This information is current as
of July 31, 2017.
IL-12 Decreases Activation-Induced Cell
Death in Human Naive Th Cells Costimulated
by Intercellular Adhesion Molecule-1. I.
IL-12 Alters Caspase Processing and Inhibits
Enzyme Function
Ellen M. Palmer, Lili Farrokh-Siar, Jean Maguire van
Seventer and Gijs A. van Seventer
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 167:749-758; ;
doi: 10.4049/jimmunol.167.2.749
http://www.jimmunol.org/content/167/2/749
IL-12 Decreases Activation-Induced Cell Death in Human
Naive Th Cells Costimulated by Intercellular Adhesion
Molecule-1. I. IL-12 Alters Caspase Processing and Inhibits
Enzyme Function1
Ellen M. Palmer,2* Lili Farrokh-Siar,† Jean Maguire van Seventer,2*‡ and
Gijs A. van Seventer2,3*‡
N
aive Th cell activation leading to proliferation and clonal
expansion requires not only Ag-specific recognition by
the TCR-CD3 complex, but also costimulatory signals
provided by the APC. These costimulatory or second signals are
generally generated as a result of interaction between T cell surface-expressed receptors with their counter structures on the APC.
Although the LFA-1/ICAM-1 pathway is capable of providing costimulatory signals sufficient for the induction of naive Th cell
proliferation (1– 4), the CD28/B7 family pathway has been shown
to additionally provide costimulatory signals that lead to clonal
expansion (5). A critical event that can prevent effective clonal
expansion is activation-induced cell death (AICD),4 which results
in a lack of cell viability after the first few rounds of proliferation.
AICD in primary Th cells is an apoptotic process that can be me*Committee on Immunology, and Departments of †Ophthalmology and Visual Sciences and ‡Pathology, University of Chicago, Chicago, IL 60637
Received for publication January 29, 2001. Accepted for publication May 9, 2001.
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 Grant AI44209 from the National Institutes of Health
(to G.A.v.S.).
2
Current address: Department of Environmental Health, Boston University School of
Public Health, Boston, MA 02118.
3
Address correspondence and reprint requests to Dr. Gijs A. van Seventer at the
current address: Department of Environmental Health, Boston University School of
Public Health, 715 Albany Street, Talbot 2E, Boston, MA 02118. E-mail address:
[email protected]
4
Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas
ligand; ⌬␺m, mitochondrial membrane potential; ROS, reactive oxygen species;
DiOC6(3), 3,3⬘-dihexyloxacarbocyanine iodide; PI, propidium iodide; PS, phosphatidylserine; IAP, inhibitor of apoptosis.
Copyright © 2001 by The American Association of Immunologists
diated by the Fas ligand (FasL)/Fas pathway (6) or by a pathway
that results in the production of reactive oxygen species (ROS) (7).
Loss of mitochondrial membrane potential (⌬␺m) across the inner
membrane of the mitochondria is one early indicator of apoptosis
that can occur in many death pathways, including the FasL/Fas and
ROS pathways (7–10). Caspases, a family of cysteine proteases
(11), are downstream effectors of both the FasL/Fas and ROS pathways that promote DNA fragmentation. In the Fas/FasL death
pathway, caspases are activated either directly or indirectly following a loss of mitochondrial integrity. The proteolytic activity of
caspases is tightly regulated. Effector caspases exist as inactive
procaspases that require specific cleavage by upstream initiator
caspases to initiate processing that will result in catalytic activity
(12). The effectiveness of the CD28/B7 pathway in costimulation
has been attributed, in part, to the induction of the anti-apoptotic
Bcl-xL protein, which is involved in maintaining ⌬␺m (13, 14).
Biologically active IL-12 (p35/p40) has a critical role in promoting the differentiation of naive Th cells into Th1 cells (15). In
addition, IL-12 is known to have a strong adjuvant effect in promoting T cell-mediated immune responses (16). This adjuvant effect is likely due, in part, to the capacity of IL-12 to enhance IL-2
secretion and responsiveness (17, 18), which can augment proliferation, and/or to induce IFN-␥ secretion, which can up-regulate
expression of APC costimulatory molecules, such as B7 family
members (19) and ICAM-1 (20). Using an in vitro model of Th cell
costimulation by accessory cells, we have shown that IL-12 can
promote ICAM-1-mediated, CD28-independent costimulation of
human naive Th cells by preventing cell death and enhancing cell
expansion (E. Palmer and G. van Seventer, unpublished observations, and Ref. 21). Similarly, a regulatory role for IL-12 in
0022-1767/01/$02.00
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Th cells can receive costimulatory signals through the LFA-1/ICAM-1 accessory pathway that are sufficient to induce early Th cell
proliferation, but not subsequent cell expansion and maintenance of cell viability. To investigate the regulatory role for IL-12 in
ICAM-1-mediated costimulation, human naive Th cells were stimulated with coimmobilized anti-CD3 mAb and ICAM-1 Ig in the
presence or absence of IL-12. The ICAM-1-mediated costimulatory signals in this model resulted in early Th cell proliferation
followed by cell death that was partially mediated by Fas and involved loss of mitochondrial membrane potential, processing of
procaspase-9 and -3, and activation of caspase-3. Addition of IL-12 prevented activation-induced cell death and promoted late
proliferation. ICAM-1 ⴙ IL-12-costimulated Th cells were resistant to Fas-mediated cell death through a mechanism that did not
appear to involve a decrease in either Fas or Fas ligand expression. IL-12 did not inhibit the loss of mitochondrial membrane
potential induced by ICAM-1-mediated costimulation, and this finding was consistent with the inability of IL-12 to increase
expression of the antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-xL. Interestingly, IL-12 promoted an altered processing of
procaspase-9 and -3 and a decrease in the percentage of cells displaying caspase-3 catalytic function. In conclusion, we now
describe a novel regulatory function for IL-12 in preventing Th cell death and, as a result, in greatly increasing Th cell viability
and expansion. Together, our findings indicate that IL-12 may perform this regulatory role by preventing Fas-mediated activationinduced cell death through inhibition of caspase-3 enzyme activity. The Journal of Immunology, 2001, 167: 749 –758.
