The Journal of Immunology
TRAIL+Human Plasmacytoid Dendritic Cells Kill Tumor
Cells In Vitro: Mechanisms of Imiquimod- and
IFN-a–Mediated Antitumor Reactivity
Madeleine L. Kalb, Astrid Glaser, Georg Stary, Frieder Koszik, and Georg Stingl
Dendritic cells (DCs) not only exhibit the unique capacity to evoke primary immune responses, but may also acquire TLR-triggered
human peripheral blood express the effector molecule TRAIL. The exact mechanisms through which pDCs acquire and elicit their
cytotoxic activity are still not clear. We now show that in the absence of costimulators, TRAIL induction on pDCs occurs with
agonists to intracellular TLRs only and is accompanied by a phenotypic as well as functional maturation, as evidenced by a com-
paratively superior MLR stimulatory capacity. pDCs acquired TRAIL in an IFN-a/b–dependent fashion and, notably, TRAIL
expression on pDCs could be induced by IFN-a stimulation alone. At a functional level, both TLR7/8- (imiquimod [IMQ]) and
TLR9-stimulated (CpG2216) pDCs lysed Jurkat T cells in a TRAIL- and cell contact-dependent fashion. More importantly, IFN-
a–activated pDCs acquired similar cytotoxic properties, independent of TLR stimulation and maturation. Both IMQ- and IFN-a–
activated pDCs could also lyse certain melanoma cell lines in a TRAIL-dependent fashion. Interestingly, suboptimal doses of IMQ
and IFN-a exhibited synergistic action, leading to optimal TRAIL expression and melanoma cell lysis by pDCs. Our data imply
that tumor immunity in patients receiving adjuvant IMQ and/or IFN-a may involve the active participation of cytotoxic
pDCs. The Journal of Immunology, 2012, 188: 1583–1591.
lymph nodes (1). Among the different Ag recognition strategies
employed by DCs, TLRs are of particular importance as they
detect unique molecular patterns conserved throughout entire
classes of pathogens (2). TLRs 1, 2, 4, 5, and 6 are located at the
extracellular surface membrane and specialize in the recognition
of bacterial products. TLRs 3, 7, 8, and 9, in contrast, are situated
intracellularly (i.e., in endosomal membranes), where they detect
viral and bacterial nucleic acids that have gained access to the
intracellular compartments (2). The TLR-mediated activation of
DCs triggers an antimicrobial and inflammatory response via the
downstream activation of NF-kB and other transcription factors,
culminating in the production of proinflammatory cytokines and
type I IFNs and the upregulation of costimulatory molecules (1,
3). Together, these factors initiate and direct the development of
an adaptive immune response.
endritic cells (DCs) are characterized by their unique
ability to evoke primary immune responses by present-
ing Ags to naive T cells after trafficking to the draining
Human peripheral blood contains five nonoverlapping lineage2
HLA-DR+subsets: hematopoietic CD34+precursors; CD11c+
myeloid DCs (mDCs), which can be further subdivided according
to their expression of blood DC Ag (BDCA)-1 (CD1c), BDCA-3
(CD141), and CD16; and, finally, BDCA-2+(CD303) plasmacy-
toid DCs (pDCs) (4). PDCs exhibit TLR7, TLR9, and, perhaps,
low levels of TLR1 and TLR6 (5–7). Their antiviral function is
probably linked to their high expression of TLR7 and TLR9 (5, 6).
After sensing viral nucleic acids, they produce large amounts of
type I IFNs due to their constitutional expression of the tran-
scription factor IFN regulatory factor-7 and so are essential in the
induction of a robust antiviral immune response (3).
Recent evidence exists that TLR activation can endow pDCs
with unique cytotoxic properties, challenging our current concept
of DC biology. HIV, human T cell leukemia virus-1, and influenza
virus, all natural ligands of TLR7, trigger a pDC innate immune
response that not only involves the massive production of IFN-a,
but also the upregulation of functionally active TRAIL (TRAIL/
Apo-2L/TNFSF10) (8–10). TRAIL is also expressed by HIV-
infected pDCs (8, 11, 12), and, notably, TRAIL+pDCs may kill
CD4+T cells isolated from HIV-infected patients (12) and thus
perhaps contribute to T cell depletion during HIV viremia. In ad-
dition to their role in viral immunity, several studies propose a di-
rect role for cytotoxic pDCs in antitumor immunity. The synthetic
TLR7/8 agonist imiquimod (IMQ) is a highly effective topical
treatment for epithelial skin tumors such as basal cell carcinomas,
viral acanthomas, and even melanomas (13). Regressing cancers
were surrounded and heavily infiltrated by inflammatory-type DCs
(14, 15), some of which expressed lytic molecules (15). In vitro
studies further showed that IMQ induces the expression of perforin
and granzyme B on mDCs and of TRAIL on pDCs (8, 15) and that
TRAIL+pDCs and granzyme B+perforin+mDCs can effectively
lyse appropriate tumor targets (9, 15).
