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IMMUNOBIOLOGY
Activated human NK and CD8⫹ T cells express both TNF-related
apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are
resistant to TRAIL-mediated cytotoxicity
Prisco Mirandola, Cristina Ponti, Giuliana Gobbi, Ivonne Sponzilli, Mauro Vaccarezza, Lucio Cocco, Giorgio Zauli,
Paola Secchiero, Francesco Antonio Manzoli, and Marco Vitale
The expression of tumor necrosis factor
(TNF)–related apoptosis-inducing ligand
(TRAIL) and TRAIL receptors was investigated in resting and cytokine-activated
purified primary human natural killer (NK)
and CD8ⴙ T cells. Resting NK and CD8ⴙ T
cells expressed the mRNA for all TRAIL
receptors, but TRAIL-R4 was the only
receptor clearly detectable on the surface
of both cell types. NK cells were activated
by interleukin 2 (IL-2) or IL-15, whereas
CD8ⴙ T cells were activated by phytohemagglutinin (PHA) ⴙ IL-2 followed by IL-2
alone for up to 10 days. On activation,
both cell types rapidly expressed
TRAIL-R2 and TRAIL-R3, whose expression peaked at day 10 of culture. TRAILR1, however, was never expressed at
any time point examined, whereas the
expression of TRAIL-R4, which showed a
progressive increase in CD8ⴙ T cells,
remained constant in NK cells. Notwithstanding the expression of TRAIL-R2, recombinant TRAIL did not show any cytotoxic activity on either NK or CD8ⴙ T cells.
Both resting and activated NK and CD8ⴙ
T cells were found to express high levels
of the 2 isoforms of c-FLIP (cellular Fas-
associated death domain protein [FADD]–
like IL-1–converting enzyme [FLICE]–
inhibitory protein). Small interference
RNA-mediated inhibition of c-FLIP expression in NK cells abrogated their resistance to the apoptotic effect of soluble
TRAIL. Thus, once activated the major
cytotoxic effector cells are potentially sensitive to TRAIL but are physiologically
protected from its apoptotic action by
intracellular level of c-FLIP. (Blood. 2004;
104:2418-2424)
© 2004 by The American Society of Hematology
Introduction
Natural killer cells provide a line of defense against autologous
tumors and virus-infected cells that have down-modulated their
major histocompatibility complex (MHC) class I expression. Such
tumors can evade the MHC class I–restricted immune recognition
and cytotoxic response mediated by CD8⫹ cytotoxic T lymphocytes (CTLs) but are not able to trigger the inhibitory receptors
present on natural killer (NK) cells and necessary to prevent their
activation. NK cells can use 2 different mechanisms to kill their
targets, either by cytotoxic granule exocytosis or by induction of
death receptor–mediated apoptosis. The secretory pathway is
active against relatively rare leukemia cell lines, whereas the
nonsecretory killing pathway is active against a variety of tumor
cell lines. Death ligands belong to the tumor necrosis factor (TNF)
family of ligands, a large family of pleiotropic, membrane-bound,
and soluble molecules, that include TNF-␣, FasL, CD40L, lymphotoxin␣ (LT␣) and LT␤, receptor activator of nuclear factor-kappa B
ligand (RANKL), and TNF-related apoptosis-inducing ligand
(TRAIL). A family of complementary molecules, TNF family
receptors, has been identified and described (reviewed in LeBlanc
and Ashkenazi1). Most of the death ligands are also known in
soluble forms that can be secreted.
Primary human NK cells express several TNF family death
ligands2 and use them to induce apoptosis in various tumor cells.3-5
Moreover, expression studies in mice have demonstrated the
relevant role played by TRAIL expressed by tissue-resident NK
cells in the control of metastatic growth.6 However, activated NK
cells have been shown to express some of the death receptors, such
as CD95,7,8 that have been implicated in the negative control of NK
cell function. Moreover, it has recently been reported that CD8⫹
NK cells undergo apoptosis by triggering of CD8 with its natural
ligands through a Fas-mediated pathway.9 Interestingly, members
of the NK inhibitory receptor superfamily inhibit this apoptosis,
acting, therefore, as survival receptors for NK cells. Although
TRAIL is known to induce apoptosis in several cancer cell lines
and primary tumors, it has been reported to play, similarly to
Fas/FasL,10 an active inhibitory role in the physiologic control of
erythropoiesis11 and in the thymic deletion induced by T-cell
receptor ligation.12 However, contrasting data have been reported
From the Department of Anatomy, Pharmacology, and Forensic Medicine,
Human Anatomy Section, University of Parma, Italy; the Department of
Anatomical Sciences, Cellular Signalling Laboratory, University of Bologna,
Italy; the Department of Normal Human Morphology, University of Trieste, Italy;
the Department of Morphology and Embriology, Human Anatomy Section,
University of Ferrara, Italy; and the Istituto per i Trapianti d’organo e
Immunocitologia-Consiglio Nazionale delle Ricerche, Bologna Unit, Research
Institute “Codivilla-Putti,” Istituti Ortopedici Rizzoli, Bologna, Italy.