750
IL-12 DECREASES AICD IN NAIVE ICAM-1-COSTIMULATED Th CELLS
ml of either 0.3 ␮g/ml of ICAM-1 Ig or anti-CD28 mAb 9.3 in PBS for 1 h
at room temperature. After being washed three times with PBS, 0.25 ⫻ 106
Th cells were added in culture medium (RPMI 1640 supplemented with
10% FBS, 20 mM L-glutamine, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin; all from BioWhittaker, Walkersville, MD), in a final volume of
0.5 ml, to coated wells with or without 1 ng/ml rIL-12. On day 4 after
initiation of culture, cells and supernatants were transferred to 12-well
plates for expansion with 1 ml fresh culture medium. T cell counts and
viability were determined by no-wash flow cytometry on days 4 –7 (see
below). To determine cell division, naive Th cells were labeled with 1.7
␮M CFSE for 10 min at room temperature, quenched with FCS, and
washed two times with culture medium before addition to the assay. Cell
division analysis was performed using Modfit software (Verity Software
House, Topsham, ME).
Cell surface flow cytometry
Flow cytometric analysis was performed on a FACScan (BD Biosciences)
as previously described (25) and was analyzed with Lysys II and CellQuest
(both from BD Biosciences) software.
No-wash flow cytometry
To determine relative cell number, Th cells were activated for 4 days and
then transferred to larger wells with supernatant as described above. Nowash flow cytometry was performed by collecting a portion of the expansion culture on days 4 –7. Events were collected on FACScan for a constant
amount of time so as to allow for calculation of cell number. Assessment
of live cells was achieved either by gating on cells based on forward scatter
and side scatter characteristics or PI exclusion (see below).
Staining cells for cell death analysis
Materials and Methods
Abs and other reagents
The following Abs and purified ligands were used to stimulate or block T
cells: anti-CD3 mAb OKT3 (IgG2a), obtained from American Type Culture Collection (Manassas, VA); anti-CD28 mAb 9.3, provided by Dr. C.
June (University of Pennsylvania, Philadelphia, PA); and recombinant human ICAM-1 Ig (extracellular domain of ICAM-1 fused to Fc portion of
IgG), a gift of P. Hoffman (ICOS, Bothwell, WA). Stimulatory Abs and
ligands were indirectly immobilized with goat anti-human IgG-coated and
sheep anti-mouse IgG-coated wells (ICN Pharmaceuticals, Aurora, OH).
Fas Abs CH-11 (agonist, cross-linking) and ZB4 (antagonist, blocking)
were purchased from Kamiya Biomedical (Seattle, WA). Isotype control
Abs MOPC21 (IgG1), MOPC195 (IgG2b), and TEPC183 (IgM) were purchased from ICN Pharmaceuticals. The following Abs to human proteins
were used for Western blotting: caspase-3 (rabbit), caspase-9 (rabbit),
Bcl-2 (mouse), Bax (mouse), and Bak (mouse), all purchased from BD
PharMingen (San Diego, CA); Bcl-xL (rabbit) Ab was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Secondary reagents antimouse-HRP, anti-rabbit-HRP, and HRP-conjugated NeutrAvidin were purchased from Pierce (Rockford, IL). Biotinylated mouse mAbs to the following molecules were used for flow cytometry: CD25 (clone B-B10;
BioSource International, Camarillo, CA) and Fas and FasL (BD PharMingen); streptavidin-PE (BD Biosciences, San Jose, CA) was used to recognize the biotinylated Abs.
Recombinant human IL-12 (p35/p40) was generously provided by Hoffman-LaRoche (Nutley, NJ). CFSE was purchased from Molecular Probes
(Eugene, OR). The following reagents were used to assess cell death: mitochondrial dye 3,3⬘-dihexyloxacarbocyanine iodide (DiOC6(3); Molecular
Probes), annexin V-PE (R&D Systems, Minneapolis, MN), propidium iodide (PI; Sigma, St. Louis, MO), PhiPhiLux-G1D2 caspase-3-like caspase
fluorescent substrate (OncoImmunin, Gaithersburg, MD).
Isolation of T cells
Human PBMC from anonymous donors (buffy coats received from LifeSource Blood Services, Glenview, IL, and United Blood Service, Chicago,
IL) were isolated by lymphocyte separation medium (Ficoll) density-gradient centrifugation. Resting CD4⫹CD45RA⫹CD45RO⫺ naive T cells
were obtained by negative selection with mouse mAbs to MHC class II,
CD19, CD16, CD14, glycophorin, CD8, and CD45RO and magnetic beads
(Polysciences, Warrington, PA) as previously described (25). Selected
CD45RA⫹ cells were consistently 99% pure by FACScan analysis.
Accessory cell-independent T cell stimulation
The 48-well plates were coated with 0.25 ml of 5 ␮g/ml each sheep antimouse Ig and goat anti-human Ig in PBS overnight at 4°C, washed three
times with PBS, and coated with 0.25 ml of 0.3 ␮g/ml OKT3 and with 0.25
To determine plasma membrane breakdown, ⬃10 ␮l of 50 ␮g/ml PI was
added to cell samples immediately before analysis in FL3 parameter on
FACScan. For ⌬␺m, 0.1-ml cultures of T cells were collected, and 0.4 ml
of 40 nM DiOC6(3) diluted in culture medium was added to the cells,
which were then incubated at 37oC for 30 min. Samples were immediately
analyzed in FL1 parameter on FACScan. To determine phosphatidylserine
(PS) exposure, T cells were collected, washed twice in bead separation
buffer, resuspended in annexin V binding buffer (10 mM HEPES, 10 mM
NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) at 1 ⫻ 106/ml, and incubated with directly labeled annexin V for 15 min at room temperature, in
the dark, according to the manufacturer’s specifications. Samples were then
diluted in annexin V binding buffer and analyzed on FACScan within 1 h
of staining. To determine caspase activity in living cells, 0.5–1 ⫻ 106 T
cells were collected in 1.5-ml Eppendorf tubes and centrifuged to remove
all supernatant. A total of 50 ␮l of 10 ␮M PhiPhiLux-G1D2 substrate and
5 ␮l of FCS were added to each sample, and cells were resuspended by
gently tapping the tube with the fingers. Open tubes were incubated in 5%
CO2, 37°C for 60 min. Subsequently, cells were washed once with 1 ml
ice-cold flow cytometry diluting buffer (provided by manufacturer) and
gently resuspended in 0.5–1 ml flow cytometry diluting buffer. Samples
were analyzed on FACScan within 60 –90 min of the final wash.
ELISA
The mAb pairs obtained from BD PharMingen were used at 1– 4 ␮g/ml in
a sandwich ELISA to measure IL-2 (sensitivity, 0.2 U/ml) in supernatants.