In this study, we attempted to gain a better understanding of the
events governing the acquisition of cytotoxic molecules by pDCs
Division of Immunology, Allergy and Infectious Diseases, Department of Dermatol-
ogy, Medical University of Vienna, Vienna 1090, Austria
Received for publication August 23, 2011. Accepted for publication December 2,
This work was supported by a research grant from the Austrian Science Fund,
Vienna, Austria (DK-W1212-B13).
Address correspondence and reprint requests to Prof. Georg Stingl, Division of
Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical
University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. E-mail ad-
The online version of this article contains supplemental material.
Abbreviations used in this article: BDCA, blood dendritic cell Ag; DC, dendritic cell;
FasL, Fas ligand; IMQ, imiquimod; mDC, myeloid dendritic cell; MFI, mean fluo-
rescence intensity; pDC, plasmacytoid dendritic cell; sTRAIL, soluble TRAIL;
skTRAIL, super killer TRAIL.
and of the molecular players involved in IMQ-induced pDC an-
titumor activity. We show in this study that IMQ endows pDCs with
the ability to lyse tumor cells, a phenomenon that is strictly TRAIL-
and IFN-a–dependent. It is of note that IFN-a–activated pDCs,
even in the absence of IMQ, can kill tumor cells—in particular also
melanoma cells—in a TRAIL-dependent fashion. This result sug-
gests that the therapeutic effect of adjuvant IMQ and/or IFN-a in
antitumor immunity may be, at least partly, mediated by killer DCs.
Materials and Methods
Cell preparations from peripheral blood
To isolate human peripheral blood pDCs, PBMCs were prepared by Ficoll-
Paque density gradient centrifugation (Ficoll-PaquePLUS; GE Healthcare)
from buffy coats purchased from the Austrian Red Cross Center (Vienna,
Austria). T cells, B cells, NK cells, hematopoietic progenitor cells,
monocytes, platelets, and erythrocytes were depleted by subsequent anti-
CD3 (UCHT1), -CD11b (Bear1), -CD16 (3G8), -CD19 (J4.119), -CD34
(581), -CD41 (SZ22), -CD56 (C218), and -CD235a (11E4B-7-6; all from
Beckman Coulter, used at 4 mg/ml each) immunolabeling and anti-mouse
IgG1immunomagnetic separation (MACS; Miltenyi Biotec). pDCs were
selected from the remaining cell fraction using anti-CD304 (BDCA-4)
microbeads (MACS Dendritic Cell Isolation Kit; Miltenyi Biotec). The
purity of the isolated pDC population was generally 95–99% and contained
,1% of other leukocyte populations tested for individually. CD3+T cells
from the same donor that served as negative controls in the cytotoxicity
assays were prepared by anti-CD3 immunolabeling and anti-mouse IgG1
immunomagnetic selection. CD3+T responder cells used in MLR and
T cell activation experiments were isolated from PBMCs by negative se-
lection using anti-CD11b, -CD11c (BU15), -CD16, -CD19, -CD34,
-CD41, -CD56, -CD235a (Beckman Coulter), and -CD123 (9F5; BD
Pharmingen; 4 mg/ml each) immunolabeling and anti-mouse IgG1immu-
nomagnetic depletion. The purity of CD3+T cells was .95% as assessed
by FACS analysis. Cell viability determined by trypan blue staining was
.99% after isolation.