2002 Fondo per gli Investimenti della Ricerca di Base; Joung Researcher Grant
of the Parma University, and 1% Sanità Alzheimer.
Submitted April 6, 2004; accepted May 26, 2004. Prepublished online as Blood
First Edition Paper, June 17, 2004; DOI 10.1182/blood-2004-04-1294.
Supported by grants from Associazione Italiana per la Ricerca sul Cancro,
Cassa di Risparmio di Parma, Cassa di Risparmio di Bologna, and Ministero
dell’Istruzione, dell’Università e della Ricerca; programmi di ricerca cofinanziati
2418
P.M. and C.P. contributed equally to this study.
An Inside Blood analysis of this article appears in the front of this issue.
Reprints: Marco Vitale, Department of Anatomy, Pharmacology and Forensic
Medicine, University of Parma, Ospedale Maggiore, Via Gramsci, 14, I-43100
Parma, Italy; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
by Simon et al13 that did not demonstrate a role for TRAIL in
antigen-induced deletion of thymocytes.
Given the potential use of TRAIL in anticancer therapy,14 here,
we investigated the expression of TRAIL and TRAIL receptors in
resting and activated primary human NK and CTL cells, the major
players of anticancer immunity. Moreover, we investigated the
functional effects of the addition of recombinant TRAIL at
different culture times on the survival of both resting and activated
NK and CD8⫹ CTLs. These biologic effects were correlated with
the expression level of c-FLIP (cellular Fas-associated death
domain protein [FADD]–like interleukin 1-converting enzyme
[FLICE]–inhibitory protein), a major regulator of cell death
induced by TRAIL and other death ligands.15
Materials and methods
Reagents
Monoclonal antibodies (MoAbs) anti-CD3 and anti-CD16 were purchased
from Becton Dickinson (San Jose, CA). Antihuman TRAIL receptors and
anti–human TRAIL monoclonal antibodies were purchased from Alexis
Biochemical (San Diego, CA). Goat anti–mouse immunoglobulin G (IgG)
phycoerythrin (PE)–labeled was purchased from Immunotech (Beckman
Coulter, Miami, FL). For double staining anti–human CD8 PE-labeled and
anti–human CD4 fluorescein isothiocyanate (FITC)–labeled, anti–human
CD3 FITC-labeled and antihuman CD16 and CD56 PE-labeled, anti–
human CD3 PE-labeled and anti–human CD19 FITC-labeled monoclonal
antibodies were purchased from Beckman Coulter. Primary cultures of
CD8⫹ T cells were stimulated with phytohemagglutinin (PHA-M; Sigma
Chemical, St Louis, MO) and grown in RPMI 1640 medium (BioWhittaker,
Walkersville, MD) plus 10% fetal calf serum (FCS; BioWittaker) with
human recombinant interleukin 2 (rIL-2) from Genzyme (Boston, MA).
Recombinant interleukin-15 (rIL-15) was purchased from Genzyme.
Recombinant Histinine6-tagged (His) TRAIL was produced in bacteria
and purified by chromatography on Ni⫹⫹ affinity resin. The absence of
endotoxin contamination in recombinant TRAIL preparation was assessed
as previously described.16,17 TRAIL-R1-Fc and TRAIL-R2-Fc chimera
cocktail was purchased from R&D (Minneapolis, MN).
NK and CD8ⴙ T-cell purification
Primary NK and CD8⫹ T cells were isolated from peripheral blood
lymphocytes (PBLs) of healthy voluntary donors by immunomagnetic
negative selection by using the NK cell isolation kit and the CD8⫹ T-cell
isolation kit (Milteny Biotech, Gladbach, Germany), according to the
manufacturer’s protocol. Briefly, PBLs were separated from buffy coats
by Ficoll-Hypaque gradient centrifugation, then magnetically labeled
with a cocktail of hapten-conjugated anti-CD3, anti-CD14, anti-CD19,
anti-CD36, and anti-IgE antibodies for NK purification; anti-CD4,
anti-CD11b, anti-CD16, anti-CD19, anti-CD36, and anti-CD56 antibodies for CD8 T-cell isolation, followed by antihapten antibody-coupled
magnetic cell sorter (MACS) microbeads. The magnetically labeled
cells were then immobilized on a depletion column in the magnetic field
of a Vario-MACS apparatus (Milteny Biotech). Purity of NK cells was
immediately checked by anti-CD16–PE and anti-CD3–FITC labeling,
whereas purity of CD8 T cells was monitored by anti-CD8–PE and
anti-CD4–FITC, anti-CD3–PE, and anti-CD19–FITC. Only samples
with a purity exceeding 95% were used.
