induced T cell proliferation stimulated by optimal
concentrations of soluble Ag.
One mechanism that could account for the described observations
would involve the ingestion of apoptotic debris by viable APC
remaining among the gamma-irradiated or staurosporine-treated
non-T cells, with subsequent presentation of components of
self-Ag to autologous T cells. To investigate a role for uptake of
cellular debris in AMLR activation, non-T cells were preincubated
with Ab to the ␣v␤3 integrin, present on monocytes and macrophages, Ab to the ␣v␤5 integrin, expressed on DC, or isotype control Ab. The non-T cells were then gamma irradiated, the Abs were
washed away, and the non-T cells were used in AMLR cultures.
As demonstrated in Fig. 8, the Ab specific for ␣v␤5, but not the
anti-␣v␤3 or control Abs, inhibited induction of T cell proliferation
in the AMLR. To directly test the role of DC in the presentation of
apoptotic material to autologous T cells, macrophage-derived DCs
were generated in vitro with GM-CSF and IL-4. At 24 h after the
initiation of the DC induction cultures, autologous non-T cells that
had been maintained at 37°C either untreated or after gamma irradiation with 1000 rad were added to the differentiating and ma-
FIGURE 8. Blockade of ␣v␤5 integrin on non-T cells inhibits T cell proliferation induced by autologous stimulator cells. Peripheral blood non-T cells
were preincubated with 50 ␮g/ml anti-␣v␤5, anti-␣v␤3, or control anti-TNP
mAbs for 1 h, gamma irradiated (XR) with 1000 rad, washed, and then cocultured with autologous T cells at a 1:1 (f) or 2:1 (u stimulator-responder cell
ratio (S:R). Mean cpm ⫾ SEM of four experiments is shown.
turing DCs to permit uptake of those cells. After 6 –7 days of
culture, the macrophage-derived DC were CD14 negative and expressed the typical phenotype of DC, with CD83, HLA-DR, and
␣v␤5 integrin present on a high proportion of the cell population
(Fig. 9A). DC derived in the presence of gamma-irradiated apoptotic non-T cells were superior to DC exposed to nonirradiated
non-T cells or DC alone in their capacity to stimulate proliferation
of autologous T cells, even when used at a 0.2:1 stimulator-responder cell ratio, conditions in which the number of fresh gammairradiated non-T cells is not sufficient to stimulate T cell proliferation (Fig. 9, B and C). Moreover, 10 ␮g/ml anti-␣v␤5, but not
anti-␣v␤3, Ab, when added to differentiating DC together with
gamma-irradiated autologous non-T cells, abrogated the increased
capacity of these DC to stimulate autologous T cells. Taken together, these data, generated in an entirely autologous system, support an important role for DC in the uptake and presentation of
caspase-modified self-Ags to T cells.
Discussion
The data reported here elucidate the requirements for activation of
T cells by autologous non-T cells in the in vitro AMLR and have
implications for understanding similar cellular interactions in vivo.
We demonstrated that induction of autologous T cell proliferation
requires apoptotic events among the non-T stimulator cell population. Both gamma irradiation and staurosporine effectively induced both non-T cell apoptosis and augmented AMLR stimulatory capacity, and the dose response for these two effects was
similar. Doses of 800-1000 rad gamma irradiation were optimal for
inducing both apoptosis and T cell activation, with higher doses of
irradiation proving less efficacious. As expected, caspases were
required for non-T cell apoptosis, but remarkably, induction of
autologous T cell proliferation was also dependent on non-T cell
caspase activity. In addition, T cell proliferation induced by low
concentrations of the soluble Ag TT was also sensitive to treatment
of Ag-pulsed non-T cells with CI. Although the CI used in this
study are only relatively selective for members of the inducer and
effector caspase families, the capacity of Z-IETD.fmk and
Z-DEVD.fmk to inhibit the induction of T cell proliferation by
autologous non-T cells suggests a role for the effector caspases,
caspase-3, -6, and -7, in this function (38, 39). An inhibitor of
caspase-1 had no effect on apoptosis or AMLR. Multiple caspases
are involved in the cellular pathways that mediate apoptosis triggered by various stimuli, including death receptors, gamma irradiation, and chemical apoptosis, such as that induced by staurosporine (39). These experiments were not designed to definitively
identify the caspases that are required for AMLR stimulation, but
the data strongly support a role for activation of at least some of
the effector caspases in modifying self-components among the
non-T preparation, and possibly foreign soluble Ags, such that
they acquire the capacity to induce autologous T cell proliferation.