Avidin-peroxidase and ABTS substrate were purchased from Sigma and
used as described in the BD PharMingen protocol.
Cell lysates
Stimulated T cell were collected at various time points, washed one time in
PBS, and snap-frozen in LN2 and stored at ⫺80°C until detergent lysis.
Cell pellets were thawed on ice and lysed in either buffer containing 1%
Triton X-100, 50 mM Tris, 300 mM NaCl, 10 ␮g/ml aprotinin, 5 ␮g/ml
leupeptin, 1 mM pefabloc, 1 mM sodium orthovanadate, 2 mM EDTA, and
10 mM NaF, or in modified Laemmli buffer containing 60 mM Tris (pH
6.8), 10% glycerol, and 2% SDS, a buffer reported to prevent procaspase
processing as result of cell lysis (26). Cell membranes were pelleted by
microcentrifugation, and the protein content of each lysate-containing supernatant was determined by bicinchoninic acid protein assay (Pierce).
Next, 5⫻ loading dye (125 mM Tris (pH 6.8), 25% glycerol, 4% SDS, 10%
2-ME, and 0.5% bromophenol blue) was added to samples before boiling
and loading, or boiling, snap-freezing, and storage at ⫺80°C.
SDS-PAGE and Western blotting
Equal microgram amounts (30 –50 ␮g) of cell lysate protein were separated
by SDS-PAGE through a 15% polyacrylamide gel and were electroblotted
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preventing apoptosis has also been reported by other investigators
studying different models, including HIV-induced Th cell apoptosis (22), Fas-mediated apoptosis in murine and human Th cells (22,
23), and ␥-irradiation-induced apoptosis in monocytes (24). However, the mechanism by which IL-12 can prevent apoptosis in all
these models remains unknown.
The goal of this study was to characterize the mechanism(s) by
which IL-12 prevents AICD in human naive Th cells costimulated
by ICAM-1 in the absence of CD28-mediated costimulation. To do
this, we used an in vitro model of costimulation by ICAM-1 in the
absence of CD28-dependent costimulation to both measure the effects of IL-12 alone in preventing AICD and to directly compare
the effects of IL-12- and CD28-mediated costimulation on regulating AICD. Using this model system, we found that the AICD
following ICAM-1 costimulation of naive Th cells correlated with
a loss of ⌬␺m, cleavage and processing of procaspase-9 and -3, and
generation of caspase-3 catalytic activity. This cell death was partially inhibited by agonistic anti-Fas mAb. Addition of IL-12
strongly promoted the expansion and viability of ICAM-1-costimulated Th cells through effects that predominantly, if not exclusively, inhibited Fas signaling without restoring mitochondrial
integrity. Indeed, the most striking effects of IL-12 on apoptotic
pathways were the induction of altered processing of procaspase-9
and -3 and inhibition of caspase-3 catalytic activity.
The Journal of Immunology
on to polyvinylidene difluoride membranes. Membranes were blocked with
5% milk in TBST for 1 h at room temperature and blotted overnight at 4°C
with primary Abs diluted, as recommended by the manufacturer, in 1%
BSA in TBST. After washing, membranes were incubated with appropriate
HRP-conjugated secondary reagent, and chemiluminescence detection was
performed according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometry analysis was done with ImageQuant (Molecular Dynamics, Sunnyvale, CA).
Results
IL-12 maintains the proliferation of ICAM-1-costimulated naive
T cells
until day 5 or 6, whereas both ICAM-1 ⫹ IL-12- and anti-CD28costimulated Th cells continued to proliferate until day 7 (Fig. 1B).
The Th cell proliferation data suggested that ICAM-1 ⫹ IL-12
and anti-CD28 costimulation may induce Th cells to produce levels of autocrine growth factors that support late proliferation. Consequently, we determined the amount of the T cell growth factor,
IL-2, detectable in the culture supernatant at day 4 and the expression levels of the inducible IL-2R ␣-chain (CD25) induced by each
costimulatory condition on days 4 – 6. We found that the differences in late expansion of the various costimulatory populations
could not be explained by differences in the amounts of IL-2 secreted or CD25 expressed. In all populations, similar levels of IL-2
were detected in supernatants on day 4, and comparable levels of
CD25 were expressed on days 4, 5, and 6 (data not shown). Together, these results show that ICAM-1, ICAM-1 ⫹ IL-12, and
anti-CD28 costimulation induced similar levels of early naive Th
cell expansion and IL-2 production, and that IL-12 enhanced late
proliferation of Th cells costimulated with ICAM-1.
It should be noted that Th cells costimulated with ICAM-1 ⫹
IL-12 increased expression levels of CD28 ligands (CD80 and
CD86) compared with ICAM-1-costimulated Th cells (data not
shown). However, blocking studies with a combination of antiCD80 and anti-CD86 mAbs demonstrated that CD28-mediated costimulation did not contribute to the IL-12-mediated effects on Th
cell viability and late proliferation (data not shown).
IL-12 decreases the portion of ICAM-1-costimulated T cells
that die
In other model systems, ICAM-1 costimulation has been shown to
induce death in T cells (4, 28). Thus, we were interested in determining whether ICAM-1 costimulation also induced Th cell death
in our model and whether IL-12 may affect the viability of ICAM1-costimulated T cells. To address these issues, the death of CFSElabeled Th cells was assessed by PI exclusion after stimulation
with ICAM-1, ICAM-1 ⫹ IL-12, and anti-CD28. The results of
no-wash flow cytometry showed that, after the cells have divided
multiple times, cell death occurs in all populations, although to
varying extents (Fig. 2). In particular, the percentage of PI⫹ dead
cells (Fig. 2, upper sections of dotplots) increased in ICAM-1costimulated cultures throughout expansion, resulting in the death
of the majority of cells by day 7. In contrast, although ICAM-1 ⫹
IL-12 and anti-CD28 costimulation cultures also accumulated dead
cells after a few rounds of cell division, these costimulatory signals
induced cell death to a much lesser extent than ICAM-1-costimulatary signals. Taken together with our previous results, these data
indicate that although ICAM-1 can costimulate the early activation
and expansion of Th cells, it cannot sustain late proliferation or Th
cell survival but, rather, results in Th cell death. Furthermore, they
show that the addition of IL-12 to ICAM-1-costimulated Th cells
can rescue late proliferation and promote cell survival, resulting in
a comparable viable Th cell recovery at day 7 as with anti-CD28
costimulation. We found that these effects of IL-12 on ICAM-1costimulated Th cells are dose dependent and optimal at concentrations of 1 ng/ml (data not shown). In addition, the effects are
obtained if IL-12 is added to the culture as late as day 3 of stimulation, but they do not occur with the addition of IL-12 on day 4
of culture or later (data not shown).