Cell cultures and treatment
Primary human NK cells were cultured up to 15 days in RPMI medium
supplemented with 10% FCS and 100 U/mL human recombinant rIL-2 at an
optimal cell density of 1 ⫻ 106 cells/mL. In some experiments NK cells
were cultured with rIL-15 (10 ng/mL) at the same cell density. Primary
human CD8⫹ T cells were cultured in RPMI 1640 medium supplemented
with 10% FCS, 5 ␮g/mL purified PHA-M plus 20 U/mL human rIL-2. After
3 days of cultures, cells were washed twice and seeded again in complete
TRAIL IN NK CELLS AND CTL
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medium plus 20 U/mL human rIL-2 alone, which was re-added every 4
days, as previously described,16,18 up to 15 days.
Human natural killer cell line NK 3.3 was routinely cultured in RPMI
1640 medium supplemented with 15% FCS and 100 U/mL human
recombinant IL-2 at an optimal cell density of 1 ⫻ 106 cells/mL. HL-60
promyelocytic and Jurkat T human cell lines were grown in RPMI 1640
medium plus 10% FCS at a density of 1 ⫻ 106 cells/mL. Human adenocarcinoma MCF-7 cells were grown adherently and maintained in RPMI 1640
medium containing 10% FCS. Cells were passaged 2 or 3 times weekly at a
ratio between 1:5 and 1:10.
In cytotoxicity experiments, 1 ␮g/mL recombinant TRAIL was added
overnight to primary cell cultures at day 1 or day 10 of activation, and to
HL-60 and Jurkat cells as controls.
For neutralization experiments, the supernatant of cells treated with
recombinant TRAIL was preincubated with TRAIL-R1-Fc and TRAILR2-Fc chimera cocktail according to the supplier’s instructions (R&D), as
previously reported.17
Flow cytometric analysis
Aliquots of 0.5 ⫻ 106 cells/experimental point were single- or multiplelabeled by a panel of anti–TRAIL-Rs monoclonal antibodies (MoAbs), as
described previously.16 Expression of TRAIL-R1, TRAIL-R2, TRAIL-R3,
and TRAIL-R4 was analyzed by indirect staining by using 1 ␮g HS101
anti–human TRAIL-R1, HS201 anti–human TRAIL-R2, HS301 anti–
human TRAIL-R3, and HS401 anti–human TRAIL-R4 monoclonal antibody, followed by PE-labeled goat anti–mouse IgG. Surface expression of
TRAIL was analyzed by indirect staining with use of 1 ␮g 5D5 anti–human
TRAIL (Alexis Biochemicals), followed by PE-labeled goat anti–mouse
IgG. Aspecific fluorescence was assessed by using isotype-matched irrelevant mAb followed by the same secondary reagent. Analysis was
performed by an Epics XL flow cytometer (Beckman Coulter) and the Expo
ADC software (Beckman Coulter). Data collected from 10 000 cells are
reported as either percentage of positive cells or mean fluorescence
intensity (MFI) values.
Reverse transcription–polymerase chain reaction (RT-PCR)
Total RNA was isolated from each cell sample (1 ⫻ 106 cells) by using the
High pure RNA isolation kit (Roche Diagnostics S.p.A, Monza, Italy).
cDNA was synthesized from 50 ng total RNA by using 50 U MuLV Reverse
Transcriptase (Roche) and random hexamers (2.5 ␮M) in a 20-␮L volume.
RT reaction was performed at 42°C for 60 minutes followed by 5 minutes at
95°C and 5 minutes at 4°C. PCR was performed with 20 ␮L cDNA in a total
volume of 100 ␮L, using AmpliTaq DNA Polymerase (Roche) according to
manufacturer’s instruction. FLIPLong and FLIPShort mRNAs were detected
by FLIP primers 5⬘-GGA GGC TTA TGT CTG CTG AAG TCA TC and
3⬘-GGG GAA TTC CTT CTG ATT CCT GAA TGG. In particular (1)
FLIPLong forward primer 5⬘-CGA GGC AAG ATA AGC AAG GA-3⬘ and
reverse primer 5⬘-TGA CTG GTT CTT GTT GAG CG-3⬘, (2) FLIPShort
forward primer 5⬘-TCA GGA ACC CTC ACC TTG TT-3⬘ and reverse
primer 5⬘-ATC AGG ACA ATG GGC ATA GG-3⬘ were used to discriminate
the long and the short form of FLIP mRNA.19
The FLIPLong (327 bp) and FLIPShort (386 bp) products were amplified
in PCR buffer (500 mM KCl, 100 mM Tris (tris(hydroxymethyl)aminomethane)–HCl, pH 8.3) with 2 mM MgCl2 for 40 cycles. Optimal PCR thermal
profile was 1 minute at 94°C, 1 minute at 58°C followed by 30 seconds at
72°C. The PCR products were separated by electrophoresis in a 3% agarose
gel and stained in ethidium bromide for 20 minutes before visualization
under UV light.