Isolated pDCs were resuspended in RPMI 1640 medium supplemented with
10% (v/v) heat-inactivated FCS, penicillin (100 IU/ml), and streptomycin
(100 mg/ml; all from Invitrogen) at 5–10 3 105cells/ml in 96-well plates
and cultured overnight at 37˚C in 5% CO2. The following TLR agonists
were used for stimulation: Pam3CSK4 (TLR2/1, 0.1–1 mg/ml), FSL-1
(TLR2/6, 0.1–1 mg/ml), polyinosinic-polycytidylic acid (TLR3, 5 mg/
ml), ultrapure Escherichia coli LPS (TLR4, 1 mg/ml), recombinant
FLA-ST (flagellin; TLR5, 0.1 mg/ml), IMQ (TLR7/8, 5 mg/ml), CL-075
(TLR7/8, 2.5 mg/ml), ssPolyU/LyoVec (TLR8, 2.5 mg/ml) and CpG2216
(TLR9, 2.5 mM; all purchased from InvivoGen). Cell viability before and
after culture was checked by trypan blue staining and was generally
.90%. The effect of other maturation stimuli on lytic molecule induction
was tested by stimulating freshly isolated pDCs with IL-3 (300 IU/ml;
PeproTech), TNF-a (50 IU/ml; Strathmann), and CD40L (1 mg/ml; Enzo
Life Sciences). To determine the role of type I IFNs in the induction of
cytotoxic molecules, pDCs were preincubated with 10 mg/ml each neu-
tralizing anti–IFN-a (MMHA-1) and anti–IFN-b (MMHB-12; PBL Bio-
medical Laboratories) Abs or an IgG1isotype (MOPC-21; Sigma-Aldrich)
30 min before addition of TLR agonists, or directly stimulated with 50–
50,000 IU/ml human rIFN-a2a (PBL Biomedical Laboratories) and then
subjected to FACS analysis or used in cytotoxicity assays. pDCs that were
used to lyse melanoma cells were activated with Roferon-A (rIFN-a2a;
Roche; 25,000 IU/ml; low dose: 250 IU/ml) or IMQ (5 mg/ml; low dose:
T cell stimulation
For analysis of TRAIL and TRAIL-R expression, T cells were stimulated
with plate-bound anti-CD3 (5 mg/ml) and soluble anti-CD28 (2 mg/ml,
L293; BD Biosciences) for up to 5 d and then analyzed by flow cytom-
etry. Fas ligand (FasL) expression on CD3+T cells was determined by flow
cytometry using PBMCs that had been stimulated with PMA (25 ng/ml) and
ionomycin (2 mg/ml; both from Sigma-Aldrich) for 4 h.
Flow cytometric analysis of blood-derived pDCs before and after culture
with TLR agonists or cytokines was performed on an FACSCalibur (BD
Biosciences). The purity of isolated pDC populations was determined using
lineage marker combinations of anti–HLA-DR (PerCP, L234; BD Bio-
sciences) and anti-CD303/BDCA-2 (allophycocyanin, AC144; Miltenyi
Biotec). The absence of other leukocyte subpopulations in freshly isolated
DCs was checked by quadruple stainings with Abs against CD14 (FITC;
MfP9), CD56 (PE; mY31), CD19 (PerCP; SJ25C1), and CD3 (allophy-
cocyanin, SK7; all from BD Biosciences). TRAIL expression was visu-
alized using the PE-labeled Ab clones 75402 (R&D Systems) and RIK-2
(BioLegend), both yielding similar results. All FACS plots shown in this
paper were generated using clone RIK-2. FasL expression was evaluated
using a purified anti-CD178 mAb (NOK-1; BD Pharmingen) followed
by Alexa Fluor 488 goat anti-mouse IgG (H+L) (Molecular Probes, Invi-
trogen) staining. Positive control stainings were performed using PMA/
ionomycin-stimulated PBMCs and gating on CD3+T cells. Intracellular
cytotoxic molecules were visualized with anti-granzyme B PE (CLB-
GB11; PeliCluster Sanquin), anti-granulysin PE (DH-2; BioLegend),
anti-perforin FITC (dG9; BD Biosciences), and anti-lysozyme FITC (LZ-
2; An Der Grub). Intracellular stainings were performed using a cell
permeabilization kit (Fix&Perm; An Der Grub) after extracellular staining
of pDCs with lineage markers. Positive control stainings for intracellular
lytic molecules were performed using freshly isolated PBMCs and gating
on NK cells (CD56+CD32) or monocytes (scatter characteristics). Co-
stimulatory molecules were stained using anti-CD80 FITC (MAB104;
Beckman Coulter), -CD40 FITC (5C3), -CD83 PE (HB15e), and -CD86
PE (2331; all from BD Biosciences). TRAIL-R expression on pDCs,
T cells, and tumor target cells was determined using anti–TRAIL-R1 PE
(69036), –TRAIL-R2 PE (71908), –TRAIL-R3 PE (90906), and –TRAIL-
R4 PE (104918; all from R&D Systems). The activation status of T cells
was checked using anti-CD69 FITC (FN50; BD Pharmingen). Type I IFNR
expression was examined by anti-IFNAR2 PE (MMHAR-2; PBL Bio-
medical Laboratories). Corresponding isotype-matched controls were used
for all stainings. After incubation with the respective Abs for 15 min at 4˚
C, cells were washed twice and then subjected to flow cytometric analysis.