Fas-mediated signals contribute to ICAM-1-costimulated death
Our observations thus far corroborated the reports of others (4, 28)
indicating that costimulation with ICAM-1 results in Th cells undergoing cell death. However, it was not known whether the Fas/
FasL death pathway, which is involved in some forms of Th cell
AICD (6), was also involved in the Th cell death that follows
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To study the regulation of T cells by LFA-1-mediated costimulatory signals, we stimulated peripheral blood-derived human naive
(CD45RA⫹CD45RO⫺) CD4⫹ Th cells with coimmobilized antiCD3 mAb and ICAM-1 Ig fusion protein in the absence or presence of recombinant human IL-12, added on day 0. Th cells stimulated with coimmobilized anti-CD3 and anti-CD28 mAbs with or
without IL-12 were used as a positive control for cell expansion.
The concentrations of anti-CD3 mAb, ICAM-1 Ig (ICAM-1), and
anti-CD28 mAb used were chosen to provide consistent and comparable activation of naive Th cells (between both costimulatory
conditions and different donors), as determined by [3H]thymidine
incorporation at day 4 (data not shown). Th cells were stimulated
for 4 days in the presence of Ab and fusion protein (activation
phase of the assay) and then were transferred to a larger well to
expand for an additional 3 days (expansion phase of the assay) in
the absence of Ab and fusion protein.
The relative number of live naive Th cells obtained under the
various costimulatory conditions was determined by PI exclusion
in a no-wash flow cytometric analysis on days 4 –7 of culture. The
results are summarized in Fig. 1A. These results indicated that up
to day 5 of culture, there was equivalent cell expansion among all
four costimulatory conditions (ICAM-1, ICAM-1 ⫹ IL-12, antiCD28, and anti-CD28 ⫹ IL-12). However, after day 5, the relative
number of live ICAM-1-costimulated Th cells began to plateau,
and by day 7, these numbers actually declined to levels significantly lower than that of ICAM-1 ⫹ IL-12-, anti-CD28-, or antiCD28 ⫹ IL-12-costimulated Th cells. On day 7 of culture, there
was no significant difference in the relative number of live Th cells
occurring under ICAM-1 ⫹ IL-12 and anti-CD28 costimulatory
conditions, and both these costimulatory conditions generated approximately four times more live cells than ICAM-1 costimulation,
whereas anti-CD28 ⫹ IL-12 costimulation generally exceeded the
viable cell recoveries of ICAM-1 ⫹ IL-12 or anti-CD28. It should
be noted that viable cell recovery following stimulation with antiCD3 alone, IL-12 alone, or anti-CD3 plus IL-12 did not differ
significantly from that of unstimulated Th cells (data not shown).
To further examine the increase in expansion induced by the
addition of IL-12 to ICAM-1-costimulated Th cells, we analyzed
the response of individual Th cells to ICAM-1 and ICAM-1 ⫹
IL-12 costimulation. This was done by labeling naive Th cells with
the fluorescent vital dye CFSE, which segregates equally between
daughter cells upon cell division such that loss of fluorescence
corresponds with cell division (27). The proliferative responses of
CFSE-labeled Th cells to ICAM-1, ICAM-1 ⫹ IL-12, and antiCD28 costimulation were compared by performing no-wash flow
cytometry on CFSE-stained cells to determine the relative number
of live Th cells (based on forward scatter vs side scatter characteristics) in each generation during the expansion phase of the
assay. The representative CFSE experiment depicted in Fig. 1B
shows that, under all three costimulatory conditions, most naive Th
cells entered the cell cycle and underwent a similar pattern of cell
division while in contact with stimulating Ab and ligand (day 4).
However, ICAM-1-costimulation maintained proliferation only
751
752
IL-12 DECREASES AICD IN NAIVE ICAM-1-COSTIMULATED Th CELLS
Expression of Bcl-2 family members does not correlate with
survival of ICAM-1 ⫹ IL-12-costimulated Th cells
FIGURE 1. IL-12 increases the proliferation and yield of ICAM-1-responsive naive Th cells. A, The relative number of live Th cells was determined by PI exclusion in a no-wash flow cytometric analysis on days
4 –7 for unstimulated (䡺), ICAM-1- (F), ICAM-1 ⫹ IL-12- (f), antiCD28- (Œ), and anti-CD28 ⫹ IL-12- (䉬) costimulated Th cells. Data represent the mean and SEM of results derived from five donors. B, Naive Th
cells were labeled with CFSE and stimulated with coimmobilized anti-CD3
mAb and ICAM-1 Ig with (䡺) or without (f) IL-12 or with coimmobilized
anti-CD3 and anti-CD28 mAbs (o). Th cells were transferred to new wells
with supernatant and fresh culture medium on day 4, and Th cells were
collected for no-wash flow cytometry on days 4 –7. Analysis of cell division was performed on live cells based on forward and side scatter characteristics. The number of live events collected in each generation was
Antiapoptotic Bcl-2 family members are effective in inhibiting
Fas-mediated death in certain types of T cells (10). Bcl-2 family
members regulate both the loss of ⌬␺m and release of cytochrome
c from mitochondria in cells undergoing apoptosis (14, 30, 31).
Consequently, we investigated whether IL-12 may inhibit ICAM1-costimulated T cell death by up-regulating expression of the antiapoptotic Bcl-2 family members, Bcl-xL and Bcl-2, and/or by
down-regulating expression of the proapoptotic members, Bax and
Bak. As shown in Fig. 4A, Bcl-xL was detected at low levels in
ICAM-1-costimulated Th cells and at high levels in anti-CD28costimulated Th cells at 48 h of stimulation. However, the results
also show that addition of IL-12 to ICAM-1-costimulated T cells
determined by no-wash flow cytometric analysis over 1 min, followed by
further analysis using Modfit software. Undivided cells are represented in
the parent (P) generation. Results depict the mean and SE of duplicate
cultures derived from one representative of six tested donors different from
those donors in A.