TRAIL receptor mRNA transcripts were amplified by RT-PCR with the
following specific primers: 5⬘-CTG AGC AAC GCA GAC TCG CTG TCC
AC-3⬘ and 5⬘-TCC AAG GAC ACG GCA GAG CCT GTG CCA T-3⬘ for
TRAIL-R1, 5⬘-GCC TCA TGG ACA ATG AGA TAA AGG TGG CT-3⬘ and
5⬘-CCA AAT CTC AAA GTA CGC ACA AAC GG-3⬘ for TRAIL-R2,
5⬘-GAA GAA TTT GGT GCC AAT GCCACT G-3⬘ and 5⬘-CTC TTG GAC
TTG GCT GGG AGA TGT G-3⬘ for TRAIL-R3, and 5⬘-CTT TTC CGG
CGG CGT TCATGT CCT TC-3⬘ and 5⬘-GTT TCT TCC AGG CTG CTT
CCC TTT GTA G-3⬘ for TRAIL-R4. As control, we amplified ␤-actin
mRNA with the following primers: 5⬘-TGA CGG GGT CAC CCA CAC
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
MIRANDOLA et al
TGT GCC CAT CTA-3⬘ and 5⬘-CTA GAA GCA TTT GCG GTG GAC GAT
GGA GGG-3⬘. Resultant PCR products were resolved in 2% agarose gels
and visualized by ethidium bromide under UV light.17,18
0.5 ⫻ 106 cells were harvested by centrifugation at 200g for 10 minutes at
4°C and incubated in Ca2⫹ and PI staining buffer following manufacturer’s
protocol. Flow cytometry analysis of the samples was performed as
described in “Flow cytometric analysis.”
Semiquantitative RT-PCR
As described in “Reverse transcription–polymerase chain reaction (RTPCR),” 1 ␮g total RNA was reverse transcribed, and progressive dilutions
(1/8, 1/20, 1/100, 1/500, 1/2500, 1/12.500) were subjected to PCR
amplification to generate ␤-actin, TRAIL-R1, TRAIL-R2, TRAIL-R3,
TRAIL-R4, FLIPLong, and FLIPShort specific sequences.
Small interference RNA
Double-strand siRNAs (dsRNA) were designed to target sequences corresponding to nt 472 to 492 (siRNA-F1) and 908 to 928 (siRNA-F2) of
human c-FLIP mRNA (U97074). The target sequences, which have been
designed starting from siRNA sequences previously developed by Siegmund at al,20 were screened against all known human genes by using a
BLAST (Basic Local Alignment Search Tool) search to confirm that only
human c-FLIP mRNA would be targeted. The respective sense and
antisense RNA sequences were synthesized by Silencer siRNA Construction Kit (Ambion, Austin, TX). To achieve an efficient transfection of
human primary NK cells, siRNA-F1 and siRNA-F2 (300 nM each) were
delivered using the Amaxa nucleofection technology (Amaxa, Koeln,
Germany) according to manufacturer’s protocols. Nucleofection efficiency
was assayed by transfecting a green fluorescence protein (GFP) expression
vector, obtaining a number of GFP⫹PI⫺ (propidium iodide) cells more than
20% of total primary T cultures.
Western blot
Cultured cells were counted, and 5 ⫻ 106 cells were collected at specific
time points, washed in PBS, and centrifuged at 200g for 10 minutes. Pellets
were resuspended in RIPA buffer supplemented with fresh protease
inhibitors and protein concentration determined by using Bradford assay
(Pierce, Rockford, IL). Proteins (60 ␮g) from each sample were then
migrated in 12% sodium dodecyl sulfate (SDS)–acrylamide gels and
blotted onto nitrocellulose filters. Blotted filters were blocked for 60
minutes in a suspension of 3% dried skimmed milk and 2% bovine serum
albumin (BSA) in phosphate-buffered saline (PBS). Filters were then
incubated overnight at 4°C with rabbit 1:1000 polyclonal anti-FLIP
antibody (Exalpha Biologicals, Boston, MA) directed against the Nterminal portion of FLIP (55 KDa) in a suspension of 3% dried skimmed
milk in PBS–0.05% Tween 20. Filters were washed and further incubated
for 1.5 hours at room temperature with 1:10 000 peroxidase-conjugated
anti–rabbit IgG (Pierce, Rockford, IL) in a suspension of 3% dried skimmed
milk in PBS–0.05% Tween 20 at room temperature. Specific reactions were
revealed with the enhanced chemiluminescence (ECL) Supersignal West
Pico Chemiluminescent Substrate detection system (Pierce).
Assessment of apoptosis
Recombinant human TRAIL (1 ␮g) was added overnight to primary cell
cultures at day 1 or day 10 of activation and to TRAIL-sensitive HL-60 and
Jurkat cells as controls.
Viability was then assessed by trypan blue exclusion. Apoptosis was
evaluated by PI staining of permeabilized cells and by staining extracellular
phosphatidylserine with Annexin V in nonpermeabilized cells. Briefly,
samples containing 0.5 ⫻ 106 cells were harvested by centrifugation at
200g for 10 minutes at 4°C, fixed, permeabilized with cold 70% ethanol for
at least 1 hour at 4°C, and treated as previously described.21 Cells were then
stained with 40 ␮g/mL PI in the presence of RNAse; analysis of PI
fluorescence was performed by an Epics XL flow cytometer (Beckman
Coulter) and the Expo ADC software (Beckman Coulter). For quantitative
evaluation of apoptosis, the subdiploid (⬍ 2n) DNA content was calculated
as described21 and expressed as percentage of apoptotic versus nonapoptotic cells, regardless of the specific cell cycle phase.
