Inhibitors of apoptosis proteins (IAPs) are required for effective T-cell

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Regular Article
IMMUNOBIOLOGY
Inhibitors of apoptosis proteins (IAPs) are required for effective T-cell
expansion/survival during antiviral immunity in mice
1
Ian E. Gentle,1 Isabel Moelter,2,3 Nadja Lechler,2,3 Sarah Bambach,2,3 Smiljka Vucikuja,2 Georg Hacker,
¨
and Peter Aichele2
1
Institute of Microbiology and Hygiene, 2Institute of Immunology, Department of Medical Microbiology and Hygiene, and 3Faculty of Biology, University of
Freiburg, Freiburg, Germany
Inhibitors of apoptosis proteins (IAPs) were originally described as regulating apoptosis
by direct binding to caspases. More recently, IAPs have been identified as important
• IAPs are required for survival modulators of canonical and noncanonical nuclear factor kB signaling via their ubiquitinE3 ligase activity. IAPs are therefore, not only gatekeepers of cell death, but are probably
and expansion of activated
also involved in the regulation of inflammation, as well as innate and adaptive immunity.
T cells.
In this study, we analyzed the role of IAPs in T-cell immunity during lymphocytic cho• IAP antagonists sensitize
riomeningitis virus (LCMV) infection by pharmacological targeting with an IAP antagonist/
to tumor necrosis factor
second mitochondria-derived activator of caspase-mimetic. Expansion of virus-specific
(TNF)-induced cell death of
CD8 T cells was drastically reduced in LCMV-infected mice exposed to IAP antagonists.
activated T cells during viral
Accordingly, virus control was substantially impaired, indicated by high virus titres in the
infection.
spleen and the spread of LCMV to peripheral organs. The profound negative effect of IAP
antagonists on T-cell immunity was partially linked to tumor necrosis factor–mediated cell
death of activated T cells and required inhibition of X-linked inhibitor of apoptosis, as well as cellular IAP-1. Thus, IAPs play an important
role in T-cell expansion and survival in the context of a highly inflammatory environment such as a virus infection, indicating that IAP
antagonists may interfere with immune responses. (Blood. 2014;123(5):659-668)
Key Points
Introduction
Inhibitors of apoptosis proteins (IAPs) were originally identiﬁed as
apoptosis inhibitors1 and as components of the signaling complex
of tumor necrosis factor receptor 2 (TNF-R2).2 Based on their
homology to Drosophila IAPs and their caspase-binding properties, mammalian IAPs were initially considered broad-spectrum
apoptosis inhibitors. However, only X-linked inhibitor of apoptosis
(XIAP) has direct caspase-inhibiting activity at physiological levels.3
Recent work has shown that IAPs regulate signaling through TNFfamily receptors, where they are involved in the activation of nuclear
factor kB (NF-kB), which regulates the expression of genes important
in inﬂammation, innate and adaptive immunity, and cell survival.4
The structurally related IAPs, cellular IAP-1 (cIAP1), cellular
IAP-2 (cIAP2), and XIAP, contain three baculovirus IAP-like repeat
domains and one really interesting new gene-ﬁnger domain that
functions as an ubiquitin-E3 ligase.5 cIAP1 and cIAP2 also contain
caspase activation and recruitment domains. IAPs regulate the activity
of large signaling complexes involved in NF-kB regulation. This has
been shown for the TNF-R1-signaling complex, whose activity is
regulated through IAP-mediated ubiquitylation of receptor-interacting
protein kinase 1 (RIPK1) and other targets, thereby determining
the activity of the canonical NF-kB pathway,6,7 and preventing
caspase-dependent and -independent cell death.8,9 IAPs also
regulate noncanonical NF-kB activation, together with TNF receptor
associated factors 2 and 3 via ubiquitylation and proteasomal degradation of NF-kB–inducing kinase.10-12
High levels of IAPs are seen in a number of human tumor cells
and are sometimes associated with a poor prognosis.13,14 Because of
this correlation, pharmaceutical companies have developed IAP
antagonists. The development of these compounds has been guided by
the IAP-binding and inhibiting activity of the mitochondrial protein
second mitochondria-derived activator of caspase/direct IAP binding
protein with low pI.15,16 IAP antagonists/second mitochondria-derived
activator of caspase-mimetics cause the ubiquitin-mediated degradation of cIAP1 and cIAP217,18; and although they don’t cause its
degradation, they inhibit XIAP with varying efﬁciencies.
IAP antagonists sensitize tumor cells to death induced by TNF in
vitro. Some cancer cells die in response to IAP antagonists due to
autocrine TNF production as a result of NF-kB activation.10,11,19 Some
of these substances have entered clinical trials as cancer therapeutics.20
However, IAPs are also widely expressed in cells of the immune system,
and targeting of IAPs is therefore likely to affect immune functions. A
number of pathways critically involved in immune defense are regulated by IAPs, most notably NF-kB pathways. IAPs are also involved
in the control of signaling downstream of the toll-like receptor-adapter,
Submitted January 23, 2013; accepted December 2, 2013. Prepublished
online as Blood First Edition paper, December 12, 2013; DOI 10.1182/blood2013-01-479543.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
I.E.G. and I.M. contributed equally to this study.