FACS plots depict mean fluorescence intensity (MFI) values of Ab stain-
ings after subtraction of the MFI of the respective isotype.
The immunostimulatory capacity of killer molecule-expressing pDCs was
assessed in allogeneic MLRs. Purified pDCs that had been stimulated with
IMQ or left unstimulated overnight were incubated with 105purified al-
logeneic CD3+T cells in round-bottom 96-well plates. The effects of type I
IFNs and TRAIL on pDC-induced T cell proliferation were investigated by
adding 5 mg/ml each azide-free neutralizing anti-TRAIL (75411; R&D
Systems), anti–IFN-a, and anti–IFN-b, or 5 mg/ml IgG1isotype to the
plate. T cell proliferation was measured by the uptake of [3H]thymidine (1
mCi/well; 6.7 Ci/mM [247.9 GBq/mM]; PerkinElmer), which was added to
the culture on day 5. After 16 h of incubation, cells were harvested onto
glass fiber filter paper with an automated harvester (Tomtec Harvester 96;
Tomtec), and [3H]thymidine incorporation was measured by liquid scin-
tillation spectroscopy (1205 Betaplate; Wallac). The T cell response is
given as mean cpm 6 SD for duplicate wells.
Cell lines and culture conditions
The TRAIL-sensitive human leukemia T cell line Jurkat (obtained from
American Type Culture Collection) and melanoma cell lines SKMel2
(American Type Culture Collection) and WM793 (Coriell Institute for
Medical Research) were maintained in RPMI 1640 medium supplemented
with 10% (v/v) heat-inactivated FCS, penicillin (100 IU/ml), and strep-
tomycin (100 mg/ml). WM793 cells were grown in collagen-coated flasks
(rat tail collagen type I; BD Biosciences), and both melanoma lines were
used when cells were 70–80% confluent.
The ability of pDCs to kill tumor cells was assessed in a classic Europium-
protocol and as previously described (16). Briefly, target cells were labeled
with the fluorescence-enhancing ligand BATDA, which is released into
the supernatant after cytolysis. The supernatant is harvested and incu-
bated with Europium solution to form a stable fluorescent chelate. Data
were obtained using the 1234 DELFIA Luminometer (Wallac) and are
expressed as the percentage of specific lysis calculated by the following
formula: specific lysis (%) = (experimental release 2 spontaneous release)/
(maximum release 2 spontaneous release) 3 100. We used the cell lines
Jurkat, SKMel2, and WM793 as targets, as well as unstimulated CD3+
T cells isolated from the same donor as the effector pDC population.
Purified pDCs that had been stimulated with TLR agonists or IFN-a
overnight or had been left unstimulated were incubated with 2–5 3 103
1584 MECHANISMS OF IMQ- AND IFN-a–INDUCED pDC CYTOTOXICITY
target cells as duplicates in 96-well plates at E:T ratios ranging from 20:1
to 2.5:1. Target cell lysis was measured after 2 h and, in the case of
melanoma cells, after 4–6 h of incubation with effector pDCs. Inhibition
experiments were performed by preincubating pDCs with neutralizing
anti–IFN-a (10 mg/ml), anti–IFN-b (10 mg/ml), and anti-TRAIL (10 mg/
ml) Abs or an isotype Ab for 30 min prior to addition of target cells. The
maximum possible level of TRAIL-induced lysis of melanoma cells was
evaluated after adding 100 ng/ml super killer TRAIL (skTRAIL; soluble
human recombinant; Enzo Life Sciences) to melanoma target cells. To
evaluate the contribution of soluble TRAIL (sTRAIL) present in the su-
pernatant of pDC cultures to pDC killer activity, we performed cytotox-
icity assays using supernatants of IMQ-activated pDCs (cultured at 5 3
105cells/ml) that were added to 2 3 103Jurkat target cells (v:v/1:1). The
baseline cytotoxicity of supernatants from unstimulated pDCs was sub-
tracted from that of TLR-activated pDCs to correct for influences of other
secreted molecules and effects of factor consumption.