Recent publications have focused on potential activities for
caspases beyond their well-documented role in mediating programmed cell death. Caspase activity in T cells was implicated in
the proliferative response of those cells to mitogens and anti-CD3
mAbs (36, 37). In those experiments, T and non-T cell populations
were not compared for effects of CI, and in one of the studies (36),
the inhibitors were routinely used at 100 ␮M. The data presented
here indicate that a broad spectrum CI, Z-VAD.fmk, inhibits non-T
cell-stimulatory capacity at concentrations as low as 6.25 ␮M, and
more selective CI used at the relatively low 25 ␮M dose also act
directly on the non-T cell population. These CI had at most a
modest direct effect on the capacity of T cells to proliferate in
response to autologous non-T cells, mitogens, or optimal concentrations of soluble Ag. There are persuasive data in the literature
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DC mediate the augmented AMLR induced by apoptotic non-T
cells
1247
1248
AUTOLOGOUS T CELL ACTIVATION BY APOPTOTIC CELLS
indicating that caspase activation within T cells is associated with
T lymphocyte activation (36, 37, 40), but we contend that those
caspases are not required for T cell proliferation, and in the AMLR
the major site of caspase action contributing to T cell proliferation
is in the non-T stimulator population.
These experiments do not define the stimulatory epitopes recognized by T cells in the AMLR, but the clear link of non-T cell
apoptosis and caspase activity to T cell proliferation suggests that
cellular components present in the non-T cell fraction are enzymatically modified such that innocuous cells are rendered stimulatory to cocultured T cells. Enzymatic cleavage of a large number
of self-proteins is mediated by caspases, as well as by granzymes,
in the setting of apoptosis (26, 27). PARP cleavage was documented in our experiments, but many other proteins are likely to be
fragmented, potentially exposing previously cryptic epitopes.
Other modifications might also be mediated by caspase activity,
resulting in induction of T cell activation. Apoptosis might modify
self-Ags by increasing APC acidity or prolonging residence of
self-proteins in the Ag-loading compartments (41– 43). Release of
reactive oxygen species in the course of apoptosis might biochemically modify cell proteins and support a form of polyclonal T cell
activation that was first reported several decades ago using sodium
periodate (44 – 45). Newly phosphorylated proteins might be recognized, or exposure of normally cryptic membrane lipids on apoptotic
cells might provide a stimulus for T cell activation based on presentation of lipid moieties on nonclassical MHC molecules (44, 46).
Because the AMLR has been documented to be predominantly an
MHC class II-restricted response of CD4⫹ T cells (17, 18, 23), it
seems most likely that the relevant caspase-mediated modification
would involve generation of MHC class II-associated peptides. T
cells reactive with those peptides and capable of a low magnitude
proliferative response are proposed to be present in the AMLR
culture. An additional interpretation of these data is that caspasedependent generation of apoptotic cells induces maturation or altered function of APC, such as DC. Previous reports showed induction of MHC class II and CD86 expression on DC incubated
with apoptotic cells at a 5:1 ratio or with CD40 ligand⫹ apoptotic
cells (33–34). However, other studies indicate that ingestion of
apoptotic cells by DC inhibits the maturation of those cells (47,
48). In our experiments, MHC class II expression on non-T cells
was not increased by irradiation-induced apoptosis, and the apoptotic cells were unlikely to have achieved a 5:1 ratio with DC or
to have been enriched in CD40 ligand (because they were non-T
cells).