The online version of this article contains a data supplement.
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© 2014 by The American Society of Hematology
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BLOOD, 30 JANUARY 2014 x VOLUME 123, NUMBER 5
GENTLE et al
toll-receptor-associated activator of interferon,21 and the pattern
recognition receptors (PRR), nucleotide oligomerization domain 1 and
2.22,23 IAP antagonists have further been shown to kill isolated human
monocytes and to have some NF-kB–activating and maturationinducing capacity in isolated dendritic cells.24
In B cells, signaling through the TNFR-family receptors, CD40
and B-cell–activating factor-receptor, is critical for activation and
survival. These receptors activate NF-kB via the noncanonical
pathway.12,25 A recent study showed that genetic loss of cIAP1
and cIAP2 removes B-cell–activating factor-dependency for
survival, and causes the accumulation of B cells in mice.26 In
activated T cells, a costimulatory effect of IAP-loss has been
reported.27 In vitro, mouse and human T cells secreted higher
levels of IL-2 during stimulation with CD3/CD28 antibodies when
an IAP antagonist was present. In vivo, coadministration of an IAP
antagonist with a cellular tumor vaccine enhanced the immune
response and reduced tumor growth.27
These observations suggest that IAPs play a signiﬁcant role
during T-cell– mediated immunity, and the loss of IAP function may
have profound effects on complex immune reactions such as during
antimicrobial host defense. When treatment of cancer patients, many of
whom may already be immune compromised, with IAP antagonists is
considered, potential alterations of the immune response are a major
issue. To test the role of IAPs during antiviral immunity and any
negative effects of IAP antagonist treatment on the immune system,
we analyzed the T-cell–immunity of mice treated with LBW242
in the context of lymphocytic choriomeningitis virus (LCMV) infection.
Our results showed a drastic reduction of CD8 T-cell expansion and
impaired virus control when mice were exposed to an IAP antagonist.
The results indicated an important role for IAPs in T-cell activation and
survival, and consequently a strong immunomodulatory function of IAP
antagonists, which may be linked to TNF-mediated induction of cell
death in activated T cells.
Materials and methods
Mice and viruses
C57BL/6J mice were obtained from Janvier (Le Genest Saint Isle, France),
kept under speciﬁc pathogen-free conditions, and analyzed between 8 to 14
weeks of age. XIAP2/2 mice were obtained from David Vaux (Melbourne,
Australia).28 TNF2/2 mice were obtained from Andreas Diefenbach (Freiburg,
Germany) who received them from Prof J. Sedgwick (Sydney, Australia).29
Mouse experiments were approved by the Regierungspraesidium, Freiburg.
The LCMV-WE was originally obtained from Dr F. Lehmann-Grube (Hamburg,
Germany), and quantiﬁed in organs using a standard focus-forming assay. Mice
were treated daily with 5, 10, or 50 mg/kg body weight LBW242 (provided by
Dr Brant Firestone, Novartis) dissolved in phosphate-buffered saline.
In vitro proliferation
Carboxyﬂuorescein diacetate succinimidyl ester (CFSE)-labeled splenocytes
were stimulated with plate-bound aCD3 antibody (clone 17A2; 1 mg/mL),
and incubated with the indicated concentrations of LBW242 or solvent. For
analysis, cultured cells were harvested completely, resuspended in an equal
volume ﬂuorescence-activated cell sorter (FACS) buffer, and counted for
a deﬁned time-interval on a FACSCalibur (BD Biosciences). Cell death
was analyzed by staining cells for CD8 and CD4 populations in addition
to Annexin V-FITC (BD Biosciences) and LIVE/DEAD Fixable Far Red
stain (Life Technologies).
Antibodies and intracellular staining
Antibodies were purchased from eBioscience, BD Biosciences, BioLegend,
or Bio X Cell. For intracellular cytokine staining, 106 lymphocytes were
stimulated with 1027 M LCMV GP33-peptide (KAVYNFATM) for 4
hours in the presence of Brefeldin A. Cells were then surface-stained with
anti-CD8a antibody, followed by a ﬁxation/permeabilization step using
Cytoﬁx/Cytoperm kit (BD Biosciences) and anti-IFNg, or anti-TNF antibody
staining. LCMV-speciﬁc CD8 T cells were detected by ﬂuorochrome-labeled
H-2Db tetramers complexed with GP33-peptide. Cell samples were analyzed
using FACSCalibur and FlowJo software (Tree Star).
Chromium release assay
Cytolytic activity of T cells was analyzed in a standard 51 Chromiumrelease assay using GP33-41, NP396-404, and adeno-peptide–loaded
EL-4 target cells. Splenocytes from LCMV-infected mice and peptideloaded EL-4 target cells were incubated for 5 hours at 37°C. Duplicate
wells were assayed for each effector to target (E:T) ratio and percentages
of speciﬁc lysis were calculated.