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activation by ICAM-1 costimulation. Consequently, we performed
experiments to examine the contribution of Fas signaling to the
death of ICAM-1-costimulated Th cells. Our initial studies focused
on determining the expression of Fas and FasL on the surface of
Th cells costimulated with ICAM-1, ICAM-1 ⫹ IL-12, or antiCD28. We found that expression of Fas on the vast majority of
cells in each costimulatory condition persisted throughout expansion (days 4 –7) (Fig. 3A). Furthermore, the percentage of cells
expressing low levels of FasL was comparable on day 4, after
which it decreased similarly for all populations (Fig. 3B). Together, these results indicated that there was the potential for Fasmediated death in all culture conditions. In addition, coculture with
IL-12 did not appear to either lower the percentage of cells expressing Fas or FasL (Fig. 3, A and B) or the intensity of Fas and
FasL staining (data not shown). Interestingly, the expression of
FasL rapidly decreases in all conditions after day 5, a point at
which the percentage of nonviable cells is still increasing in the
ICAM-1 costimulatory condition.
To determine whether signaling through Fas might actually contribute to the death of ICAM-1-costimulated Th cells, the Fasblocking (antagonistic) mAb ZB4 was added to the various costimulatory cultures on day 4, at the time of Th cell transfer to
expansion cultures. Induction of cell death was assessed in live Th
cells on day 6 by staining cells with annexin V, a protein that binds
to the PS that moves to the outer membrane of cells undergoing
apoptosis (29). The amount of ZB4 mAb added to cultures was
saturating, as it was able to completely block agonistic Fas Ab
CH-11-induced annexin V binding to Th cells in all culture conditions (data not shown). The results shown in Fig. 3C confirm that
ICAM-1-costimulated Th cells undergo apoptosis, as demonstrated by binding of annexin V. ZB4 mAb inhibited the percentage of live Th cells positive for PS by ⬃35% in ICAM-1-costimulated cultures compared with cultures containing isotype control
Ab, and the mAb had no inhibitory effect on the PS expression of
live Th cells costimulated with ICAM-1 ⫹ IL-12 (Fig. 3C). Thus,
although Fas-blocking experiments indicate that Fas signaling contributes to the death of ICAM-1-costimulated Th cells, these combined results (Fig. 3, B and C) also suggest that other death mechanisms must be involved in the induction of ICAM-1-dependent
AICD. Furthermore, the data indicate that the major, if not only,
mechanism by which IL-12 inhibits the death of ICAM-1-costimulated Th cells is by inhibition of Fas-mediated death.
The Journal of Immunology
753
FIGURE 2. IL-12 decreases the portion
of ICAM-1-costimulated T cells that die. Naive Th cells were labeled with CFSE and
were left unstimulated or stimulated as described in Fig. 1. Cells were collected for
no-wash flow cytometry on days 4 –7. PI was
added immediately before flow cytometric
analysis. CFSE (x-axis) vs PI (y-axis) staining of total cells is shown in dot plots. Decreasing CFSE staining indicates cells undergoing an increasing number of divisions. The
percentage of PI⫹ cells is noted in each dot
plot. One representative of 20 donors tested
is shown.
Induction of loss of ⌬␺m is similar in ICAM-1- and ICAM-1 ⫹
IL-12-costimulated T cells
Loss of ⌬␺m across the inner membrane of the mitochondria is an
early indicator of apoptosis generated by many death stimuli (8, 9),
including some types of AICD-inducing stimuli (10, 31). We investigated whether the death of ICAM-1-costimulated T cells
might correlate with a loss of ⌬␺m during expansion, despite the
enhanced expression of Bcl-2 and Bcl-xL during this period. Loss
of ⌬␺m was determined by staining with the lipophilic cationic dye
DiOC6(3). High DiOC6(3) staining is detected in cells in which
⌬␺m is intact, whereas low staining is detected in cells in which
⌬␺m has dissipated. Live cells were analyzed to determine whether
the ICAM-1-costimulatory signal induced a loss of ⌬␺m before the
ability to exclude PI was lost. We found that 20 –30% of live
ICAM-1- and ICAM-1 ⫹ IL-12-costimulated Th cells stained low
for DiOC6(3) on days 4, 5, and 6, whereas negligible staining was
observed in anti-CD28-costimulated live Th cells (Fig. 5). These
results indicate that ICAM-1 costimulation initiated a loss of ⌬␺m
regardless of the addition of IL-12, whereas CD28 costimulation
did not. Based on these results, dead cells would be predicted to
accumulate in both ICAM-1- and ICAM-1 ⫹ IL-12-costimulated
Th cell cultures. However, the results depicted in Fig. 2 show that
only ICAM-1-costimulated Th cells accumulate a majority of dead
cells. These combined results suggest that IL-12 can inhibit death
pathways downstream of mitochondria in ICAM-1-costimulated
Th cells.
Procaspase-9 and procaspase-3 are differentially processed in
ICAM-1- and ICAM-1 ⫹ IL-12-costimulated Th cells
To determine the potential downstream effectors of Fas signaling
that may be affected by mitochondrial dysregulation in ICAM-1costimulated Th cells, we investigated the cleavage of initiator
caspase-9 and effector caspase-3 in the costimulated Th cell populations. Caspases are synthesized as inactive proenzymes that require cleavage and subsequent heterodimerization of large and
small subunits for activity (11). Procaspase-9 processing results
from the interaction of Apaf-1 with mitochondria-derived cytochrome c (32). Western blot analysis of cellular lysates collected
on day 6 demonstrated that although a portion of caspase-9 is
processed in all populations, the resulting fragments of caspase-9
are unique depending on the stimulus (Fig. 6A). Two aspartic acid
residues near the end of the large subunit of caspase-9 result in
cleavage fragments sized 35 kDa (p35) and 37 kDa (p37), both of
which include the prodomain and large subunit (33). In Th cells
costimulated with ICAM-1, only p35 was generated (Fig. 6A, lane
1). In contrast, ICAM-1 ⫹ IL-12 costimulation resulted in the
generation of predominantly p37 (Fig. 6A, lane 2). p35 was generated in anti-CD28-costimulated Th cells, although substantial
levels of uncleaved procaspase-9 remained (Fig. 6A, lane 3). We
also investigated the effect of IL-12 on caspase-9 processing in
anti-CD28-costimulated T cells and found that, similar to its
effects on ICAM-1-costimulated Th cells, IL-12 promoted the
processing of procaspase-9 to primarily the p37 fragment (Fig. 6A,
lane 4).
