Human Dendritic Cells Mediate Cellular Apoptosis via Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (Trail)

Abstract

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family that induces apoptosis in a variety of cancer cells. In this study, we demonstrate that human CD11c(+) blood dendritic cells (DCs) express TRAIL after stimulation with either interferon (IFN)-gamma or -alpha and acquire the ability to kill TRAIL-sensitive tumor cell targets but not TRAIL-resistant tumor cells or normal cell types. The DC-mediated apoptosis was TRAIL specific, as soluble TRAIL receptor blocked target cell death. Moreover, IFN-stimulated interleukin (IL)-3 receptor (R)alpha(+) blood precursor (pre-)DCs displayed minimal cytotoxicity toward the same target cells, demonstrating a clear functional difference between the CD11c(+) DC and IL-3Ralpha(+) pre-DC subsets. These results indicate that TRAIL may serve as an innate effector molecule on CD11c(+) DCs for the elimination of spontaneously arising tumor cells and suggest a means by which TRAIL-expressing DCs may regulate or eliminate T cells responding to antigen presented by the DCs.

Full text

1155
J. Exp. Med.

The Rockefeller University Press • 0022-1007/99/10/1155/10 $5.00
Volume 190, Number 8, October 18, 1999 1155–1164
http://www.jem.org
Human Dendritic Cells Mediate Cellular Apoptosis via
Tumor Necrosis Factor–related Apoptosis-inducing
Ligand (TRAIL)
By Neil A. Fanger,
*
Charles R. Maliszewski,
*
Ken Schooley,
‡
and Thomas S. Griffith
§
From the
*
Department of Discovery Research,
‡
Department of Molecular Biology, and
§
Department of
Immunobiology, Immunex Corporation, Seattle, Washington 98101
Summary
TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family that induces
apoptosis in a variety of cancer cells. In this study, we demonstrate that human CD11c
1
blood
dendritic cells (DCs) express TRAIL after stimulation with either interferon (IFN)-
g
or -
a
and
acquire the ability to kill TRAIL-sensitive tumor cell targets but not TRAIL-resistant tumor
cells or normal cell types. The DC-mediated apoptosis was TRAIL specific, as soluble TRAIL
receptor blocked target cell death. Moreover, IFN-stimulated interleukin (IL)-3 receptor
(R)
a
1
blood precursor (pre-)DCs displayed minimal cytotoxicity toward the same target cells,
demonstrating a clear functional difference between the CD11c
1
DC and IL-3R
a
1
pre-DC
subsets. These results indicate that TRAIL may serve as an innate effector molecule on
CD11c
1
DCs for the elimination of spontaneously arising tumor cells and suggest a means by
which TRAIL-expressing DCs may regulate or eliminate T cells responding to antigen pre-
sented by the DCs.
Key words: TRAIL • apoptosis • tumor • dendritic cells • human
D
endritic cells (DCs)
1
are bone marrow–derived cells
that specialize in the uptake, processing, and presenta-
tion of foreign and self-antigens (1, 2). In humans, two dis-
tinct peripheral blood DC subsets have been described (3–
7). One subset is characterized by a multilobulated nucleus,
the absence of CD3, CD14, CD19, and CD56, the pres-
ence of the myeloid-associated antigen CD33, moderate
levels of CD11c and CD4, and high levels of MHC class
II (HLA-DR). This DC subset, hereafter referred to as
CD11c
1
DC, also constitutively expresses the high- and
low-affinity IgG receptors (Fc
g
RI/CD64 and Fc
g
RII/
CD32, respectively), which help mediate the uptake of im-
mune complexes (6, 7). DCs with a similar morphology
and phenotype are dispersed throughout the germinal cen-
ter dark and light zones of human tonsils, spleens, and
lymph nodes (8). The second blood DC subset, hereafter
referred to as IL-3R
a
1
pre-DC, is distinguished by an im-
mature appearance containing an oval nucleus, low levels
of CD33, and absence of CD3, CD14, CD19, CD56, and
CD11c antigens. These DCs express CD4, high levels of
HLA-DR and IL-3R
a
, low levels of Fc
g
RII/CD32, and
no detectable levels of Fc
g
RI/CD64 (3, 7). A precursor
DC with similar phenotype and morphology resides in the
extrafollicular T cell–rich regions of the tonsils and lymph
nodes (9, 10). Another difference between these two DCs
is the expression of CD83, which is detected on CD11c
1
DCs but not on IL-3R
a
1
pre-DCs or monocytes (M
f
) af-
ter short-term culture (7, 11, 12).