A role for DC in the induction of autologous T cell proliferation
is supported by the data presented here, along with substantial data
in the literature (49 –53). Recent work has characterized the role of
immature DC in phagocytosing and processing components of apoptotic cells. Once matured, the DC present Ags derived from the
apoptotic cells to CD8⫹ T cells, inducing activation, proliferation,
and effector function, a phenomenon termed cross-priming (54 –
57). DC use CD36, ␣v␤5 integrin, and perhaps other cell surface
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FIGURE 9. DC process gamma-irradiated autologous
non-T cells and induce augmented autologous T cell proliferation. DCs were generated by incubation of peripheral
blood non-T cells with GM-CSF and IL-4. Twenty-four
hours after the initiation of DC-induction cultures, those
cells were supplemented with culture medium alone, nonirradiated autologous non-T cells (nT), or autologous
non-T cells gamma irradiated with 1000 rad (XRnT) at a
1:1 ratio of added non-T cells to maturing non-T cells. At
days 6 –7, DC cultures were assessed by immunofluorescence for expression of CD83, HLA-DR, and ␣v␤5 and
had a typical phenotype of maturing DCs (A). The DCs
were collected, washed, adjusted to 1 ⫻ 106/ml, and used,
along with fresh gamma-irradiated autologous non-T cells
as a control, as stimulators in AMLR at the indicated stimulator-responder cell ratios (S:R) (B). T cells were cultured with fresh non-T cells gamma irradiated with 1000
rad (F), DCs generated in medium alone (E), DCs generated in the presence of nonirradiated non-T cells (Œ), or
DCs generated in the presence of non-T cells gamma irradiated with 1000 rad. The mean cpm ⫾ SEM for two
representative experiments of four performed is shown. C,
mAbs to ␣v␤3 or ␣v␤5 integrin (10 ␮g/ml) were added to
DC induction cultures, together with nonirradiated (nT) or
gamma-irradiated non-T cells (XRnT), 24 h after the initiation of culture. At days 6 –7, DCs were collected,
washed, and used, along with fresh gamma-irradiated
non-T cells (XRnT), as stimulators in AMLR at various
stimulator-responder cell ratios. T cell proliferation was
assessed by incorporation of [3H]thymidine added at day
5 of culture. The experiment is representative of three
similar experiments performed.
The Journal of Immunology
References
1. Faherty, D. A., D. R. Johnson, and M. Zauderer. 1985. Origin and specificity of
autoreactive T cells in antigen-induced populations. J. Exp. Med. 161:1293.
2. Kotani, H., H. Mitsuya, E. Benson, S. P. James, and W. Strober. 1988. Activation
and function of an autoreactive T cell clone with dual immunoregulatory activity.
J. Immunol. 140:4167.
3. Sun, J.-B., T. Olsson, W.-Z. Wang, B.-G. Xiao, V. Kostulas, S. Fredrikson, H.-P.
Ekre, and H. Link. 1991. Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur. J. Immunol. 21:1461.
4. Link, H., O. Olsson, J.-B. Sun, W.-Z. Wang, G. Anderson, H.-P. Ekre,
T. Brenner, O. Abramsky, and T. Olsson. 1991. Acetylcholine receptor-reactive
T and B cells in myasthenia gravis and controls. J. Clin. Invest. 87:2191.
5. Liblau, R., E. Tournier-Lasserve, J. Maciazek, G. Dumas, O. Siffert, G. Hashim,
and M.-A. Bach. 1991. T cell response to myelin basic protein epitopes in multiple sclerosis patients and healthy subjects. Eur. J. Immunol. 21:1391.
6. Rider, B. J., E. Fraga, Q. Yu, and B. Singh. 1996. Immune responses to self
peptides naturally presented by murine class II major histocompatibility complex
molecules. Mol. Immunol. 33:625.
7. Pender, M. P., P. A. Csurhes, J. M. Greer, P. D. Mowat, R. D. Henderson,
K. D. Cameron, D. M. Purdie, P. A. McCombe, and M. F. Good. 2000. Surges
of increased T cell reactivity to an encephalitogenic region of proteolipid protein
occur more often in patients with multiple sclerosis than in healthy subjects.
J. Immunol. 165:5322.
8. O’Brien, R. M., D. S. Cram, R. L. Coppel, and L. C. Harrison. 1990. T cell
epitopes on the 70-kDa protein of the (U1) RNP complex in autoimmune rheumatologic disorders. J. Autoimmun. 3:747.
9. Okubo, M., K. Yamamoto, T. Kato, N. Matsuura, T. Nishimaki, R. Kasukawa,
K. Ito, Y. Mizushima, and K. Nishioka. 1993. Detection and epitope analysis of
autoantigen-reactive T cells to the U1-small nuclear ribonucleoprotein A protein
in autoimmune disease patients. J. Immunol. 151:1108.