Results
In vitro activated CD4 and CD8 T cells are sensitive to
IAP antagonists
To evaluate the impact of the IAP antagonist LBW242 on the
proliferation and survival of T cells after in vitro stimulation, CFSElabeled splenocytes were activated by plate-bound anti-CD3 antibodies and exposed to titrated concentrations of LBW242. For
analysis, splenocyte cultures were harvested completely, and cells
were resuspended and counted in the ﬂow cytometer for a deﬁned
time interval. The area under each curve is a measure for T-cell
expansion and the dilution of CFSE ﬂuorescence represents the
number of cell divisions. After 45 hours and 69 hours stimulation,
CD4 and CD8 T cells exposed to 10 mM LBW242 failed to proliferate, when compared with the solvent control culture (Figure 1A),
indicating a profound negative effect of the IAP antagonist on
activated T cells. During exposure to 5 mM LBW242, T cells initially
proliferated (after 45 hours), although slightly less than controls, but
were drastically decreased in cell numbers when analyzed after 69
hours (Figure 1A). At 0.5 mM LBW242, T-cell proliferation and
accumulation were comparable to the solvent control (Figure 1A).
Importantly, 5 mM LBW242 during in vitro culture had no signiﬁcant
negative effect on unstimulated T cells (Figure 1A), indicating that the
IAP antagonist mainly affects activated T cells. To further determine
that LBW242 targets activated, proliferating T cells, we performed
bromodeoxyuridine (BrdU) incorporation experiments. A clear dosedependent reduction of BrdU-positive CD4 and CD8 T cells were
detectable under LBW242 treatment. Only T cells that entered the cell
cycle and stained positive for BrdU were LBW242-sensitive, thereby
conﬁrming the CFSE experiments results (see supplemental Figure 1,
available on the Blood Web site). To demonstrate that the effect of
IAP antagonists on T cells is independent of the mode of activation,
we stimulated splenocytes with combinations of different stimuli.
Expansion of CD4 and CD8 T cells was heavily impaired under
LBW242 treatment with all the distinct stimulation protocols
(supplemental Figure 2), indicating that activated T cells are sensitive
to LBW242 treatment, independent of the mode of stimulation. Thus,
LBW242 had a profound negative effect on the proliferation and
survival of in vitro-stimulated T cells in a concentration range
that did not affect resting T cells to the same extent.
IAPs regulate TNF-signaling, and IAP antagonists are able to alter
signaling from TNF-R1 to a pro-death signal.11 Enhanced TNF-killing
seen by IAP antagonist sensitization can be blocked by necrostatin, an
inhibitor of RIPK1 activity.30 To analyze whether LBW242-induced
loss of activated CD8 T cells is due to a RIPK1-dependant mechanism,
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Figure 1. IAP antagonist inhibits T cell proliferation in
vitro. (A) A total of 3 3 105 CFSE-labeled wild-type (WT)
splenocytes were stimulated with plate-bound anti-CD3 antibody. Splenocytes were exposed to LBW242 (red line) or
solvent (green line). After 45 hours (left column) and 69 hours
(right column), cultures were harvested completely and
stained with anti-CD4 and anti-CD8 antibodies. Cells were
resuspended in equal volumes of FACS buffer and counted in
the flow cytometer for a defined time interval. The area under
each curve indicates the total number of cells collected and
the dilution steps of CFSE fluorescence represent the number
of cell divisions. Red numbers represent a statistical analysis
of the proportion of total T cells exposed to IAP antagonist
relative to solvent control from 5 experiments. (B) Assessment
of cell death in ConA/IL-2 stimulated CD8 T cells. Splenocytes
were stimulated with 2 mg/ml ConA and 10 IU/ml IL-2 for 4
days in the presence of LBW242 and/or 10 mM necrostatin-1.
CD8 T cells were analyzed for Annexin V exposure and Live/
Dead FACS staining. Annexin V positive cells were classified
as apoptotic and cells single positive for Live/Dead stain were
classified as necrotic. Dot plots show a representative
experiment and column graph shows the mean of 2
experiments.
we pretreated mouse splenocytes with necrostatin prior to the addition
of LBW242. T cells were stimulated with concanavalin A (ConA)/IL-2,
followed by assessing cell death with Annexin V and Live/Dead
stain. Necrostatin substantially protected CD8 T cells treated with
low-to-mid range doses of LBW242 from cell death, but was less
effective at higher doses (Figure 1B). Cell death induced under these
conditions showed an apoptotic signature with positive Annexin V
staining. Of note, CFSE dilution revealed that those T cells rescued by
necrostatin treatment, cycled largely normally and proliferated to the
same extent as solvent-treated T cells, supporting the idea that the
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662
GENTLE et al
phenotype is due to excessive T-cell death, rather than blockage of
proliferation (data not shown).