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does not alter Bcl-xL expression. Thus, the survival advantage of
costimulation with ICAM-1 ⫹ IL-12 did not correlate with early
enhancement of Bcl-xL expression. Surprisingly, IL-12 was found
to inhibit expression of Bcl-xL in Th cells costimulated with either
ICAM-1 or anti-CD28 for 96 h (Fig. 4B). Furthermore, this negative influence of IL-12 on Bcl-xL expression in ICAM-1-costimulated cells was maintained through days 4 –7 of culture, during
which time a similar IL-12-inhibitory effect was found for Bcl-2
expression (Fig. 4C).
Western analysis for the proapoptotic Bcl-2 family members,
Bak and Bax, showed mixed results. In both ICAM-1- and
ICAM-1 ⫹ IL-12-costimulated Th cells, the expression of both
Bak and Bax proteins consistently increased with culture time (Fig.
4C). However, whereas coculture with IL-12 was found to actually
up-regulate Bak expression in all experiments, the expression of
Bax was decreased in the majority of experiments (4 of 7; Fig. 4C).
However, the IL-12-induced relative decrease in expression of the
antiapoptotic Bcl-2 family members, Bcl-xL and Bcl-2, was observed to be greater than the IL-12-induced relative decrease in
Bax expression (Fig. 4C). Thus, together, these data strongly suggest that IL-12 does not inhibit AICD in ICAM-1-costimulated
human naive Th cells through effects on the expression of Bcl-2
family members.
754
IL-12 DECREASES AICD IN NAIVE ICAM-1-COSTIMULATED Th CELLS
Effector caspases, such as caspase-3, are activated by initiator
caspases and, once activated, the effector caspases cleave cellular
substrates, resulting in cell death (12). Effector caspases are characterized by short prodomains. Two aspartic acid residues are associated with the prodomain of procaspase-3, one contained in the
prodomain at residue 9 and the other at the junction between the
prodomain and the large subunit (11). Investigation of caspase-3
cleavage from its proform (p32) to its large (p17) and small (p12)
subunits revealed that although procaspase-3 was processed in every T cell population, the relative proportion of the p21 (prodomain plus large subunit), p19 (partial prodomain plus large subunit), p17, and p12 fragments differed depending on the stimulus.
Significantly, the putatively active subunits of caspase-3, p17, and
p12, were the most prevalent fragments formed only in ICAM-1costimulated Th cells (Fig. 6B, lane 1). Addition of IL-12 to
ICAM-1-costimulated Th cells did not inhibit the processing of
procaspase-3 (p32) but, rather, it inhibited the generation of the
p17 and p12 subunits in favor of the p21 subunit (Fig. 6B, lane 2).
CD28-derived signals generated primarily the p21 fragment, with
little or no detectable p17 and p12 active subunits, while retaining
a substantial portion of procaspase-3 (Fig. 6B, lane 3). It should be
noted that, despite equal loading in each lane (as determined by
micrograms of protein in the cell lysate), the anti-CD28 lane consistently showed a stronger total caspase (procaspase plus caspase
fragments) signal than with the other costimulatory conditions.
Nevertheless, the anti-CD28 lane always showed a very weak signal for the p17 fragment and an undetectable signal for the p12
fragment, consistent with the lack of significant cell death found
with this costimulation condition. Interestingly, the addition of IL-12
to anti-CD28-costimulated Th cells resulted in an alteration of procaspase-3 processing such that nearly all procaspase-3 was processed to the p19 and p21 caspase-3 fragments (Fig. 6B, lane 4).
In summary, these results show that IL-12 can alter processing
of procaspase-9 and -3 in both ICAM-1- and anti-CD28-
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FIGURE 3. Fas-mediated signals contribute to ICAM-1-costimulated T
cell death. Naive Th cells were stimulated with coimmobilized anti-CD3
mAb and either ICAM-1 Ig with or without IL-12 or anti-CD28 mAb. Cells
were stained for cell surface expression of Fas (A) and FasL (B) on the day
indicated. The percentage of cells positive for cell surface proteins is plotted for each stimulation condition. The mean and SEM of results derived
from three or more donors is shown. C, Fas blocking during expansion.
Naive Th cells were stimulated as described above. On day 4, expansion
cultures were distributed over microtiter wells in 100-␮l aliquots after addition of fresh medium, and 3 ␮g of MOPC21 (isotype control) or ZB4
(Fas antagonist Ab) was added to each culture. The percentage of annexin
V-PE-binding live cells was determined by forward vs side scatter characteristics on day 6. The mean and SEM of results from three donors tested
is shown.
FIGURE 4. Anti- and proapoptotic Bcl-2 family member expression in
ICAM-1-, ICAM-1 ⫹ IL-12-, anti-CD28, and anti-CD28 ⫹ IL-12-costimulated T cells. Naive Th cells were stimulated with coimmobilized anti-CD3
mAb and either ICAM-1 Ig or anti-CD28 mAb with or without IL-12. Cells
were collected at 48 h (A), 96 h (B), or days 4 –7 (C), lysed with 1% Triton
X-100 lysis buffer, and lysates were analyzed by anti-human Bcl-xL, antihuman Bcl-2, anti-human Bax or, anti-human Bak in a Western analysis.
For Bcl-xL, Bcl-2, and Bax, one representative of at least three donors
tested is shown. The experiment using Bak is representative of two donors
tested. In Fig. 4C, the level of expression of Bcl-2 family members (as
determined by densitometric analysis of Western blot autoradiographs) following ICAM-1 and ICAM-1 ⫹ IL-12 costimulation relative to expression
occurring upon costimulation with ICAM-1 is depicted beneath the Western blot at each time point.
The Journal of Immunology
755
FIGURE 5. Evidence of loss of ⌬␺m in ICAM-1- and ICAM-1 ⫹ IL12-costimulated T cells. Naive Th cells were stimulated with coimmobilized anti-CD3 mAb and either ICAM-1 Ig with or without IL-12 or antiCD28 mAb. On the indicated day, Th cells were stained with 40 nM
DiOC6(3) for 30 min at 37°C and immediately analyzed for DiOC6(3)
staining by flow cytometry. The percentage of live cells demonstrating low
DiOC6(3) staining (DiOC6(3)low), indicative of a loss of ⌬␺m, is plotted for
each stimulation condition. The mean and SEM of results derived from
three donors is shown.