10. Hoffman, R. W., Y. Takeda, G. C. Sharp, D. R. Lee, D. L. Hill, H. Kaneoka, and
C. W. Caldwell. 1993. Human T cell clones reactive against U-small nuclear
ribonucleoprotein autoantigens from connective tissue disease patients and
healthy individuals. J. Immunol. 157:6460.
11. Crow, M. K., G. DelGiudice-Asch, J. B. Zehetbauer, J. L. Lawson, N. Brot,
H. Weissbach, and K. B. Elkon. 1994. Autoantigen-specific T cell proliferation
induced by the ribosomal P2 protein in patients with systemic lupus erythematosus. J. Clin. Invest. 94:345.
12. Fenning, S., G. Wolff-Vorbeck, W. Hackl, U. Krawinkel, R. Luhrmann,
W. Northemann, H. H. Peter, and M. Schlesier. 1995. T cell lines recognizing the
70-kD protein of U1 small nuclear ribonucleoprotein (U1snRNP). Clin. Exp.
Immunol. 101:408.
13. Desai-Mehta, A., C. Mao, S. Rajagopalan, T. Robinson, and S. K. Datta. 1995.
Structure and specificity of T cell receptors expressed by potentially pathogenic
anti-DNA autoantibody-inducing T cells in human lupus. J. Clin. Invest. 95:531.
14. Holyst, M. M., D. L. Hill, S. O. Hoch, and R. W. Hoffman. 1997. Analysis of
human T cell and B cell responses against U-small nuclear ribonucleoprotein
70-kD, B, and D polypeptides among patients with systemic lupus erythematosus
and mixed connective tissue disease. Arthritis Rheum. 40:1493.
15. Voll, R. E., E. A. Roth, I. Girkontaite, H. Fehr, M. Herrmann, H.-M. Lorenz, and
J. R. Kalden. 1997. Histone-specific Th0 and Th1 clones derived from systemic
lupus erythematosus patients induce double-stranded DNA antibody production.
Arthritis Rheum. 40:2162.
16. Ito, Y., M. Nieda, Y. Uchigata, M. Nishimura, K. Tokunaga, S. Kuwata, F. Obata,
K. Tadokoro, Y. Hirata, and Y. Omori. 1993. Recognition of human insulin in the
context of HLA-DRB1*0406 products by T cells of insulin autoimmune syndrome patients and healthy donors. J. Immunol. 151:5770.
17. Opelz, G., M. Kiuchi, M. Takasugi, and P. Terasaki. 1975. Autologous stimulation of human lymphocyte subpopulations. J. Exp. Med. 142:1327.
18. Kuntz, M. M., J. B. Innes, and M. E. Weksler. 1976. Lymphocyte transformation
induced by autologous cells. J. Exp. Med. 143:1042.
19. Sakane, T., and I. Green. 1978. Specificity and suppressor function of human T
cells responsive to autologous non-T cells. J. Immunol. 123:1889.
20. Smith, J. B., and R. P. Knowlton. 1979. Activation of suppressor T cells in human
autologous mixed lymphocyte culture. J. Immunol. 123:419.
21. Innes, J. B., M. M. Kuntz, Y. T. Kim, and M. E. Weksler. 1979. Induction of
suppressor activity in the autologous mixed lymphocyte reaction and in cultures
with concanavalin A. J. Clin. Invest. 64:1608.
22. Rosenkrantz, K., B. Dupont, D. Williams, and N. Flomenberg. 1987. Autocytotoxic and autosuppressor T-cell lines generated from autologous lymphocyte cultures. Hum. Immunol. 19:189.
23. Clayberger, C., R. H. DeKruyff, and H. Cantor. 1984. Immunoregulatory activities of autoreactive T-cells: an Ia-specific T cell clone mediates both help and
suppression of antibody responses. J. Immunol. 132:2237.
24. Bell, D. A., and B. Morrison. 1991. The spontaneous apoptotic cell death of
normal human lymphocytes in vitro: the release of, and immunoproliferative
response to, nucleosomes in vitro. Clin. Immunol. Immunopathol. 60:13.