Recent reports demonstrate that human DCs can effi-
ciently present antigens derived from apoptotic cells and
can cross-present tumor, viral, transplantation, and self-
antigens to CD8
1
T cells in vitro (13, 14). This may corre-
spond to the in vivo
phenomenon of cross-priming, where
antigens derived from dying tumor cells or transplanted tis-
sue are presented by host APCs to antigen-specific CD8
1
cytotoxic T cells (15–17). Expression of CD36 and the
a
v
b
5
integrin on the DC appears to be critical for the up-
take of antigens from apoptotic cells (14), perhaps through
the binding of a bridging molecule such as thrombospon-
din (18). The fact that DCs can act as efficient APCs in this
setting has suggested that these cells may act as “adjuvants”
for MHC class I–restricted antitumor immunity (19). Such
a protective antitumor immune response can be induced in
vivo with DCs that have been incubated in vitro with tu-
mor antigens or peptides derived from tumor antigens (20–
22). It remains unclear, however, whether DCs can induce
1
Abbreviations used in this paper:
DCs, dendritic cells; FLIP, FLICE (Fas-
associated death domain–like IL-1
b
–converting enzyme)-inhibitory pro-
tein; L, ligand; LZ, leucine zipper; M
f
, monocytes; RT, reverse tran-
scriptase; TRAIL, TNF-related apoptosis-inducing ligand.

1156
Dendritic Cells Induce Apoptosis via TRAIL
cellular apoptosis and selectively process and present anti-
gens from apoptotic bodies in vivo. One possible scenario
would involve DCs directly inducing apoptotic cell death
of tumor cells or cells within transplanted tissue followed
by the uptake of cellular fragments, antigen processing, and
eventual cross-priming of naive CD8
1
T cells.
Among the molecules known to induce the apoptotic
cell death of tumor cells, TRAIL (TNF-related apoptosis-
inducing ligand) has received great attention (23). Recom-
binant, soluble forms of TRAIL are potent mediators of
tumor cell apoptosis, while demonstrating little or no cyto-
toxicity toward normal cells and tissues in vitro and in vivo
(24–27). As with the other death-inducing members of the
TNF family (i.e., FasL [ligand] and TNF), cells undergoing
TRAIL-induced death exhibit many of the hallmarks of
apoptosis, including zeiosis and apoptotic body release,
chromatin condensation and DNA fragmentation, expres-
sion of prophagocytic signals (i.e., phosphatidylserine) on
the cell membrane, and cleavage of multiple intracellular
proteins by caspases (23, 24, 28, 29). Soluble TRAIL is tu-
moricidal for approximately two-thirds of the more than
30 hematopoietic and nonhematopoietic tumor cell lines
tested in vitro, suggesting that TRAIL could be a broad-
spectrum antitumor molecule in vivo (23, 24, 30, 31). Al-
though a normal biological function for TRAIL remains to
be determined, it has been suggested that TRAIL may be
important in the activation-induced cell death (AICD) of
T cells during HIV infection (32, 33). Peripheral blood
human T cells express TRAIL after CD3 cross-linking com-
bined with type I IFN stimulation, perhaps also contribut-
ing to the AICD of T cells in the natural setting (34). In
addition, human M
f
express TRAIL after IFN stimula-
tion, transforming them into potent killers of tumor cells
(27).
The aim of these studies was to determine if human DCs
are able to induce apoptosis via TRAIL. We demonstrate
that human CD11c
1
DCs, but not IL-3R
a
1
pre-DCs, ex-
press TRAIL after stimulation with IFN and are able to in-
duce cellular apoptosis in TRAIL-sensitive cells.
Materials and Methods
Reagents and mAbs.
Reagents and sources were as follows:
GM-CSF and leucine zipper (LZ)-CD40L (100 ng/ml; Immu-
nex Corp.); IFN-
a
and -
g
(100 ng/ml; Genzyme Corp.); LPS (5
ng/ml; Difco Labs., Inc.); MOPC-21, nonspecific IgG1 isotype
control; M181, IgG1 anti-TRAIL (Immunex Corp.); 7G3,
IgG2a anti–IL-3R
a
–biotin; G155-178, IgG2a–biotin isotype
control (PharMingen); 3.9, IgG1 anti-CD11c–PE; TUK4, IgG2a
anti-CD14–FITC; 4D3, IgG2b anti-CD33–FITC; TU39, IgG2b
anti–HLA-DR–FITC; IgG1–PE isotype control; IgG2a–FITC
isotype control; IgG2b–FITC isotype control; and IgG2b–biotin
isotype control (Caltag Labs., Inc.). HB-15a, IgG2b anti-CD83 (a
gift of Dr. Thomas F. Tedder, Duke University Medical Center,
Durham, NC). The soluble fusion proteins TRAILR2–Fc, Fas–
Fc, and TNFR–Fc were produced at Immunex Corp. The LZ-
huTRAIL expression plasmid and the production and purifica-
tion of LZ-huTRAIL (TRAIL) have been previously described
(26).
Cell Lines.