Staining of extracellular phosphatidylserine in nonpermeabilized cells
was performed by FITC conjugate Annexin V (ACTIPLATE; Valter
Occhiena, Torino, Italy) in Ca2⫹ and PI staining buffer. Samples containing
Results
Differential expression of TRAIL-Rs and TRAIL in resting
and activated NK and CD8ⴙ CTLs
Purified NK cells were cultured for 15 days with IL-2 or with
IL-15, whereas purified CD8⫹ CTLs were cultured for 3 days in
the presence of PHA and cultured for the subsequent 12 days
with IL-2. In the first group of experiments the mRNA for
TRAIL receptors was examined by RT-PCR analysis both in
resting and activated NK cells. As shown in Figure 1, the mRNA
of all TRAIL receptors (R1, R2, R3, R4) was clearly detectable
in both resting and activated NK cells. Similar data were
obtained with purified CD8⫹ CTLs.
The expression profile of TRAIL receptors was next examined
by flow cytometry (Figure 2A-C).
Despite the expression of the mRNA for all receptors, resting
NK and CD8⫹ T cells did not show any significant surface
expression of R1, R2, or R3. However, a variable proportion of
freshly isolated NK and CD8⫹ T cells did express R4. The surface
expression of TRAIL-Rs was next examined in both populations at
different time points on stimulation. R1 was never expressed up to
day 15 of culture, whereas R2 and, to a lower extent, R3 expression
was up-regulated as soon as 36 hours after stimulation, reaching a
peak around day 10. Moreover, although the expression of surface
R4 remained constant in resting and activated NK cells, it showed a
significant increase in activated CD8⫹ T cells.
The NK-like cell line NK 3.3 showed surface expression of
TRAIL-R2 similar to that of primary NK cells but no expression of
TRAIL-R3 or R4 (Figure 2D).
In parallel experiments, we examined the surface expression of
membrane-bound TRAIL, which was undetectable in resting NK
cells and CTLs (data not shown), but it became clearly detectable in
activated NK cells and CTLs (Figure 2E).
To quantify the effect of cytokine induction on TRAIL receptor
mRNA expression, we performed a semiquantitative RT-PCR
analysis (Figure 3). In NK cells, IL-2 promoted ␤-actin transcription, raising the amount of intracellular mRNA up to 26.2- ⫾ 5.6fold; similarly TRAIL-R2 gene transcription was induced up to
26.1- ⫾ 3.1-fold. No induction of TRAIL-R1, TRAIL-R3, or
TRAIL-R4 transcription was observed.
Figure 1. RT-PCR analysis of TRAIL-Receptors mRNA expression in resting
and IL-2–stimulated primary human NK cells. Lanes 1, 3, and 5 show resting NK
cells. Lanes 2, 4, 6, and 7 show IL-2–treated NK cells for time intervals ranging from
12 hours to 7 days. K indicates positive control (K562 cells).
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
TRAIL IN NK CELLS AND CTL
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Figure 2. Flow cytometry analysis of the surface expression of TRAIL-Receptors and TRAIL ligand in NK and CTL cells. (A) IL-2–stimulated NK cells. (B)
IL-15–stimulated NK cells. (C) CD8⫹ T cells, at different times of activation. (D) Surface expression of TRAIL-Rs by the NK-like cell line NK 3.3. The cell line was routinely
cultured in RPMI 1640 ⫹ 15% FCS ⫹ 100 U/mL rIL-2. (E) Expression of surface TRAIL-ligand (TRAIL-L) by activated (day 10) NK and CD8⫹ T cells. Empty histograms indicate
isotype-matched irrelevant antibody, negative controls.
The increased amount of surface TRAIL-R2 protein, observed
by flow cytometry on cytokine stimulation of NK cells, correlated
with its increased gene expression, whereas TRAIL-R3 phenotypic
up-regulation did not, suggesting a posttranscriptional regulation of
TRAIL-R3 expression.