The concentration of sTRAIL in the supernatants of TLR-stimulated pDCs
cultured at 5 3 105cells/ml was determined by ELISA (eBioscience)
according to the manufacturer’s instructions. The concentrations of IFN-a,
TNF-a, and IFN-g in the supernatants of TLR-stimulated pDCs and PMA/
ionomycin-activated PBMCs, both cultured at 1 3 106cells/ml overnight,
were determined by FlowCytomix (eBioscience). Data analysis was per-
formed using FlowCytomix Pro 2.4 software (eBioscience).
Data are expressed as mean 6 SD or SEM. Statistical differences were
calculated using the two-tailed Student t test, and p values ,0.05 were
considered statistically significant.
pDCs express granzyme B and acquire TRAIL upon TLR7/8
and TLR9 stimulation
In a first series of experiments, we comparatively assessed the
expression of different cytotoxic molecules on purified pDCs (Fig.
1A) that had been either left untreated or stimulated with agonists
to TLR1–9. Fig. 1B shows representative histograms of lytic
molecule expression and Fig. 1C the quantitative analysis of the
overall percentage of pDCs expressing lytic molecules after
stimulation with TLR agonists. In agreement with previous
observations, we found that TLR7/8- and TLR9-stimulated pDCs,
as opposed to their nonstimulated counterparts, express high levels
of TRAIL (Fig. 1B). Kinetic analysis showed that TRAIL is de-
tectable on the cell surface as early as 3 h after stimulation, peaks
after 12 h, and is maintained for .24 h after stimulation (Fig. 1D).
As pDCs do not express TLR8 (5–7), we did not find TRAIL
expression using the selective TLR8 agonist ssPolyU as stimulus
(Fig. 1C). Even though pDCs also express transcripts for the
extracellular TLRs 1 and 6 that are involved in the recognition
of bacterial products (6, 7), none of the extracellular TLR ago-
nists used, including the respective TLR1 and TLR6 agonists
Pam3CSK4 and FSL-1, caused lytic molecule expression (Fig.
1C). Notably, these agonists were functional in stimulating mDC
maturation (data not shown).
We next asked whether TLR7–9 agonists induce also lytic mol-
ecules other than TRAIL in freshly isolated pDCs. Positive controls
were generated using NK cells (perforin, granulysin), monocytes
(lysozyme), and T cells (FasL). Data obtained show that unstimu-
lated, and, to a lesser extent, TLR7/8- and TLR9-stimulated pDCs
express granzyme B (Fig. 1B, 1C). Granulysin, perforin, or FasL
were consistently absent (Fig. 1E). In contrast to what has been
reported previously (17), we were unable to detect lysozyme ex-
pression (Fig. 1E), even in the CD2highpDC subset (data not shown).
DC activation and maturation, and therefore possibly also
TRAIL expression, can be induced by a multitude of different
stimuli. To investigate whether TRAIL induction is exclusively
linked to TLR activation, we treated freshly isolated pDCs with
different cytokines and then evaluated TRAIL expression. Inter-
estingly, IL-3 not only caused DC maturation, measured by the
but also induced TRAIL on up to 30% of cells after 24 h (Fig. 1F).
The levels of IL-3–induced TRAIL, however, were much lower
when compared with those measured after IMQ stimulation. No-
tably, pDCs treated with TNF-a and CD40L exhibited signs of
maturation (upregulation of CD83, data not shown) but did not
express TRAIL (Fig. 1F). These findings imply that TRAIL in-
duction is not a general attribute of DC activation or maturation,
but is confined to distinct pDC stimuli. Taken together, our results
demonstrate that, with the exception of intracellular granzyme B,
human blood-derived pDCs do not react with Abs against the
major lytic molecules identified so far. They can, however, be
stimulated to express TRAIL, the induction of which is exclu-
sively linked to the occupancy of cytoplasmic rather than extra-
cellular membrane-bound TLRs.
Killer molecule-expressing pDCs are phenotypically and
The capacity of DCs to induce a productive response in naive
T cells is critically linked to the expression of certain costimulatory
molecules (1). We asked whether TLR-mediated killer molecule
expression on pDCs interferes with them acquiring such moieties.
Results obtained show that, with the exception of low-level CD86
expression (∼40%), freshly isolated pDCs are essentially devoid
of activating costimulatory molecules (Fig. 2A). After TLR7/8
(IMQ, CL-075) and TLR9 (CpG2216) stimulation, the expres-
sion of killer molecules was accompanied by a marked upregu-
lation of CD40, CD80, CD83, and CD86 as well as HLA-DR in
the vast majority of pDCs, as indicated by the increase in MFI
(Fig. 2A) and the overall percentage of positive cells (Fig. 2B).