IL-12 prevents the generation of catalytic active caspase-3 in
ICAM-1-costimulated Th cells
The results shown in Fig. 6B suggested that ICAM-1 costimulation
may lead to the generation of catalytically active caspase-3,
whereas addition of IL-12 may promote incomplete processing of
procaspase-3 and the generation of an enzymatically inactive
caspase-3 form. To directly determine whether this might be the
case, ICAM-1- and ICAM-1 ⫹ IL-12-costimulated Th cells were
loaded with PhiPhiLux-G1D2, a substrate for caspase-3-like enzymes that contains the DEVD sequence. The fluorescence of the
PhiPhiLux substrate increases when it is cleaved by caspase-3. The
results in Fig. 7 show that on day 7, ⬃74% of ICAM-1-costimulated Th cells demonstrated caspase-3-like enzyme activity,
whereas only ⬃21% of ICAM-1 ⫹ IL-12-costimulated Th cells
exhibited increased fluorescence associated with the PhiPhiLux
substrate (Fig. 7, right quadrants). Simultaneous PI staining revealed that virtually all cells positive for caspase activity were also
in the late stages of death, as demonstrated by their inability to
exclude PI (Fig. 7, upper right quadrants). Similar PI exclusion
results were observed on days 5 and 6 (data not shown). These
results indicate that caspase-3-like enzyme activation is associated
with the late stages of death in primary human Th cells costimulated with ICAM-1. Furthermore, they indicate that IL-12 can inhibit caspase-3-like enzyme activity and cell death in ICAM-1costimulated Th cells.
Discussion
The studies we now present show that although naive human Th
cell costimulation through the LFA-1/ICAM-1 accessory pathway
leads to early proliferation, it cannot support cell expansion by
means of late proliferation and maintenance of cell viability. Furthermore, our findings demonstrate that addition of IL-12 to cultures of ICAM-1-costimulated naive Th cells will promote cell
proliferation and viability resulting in cell expansion. In addition,
FIGURE 6. Procaspase-9 and procaspase-3 are differentially processed
in ICAM-1- and ICAM-1 ⫹ IL-12-costimulated Th cells. Naive Th cells
were stimulated with coimmobilized anti-CD3 mAb and either ICAM-1 Ig
or anti-CD28 mAb with or without IL-12. Cells collected on days 6
(caspase-9) and 7 (caspase-3) were lysed in modified Laemmli buffer, and
lystaes were analyzed by anti-human caspase-9 (A) or caspase-3 (B) Western analysis. Results derived from one representative of three donors tested
are depicted.
they establish that this effect of IL-12 on cell expansion is accomplished, at least in part, by preventing AICD through a mechanism
that involves suppression of caspase-3 activation.
We found that coengagement of the TCR and LFA-1 with antiCD3 mAb and ICAM-1 Ig is capable of stimulating naive Th cells
to secrete high levels of IL-2 and of inducing Th cell division (data
not shown and Fig. 1). This was a result inferred from prior studies
using [3H]thymidine incorporation to measure proliferation in both
murine and human Th cells costimulated by ICAM-1 (1–3, 34, 35).
Consistent with reports from Damle et al. (28), we also showed
that ICAM-1 costimulation leads to the death of human Th cells.
Results obtained with a murine ICAM-1 costimulation model
differed from those presented here in that, in the absence of exogenous IL-2, there was little IL-2 production or cell division in
murine naive TCR-transgenic Th cells stimulated with antigenic
peptide and ICAM-1-positive (B7-1- and B7-2-negative) APC, although there was extensive cell death (4, 36). The nature and
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costimulated Th cells. Specifically, IL-12 can induce the processing of procaspase-9 to the p37 fragment and inhibit the generation
of the active p12 and p17 subunits of procaspase-3 while increasing the generation of a p21 fragment. Taken together, the results
suggest the possibility that IL-12 may inhibit the occurrence of
death in ICAM-1-costimulated Th cells by inducing the generation
of a caspase-9 p37 fragment and inhibiting the generation of the
catalytically active p12 and p17 subunits of caspase-3.
756
IL-12 DECREASES AICD IN NAIVE ICAM-1-COSTIMULATED Th CELLS
FIGURE 7. IL-12 prevents caspase-3-like enzyme activity in intact Th cells. Naive Th cells
were stimulated with coimmobilized anti-CD3
mAb and ICAM-1 Ig with or without IL-12. Total
Th cells collected on day 7 were analyzed by flow
cytometry for caspase-3-like activity using PhiPhiLux G1D2 substrate (FL1, x-axis) and for PI exclusion (FL3, y-axis). The percentage of cells positive
for both caspase substrate cleavage and PI is noted
in each dot plot. A total of 5000 events are shown
for each condition. Results derived from one representative of three donors tested are shown.
Bcl-xL expression (Fig. 4C). Therefore, these observations, in
combination with results showing that a loss of ⌬␺m was not inhibited by IL-12 (Fig. 5), suggest that IL-12 induces a survival
factor acting downstream of the mitochondria.
The strongest indications that IL-12 actively inhibited the death
of ICAM-1-costimulated Th cells are evident in the ability of
IL-12 to inhibit Fas-mediated death and to prevent death events
downstream of the loss of ⌬␺m. Although one-third of ICAM-1costimulated Th cells were susceptible to Fas-mediated signals,
ICAM-1-costimulated Th cells exposed to IL-12 showed no evidence of Fas-mediated death (Fig. 3C). Furthermore, we found that
IL-12 altered ICAM-1-mediated processing of procaspase-9 and
-3, (Fig. 6, A and B) and prevented both the activation of procaspase-3 and the accumulation of dead cells (Fig. 7). Loss of ⌬␺m
is an early indicator of apoptosis (39). Cytochrome c release from
the mitochondria is an event that is known to promote caspase
activation and, thus, lead to cell death (32), and to take place independently of but coordinated with loss of ⌬␺m (31, 40). Although we do not know the status of cytochrome c in ICAM-1costimulated Th cells, it is possible that IL-12 did not prevent a
loss of ⌬␺m but did prevent the release of cytochrome c from the
mitochondria of ICAM-1-costimulated Th cells in a Bcl-2/Bcl-xLindependent fashion. Alternatively, we believe it is more likely
that cytochrome c was released from the mitochondria in all cells
costimulated with ICAM-1, and that IL-12 altered the procaspase-9 processing promoted by this cytochrome c and possibly
altered procaspase-3 processing, as well, in a manner that prevented caspase-3 activation and cell death.