The ovarian carcinoma cell line OVCAR3 was
obtained from Dr. Richard F. Camalier (Developmental Thera-
peutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute, Bethesda, MD). The human prostate
carcinoma cell line PC-3 was obtained from Dr. Michael Cohen
(University of Iowa, Iowa City, IA). The human melanoma cell
lines WM 793 and 164 were obtained from Dr. M. Herlyn (Wistar
Institute, Philadelphia, PA). The Jurkat cell line was purchased
from American Type Culture Collection. All tumor cell lines
were cultured as directed. Normal lung fibroblasts, lung micro-
vascular endothelial cells, and skeletal muscle cells were pur-
chased from Clonetics Corp. and cultured as directed.
Isolation of Human DCs and Monocytes.
Peripheral blood DCs
were enriched using countercurrent elutriation. Cells from leu-
kopheresis packs obtained from healthy volunteers were loaded
onto a JE-5 elutriator (Beckman Instruments, Inc.), and 50-ml
fractions were collected while increasing the flow rate from 65 to
85 ml/min at 2,000 rpm. Fractions containing the highest blood
DC percentages were further enriched using magnetic bead se-
lection. The DC-positive fractions were pooled and incubated
with anti-CD3 (OKT3), anti-CD14 (MY23), anti-CD16 (3G8),
anti-CD19 (B43), and anti-CD56 (B159) mAbs for 10 min at
20
8
C. Cells binding these mAbs were then removed using Dynal
goat anti–mouse Ig–coated magnetic beads. Remaining cells
were recovered and incubated with anti-CD7 (T3-3AI), anti-
CD8 (OKT8), anti-CD11b (OKM1), anti-CD34 (MY10), and
antiglycophorin A (10F7MN). Cells binding these mAbs were
removed using the goat anti–mouse Ig–coated magnetic beads.
To separate CD11c
1
DCs from IL-3R
a
1
DCs, the remaining
cells were stained with a PE-labeled anti-CD11c and biotin-
labeled anti–IL-3R
a
, followed by APC-labeled streptavidin, and
sorted on a FACStar
PLUS™
(Becton Dickinson) into CD11c
1
and
IL-3R
a
1
populations. Peripheral blood M
f
were enriched in
elutriated fractions generated from a flow rate
.
75 ml/min. The
fractions were
.
93% CD14
1
M
f
, as assessed by flow cytometric
analysis. Complete medium used for DC and M
f
culturing and
functional assays consisted of RPMI 1640 (GIBCO BRL) supple-
mented with penicillin–streptomycin–glutamine and 10% pooled
human serum (Immunex Corp.).
Flow Cytometry.
Cell analysis was performed on a FAC-
Scan™ (Becton Dickinson), with
.
5,000 cells analyzed per sam-
ple. For multicolor cell analysis, samples consisting of 20
m
l cells
were combined in a 96-well flat-bottom plate (Costar Corp.)
with 20
m
l human IgG (12 mg/ml; Sigma Chemical Co.) to
block Fc binding of the mAbs and 20
m
l each of the direct PE-
labeled, FITC-labeled, and biotin-labeled mAbs (60
m
g/ml).
Cells were then incubated on a rotator at 4
8
C for 1 h. After three
washes with 200
m
l PBS containing 2 mg/ml BSA, 40
m
l of
APC-labeled streptavidin (1:100 dilution; Molecular Probes, Inc.)
was added for an additional 1 h. Cells were analyzed immediately
after staining or fixed in 1% paraformaldehyde until analysis.
Morphological Analysis.
Cytospins were performed by centri-
fuging 2
3
10
5
sorted DCs at 500 rpm for 5 min onto slides. The
DCs were stained with a Hemacolor Stain Set (EM Diagnostic
Systems), and photomicrographs were recorded using a Nikon
Diaphot microscope with a 40
3
objective (Carl Zeiss, Inc.).
DC-mediated Killing of Human Tumor Cells.
DCs were cul-
tured for 12 h in medium alone, GM-CSF, LZ-CD40L, LPS,
IFN-
g
, or IFN-
a
, washed, and resuspended in complete me-
dium. Tumor cells were labeled with 100
m
Ci of
51
Cr for 1 h at
37
8
C, washed three times, and resuspended in complete medium.
To determine TRAIL-induced death,
51
Cr-labeled tumor cells
(10
4
cells/well) were incubated with varying numbers of DC ef-

1157
Fanger et al.
fector cells for 8 h. As a positive control, soluble TRAIL was
added to the target cells. M
f
used for comparison were cultured
and stimulated under conditions identical to those described for
DCs. In some cultures, TRAILR2–Fc, Fas–Fc, or TNFR–Fc
(20
m
g/ml) was added to the DCs 15 min before adding tumor
cell targets. All cytotoxicity assays were performed in 96-well
round-bottom plates, and the percent specific lysis was calculated
as: 100
3
(experimental cpm
2
spontaneous cpm)/(total cpm
2
spontaneous cpm). Spontaneous and total
51
Cr release values
were determined in the presence of either medium alone or 1%
NP-40, respectively. The presence of TRAILR2–Fc, Fas–Fc, or
TNFR–Fc during the assay had no effect on the level of spon-
taneous release by the target cells. Apoptotic cell death of tu-
mor cells was measured by flow cytometry using FITC-conju-
gated annexin V and propidium iodide (apoptosis detection kit;
R & D Systems, Inc.) as per the manufacturer’s protocol. Light
scatter characteristics were used to distinguish the tumor cells
from the DCs.