We next examined the capacity of cytotoxic molecule-expressing
pDCs to induce T cell proliferation in an allogeneic MLR. Over-
all, pDCs exhibited a low stimulatory capacity compared with
mDCs (M.L. Kalb, unpublished observations). Notably, IMQ-
stimulated pDCs were more effective stimulators compared with
the nonactivated pDC population, despite their cytotoxic molecule
expression (Fig. 2C). These results were somewhat surprising, as
one may have expected that IFN-a produced by pDCs would exert
an antiproliferative effect and that killer molecule-expressing
pDCs would lyse activated T cells (12). Indeed, we found that
anti–CD3-/CD28-activated, but not freshly isolated CD3+T cells
express the proapoptotic receptor TRAIL-R1 (DR4/TNFRSF10A)
(Fig. 2D). Fig. 2C shows, however, that neutralizing Abs against
TRAIL and/or IFN-a do not alter the extent of pDC-induced
T cell proliferation. These data demonstrate that TLR7/8 ago-
nists, in particular IMQ, not only induce lytic molecules in pDCs
but also enhance the immunostimulatory capacity of these cells.
Expression of TRAIL on TLR7/8- and TLR9-activated pDCs is
type I IFN dependent
pDCs are known to produce vast amounts of type I IFNs upon
encounter of natural (viruses) and synthetic TLR7 ligands (3).
TRAIL is an IFN-stimulated gene (18), and the same TLR7
ligands that induce IFN-a production can also induce TRAIL
expression on pDCs (8, 9). In agreement with these observations,
we found that TRAIL expression on pDCs induced by TLR7/8
(IMQ, CL-075) and also TLR9 (CpG2216) agonists was paralleled
by an increase of IFN-a and TNF-a, but not IFN-g in the su-
pernatant (Fig. 3A). In contrast to pDCs, we found that PMA/
ionomycin-treated PBMCs produce large amounts of IFN-g as
well as TNF-a, but not IFN-a. When we investigated the role
of type I IFNs in the development of a cytotoxic phenotype, we
The Journal of Immunology 1585
found that: 1) the IFN-a/b receptor expressed on nonstimulated
pDCs (Fig. 3B); and 2) pDC stimulation with TLR7/8 and TLR9
agonists in the presence of anti–IFN-a/b Abs prevents their
TRAIL expression (Fig. 3C). To test whether TLR stimulation is
required or, alternatively, whether IFN-a alone may be sufficient
for TRAIL induction, we stimulated pDCs with different amounts
of IFN-a overnight. Fig. 3D shows that IFN-a stimulation alone
induces TRAIL expression on pDCs (Fig. 3D, upper panel), albeit
at a lesser extent than what is seen with IMQ (Fig. 1B). It should
also be noted that IFN-a, in contrast to IMQ stimulation, does not
lead to an upregulation of costimulatory molecules, not even at
high doses (Fig. 3E). These findings suggest that TLR agonists
activate signaling pathways in addition to those engaged by IFN-a.
To investigate this hypothesis, we added minimal doses of IMQ
(0.05 mg/ml) that alone did not induce relevant levels of TRAIL
to pDCs cultured with IFN-a and, indeed, found that TRAIL
expression was significantly enhanced under such conditions
(Fig. 3D, lower panel). We further observed that in the presence of
suboptimal IFN-a concentrations (250 IU/ml), agonists to TLR2/1
(Pam3CSK4) and TLR2/6 (FSL-1) that were unable to stimulate
TRAIL expression when used alone now induced low-level
TRAIL expression (Supplemental Fig. 1).