Certain caspase processing patterns are correlated with death,
such as the generation of the p35 fragment of caspase-9 and the
p17 and p12 fragments of caspase-3. We detected all of these
caspase fragments in ICAM-1-costimulated T cells (Fig. 6, A and
B), thus indicating that cell death in our model is caspase-mediated. However, in ICAM-1-costimulated cells exposed to IL-12
during activation, caspase-9 was processed to primarily a p37 fragment and caspase-3 to primarily a p21 fragment, while the cells
continued to proliferate. The significance of these alternative
cleavage patterns is not known. These cleavage products may have
no function as incompletely processed enzymes. Alternatively, if
they are capable of pairing with fully processed subunits, they may
act as decoys that limit the amount of active enzyme generated. It
is also possible that the alternative cleavage products may have a
function in promoting cellular proliferation, as has been shown for
certain caspases in early Th cell proliferation (41– 43).
In addition to inhibiting apoptosis in ICAM-1-costimulated Th
cells, IL-12 has been shown to inhibit Fas-mediated death in T
cells from HIV⫹ donors (22), both ␥ irradiation-induced and Fasmediated death in macrophages (24), and death of a human Th1
clone resulting from either IL-2 withdrawal or treatment with HIV
gp120 (44). In all of these scenarios, death is executed by effector
caspases. The ability of IL-12 to block many different death stimuli
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strength of the TCR- and LFA-1-derived signals may account for
at least some of the differences between our model and the TCRtransgenic model. Nevertheless, despite differences in IL-2 secretion and early proliferation, it is evident that ligation of LFA-1 by
ICAM-1 leads to death in both human and murine naive Th cells.
The activation-dependent cell death seen upon naive Th cell
costimulation with ICAM-1 occurs as a consequence of a primary
stimulation. The mechanism(s) involved in this type of activationdependent cell death may therefore differ from that of the AICD
that takes place after restimulation of previously activated Th cells.
Our data are consistent with a model of activation-dependent Th
cell death upon ICAM-1 costimulation in which downstream
events resulting from activation of the FasL/Fas pathway and some
other apoptotic pathway contribute to Th cell death. Specifically,
Fas-blocking studies indicated that ⬃35% of the death seen in
naive Th cells costimulated with ICAM-1 can be attributed to
FasL/Fas interaction.
Addition of IL-12 to ICAM-1-costimulated Th cells results in an
increase in the number of cell divisions in cycling Th cells, a lower
percentage of dead cells, and ultimately, four times more live Th
cells than with ICAM-1 costimulation alone (Figs. 1 and 2). These
results suggest that IL-12 acts in a manner similar to CD28 in
sustaining proliferation and enhancing cell survival (5). Does
IL-12 promote proliferation, survival, or both in ICAM-1-costimulated Th cells? Our results show that IL-12 enhanced the proliferative capacity of ICAM-1-costimulated Th cells just as it is reported to enhance the proliferation of anti-CD28-costimulated T
cells (37, 38). It is difficult to separate proliferation and survival in
our system, because they are always linked. Thus, it is possible that
IL-12 may induce the outgrowth of Th cells that survive ICAM-1
costimulation, because the percentages of PI⫹ cells differed greatly
between ICAM-1- and ICAM-1 ⫹ IL-12-costimulated Th cells,
although the number of PI⫹ cells did not (data not shown). However, our analysis of death indicators supports the notion that IL-12
actively inhibits death induced by ICAM-1 costimulation while
promoting proliferation.
Our investigation of the expression of antiapoptotic and proapoptotic Bcl-2 family members during Th cell activation and expansion in our costimulatory models revealed that changes in the
relative expression levels of these proteins are unlikely to explain
the increased survival of ICAM-1 ⫹ IL-12-costimulated Th cells.
We found that the relative expression of the antiapoptotic Bcl-2
family members, Bcl-2 and Bcl-xL, in Th cells costimulated with
ICAM-1 ⫹ IL-12 is decreased compared with Th cell costimulated
with ICAM-1 only (Fig. 4C). Furthermore, the expression of the
proapoptotic Bcl-2 family member Bak is actually up-regulated
following IL-12 coculture (Fig. 4C). The expression of the proapoptotic Bcl-2 family member Bax is often (although not always)
down-regulated by IL-12 coculture and is not likely to influence
the overall outcome, because the relative decrease in expression of
Bax is much less than the IL-12-induced decreases in Bcl-2 and
The Journal of Immunology
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suggests a model in which IL-12 is capable of modifying apoptotic
signals at a distal stage in multiple death pathways. Our results
demonstrating an inhibitory effect of IL-12 on caspase-3 activity
are consistent with this model.
One way in which IL-12 may influence caspase processing at a
distal stage of multiple death pathways is by inducing expression
of an inhibitor of apoptosis (IAP), a family of proteins that can
inhibit apoptosis through direct interaction with caspases (45). In
support of such a mechanism of IL-12 function, IAP family members have been shown to interact with caspase-3, -7, and -9 and to
be expressed in activated T cells (46, 47). Furthermore, similar to
the effects of IL-12, certain IAPs, such as survivin, are involved in
promoting cell division (48).
In conclusion, we have characterized a mechanism by which
IL-12 prevents a form of AICD in ICAM-1-costimulated Th cells.
We believe that the significance of this novel regulatory role of
IL-12 is clearly beyond its role in our in vitro ICAM-1 costimulation in the absence of CD28. We speculate that the regulatory
role of IL-12 in preventing cell death and promoting cell expansion
is, for example, a crucial component of the previously recognized
adjuvant effect of IL-12 seen in vivo (16). Consistent with this
notion, Marth et al. (23) have recently shown that IL-12 can promote the clonal expansion phase of a physiological immune response. Moreover, they demonstrated that this positive effect of
IL-12 was lost in mice deficient for the FasL/Fas pathway. Our
results would predict that the expansion effect of IL-12 observed
by Marth and colleagues is due to the capacity of IL-12 to prevent
FasL/Fas-mediated cell death through inhibition of caspase-3 activity. In addition to its beneficial effect on immune responses, the
novel regulatory role of IL-12 in promoting cell expansion can also
be envisioned to have detrimental effects. The expression of IL-12
in gut-associated tissues of patients with inflammatory bowel disease has, thus far, been correlated mainly with the presence of
Th1-type cytokines (49). Our findings suggest another way in
which IL-12 may influence normal gut homeostasis; specifically,
by inhibiting cell death and, thus, promoting the local expansion of
inflammatory cytokine-secreting Th1 cells in the gut, IL-12 may
contribute significantly to the pathogenesis of inflammatory bowel
disease.
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IL-12 DECREASES AICD IN NAIVE ICAM-1-COSTIMULATED Th CELLS
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