Reverse Transcriptase–PCR.
Total RNA was isolated from
DCs with TRIzol reagent (Life Technologies) as per the manu-
facturer’s instructions. RNA samples (1
m
g each) were tested for
DNA contamination by 30 cycles of PCR with human
b
-actin
primers. After it was shown that there was no DNA contamina-
tion, cDNA synthesis was performed using an RNA PCR kit
(Perkin-Elmer Corp.) with the supplied oligo d(T)
16
primer. Re-
verse transcription was performed using a thermal program of
25
8
C for 10 min, 42
8
C for 30 min, and 95
8
C for 5 min. PCR
reactions were performed using the following primers: human
b
-actin (forward: 5
9
-GAAACTACCTTCAACTCCATC-3
9
,
reverse: 5
9
-CGAGGCCAGGATGGAGCCGCC-3
9
); and hu-
man TRAIL (forward: 5
9
-CAACTCCGTCAGCTCGTTA-
GAAAG-3
9
, reverse: 5
9
-TTAGACCAACAACTATTTCTAG-
CACT-3
9
), giving products of 219 and 443 bp, respectively.
b
-actin PCR cycle conditions were 95
8
C for 45 s, 55
8
C for 1 min,
and 72
8
C for 45 s for 30 cycles. TRAIL cycle conditions were
95
8
C for 45 s, 55
8
C for 45 s, and 72
8
C for 45 s for 30 cycles.
Samples were resolved on a 2% agarose gel and visualized with
ethidium bromide.
Results
Characterization of Blood DC Subsets.
The enriched “bulk”
DCs comprised two distinct subsets distinguishable by the
surface expression of CD11c and IL-3R
a
(Fig. 1 A). The
ratio of these two cell populations varied by donor, with
some donors demonstrating as high as 70% CD11c
1
DCs
and 30% IL-3R
a
1
pre-DCs, whereas others demonstrated
the reverse percentages. Neither CD11c
1
DCs nor IL-3Ra
1
pre-DCs expressed CD14, but they did differentially ex-
press CD33 and HLA-DR. After 12 h of culture in com-
plete medium, the levels of CD11c and CD33 remained
relatively constant, whereas CD83 expression was detected
on the majority (.90%) of CD11c
1
DCs (Fig. 1 B). In con-
trast, the IL-3Ra
1
pre-DCs expressed lower levels of CD33
and HLA-DR and failed to express CD83 after 12-h incu-
bation under identical conditions (Fig. 1, A and B). Distinct
morphological differences were also observed between the
two DC subsets. The CD11c
1
DCs exhibited a characteris-
tic multilobulated nucleus, in contrast to the IL-3Ra
1
pre-
DCs, which displayed an immature appearance consisting
of an oval nucleus (Fig. 2, A and B). Highly pure CD11c
1
DCs and IL-3Ra
1
pre-DCs were rapidly obtained using
this method for functional analysis of TRAIL.
IFN-stimulated DCs Kill Tumor Cells via a TRAIL-depen-
dent Mechanism. Previous reports have demonstrated that
a variety of lymphoid and myeloid cells (T cells, NK cells,
Mf) can express TRAIL and kill TRAIL-sensitive target
cells under certain circumstances (27, 34, 35). To deter-
mine if DCs also exhibit tumoricidal activity, unsorted
Figure 1. Surface phenotype of
freshly isolated CD11c
1
and
IL3Ra
1
pre-DC subsets. (A) The
two blood DCs were distinguishable
based on CD11c and IL-3Ra ex-
pression as determined by multicolor
flow cytometry. The CD11c
1
and
IL-3Ra
1
pre-DCs were compared
with Mf for CD14, CD33, and
HLA-DR expression. Filled histo-
grams represent staining with specific
antibody; open histograms represent
isotype-matched controls. (B)
CD11c
1
DCs, but not IL-3Ra
1
pre-DCs, express CD83 after 12-h
incubation in complete medium.
Histograms and contour plots repre-
sent 10
4
gated DCs, and viability was
.95% as assessed by propidium io-
dide exclusion.

1158 Dendritic Cells Induce Apoptosis via TRAIL
“bulk” DCs were cultured for 12 h with IFN-g, IFN-a,
GM-CSF, CD40L, or LPS and cultured for an additional 8 h
in the presence of the TRAIL-sensitive human tumor cell
lines OVCAR3, an ovarian carcinoma cell line, and WM
793, a melanoma cell line, at a 50:1 DC/target ratio.
Whereas unstimulated, GM-CSF-, CD40L-, and LPS-
stimulated DCs demonstrated little or no tumoricidal activ-
ity toward the tumor cell targets, DCs stimulated with
IFN-g or -a were potent killers of each of these tumor
cells (Fig. 3). The cytotoxic activity of unstimulated, IFN-g-,
and IFN-a-stimulated Mf is presented for comparison.