IAP-antagonism inhibits virus-specific T cell responses and
impairs virus control in vivo
To investigate the impact of an IAP antagonist on antiviral T-cell
responses, we used the LCMV infection model. C57BL/6 mice were
treated intraperitoneally with 50 mg/kg LBW242 daily, a dose successfully administered in mouse tumor models.31,32 Some studies
have also reported an oral application of LBW242 using similar
doses.19,27 To conﬁrm this, 50 mg/kg LBW242 were administered
either intraperitoneally or orally (supplemental Figure 3). Targeting of
cIAPs by LBW242 was demonstrated on whole cell lysates from
spleens and thymi by immunoblotting for cIAP1 and XIAP expression
(supplemental Figure 3A; cIAP2 is not detectable by immunoblotting in
mouse cells). The results demonstrated the loss of cIAP1 after treatment
of mice with titrated doses of LBW242 either intraperitoneally or orally,
indicating that the IAP antagonist is active over a wide concentration
range in vivo. There was no loss of XIAP at these concentrations, as
expected.33
Analysis of the immune response on day 8 after LCMV infection
showed a reduction of spleen cellularity in LBW242-treated mice
when compared with solvent controls (Figure 2A). The proportions of
CD8 T cells were about 20% in the LBW242 group compared with
;60% in the solvent group. In absolute numbers, CD8 T cells were
reduced 5- to 10-fold in the spleen of LBW242-treated mice compared
with control mice (Figure 2A). Lower absolute numbers of CD8 T cells
speciﬁc for the immunodominant LCMV epitopes GP33 (Figure 2A)
and NP396 (data not shown) were detectable in LBW242-treated mice.
Thus, in the presence of an IAP antagonist, CD8 T cells are profoundly
limited in their expansion during an antiviral immune response.
The impact of IAP-antagonist treatment on differentiation of CD8
effector T cells into short-lived effector cells (KLRG-1hi/CD127lo)
and memory precursor effector cells (KLRG-1lo/CD127hi)34 was analyzed. CD8 T cells from LBW242-treated mice were severely impaired in KLRG-1 upregulation, as shown in relative and absolute
numbers (a 20- to 30-fold reduction) (Figure 2B). Accordingly, the
proportion of short-lived effector cells in the CD8 T-cell compartment
was reduced in the LBW242 group (Figure 2B). Equal percentages of
CD44hi/CD62Llo CD8 effector T cells were detectable in LBW242and solvent-treated mice, although the total number of CD44hi/CD62Llo
CD8 T cells was decreased in the LBW242 group due to the limited
expansion of virus-speciﬁc T cells (Figure 2C). This indicates that
under IAP antagonist exposure, the remaining LCMV-speciﬁc CD8
effector T cells exhibit a highly activated phenotype.
Effector T cells from LBW242-treated mice were assessed for
function by analysis of cytokine expression after short-term restimulation. An equal proportion of CD8 T cells (30% to 40%) produced IFNg in LBW242- and solvent-treated mice. Nevertheless,
a drastic reduction in absolute numbers of IFNg-expressing CD8 T cells
was observed under LBW242 treatment, reﬂecting the limited T-cell
expansion (Figure 2D). In contrast, TNF-expressing T cells were
signiﬁcantly reduced in relative numbers in LBW242-treated mice as
well (Figure 2E). Ex vivo cytolytic activity assayed on GP33- and
NP396-loaded EL-4 target cells was reduced in LBW242-treated
animals (Figure 2F). To examine the effect of the observed impairment
of T-cell activation on viral replication, virus titres from various organs
were examined. Elevated virus titres were detectable in the spleens of
treated mice compared with control mice. Additionally, the spread of
LCMV in peripheral organs like the liver, kidney, and lung was observed
under LBW242 treatment, indicating a loss of viral control (Figure 2G).
BLOOD, 30 JANUARY 2014 x VOLUME 123, NUMBER 5
IAP antagonists do not affect na¨ıve T cells and the antigen
presenting cell (APC) function of the spleen in vivo
To analyze whether IAP antagonists affect naive T cells, WT mice
were ﬁrst treated with LBW242 for 7 days, treatment was then
discontinued, and the mice were infected with LCMV. Total
lymphocyte counts in the spleen were comparable in LBW242 and
solvent pretreated groups. Expansion of total CD8 or GP33-speciﬁc
CD8 T cells was not affected (Figure 3A). T-cell differentiation was
identical in both experimental groups, and KLRG-1 upregulation
was not impaired in mice pretreated with LBW242. No differences
in the absolute numbers of IFNg- and TNF-producing T cells could
be detected, and complete virus elimination was observed by day 8 in
mice of both experimental groups (Figure 3A).
One possible explanation for the limited antiviral immune
response in LBW242-treated mice may be due to a defect in the
function of APCs. To test for this, splenocytes of day 4 LCMVinfected mice treated with or without LBW242 were used as
stimulator cells in an in vitro proliferation assay. CFSE-labeled
GP33-speciﬁc P14 T cells were cultured with the two-stimulator
cell preparations. Preliminary results indicated no signiﬁcant differences in P14 T-cell proliferation or survival when titrated numbers
of LCMV-infected splenocytes from LBW242- or solvent-treated
mice were used as APCs (Figure 3B). Although we did not test
other APC features such as expression of costimulatory molecules, this assay showed no indication of a rate-limiting function
of APCs.