25. Casciola-Rosen, L. A., G. Anhalt, and A. Rosen. 1994. Autoantigens targeted in
systemic lupus erythematosus are clustered in populations of surface structures on
apoptotic keratinocytes. J. Exp. Med. 179:1317.
26. Greidinger, E. L., L. Casiola-Rosen, S. M. Morris, R. W. Hoffman, and A. Rosen.
2000. Autoantibody recognition of distinctly modified forms of the U1–70-kD
antigen is associated with different clinical disease manifestations. Arthritis
Rheum. 43:881.
27. Casciola-Rosen, L., F. Andrade, D. Ulanet, W. B. Wong, and A. Rosen. 1999.
Cleavage by granzyme B is strongly predictive of autoantigen status: implications
for initiation of autoimmunity. J. Exp. Med. 190:815.
28. Moudgil, K. D., and E. E. Sercarz. 1993. Dominant determinants in hen egg white
lysozyme correspond to the cryptic determinants within its self-homologue,
mouse lysozyme: implications in shaping of the T cell repertoire and autoimmunity. J. Exp. Med. 178:2131.
29. Kirou, K. A., R. K. Vakkalanka, M. J. Butler, and M. K. Crow. 2000. Induction
of Fas ligand-mediated apoptosis by interferon-␣. Clin. Immunol. 95:218.
30. Li, X., and Z. Darzynkiewicz. 2000. Poly(ADP-ribose) polymerase measured in
situ in individual cells: relationship to DNA fragmentation and cell cycle position
in apoptosis. Exp. Cell Res. 255:125.
31. Payne, C. M., C. G. Bjore, Jr., and D. A. Schultz. 1992. Change in the frequency
of apoptosis after low- and high-dose x-irradiation of human lymphocytes. J. Leukocyte Biol. 52:433.
32. Philippe, J., H. Louagie, H. Thierens, A. Vral, M. Cornelissen, and L. De Ridder.
1997. Quantification of apoptosis in lymphocyte subsets and effect of apoptosis
on apparent expression of membrane antigens. Cytometry 29:242.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
receptors to bind and internalize apoptotic cells, and peptides
derived from those cells are then presumably expressed with MHC
molecules on the DC surface for presentation to T cells (58 – 60).
However, in contrast to the present report, in those studies the
source of apoptotic cells was not autologous to the responding T
cell. An important role for DC in the uptake and presentation of
autologous apoptotic material to T cells is demonstrated by our
data and is supported by studies in the murine and rat systems (52,
61). An Ab that blocked the DC surface integrin, ␣v␤5, implicated
in the uptake by DC of apoptotic cellular debris (52, 58), strongly
inhibited AMLR stimulation. In contrast, an Ab that blocked ␣v␤3
integrin, typically expressed on monocyte-macrophages, had no
effect on AMLR stimulation. The capacity of DC to present autologous apoptotic material to T cells was directly demonstrated
using macrophage-derived DC matured in the presence of gammairradiated non-T cells. These results suggest a scenario in which
DC engulf autologous cells undergoing caspase-mediated apoptosis and present peptides derived from that cell debris or modified
by the actions of caspases to autoreactive T cells. We propose that
the AMLR reflects such a scenario and that a similar set of events
may routinely occur in vivo.
Abundant data from earlier studies indicate that the CD4⫹ T
cells activated in the AMLR generate suppressor activity, perhaps
equivalent to that of the T-regulatory cells that have recently garnered attention (2, 19 –23, 62). Additional study will be required to
relate the T cells activated by caspase-modified autologous non-T
cells to the proposed phenotype of T-regulatory cells. If apoptotic
cells provide a stimulus for activation of autologous T cells, including T-regulatory cells, a question arises regarding the fate of T
cells that interact with the abundant caspase-cleaved self-proteins
encountered in the thymus during T cell repertoire development.
Although it is likely that many high affinity self-peptide reactive
thymocytes are deleted, some thymic CD4⫹ T cells may be sensitized to peptides derived from caspase-cleaved self-proteins and
survive to become part of the peripheral T cell compartment. Once
in the periphery, those cells would have the capacity to become
activated, undergo proliferation, and differentiate to become regulatory/suppressor cells when re-exposed to their cognate peptide,
generated in the setting of apoptosis induced by any number of
cellular triggers (63, 64). In addition, the patterns of caspase-mediated cleavage may differ between the thymus and cells in the
peripheral compartment. In that regard, it is interesting to note
recent observations indicating that caspase-3 is required for many
forms of apoptosis by peripheral lymphocytes but is not required
for thymocyte apoptosis induced by some stimuli (65).