Both TLR7/8- and IFN-a–activated pDCs elicit their killing via
To investigate whether TRAIL is functionally active on pDCs, we
performed 2-h Europium-TDA release cytotoxicity assays. TDA
release from target cells occurs early and rapidly, allowing for the
assessed by flow cytometry using anti–BDCA-2 and anti–HLA-DR stainings. B–F, The expression of different cytotoxic molecules on freshly isolated and
purified pDCs before and after stimulation with TLR agonists or cytokines was assessed by flow cytometry and by gating on BDCA-2+HLA-DR+cells. B
and C, Freshly isolated pDCs were incubated with the TLR agonists Pam3CSK4 (TLR2/1), FSL-1 (TLR2/6), polyinosinic-polycytidylic acid (Poly I:C,
TLR3), LPS (TLR4), flagellin (TLR5), IMQ (TLR7/8), CL-075 (TLR7/8), ssPolyU (TLR8), and CpG2216 (TLR9). Cells cultured with IL-3 (300 IU/ml) or
medium alone served as controls. After overnight culture, the expression of cytotoxic molecules was assessed using extracellular anti-TRAIL and intra-
cellular anti-granzyme B stainings. Representative histograms of membrane TRAIL and intracellular granzyme B expression are shown in B. Data of
quantitative analysis (mean 6 SD) of pDCs expressing lytic molecules are shown in C (n = 4). D, The kinetics of TRAIL expression on IMQ-stimulated (5
mg/ml) pDCs were assessed over a period of 48 h using anti-TRAIL stainings. One representative example is shown (n = 3). E, The expression of other
cytotoxic molecules by pDCs was assessed on freshly purified pDCs and after overnight stimulation with IMQ (5 mg/ml) using Abs against intracellular
granulysin, perforin, and lysozyme as well as extracellular FasL. Stainings using freshly isolated PBMCs and gating on NK cells (CD56+CD32) or
monocytes (scatter characteristics) and PMA/ionomycin-stimulated PBMCs and gating on T cells (CD3+) served as positive controls. Representative
histograms are shown (n = 3). F, The expression of surface TRAIL on pDCs was assessed after stimulation with IMQ (5 mg/ml), IL-3 (300 IU/ml), TNF-a
(50 IU/ml), and CD40L (1 mg/ml) using anti-TRAIL stainings. Data represent mean percentages of pDCs expressing lytic molecules 6 SD (n = 3).
Expression of cytotoxic molecules on human peripheral blood-derived pDCs. A, The purity of pDCs after isolation and purification was
1586 MECHANISMS OF IMQ- AND IFN-a–INDUCED pDC CYTOTOXICITY
detection of cell death already after shorter periods of culture
compared with other commonly used cell death assays (Supple-
mental Fig. 2). pDCs were incubated with the leukemic T cell
line Jurkat, which expresses the proapoptotic receptor TRAIL-
R2 (DR5/TNFRSF10B) (Fig. 4A) and is highly susceptible to
TRAIL-mediated killing. We found that pDCs that had been
stimulated overnight with IMQ, but not untreated pDCs, kill
Jurkat cells in a TRAIL-dependent fashion, as demonstrated by
the lack of killing in the presence of neutralizing anti-TRAIL Abs
(Fig. 4B). In keeping with earlier observations (Fig. 2D), TRAIL-
expressing pDCs were unable to lyse unstimulated CD3+T cells
that had been isolated from the same donor as the pDC effector
population (Supplemental Fig. 3). Having shown that TRAIL is
also induced through TLR9 (Fig. 1B), we performed cytotoxicity
assays using pDCs that had been activated with CpG2216 over-
night. Cytotoxic activity of TLR9-activated pDCs was less pro-
nounced than that of their TLR7/8-activated counterparts isolated
from the same donor (Fig. 4C). This could, but must not neces-
sarily, be due to a lower expression of TRAIL (Fig. 1B, upper
Because IFN-a itself (i.e., in the absence of TLR ligands) can
induce TRAIL expression on pDCs (Fig. 3D, upper panel), we
performed additional cytotoxicity assays using IFN-a–activated
pDCs to test their lytic capacity. Notably, IFN-a–activated pDCs
were very efficient killers (Fig. 4D). Despite their often only
marginal expression of TRAIL (Fig. 3D, upper panel), IFN-a–
activated pDCs lysed Jurkat targets in a strictly TRAIL-dependent
fashion (Fig. 4D). pDC cytotoxicity is apparently independent of
DC maturation, as IFN-a–activated TRAIL+pDCs had not up-
regulated costimulatory molecules to the same extent as seen after
IMQ stimulation (Figs. 3E and 2A, respectively). As expected, pDCs
that had been incubated with neutralizing anti–IFN-a/b Abs prior
to IMQ stimulation, and therefore could not upregulate TRAIL,
were also not cytotoxic to Jurkat cells (Fig. 4E). In contrast, the
addition of neutralizing anti–IFN-a/b Abs during the cytotoxicity
assay did not affect tumor cell lysis (Fig. 4B), indicating that IFN-a
produced by pDCs that already express TRAIL is dispensable for
their cytotoxic function. Taken together, our data show that both
IMQ- and IFN-a–activated pDCs kill Jurkat cells in a TRAIL-de-
pendent fashion and may therefore be actively involved in clearing
TRAIL-sensitive tumor targets.