TNF is partially responsible for T-cell dysfunction during LCMV
infection in LBW242-treated mice
Direct sensitization to TNF-induced cell death has been reported
for tumor cells exposed to IAP antagonists. In some cells, IAP
antagonists induce the production of TNF by activation of the
noncanonical NF-kB signaling pathway; and autocrine TNF
signaling via TNF-R1 leads to cell death. The loss of TNFexpressing T cells after LCMV infection in LBW242-treated mice
also suggests a role for TNF in the phenotype. To test whether
TNF-mediated killing of T cells is responsible for their reduced
expansion in vitro, TNF2/2 splenocytes were used in proliferation
assays. Interestingly, there was a clear protection of TNF2/2
T cells exposed to 5 mM IAP antagonist as compared with WT
controls (Figure 4A). However, a reduction of dividing T cells was
also observed in cultures of TNF2/2 T cells at high concentrations
of LBW242. Thus, the protection seen in TNF2/2 T cells seems to
be more reliable at lower doses of LBW242, reﬂecting the results
seen with necrostatin, which also protects mainly at lower doses of
LBW242.
To test whether this TNF-mediated cell death pathway is a
mechanistic basis for the limited T-cell expansion observed in IAP
antagonist-treated animals, we examined CD8 T cell responses in
TNF2/2 mice during LCMV infection. The impact of LBW242
treatment in relation to the solvent group was calculated as ratio for
WT and TNF2/2 mice, respectively. Total leukocyte counts in the
spleens of LBW242-treated WT mice were reduced by a factor of
;3.6 when compared with solvent controls. Interestingly, only a 1.7fold reduction was observed in LBW242-treated TNF2/2 mice
(Figure 4B). Total numbers of CD8 T cells in WT mice under
LBW242 treatment were reduced by a factor of ;9.3, whereas the
same treatment caused only a threefold reduction in TNF2/2 mice
(Figure 4C). Correspondingly, a 2.5-fold reduction in the number
of GP33-speciﬁc T cells could be detected in TNF2/2 mice
compared with an eightfold reduction in LBW242-treated WT
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Figure 2. Impact of IAP antagonist on T cell immunity during LCMV infection. WT mice were infected intravenously with 200 PFU LCMV and treated daily with 50 mg/kg
LBW242 or solvent intraperitoneally, or were left untreated. (A) At day 8 post-infection, total leukocyte counts, percent and total counts of CD8 T cells, and GP33-specific
T cells were determined in the spleen. (B-C) Differentiation of effector T cell subpopulations were analyzed by FACS staining with anti-KLRG-1/anti-CD127 or anti-CD44/antiCD62L antibodies on gated CD8 T cells. (D-E) Percentages and total numbers of INFg- and TNF-expressing CD8 T cells are given. (F) Cytolytic activity of splenocytes was
determined on GP33-, NP396-, or irrelevant adenovirus peptide-loaded EL-4 target cells in a 51Cr-release assay. (G) LCMV titres were analyzed in the spleen, liver, lung, and
kidney using a focus forming assay. Dotted line represents detection levels. Symbols represent individual mice. The horizontal lines represent mean values. *P , .05; **P , .005;
***P , .001. NS, not significant (Student unpaired t test). Data are representative of 3 independent experiments.
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BLOOD, 30 JANUARY 2014 x VOLUME 123, NUMBER 5
Figure 3. Impact of IAP antagonist on na¨ıve T cells
and APC function of the spleen. (A) To evaluate the
impact of IAP antagonist on na¨ıve T cells, WT mice
were pretreated intraperitoneally with LBW242 or
solvent for 7 days. Three days after the last application,
the mice were infected intravenously with 200 PFU
LCMV. On day 8 post-infection, total leukocyte counts,
percent and total counts of CD8 T cells, and GP33specific T cells were determined in the spleen (upper
panels). KLRG-1 upregulation, as well as total numbers
of IFNg- and TNF-expressing CD8 T cells were calculated. Viral titres in the spleens were determined in a
virus focus forming assay (lower panels). (B) Antigen
presenting function of splenocytes from day 4 LCMVinfected WT mice, treated during infection with LBW242
or solvent, was tested with CFSE-labeled P14 T cells in
an in vitro proliferation assay. Data are representative
of 2 to 3 independent experiments.
mice (Figure 4D). Thus, in the absence of TNF-signaling, treatment of mice with IAP antagonists had less severe negative effects
on T-cell immunity during LCMV infection. The failure to
upregulate KLRG-1 during T-cell effector differentiation was also
less pronounced in LBW242-treated TNF2/2 (4.6-fold reduction)
compared with WT mice (;18-fold reduction) (Figure 4E). The
number of IFNg-expressing CD8 T cells was reduced ;eightfold in
WT mice compared with only a 2.5-fold reduction in TNFdeﬁcient mice (Figure 4F).