In summary, these experiments identify an essential role for
caspase activity and apoptosis in the capacity of DC to induce the
proliferation of autologous T cells. The activation and low magnitude expansion of AMLR T cells may reflect a mechanism designed to maintain immune homeostasis in a microenvironment in
which modified-self frequently arises.
1249
1250
49. Crow, M. K., and H. G. Kunkel. 1982. Human dendritic cells: major stimulators
of the autologous and allogeneic mixed leukocyte reactions. Clin. Exp. Immunol.
49:338.
50. Van Voorhies, W. C., L. S. Hair, R. M. Steinman, G. Kaplan. 1982. Human
dendritic cells: enrichment and characterization from peripheral blood. J. Exp.
Med. 155:1172.
51. Guery, J.-C., and L. Adorini. 1995. Dendritic cells are the most efficient in presenting endogenous naturally processed self-epitopes to class II-restricted T cells.
J. Immunol. 154:536.
52. Inaba, K., M. Pack, M. Inaba, H. Sakuta, F. Isdell, and R. M. Steinman. 1997.
High levels of a major histocompatibility complex II-peptide complex on dendritic cells from the T cell areas of lymph nodes. J. Exp. Med. 186:665.
53. Inaba, K., S. Turley, F. Yamaide, T. Iyoda, K. Mahnke, M. Inaba, M. Pack,
M. Subklewe, B. Sauter, D. Sheff, et al. 1998. Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. J. Exp. Med. 188:2163.
54. Kurts, C., W. R. Heath, F. R. Carbone, J. Allison, J. F. A. P. Miller, and
H. Kosaka. 1996. Constitutive class I-restricted exogenous presentation of self
antigens in vivo. J. Exp. Med. 184:923.
55. Kurts, C., H. Kosaka, F. R. Carbone, J. F. A. P. Miller, and W. R. Heath. 1997.
Class I-restricted cross-presentation of exogenous self antigens leads to deletion
of autoreactive CD8⫹ T cells. J. Exp. Med. 186:239.
56. Albert, M. L., B. Sauter, and N. Bhardwaj. 1998. Dendritic cells acquire antigen
from apoptotic cells and induce class I-restricted CTLs. Nature 392:86.
57. Albert, M. L., S. F. Pierce, L. M. Francisco, B. Sauer, P. Roy, R. L. Silverstein,
and N. Bhardwaj. 1998. Immature dendritic cells phagocytose apoptotic cells via
␣v␤5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp.
Med. 188:1359.
58. Albert, M. L., J. I. Kim, and R. B. Birge. 2000. ␣v␤5 integrin recruits the CrkIIDock180-rac1 complex for phagocytosis of apoptotic cells. Nat. Cell Biol. 2:899.
59. Ren, Y., R. L. Silverstein, J. Allen, and J. Savill. 1995. CD36 gene transfer
confers capacity for phagocytosis of cells undergoing apoptosis. J. Exp. Med.
181:1857.
60. Savill, J., N. Dransfield Hogg, and C. Haslett. 1990. Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 343:170.
61. Huang, F.-P., N. Platt, M. Wykes, J. R. Major, T. J. Powell, C. D. Jenkins, and
G. G. MacPherson. 2000. A discrete subpopulation of dendritic cells transports
apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes.
J. Exp. Med. 191:435.
62. Shevach, E. M. 2000. Regulatory T cells in autoimmunity. Annu. Rev. Immunol.
18:423.
63. Forster, I., and I. Lieberam. 1996. Peripheral tolerance of CD4 T cells following
local activation in adolescent mice. Eur. J. Immunol. 26:3194.
64. Steinman, R. M., S. Turley, I. Mellman, and K. Inaba. 2000. The induction of
tolerance by dendritic cells that have captured apoptotic cells. J. Exp. Med. 191:
411.