Both resting and activated NK cells and CTLs are resistant
to TRAIL-induced apoptosis
The simultaneous expression of TRAIL and TRAIL-R2, 1 of the 2
death receptors, in activated NK cells and CTLs prompted us to
investigate the susceptibility to TRAIL-induced cytotoxicity of
both resting and activated lymphocytes. In this group of experi-
ments, we used TRAIL-sensitive Jurkat and HL-60 cell lines as
positive controls (Figure 4). The specificity of TRAIL-induced cell
death was ascertained by using a TRAIL-R1-Fc and TRAIL-R2-Fc
chimera cocktail, which was administered to the cell cultures at the
same time as TRAIL. Figure 4A-B shows that the chimera cocktail,
by sequestering soluble TRAIL, completely inhibited TRAILinduced apoptosis on Jurkat and HL-60 cells. TRAIL did not
induce apoptosis in resting NK or CD8⫹ T cells. This was an
expected finding on the basis of the lack of surface TRAIL-R1 and
TRAIL-R2 in primary resting lymphoid cells. Notwithstanding the
expression of death receptor R2, however, TRAIL did not induce
apoptosis either in activated NK or CD8⫹ cells. Similarly, NK 3.3
cells, routinely cultured in the presence of IL-2, were resistant to
TRAIL-induced apoptosis (Figure 4B). These findings clearly
indicate that the expression of R2 is not sufficient to sensitize NK
or CD8⫹ cells to the apoptogenic potential of TRAIL. Given the
absence of decoy receptors on NK 3.3 cells, they also suggest that
intracellular mechanisms might be responsible for the resistance of
activated NK cells to TRAIL-induced apoptosis.
Therefore, to rule out the possible actors in the intracellular
death machinery of primary lymphoid cells potentially responsible for resistance to TRAIL-induced apoptosis, we next
performed experiments in which NK and CD8⫹ T cells were
pretreated with 50 ng/mL actinomycin D before adding recombinant TRAIL. As shown in Figure 5, actinomycin D strongly
sensitized activated NK cells to TRAIL-induced apoptosis,
evaluated by Annexin V–FITC. Similar results were obtained
for CD8⫹ CTLs (not shown).
Both NK cells and CTLs show a high expression of c-FLIP
Figure 3. Quantification of gene transcription by semiquantitative RT-PCR in
resting and Il-2–stimulated primary human NK cells. RT-PCR of ␤-actin, TRAILR1, TRAIL-R3, and TRAIL-R4 was performed by using the following cDNA dilutions:
1/8, 1/20, 1/100, 1/500, 1/2500 (lanes 1-5, respectively). RT-PCR of TRAIL-R2 was
performed by using the following cDNA dilutions: 1/20, 1/100, 1/500, 1/2500,
1/12 500 (lanes 1-5, respectively). Lane 6 is RT-PCR negative control.
Given the well-documented inhibitory role of FLIP on the activity
of apical caspase-8,15 we performed a series of experiments on
purified NK and CD8⫹ T cells to determine the expression of FLIP
at the mRNA and protein level. Figure 6A shows the RT-PCR for
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Figure 4. TRAIL-induced apoptosis in NK and CD8ⴙ T
cells. NK and CD8⫹ T cells were stimulated for 1 day (A)
or 10 days (B) as reported in “Materials and methods.”
Cells were incubated overnight with 1 ␮g recombinant
human TRAIL. TRAIL sensitivity of NK 3.3 cell line is also
reported (B). TRAIL chimera indicates TRAIL-R1-Fc ⫹
TRAIL-R2-Fc chimera cocktail administered to the cells
at the same time of recombinant TRAIL. TRAIL-sensitive
Jurkat and HL-60 cell lines served as positive controls.
the long and short forms of FLIP in NK and CD8 T cells after a
short (1 day) or long period (10 day) of IL-2 stimulation. Both
forms are expressed at the same level by resting and activated cells.
The NK cell line NK 3.3 expresses both forms of FLIP, as well. The
expression of FLIP short or long mRNA in CD8⫹ T cells was not
dependent on the type of stimulation used, as anti-CD3, IL-2, and
PHA did not influence gene transcription and mRNA expression
(Figure 6B).
Western blot analysis showed that both resting NK and CD8⫹ T
cells express FLIP protein, and prolonged activation with cytokines
apparently slightly enhanced the total protein amount (Figure 6C).
To better quantify the modulation of FLIP mRNA transcription
under cytokine stimulation, we performed a semiquantitative PCR
analysis. Figure 6D shows that FLIPLong messenger RNA was
expressed at the same level in resting and IL-2–activated NK cells,
whereas FLIPShort mRNA was partially down-modulated (up to
27% ⫾ 4% of resting cells) in IL-2–stimulated NK cells. Furthermore, actinomycin D impaired transcription and accumulation of
FLIP mRNA in IL-2–stimulated NK cells (Figure 6E; compare lane
5 with lane 2 and lane 6 with lane 3). However, actinomycin D did
not affect TRAIL receptors surface expression (Figure 6F).
Blockage of FLIP transcription sensitizes NK cells to TRAIL
To formally prove that FLIP levels in resting and activated NK cells are
responsible for their resistance to the apoptotic effects of TRAIL, we
designed siRNAs specific for FLIP mRNA and transfected them into
primary, IL-2–stimulated, NK cells. Figure 7A-C shows that a significant proportion of FLIP siRNA-transfected NK cells effectively die after
soluble TRAIL administration.
Figure 5. TRAIL-induced apoptosis in IL-2–treated NK cells (10 days) in the
presence of 50 ng/mL Actinomycin D (Act D). Act D was administered to the cells
at the same time of TRAIL. (A) Negative control was IL-2–treated NK cells ⫹ Act D.