TNF is expressed during LCMV infection.35 To examine if there
was a difference in the TNF serum levels under LBW242 treatment,
TNF was measured on day 8 of infection in WT mice (Figure 4G). A
signiﬁcant increase in the production of TNF was detected in
LBW242-treated mice when compared with solvent-treated mice.
This demonstrates that LBW242 triggers an upregulation in TNF
levels in LCMV-infected mice. This enhanced TNF production may,
in turn, trigger cell death of activated T cells.
XIAP inhibition is required for IAP–antagonist-induced
T-cell death
Mice treated with 10 mg/kg of LBW242 during LCMV infection
showed no signiﬁcant effect on T cell proliferation (supplemental
Figure 3B) and virus control, despite exhibiting clear degradation
of cIAP1 at this dose (supplemental Figure 3A). Thus, we observed
a discrepancy between doses of LBW242 required to degrade
cIAP1 and cIAP2, and doses required to induce a T-cell phenotype.
cIAP2 has been reported to return after IAP antagonist treatment.17
XIAP was also not degraded by LBW242. One likely explanation
for this discrepancy between cIAP1 degradation and the observed
immune phenotype is a failure of LBW242 to fully inhibit either
cIAP2 or XIAP. To conﬁrm if cIAP2 has returned in activated
T cells at both low and high doses of LBW242, human peripheral
blood mononuclear cells were stimulated in vitro due to a lack of
effective antibodies against mouse cIAP2. Stimulated human peripheral blood mononuclear cells showed detectable levels of cIAP2
24- and 48-hours after LBW242 treatment at all doses tested
(supplemental Figure 4A). By contrast, cIAP1 was degraded at all
doses, and XIAP as published previously, was not (supplemental
Figure 4A-B). Consequently, both cIAP2 and XIAP may be
driving resistance to low doses of LBW242. However, the inhibitory activity of LBW242 toward XIAP is quite low compared
with cIAP1 and cIAP2.33,36 Therefore, the dose/phenotype discrepancy could be due to the lower LBW242 dose not effectively
blocking XIAP function. To determine this and to test for off-target
toxic effects of LBW242, XIAP2/2 mice were treated with 5 mg/kg
of LBW242 (a 10-fold lower dose than used previously) during
LCMV infection. XIAP2/2 mice treated with 5 mg/kg LBW242
showed a near identical phenotype to WT mice at 50 mg/kg with
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Figure 4. Effect of IAP antagonist is reduced in TNF-deficient mice. (A) CFSE-labeled WT and TNF2/2 splenocytes were stimulated with plate-bound anti-CD3 antibody
and exposed to graded concentrations of LBW242 (red line) or solvent (green line). After 72 hours, the cultures were harvested and stained with anti-CD8 antibody. Cells were
resuspended in equal volumes of FACS buffer and counted in the flow cytometer for a defined time interval. The area under each curve represents the total number of cells
collected. WT or TNF2/2 mice were infected intravenously with 200 PFU LCMV and treated daily with LBW242 or solvent intraperitoneally. At day 8 post-infection, (B) total
counts of leukocytes, (C) CD8 T cells, and (D) GP33-specific CD8 T cells were determined in the spleen. (E) KLRG-1 upregulation was analyzed on gated CD8 T cells and
total numbers are shown. (F) Expression of intracellular INFg was quantified on CD8 T cells and total numbers are given. Numbers displayed in the panels indicate the ratio of
the different cell populations from solvent and LBW242 treated animals for WT and TNF2/2 mouse groups, respectively. (G) Serum was taken from day 8 LCMV-infected mice
and analyzed for TNF levels using enzyme-linked immunosorbent assay. Data are representative of 3 independent experiments.
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GENTLE et al
BLOOD, 30 JANUARY 2014 x VOLUME 123, NUMBER 5
Figure 5. XIAP deficiency sensitizes mice to LBW242-induced T-cell death. C57BL/6 WT and XIAP2/2 mice were infected intravenously with 200 PFU LCMV and treated
daily with 5 mg/kg LBW242 or solvent intraperitoneally. At day 8 post-infection, (A) total leukocyte counts and (B) percent and total counts of CD8 T cells were determined in
the spleen. (C) Differentiation of effector T-cell subpopulations was analyzed by staining with anti-KLRG-1. (D) At day 8 post-infection, GP33-specific T cells were determined
in the spleen. (E) Proliferation of XIAP2/2 CD8 T cells treated with LBW242. A total of 3 3 105 CFSE-labeled WT splenocytes were stimulated with plate-bound anti-CD3
antibody. Splenocytes were exposed to LBW242 (red line) or solvent (green line). After 72 hours, cultures were harvested completely and stained with anti-CD8 antibodies.