This cytotoxic activity was seen with IFN-stimulated DCs
from multiple donors and with other TRAIL-sensitive tu-
mor cells but not with the TRAIL-resistant melanoma cell
line WM 164 or several normal primary cell types (Table
I). No cytotoxic effect was observed when normal human
lung fibroblasts, microvascular endothelial cells, or skeletal
muscle cells were used as targets.
CD11c
1
DCs, but Not IL-3R
a
1
Pre-DCs, Exhibit
TRAIL-dependent Tumoricidal Activity. Once it was deter-
mined that bulk DCs can kill TRAIL-sensitive tumor cell
targets, we examined the tumoricidal activity of the
CD11c
1
DC and IL-3Ra
1
pre-DC subsets. Sorting the
bulk DCs into these subsets revealed that nearly all of
the cytotoxic activity of the IFN-g–stimulated DCs (Fig.
3) was attributable to the CD11c
1
DC subset (Fig. 4). Fur-
thermore, the observed tumoricidal activity was dependent
upon the number of IFN-g–stimulated DCs, as decreasing
the E/T ratio decreased the amount of target cell death.
The tumoricidal activity of the IFN-g–stimulated CD11c
1
DCs was nearly equivalent to that of IFN-g–stimulated
Mf. Similar results were observed with IFN-a–stimulated
DCs and Mf (data not shown). To confirm that the ob-
served cytotoxicity of the CD11c
1
DCs was specific to
TRAIL and not other apoptosis-inducing molecules (i.e.,
FasL and TNF), IFN-g–stimulated CD11c
1
DCs were
treated with recombinant soluble receptors for TRAIL
(TRAILR2–Fc), Fas (Fas–Fc), or TNF (TNFR–Fc) before
incubation with the tumor cell targets. Pretreating the
IFN-g–stimulated CD11c
1
DCs with TRAILR2–Fc re-
duced target cell death to control (unstimulated DC effec-
tor cells) levels, whereas Fas–Fc or TNFR–Fc did not alter
the cytolytic ability of the IFN-g–stimulated DCs (Fig. 5).
These results demonstrate that the TRAIL expressed on
the CD11c
1
DCs induces the killing of TRAIL-sensitive
targets.
IFN-stimulated CD11c
1
DCs Induce Apoptotic Cell Death
of Tumor Cells. The results presented in Figs. 3–5 clearly
demonstrate that IFN-stimulated CD11c
1
DCs kill tumor
cells via a TRAIL-dependent mechanism. However, these
experiments only measure the release of
51
Cr-labeled intra-
cellular proteins into the culture supernatant, which is an
event that can occur with either necrotic or apoptotic cell
death. Previous reports have shown that TRAIL induces
apoptotic death, as measured by DNA fragmentation,
caspase activation, and annexin V binding (23, 24, 28).
Thus, to demonstrate that the TRAIL-expressing CD11c
1
DCs were inducing the apoptotic cell death of the target
tumor cells, the binding of FITC-conjugated annexin V to
the tumor cells was measured (36, 37). Light scatter charac-
teristics were used to distinguish the tumor cells from the
DCs, such that only the tumor cells were counted in the
analysis. After 8-h incubation, only those tumor cells incu-
bated with soluble TRAIL or IFN-stimulated DCs (E/T
ratio, 4:1) were positive for annexin V binding (Fig. 6).
This apoptosis-inducing, tumoricidal activity of the IFN-
stimulated DCs was seen with DCs from multiple donors
and with other tumor cell targets (data not shown). These
results demonstrate that TRAIL-expressing human DCs
can kill TRAIL-sensitive tumor cells by inducing apoptosis.
IFN Stimulation Upregulates TRAIL Expression on the
CD11c
1
DCs. The results obtained thus far functionally
F
igure 2. Blood CD11c
1
DCs and IL-3Ra
1
pre-DCs exhibit distinct
m
orphological differences. Wright-Giemsa staining of freshly sorted
C
D11c
1
DCs (A) and IL-3Ra
1
pre-DCs (B). Photomicrographs were
c
aptured at a magnification of 40.