The percentage of living cells (57.4%) is indicated in the Annexin V⫺/PI⫺ population.
(B) IL-2–treated NK cells ⫹ TRAIL ⫹ Act D. The percentage of living cells (25.9%) is
indicated, as well. (C) TRAIL-induced apoptosis in IL-2–stimulated NK cells with or
without Act D. Results obtained from 3 unrelated donors are reported. Values are
expressed as means ⫾ standard deviation.
Discussion
Immune surveillance against tumors is mediated by natural killer
cells and cytotoxic T lymphocytes. Both these cell types are
equipped with receptors for MHC class I, although their molecular
nature and function are different.22 Although NK cells display a
spontaneous cytolytic activity against a variety of tumor cells, their
antitumor activity is greatly enhanced, both in terms of efficiency
and array of targets, by cytokine treatment. Antitumor CTLs
physiologically act under the proliferative/activatory action of
cytokines as well, and both NK cells and CTLs acquire new
antitumor molecular equipment when activated by cytokines.
Increasing experimental evidence suggests that TRAIL, the most
recently identified of such molecules expressed on the surface of
activated NK cells and CTLs, plays a key role in their antitumor
activity. In fact, the expression of TRAIL has been reported on the
surface of activated NK and T cells in response to several
cytokines,4,23-26 and recent evidence indicates that surface TRAIL
plays a major role in optimal graft-versus-tumor activity.26 Furthermore, Cretney et al27 recently generated a TRAIL-deficient mouse
that was found to be more susceptible to experimental and
spontaneous tumor metastasis. Studies in the mouse demonstrated
that TRAIL expression is constitutive only in liver NK cells,
playing a role in the control of liver metastasis. However, on
cytokine activation, TRAIL expression is up-regulated also in NK
cells resident in other tissues, such as spleen and lung, playing a
role, as well, in the control of tumor metastasis to these organs.6
Therefore, recombinant TRAIL protein offers great promise as a
cancer therapeutic,14 showing the unique property to kill several
types of tumor cells, sparing most normal cells and tissues. Hence,
the great expectations from the clinical trials that are about to start.
Moreover, cytokine-mediated TRAIL induction on lymphoid cells
has been proposed as a prognostic marker in multiple sclerosis,
suggesting a role for TRAIL⫹ lymphocytes in patients’ response to
cytokine therapy.28 Our data show that both activated NK and
CD8⫹ T cells express membrane-bound TRAIL. In transplantation
tolerance, these TRAIL-expressing cells might be responsible for
the “veto cell” function by killing recipient’s CD8⫹ CTL precursors
that are specific for them.29-31 Given the great importance of the
anticancer effects of TRAIL, and the use of membrane-bound
TRAIL made by antitumor lymphocytes, understanding whether
cytokine stimulation can modulate the cellular distribution of
TRAIL death receptors exposing major antitumor cytotoxic cells to
the apoptogenic action of TRAIL is of great relevance. Our data
essentially demonstrate that (1) resting NK and CD8⫹ T cells do
not express TRAIL-R1, TRAIL-R2, or TRAIL-R3 but show a
constitutive expression of the decoy receptor TRAIL-R4; (2) IL-2
stimulation induces the stable surface expression of TRAIL-R2 and
TRAIL-R3 by both cell types; (3) both resting and activated NK
cells and CTLs are insensitive to the apoptogenic effects of TRAIL;
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
TRAIL IN NK CELLS AND CTL
2423
Figure 6. Expression of the short and long forms of
FLIP in resting and activated NK cells, CD8ⴙ T cells,
and in the NK 3.3 cell line. (A) RT-PCR of FLIPShort and
FLIPLong in resting and activated (as described in “Materials and methods”) NK and CD8⫹ T cells for 1 day or 10
days. MCF7 cells served as positive controls. (B) RTPCR of FLIPShort and FLIPLong in CD8⫹ T cells activated
by anti-CD3, IL-2 alone, or PHA alone. The expression of
FLIP mRNA is not dependent on the type of stimulation
used to activate CD8⫹ CTLs. (C) Western blot analysis of
FLIP protein in resting (0 day) and stimulated (10 days)
NK cells, CD8⫹ T cells, and NK 3.3 cell line. (D)
Quantification of gene transcription by semiquantitative
RT-PCR in NK cells before and after IL-2 stimulation
(lanes 1-5, 1/8, 1/20, 1/100, 1/500, 1/2500 cDNA dilutions
were analyzed to detect FLIPShort and FLIPLong; lane 6,
RT-PCR negative control). (E) RT-PCR analysis of FLIPShort
and FLIPLong expression with or without actinomycin
D treatment (lanes 1 and 4, ␤-actin; lanes 2 and 5, FLIPShort;
lanes 3 and 6, FLIPLong RT-PCR products). (F) Flow cytometry analysis of the surface expression of TRAIL receptors in
NK cells with or without actinomycin D treatment (50 ng/mL
for 24 hours). Empty histograms indicate controls, untreated
cells. Gray histograms indicate actinomycin-treated cells.
and (4) both resting and IL-2–activated NK cells and CTLs
constitutively express both forms of FLIP (short and long) at the
mRNA and protein level that are responsible for their resistance to
the apoptotic effects of TRAIL.