Cells were resuspended in equal volumes of FACS buffer and counted in the flow cytometer for a defined time interval. The area under each curve indicates the total number
of cells collected, and the dilution steps of CFSE fluorescence represent the number of cell divisions. Red numbers represent a statistical analysis of the proportion of total
T cells exposed to IAP antagonists relative to solvent control from 4 experiments. Horizontal lines represent mean values. *P , .05; **P , .005; ***P , .001. NS, not
significant (Student unpaired t test). Data are representative of 2 independent experiments for in vivo and 4 for CFSE in vitro experiments.
signiﬁcantly reduced numbers of leukocytes, reduced CD8 T-cell
populations, reduced KLRG11 populations, as well as reduced
numbers of GP33-speciﬁc T cells (Figure 5A-D). In all cases, this
low dose of LBW242 had no signiﬁcant effect on WT T cells
(Figure 5). When splenocytes from XIAP2/2 mice were stimulated
with aCD3 antibodies in vitro, doses as low as 0.5 mM LBW242
impaired T-cell expansion, while only doses of 5 mM or above
reduced WT T-cell populations (Figure 5E). This CD8 T-cell loss
was due to apoptotic cell death as previously shown in Figure 1 and
was also observed for CD4 T cells (data not shown). These results
demonstrate that high doses of LBW242 do not exhibit an off-target
toxic side effect, but are needed to successfully block XIAP function.
Based on these results, cIAP2 is unlikely to be responsible for the
discrepancy between LBW242 doses and the T-cell phenotype.
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
BLOOD, 30 JANUARY 2014 x VOLUME 123, NUMBER 5
Discussion
A number of IAP antagonists are currently in clinical trials for the
treatment of both solid tumors and lymphomas.4 Originally designed
to antagonize the caspase-inhibiting function of IAPs, they are now
known to regulate numerous immune signaling pathways such as PRR
signaling, NF-kB, and inﬂammasome activation. Since T cells use
many of the pathways regulated by IAPs during their activation, we
assessed the impact of IAP antagonism in this situation. In vivo and in
vitro IAP antagonism had no signiﬁcant effect on resting T cells, as
mice pretreated with LBW242 for several days before subsequent
infection showed normal expansion of CD8 T cells and control of
LCMV. This is consistent with previous ﬁndings that the systemic
administration of IAP antagonists for 1 week did not alter the composition of the T- and NK-cell compartments in the spleen of mice.27 In
contrast, LCMV-infected mice treated with IAP antagonist in parallel
showed severe reductions in CD8 T-cell expansion, differentiation, and
survival. Of note, the remaining CD8 T cells exhibited a highly activated phenotype. Although ex vivo cytolytic activity of these cells
showed reduced killing activity, this reﬂected the lower number of
virus-speciﬁc T cells in these mice, since on a per cell basis no
differences in cytolytic activity and degranulation capacity could be
observed between T cells from LBW242- or solvent-treated mice
(data not shown). The impaired T-cell expansion and differentiation
resulted in the loss of viral control, with increased LCMV titres in the
spleen and the virus spreading to peripheral organs.
One possible reason for the reduced T-cell expansion could be
a limited APC function in LBW242-treated mice since IAPs are
known to be involved in PRR signaling. However, when APC
function was assessed, LBW242 appeared to have no effect since
APCs from infected mice receiving LBW242 showed normal T-cell
stimulatory capacity. This approach does not analyze in detail a
number of important APC functions; therefore, we cannot fully
exclude an effect of LBW242 on APC function using this approach.
When comparing cytokine production as a marker for activation,
the remaining live CD8 T cells from LBW242-treated mice showed
a normal upregulation of INFg expression during LCMV infection.
Conversely, there was a severe reduction in TNF-expressing CD8
T cells. Under conditions of IAP-deﬁciency, TNF can induce both
caspase-dependent and RIP kinase-dependent cell death.11,37 TNF,
therefore, may limit CD8 T-cell expansion under IAP-antagonist
exposure through the induction of cell death, and TNF-producing
T cells may die in an autocrine fashion. Indeed, when in vitro–
stimulated T cells were assessed for death, there was a strong
increase in the number of apoptotic cells detected when the cells
were stimulated in the presence of LBW242. This increase in cell
death could be partially blocked by the addition of necrostatin, an
inhibitor of RIPK1 activity. Inhibition of cell death by necrostatin
is often interpreted as a sign of necroptosis; however, necrostatin
may also block caspase-dependent cell death induced by TNF,38
and such a mechanism could be at play here. In line with this
ﬁnding, aCD3-stimulated TNF2/2 T cells exhibited improved in
vitro proliferation in the presence of low-to-mid range doses of
LBW242. This is similar to the effect of necrostatin and may
suggest that TNF plays a major role in activated T-cell death. T-cell
expansion during LCMV infection was improved in LBW242-treated
TNF2/2 mice relative to WT, but was still impaired relative to solvent
controls, suggesting that TNF is not the only contributing factor. IAPs
are also known to regulate signaling through other death receptors
such as TRAIL19 and Fas,37 and both receptors have been reported to
IAP ANTAGONISTS INHIBIT T-CELL EXPANSION
667
be engaged during T-cell responses.39,40 It is likely that death induced
through several death receptors may contribute to the impaired T-cell
expansion during LCMV infection in the absence of IAPs.