1159 Fanger et al.
describe the tumoricidal activity of IFN-stimulated CD11c
1
DCs to be via a TRAIL-dependent mechanism. However,
we wanted to correlate this functional activity with TRAIL
protein expressed on the surfaces of these cells. Thus, DCs
were cultured in medium or stimulated with GM-CSF or
IFN-g for 12 h and then analyzed for TRAIL expression
using flow cytometry. Whereas Mf express significant lev-
els of TRAIL on their surfaces after stimulation with IFN-g
(27), lower levels of TRAIL expression were detected on
CD11c
1
DCs after IFN-g stimulation (Fig. 7 A). TRAIL
was undetectable on unstimulated or GM-CSF–stimulated
CD11c
1
DCs (Fig. 7 A), as well as on cells stimulated with
CD40L or LPS (data not shown). In contrast, TRAIL was
not detected on the unstimulated or cytokine-stimulated
IL-3Ra
1
pre-DCs. Analysis of the IFN-g–stimulated
CD11c
1
DCs by reverse transcriptase (RT)-PCR revealed
that TRAIL mRNA levels increased during the culture pe-
riod as compared with unstimulated or GM-CSF–stimu-
lated CD11c
1
DCs (Fig. 7 B). Conversely, whereas TRAIL
mRNA was detected in unstimulated IL-3Ra
1
pre-DCs,
the levels were unaltered after GM-CSF or IFN-g stimula-
tion (Fig. 7 B).
Discussion
The data presented here demonstrate that human blood
DCs express the apoptosis-inducing molecule TRAIL after
stimulation with either IFN-g or IFN-a and acquire the
ability to kill TRAIL-sensitive target cells. The TRAIL-
specific activity was restricted to CD11c
1
DCs and corre-
lated with the increased levels of TRAIL mRNA and pro-
tein after IFN-g stimulation. The low number (,8 3 10
6
)
of blood CD11c
1
DCs obtained after enrichment from
each donor restricted our ability to examine TRAIL ex-
pression and cytotoxicity after stimulation with the various
cytokines. The 12-h stimulation time used in these studies
was based on previous studies where TRAIL was maxi-
mally expressed on monocytes (27). Although the TRAIL
surface expression was lower in comparison to that on
IFN-g–stimulated human Mf, the IFN-stimulated CD11c
1
Figure 3. Cytolytic activity by human DCs
after stimulation. Bulk DCs were incubated for
12 h in the absence (Unstim.) or presence of
CD40L, GM-CSF, LPS, IFN-a, or IFN-g and
then cultured for 8 h with
51
Cr-labeled
OVCAR3 or WM 793 target cells at an E/T
ratio of 50:1. As a positive control, soluble
TRAIL was added to target cells at 1 mg/ml.
For comparison, the cytolytic activity of unstim-
ulated (Unstim.), IFN-a–, or IFN-g–stimu-
lated human monocytes at 50:1 E/T is also in-
cluded. Data represent the mean of triplicate
wells, and experiments were repeated at least
three times with similar results. SD bars were
omitted from the graphs but were ,10% of the
value of all points.
Table I. Tumoricidal Activity of Cytokine-stimulated CD11c
1
DCs
Target cell
No.
donors
tested
CD11c
1
DCs*
TRAIL
‡
Unstimulated GM-CSF IFN-g IFN-a
Jurkat (T cell lymphoma) 2 0.2 6 0.1 4.3 6 0.8 33.4 6 2.6 23.0 6 2.1 35.9 6 2.5
OVCAR3 (ovarian carcinoma) 4 4.6 6 1.3 5.4 6 1.3 47.3 6 4.7 38.7 6 3.1 59.2 6 2.0
PC-3 (prostate carcinoma) 3 5.5 6 1.4 4.8 6 0.9 49.1 6 4.2 33.7 6 2.0 68.3 6 1.9
WM 164 (melanoma) 2 0.4 6 0.1 1.5 6 0.7 3.4 6 1.1 1.7 6 0.6 1.7 6 1.2
WM 793 (melanoma) 3 6.2 6 2.6 6.4 6 0.9 42.3 6 3.9 38.6 6 2.7 55.2 6 2.8
Normal lung fibroblasts 2 1.0 6 0.2 1.2 6 0.7 2.9 6 1.3 2.4 6 0.6 1.1 6 0.2
Lung microvascular endothelium 2 1.0 6 0.4 0.6 6 0.3 0.1 6 0.1 1.1 6 0.5 3.6 6 1.0
Skeletal muscle cells 2 2.1 6 0.4 1.4 6 1.2 2.4 6 0.9 1.6 6 1.1 3.9 6 0.7
Means were calculated from experiments performed with sorted CD11c
1
DCs from the indicated numbers of donors.
*Mean percent specific lysis (6SD) at 50:1 DC/target cell ratio.
‡
Mean percent specific lysis (6SD) with 1 mg/ml soluble TRAIL.

1160 Dendritic Cells Induce Apoptosis via TRAIL
DCs were able to mediate apoptosis with comparable effi-
ciency, suggesting that even low levels of membrane-bound
TRAIL result in potent tumoricidal activity. Moreover, the
cytotoxic activity of the IFN-g–stimulated CD11c
1
DCs
was completely inhibited by soluble TRAILR2–Fc and not
by Fas–Fc or TNFR–Fc. These blocking studies provide
additional evidence that the cellular apoptosis elicited by
the IFN-stimulated CD11c
1
DCs is a TRAIL-specific phe-
nomenon.