With some exceptions, such as developing erythroblasts,11
hepatocytes,32 prostate cells,33 and thymic lymphocytes,12 most of
the normal cells examined are resistant to TRAIL-mediated
apoptosis. In the TRAIL⫺/⫺ mouse model system, no specific
alteration of peripheral T-cell or NK cell homeostasis was reported
under physiologic conditions.27 However, controversial data have
been obtained from different groups of investigators examining the
response to TRAIL of primary normal T lymphocytes, as either no
effect,34 cell cycle arrest without cytotoxicity,35,36 or induction of
apoptosis 37 have been reported. However, several data suggest that
Figure 7. TRAIL-induced apoptosis of IL-2–activated NK cells (10 days) in the
presence of anti-FLIP siRNAs. siRNAs were nucleofected into NK cells 24 hours
before TRAIL addition. (A) Negative control was IL-2–treated NK cells ⫹ siRNA. The
percentage of living cells (37.5%) was calculated by selecting the events with a PI
fluorescence lower than 10° relative units. (Insert) Ten-day IL-2–stimulated control
lymphocytes. (B) IL-2–treated NK cells ⫹ siRNA ⫹ TRAIL. The percentage of living
cells was 15.0%. (C) TRAIL-induced apoptosis in IL-2–stimulated NK cells with or
without siRNA. Results obtained from 3 unrelated donors are reported. Values are
expressed as means ⫾ standard deviation.
viral infections can render lymphoid cells38-40 as well as hepatocytes41 susceptible to TRAIL-mediated cytotoxicity. Furthermore,
a reciprocal expression of membrane-bound TRAIL and FasL has
been recently suggested to play a role in the T helper 1 (Th1) versus
Th2 cell differentiation.42
Theoretically, there are at least 2 possible, not mutually
exclusive, mechanisms for NK cells and CTLs to be protected by
TRAIL-induced apoptosis.43 The first mechanism is represented by
the expression of TRAIL-R4 in both resting and activated cells, and
TRAIL-R3 exclusively in activated cells. In fact, although
TRAIL-R1 and TRAIL-R2 transduce apoptotic signals on binding
of TRAIL, TRAIL-R3 and TRAIL-R4 are homologous to
TRAIL-R1 and TRAIL-R2 in their cysteine-rich extracellular
domain but lack intracellular death domain and apoptosis-inducing
capability. Although it is not yet completely clear whether the
expression of TRAIL-R3 and/or TRAIL-R4 is a key factor in
determining the resistance or sensitivity to TRAIL cytotoxicity,
they have been proposed to function as decoy receptors, protecting
some cell types from apoptosis.44,45 However, our data show that
the second possible mechanism of protection, represented by the
high level of FLIP protein expression by NK cells and CTLs, is the
one that accounts for their resistance to the apoptotic effect of
TRAIL. A direct proof of the role for FLIP on the protection from
TRAIL activity is given by the siRNA experiments, in which
FLIP-siRNA transfected primary human NK cells became sensitive
to TRAIL and die by apoptosis. FLIPLong, the full-length 55-kDa
form of FLIP, shows overall structural homology to caspase-8,
containing 2 death effector domains that interact with FADD, and
an inactive caspase-like domain. FLIPShort, an alternative spliced
short form of FLIP, contains only the 2 death effector domains and
has lower antiapoptotic capacity.15 Our data parallel those obtained
by overexpression of FLIPLong in mouse T cells, which inhibits
FasL-induced apoptosis in vitro,46 whereas it is not able to block
activation-induced cell death.47 Although both TRAIL-R4 and
FLIP might play a role in the protection of NK and CTL cells
from TRAIL-induced apoptosis, the fact that (1) selective
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2424
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
MIRANDOLA et al
down-modulation of FLIP exposes NK cells to TRAIL-induced
apoptosis; (2) NK 3.3 cells, which are resistant to TRAIL, do
express FLIP but do not express TRAIL decoy receptors; and (3)
actinomycin D, which restores TRAIL sensitivity in NK cells,
does not affect TRAIL receptor surface expression while
decreasing FLIP expression, strongly suggest that FLIP is
primarily responsible for the resistance of NK and CD8 T cells
to the apoptotic effect of TRAIL.
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From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2004 104: 2418-2424
doi:10.1182/blood-2004-04-1294 originally published online
June 17, 2004
Activated human NK and CD8+ T cells express both TNF-related
apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant
to TRAIL-mediated cytotoxicity
Prisco Mirandola, Cristina Ponti, Giuliana Gobbi, Ivonne Sponzilli, Mauro Vaccarezza, Lucio Cocco,
Giorgio Zauli, Paola Secchiero, Francesco Antonio Manzoli and Marco Vitale
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