When LCMV-infected XIAP2/2 mice were treated with only
5 mg/kg of LBW242, they showed a similar T-cell phenotype to WT
mice treated at 50 mg/kg. This clearly indicates that XIAP plays a
critical role in activated T-cell survival in the context of loss of cIAP1
activity. The observation that cIAP2 returns even at higher doses of
LBW242, combined with the dramatic increase in LBW242 sensitivity in XIAP2/2 mice, suggests that cIAP2 is still inhibited by low
doses of LBW242 or that it plays a minor role in the observed phenotype. Thus, the poor targeting of XIAP by LBW242 likely explains the
discrepancy between doses of LBW242 required to degrade cIAP1 and
cIAP2, and doses required to induce T-cell death during viral infection.
This also indicates that the T-cell death phenotype seen in WT mice
treated with 50 mg/kg of LBW242 is not simply an off-target toxic
effect. Higher doses of LBW242 have little or no effect on resting
T cells, which argues against a nonspeciﬁc “toxic” effect.
The exact role performed by the IAPs during T-cell activation is
not clear, but it may be linked to levels of cytokine production, as
shown by the signiﬁcant increase in TNF production in LCMVinfected mice treated with 50 mg/kg of LBW242. The combined loss
of all three IAPs can have a stronger promoting effect on cytokine
production in response to various stimuli, than the loss of only one or
two41,42; and IAP antagonists that efﬁciently target all three IAPs are
much more potent inducers of cell death than those that target cIAP1
and cIAP2.43 LBW242 may induce more potent cytokine upregulation
at high doses due to combined inhibition of cIAP1 and XIAP, while
at lower doses there is enough “active” XIAP to defy the antagonist.
A recent study has demonstrated that in vivo application of IAP
antagonists can augment the potency of tumor vaccines. Mice treated
with a combination of IAP antagonists and irradiated B16 melanoma
cells exhibited better tumor protection compared with mice with single
treatments.27 The immunostimulatory effect of IAP antagonists was
correlated with an increase of tumor-speciﬁc T cells and IFNg-producing NK cells in the spleen of these mice. The conﬂicting results may
be explained by the different inﬂammatory milieu present during an
antitumor response vs an antiviral reaction. LCMV induces a strong
inﬂammatory milieu characterized by high levels of type I interferons,
IFNg, TNF, and other cytokines. Under these highly inﬂammatory
conditions, IAP expression is likely required for T-cell survival.
Our data suggests that IAPs play a crucial role in the survival of
activated T cells and that their function is to some extent redundant, as at least cIAP1 and XIAP, and probably cIAP2, must be
inhibited to kill activated T cells. T-cell death due to the loss of
IAP activity is at least partly due to TNF, and likely other TNF
super-family cytokines. It is possible that IAP antagonists may be
useful in the treatment of cancer patients; however, due to their
ability to induce or enhance cytokine production in immune cells,
inhibition of PRR signaling, sensitization to TNF super-family
cytokines, as well as the reduced T-cell–mediated antiviral responses,
it is clear that IAP antagonists can have a powerful immunomodulatory function as well. And, therefore, their effects on the immune
system should be carefully considered.
Acknowledgments
The authors thank Dr Leigh Zawel and Dr Dale Porter (Novartis, Boston,
MA) for providing LBW242. TNF2/2 mice were a kind donation from
Prof Andreas Diefenbach (Institute of Medical Microbiology and Hygiene, Freiburg, Germany). We also thank Associate Prof J. Silke for
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
668
BLOOD, 30 JANUARY 2014 x VOLUME 123, NUMBER 5
GENTLE et al
providing cIAP1 and cIAP2 antibodies and Prof D. David Vaux (both of
Walter and Eliza Hall Institute, Melbourne Australia) for XIAP2/2 mice.
This work was supported by grants from the German Federal
Ministry of Education and Research (BMBF 01 EO 0803) (P.A.), the
Deutsche Krebshilfe (G.H.), and EMBO LTF (Lt_781_2009) (I.E.G.).
Authorship
Contribution: I.E.G. designed and performed research, analyzed data,
and wrote the paper; I.M., N.L., S.B., and S.V. performed research and
analyzed data; and G.H. and P.A. designed research, analyzed data,
wrote the paper, and provided joint senior authorship.
Conﬂict-of-interest disclosure: The authors declare no competing ﬁnancial interests.
Correspondence: Peter Aichele, Department of Medical Microbiology and Hygiene, Institute of Immunology, Hermann-HerderStrasse 11, D-79104 Freiburg, Germany; e-mail: peter.aichele@
uniklinik-freiburg.de; and Georg H¨acker, Department for Medical
Microbiology and Hygiene, Institute of Microbiology, HermannHerder-Strasse 11, D-79104 Freiburg, Germany; e-mail: georg.
[email protected].
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From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
2014 123: 659-668
doi:10.1182/blood-2013-01-479543 originally published
online December 12, 2013
Inhibitors of apoptosis proteins (IAPs) are required for effective T-cell
expansion/survival during antiviral immunity in mice
Ian E. Gentle, Isabel Moelter, Nadja Lechler, Sarah Bambach, Smiljka Vucikuja, Georg Häcker and
Peter Aichele
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