In contrast to the CD11c
1
DCs, IFN-stimulated IL-
3Ra
1
pre-DCs did not demonstrate significant cytotoxic
activity against the same TRAIL-sensitive targets. We
elected to positively select for IL-3Ra
1
pre-DCs by sort-
ing with an anti–IL-3R mAb, as this appears not to alter
the functional capacity of these cells (10). We cannot rule
out the possibility, however, that IL-3R triggering by the
anti–IL-3R mAb did, in part, enhance cell viability. We
routinely observed .90% cell viability of the IL-3Ra
1
pre-DCs for prolonged periods (up to 20 h) when cultured
in the presence of human serum. This is significantly longer
than previous reports, where IL-3Ra
1
pre-DCs cultured
in the presence of FBS underwent rapid cell death (9).
Thus, the lack of IL-3Ra
1
pre-DC cytotoxicity in this
study was not due to poor viability.
It is interesting that these two blood DCs respond differ-
ently with regard to induced TRAIL expression and
TRAIL-mediated death. Previous reports have demon-
strated that these two DC subsets differ with regard to pha-
gocytic capacity, T cell stimulation capacity, and cell sur-
face phenotype before and after stimulation (9, 10; Fanger,
N.A., unpublished observations). The differential expres-
sion of TRAIL after IFN-g stimulation suggests further
that these two DC subsets have different roles in directing
antitumor responses via TRAIL. It is possible, however,
that the IL-3Ra
1
pre-DCs are able to express TRAIL
upon further maturation/differentiation or with stimuli not
examined in this study. It has been shown that when IL-
3Ra
1
pre-DCs are cultured with IL-3 6 GM-CSF and
CD40L, a phenotypically and functionally different cell re-
sults by selective proliferation and death (9). Ongoing stud-
ies will determine whether these more mature/differen-
tiated IL-3Ra
1
pre-DCs can express TRAIL after IFN
activation or other stimuli.
The melanoma cell line WM 164 and normal primary
cells were resistant to both TRAIL-mediated apoptosis and
TRAIL-expressing DCs, even though they express one
and both of the apoptosis-inducing TRAIL receptors
(TRAILR1 and -R2), respectively (29; Griffith, T.S., un-
published observation). The mechanism(s) that regulate
sensitivity and resistance to TRAIL-induced apoptosis re-
Figure 4. TRAIL-mediated tumoricidal activity of blood DCs is restricted to CD11c
1
DCs. CD11c
1
DCs, IL-3Ra
1
pre-DCs, and Mf were incu-
bated for 12 h in the absence or presence of GM-CSF or IFN-g and then cultured for 8 h with
51
Cr-labeled OVCAR3 (A–C) or PC-3 (D–F) target cells
at the indicated E/T ratios. As a positive control, soluble TRAIL was added to the target cells at the indicated concentrations. Data represent the mean of
triplicate wells, and experiments were repeated at least three times with similar results. SD bars were omitted from the graphs but were ,10% of the value
of all points.

1161 Fanger et al.
main unclear. It was initially hypothesized that the expres-
sion of the non–death-inducing, or “decoy,” TRAIL
receptors (TRAILR3 and -R4) were responsible for resis-
tance to TRAIL (38–40). However, we have examined the
expression of the four TRAIL receptors at both the
mRNA and protein level in a variety of human tumor cell
lines and found there to be no correlation between decoy
receptor expression and TRAIL sensitivity/resistance (24,
29). Likewise, additional studies have failed to clearly show
a link between decoy receptor expression and resistance
(41, 42). Thus, it is unlikely that the decoy receptor hy-
pothesis is the sole explanation for resistance to TRAIL-
induced apoptosis. One component of the cell death ma-
chinery that appears to play a role in determining sensitivity
and resistance to TRAIL is FLICE (Fas-associated death
domain–like IL-1b–converting enzyme)-inhibitory protein
(FLIP). It is believed that FLIP prevents the binding of
caspase-8 to the death domain of cross-linked death recep-
tor, inhibiting any downstream apoptotic signaling events.
Intracellular levels of FLIP are high in the TRAIL-resistant
melanoma WM 164 and in the normal cells used in this
study (24; Griffith, T.S., unpublished observations). High
FLIP levels have also been shown to correlate with resis-
tance to Fas-mediated apoptosis in naive peripheral T cells
(43, 44). Although FLIP may have a protective function in
these cells, it is likely to be one of several intracellular pro-
teins that cooperate with other proteins (both intracellular
and at the cell surface) to regulate sensitivity to TRAIL-
induced death.