65. Woo, M., R. Hakem, M. S. Soengas, G. S. Duncan, A. Shahinian, D. Kagi,
A. Hakem, M. McCurrach, W. Khoo, S. A. Kaufman, et al. 1998. Essential
contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes.
Genes Dev. 12:806.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
33. Rovere, P., C. Vallinoto, A. Bondanza, M. C. Crosti, M. Rescigno,
P. Ricciardi-Castagnoli, C. Rugarli, and A. A. Manfredi. 1998. Cutting edge:
bystander apoptosis triggers dendritic cell maturation and antigen-presenting
function. J. Immunol. 161:4467.
34. Propato, A., G. Cutrona, V. Francavilla, M. Ulivi, E. Schiaffella, O. Landt,
R. Dunbar, V. Cerundolo, M. Ferrarini, and V. Barnaba. 2001. Apoptotic cells
overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming.
Nat. Med. 7:807.
35. Boesen-de Cock, J. G. R., A. D. Tepper, E. de Vries, W. J. van Blitterswijk, and
J. Borst. 1999. Common regulation of apoptosis signaling induced by CD95. and
the DNA-damaging stimuli etoposide and ␥-radiation downstream from
caspase-8 activation. J. Biol. Chem. 274:14255.
36. Alam, A., L. Y. Cohen, S. Aouad, and R.-P. Sekaly. 1999. Early activation of
caspases during T lymphocyte stimulation results in selective substrate cleavage
in nonapoptotic cells. J. Exp. Med. 190:1879.
37. Kennedy, N. J., T. Kataoka, J. Tschopp, and R. C. Budd. 1999. Caspase activation
is required for T cell proliferation. J. Exp. Med. 190:1891.
38. Salvesen, G. S., and V. M. Dixit. 1999. Caspase activation: the induced-proximity
model. Proc. Natl. Acad. Sci. USA 96:10964.
39. Sun, X.-M., M. MacFarlane, J. Zhuang, B. B. Wolf, D. R. Green, and
G. M. Cohen. 1999. Distinct caspase cascades are initiated in receptor-mediated
and chemical-induced apoptosis. J. Biol. Chem. 274:5053.
40. Miossec, C., V. Dutilleul, F. Fassy, and A. Diu-Hercend. 1997. Evidence for
CPP32 activation in absence of apoptosis during T lymphocyte stimulation.
J. Biol. Chem. 272:13459.
41. Casciola-Rosen, L. A., G. J. Anhalt, and A. Rosen. 1995. DNA-dependent protein
kinase is one of a subset of autoantigens specifically cleaved during early apoptosis. J. Exp. Med. 182:1625.
42. Drakesmith, H., D. O’Neil, S. C. Schneider, M. Binks, P. Medd, E. Sercarz,
P. Beverley, and B. Chain. 1998. In vivo priming of T cells against cryptic
determinants by dendritic cells exposed to interleukin 6 and native antigen. Proc.
Natl. Acad. Sci. USA 95:14903.
43. Barlow, A. K., X. He, and C. Janeway, Jr. 1998. Exogenously provided peptides
of a self-antigen can be processed into forms that are recognized by self-T cells.
J. Exp. Med. 187:1403.
44. Sambrano, G. R., and D. Steinberg. 1995. Recognition of oxidatively damaged
and apoptotic cells by an oxidized low density lipoprotein receptor on mouse
peritoneal macrophages: role of membrane phosphatidylserine. Proc. Natl. Acad.
Sci. USA 92:1396.
45. Novogrodsky, A., K. H. Stenzel, and A. L. Rubin. 1977. Stimulation of human
peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differential effects of adherent cells. J. Immunol. 118:
852.
46. Utz, P. J., M. Hottelet, P. H. Schur, and P. Anderson. 1997. Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus. J. Exp. Med. 185:843.
47. Urban, B. C., N. Wilcox, and D. J. Roberts. 2001. A role for CD36 in the regulation of dendritic cell function. Proc. Natl. Acad. Sci. USA 98:8750.
48. Stuart, L. M., M. Lucas, C. Simpson, J. Lamb, J. Savill, and A. Lacy-Hulbert.
2002. Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation. J. Immunol. 168:1627.
AUTOLOGOUS T CELL ACTIVATION BY APOPTOTIC CELLS