The tumoricidal activity reported here suggests that
CD11c
1
DCs may be one of several cells responsible for
the active killing and removal of spontaneously arising tu-
mors in the body. To date, NK cells, Mf, T cells, and now
DCs have been shown to express TRAIL under certain
conditions, providing tumoricidal capability to a variety of
effector cells that patrol the body (27, 34, 35). Whereas all
these cell types may employ TRAIL to induce cell death
outright, DCs may use TRAIL to generate apoptotic bod-
ies for subsequent uptake, processing, and presentation of
Figure 7. TRAIL expression on IFN-stimulated human CD11c
1
DCs
and Mf but not IL-3Ra
1
pre-DCs. (A) Flow cytometric analysis of
TRAIL protein expression. DCs and Mf were incubated for 12 h in the
absence or presence of IFN-g or GM-CSF and then analyzed for TRAIL
expression. Open histograms represent staining by the FITC-labeled
M181 (anti-TRAIL mAb); filled histograms represent staining by the
FITC-labeled isotype control. Histograms represent 10
4
gated cells in all
conditions. (B) RT-PCR analysis of TRAIL mRNA levels in CD11c
1
and IL-3Ra
1
pre-DCs. Sorted CD11c
1
and IL-3Ra
1
pre-DCs were in-
cubated for 12 h in the absence or presence of IFN-g or GM-CSF.
b-actin was used as a control over the same time period.
Figure 5. Specificity of CD11c
1
DC cytolytic activity is restricted to
TRAIL. Inclusion of the fusion protein TRAILR2–Fc (20 mg/ml), but
not Fas–Fc (20 mg/ml) or TNFR–Fc (20 mg/ml), to 12-h IFN-g–stimu-
lated CD11c
1
DCs inhibited killing of WM 793 (A) or PC-3 (B) tumor
cell targets. Data points represent the mean of triplicate wells, and experi-
ments were repeated at least three times with similar results. For clarity,
SD bars were omitted from the graphs but were ,10% of the value of all
points. s, unstimulated; j, IFN-g; r, IFN-g 1 TR2–Fc; n, IFN-g 1
Fas–Fc; ,, IFN-g 1 TNFR–Fc.
Figure 6. Tumor cell targets
undergo apoptotic cell death
when cultured with IFN-stimu-
lated DCs as determined by
phosphatidylserine externaliza-
tion. OVCAR3 tumor cells
were cultured for 8 h in com-
plete medium alone or in the
presence of LZ-TRAIL (1 mg/
ml), unstimulated DCs, or cyto-
kine (GM-CSF, IFN-g, or IFN-a
[100 ng/ml for 12 h])-stimulated
DCs (E/T ratio 4:1). Cells were
then stained with FITC–annexin
V and analyzed by flow cytome-
try. The percentage of FITC–
annexin V–positive tumor cells is
indicated for each condition.
Histograms represent 10
4
gated
tumor cells. Similar results were
seen with DCs from three other
donors.

1162 Dendritic Cells Induce Apoptosis via TRAIL
target cell antigens to CD8
1
T cells, resulting in a stimula-
tory (i.e., CTL) or tolerogenic response. Indeed, several re-
ports have demonstrated that DCs engulf apoptotic bodies
and present antigen derived from these cell fragments in an
MHC class I–restricted fashion, resulting in CTL activity
(13, 14). T cell tolerance is important in preventing the in-
duction of autoimmunity. It can occur in the thymus, re-
sulting in the deletion of self-reactive thymocytes before
they are released into the periphery (45). Peripheral toler-
ance can also occur when specialized, tissue-specific anti-
gens are encountered (17, 46, 47). In this situation, a DC
presenting self-antigen may also be expressing TRAIL, or
some other apoptosis-inducing molecule, which would kill
the antigen-specific T cell once it came into contact with
the DC. Recent studies have demonstrated that DCs can
kill CD4
1
T cells through the expression of FasL (48, 49).
Thus, the expression of TRAIL and/or FasL on DCs may
prove to be a mechanism for deleting T cells in vivo.
The data presented here also suggest that DC TRAIL
expression may contribute to other physiologic and patho-
logic situations, such as HIV infection. DCs are one of the
first cells to encounter antigen at areas of inflammation in
mucous membranes (50), which are the major sites of initi-
ation of HIV infection. After the interaction between DCs
and the virus at these sites, the DCs migrate to the draining
lymph nodes, where they stimulate CD4
1
T cells. In the
process, the T cells become infected, leading to the replica-
tion and spread of the virus (12, 51). The viral infection
may also induce the production of various cytokines, such
as IFN-g and -a, activating an antiviral immune response.
It could be hypothesized from our results that IFN produc-
tion would stimulate the DCs to express TRAIL, which
could potentially kill any activated T cells in the area.
We thank Drs. David Cosman, Raymond Goodwin, David Lynch, Craig Smith, Michael Widmer, Steven
Wiley, and Douglas Williams for careful reading of the manuscript, Gary Carlton for figure preparation, and
Anne Aumell for editorial assistance.
Address correspondence to Dr. Thomas S. Griffith at his present address, Dept. of Urology, 428 MRC,
University of Iowa, 200 Hawkins Dr., Iowa City, IA 52242-1089. Phone: 319-335-7581; Fax: 319-356-3900;
E-mail: griffitht@mail.medicine.uiowa.edu
Submitted: 9 June 1999 Revised: 18 August 1999 Accepted: 23 August 1999
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