Mechanisms by which TRAIL+pDCs induce Jurkat cell lysis
Once distributed to the cell membrane, the type II membrane
protein TRAIL may be subject to cleavage by metalloproteases
(19). Because evidence exists that TRAIL is functional in both its
membrane-bound and soluble (sTRAIL) form, we now asked how
TRAIL mediates pDC cytotoxicity. Based on our results, we be-
lieve that Jurkat cell lysis via TRAIL+pDCs involves mainly, or
even exclusively, membrane TRAIL for two reasons: we observed
that 1) levels of sTRAIL in supernatants collected from unsti-
mulated, TLR7/8- (IMQ), and TLR9-stimulated (CpG2216) pDCs
are negligible and below those found in normal human serum (Fig.
5A); and 2) Jurkat cells express TRAIL-R2 exclusively (Fig. 4A).
The latter finding is of particular relevance as membrane TRAIL
cules and exhibit allostimulatory capacity. A and B, The expression of
costimulatory molecules on freshly purified, unstimulated pDCs and
TRAIL+pDCs generated by overnight TLR7/8 (IMQ, CL-075) or TLR9
(CpG2216) stimulation was analyzed by flow cytometry. Cells were gated
on BDCA-2 and HLA-DR. Representative histograms (A) depict the
baseline expression of costimulatory molecules on freshly isolated pDCs
and their upregulation after overnight stimulation with IMQ. MFI values
of IMQ-activated cells are shown. Quantitative analysis (B) shows mean
percentages of TRAIL-expressing cells before and after stimulation with
TLR7/8 and TLR9 agonists (n = 3). C, MLRs were performed with titrated
numbers of unstimulated or IMQ-activated pDCs incubated with 105al-
logeneic T cells per well. The effects of TRAIL and type I IFNs were
investigated using neutralizing anti-TRAIL, anti–IFN-a, anti–IFN-b (5
mg/ml each) or isotype-matched control Abs that were preincubated with
TRAIL+pDCs express high levels of costimulatory mole-
pDCs for 30 min prior to addition of responder cells. T cell proliferation on
day 6 was measured by [3H]thymidine incorporation. Data are expressed as
mean cpm of duplicate wells 6 SD. One representative example of three
independent experiments giving similar results is shown. D, Flow cyto-
metric analysis of CD69, TRAIL, and TRAIL-R1-R4 expression on freshly
isolated and anti–CD3-/CD28-activated CD3+T cells. Cells were gated on
CD3 and histograms shown are representative of three different donors
The Journal of Immunology 1587
can activate both TRAIL-R1 and -R2, whereas sTRAIL signals
only via TRAIL-R1, unless secondarily cross-linked by Abs (20).
It was therefore not unexpected that supernatants retrieved from
IMQ-activated pDCs (Fig. 5B) and pDCs separated from their
targets using a transwell (Supplemental Fig. 4) were unable to
induce significant Jurkat cell lysis. Together, these findings indi-
cate that the rapid lysis observed in our 2-h cytotoxicity assays
requires cell contact and that membrane-bound TRAIL, but not
sTRAIL, is the primary mediator of target cell lysis.
IMQ-/IFN-a–activated pDCs kill melanoma cells in a
Both in man and mice, tumor regression of melanocytic neoplasms
upon treatment with topical IMQ is associated with an influx of
pDCs (14, 21), but evidence that activated pDCs may directly
exert antimelanoma activity is lacking. To address this issue, we
performed cytotoxicity assays with the melanoma cell lines
SKMel2 and WM793 as targets. Similar to Jurkat cells, these cell
lines express TRAIL-R2, but not TRAIL-R1, as determined by
FACS analysis (Fig. 6A). After 4–6 h of coculture, IMQ-activated
pDCs effectively lysed WM793 and, less so, SKMel2 melanoma
cells in a TRAIL-dependent fashion (Fig. 6B, 6C). The degree of
melanoma cell lysis correlated roughly with their expression
levels of TRAIL-R2 (Fig. 6A–C). In general, though, the extent of
pDC-induced melanoma cell lysis was lower and required pro-
longed periods of coculture compared with Jurkat cells. This was
even true when optimal killing conditions (skTRAIL) were used
Systemic treatment with rIFN-a has shown to increase disease-
free and, when used in high doses, overall survival of stage II and
III melanoma patients (22). In keeping with our observation of
functionally active TRAIL on IFN-a–stimulated pDCs (Fig. 4D),
we found that IFN-a–activated, but not unstimulated, pDCs ef-
fectively lysed both SKMel2 and WM793 target cells after 4–6 h
(Fig. 6E, 6F) and that the levels of lysis were similar to those
observed with IMQ-activated cells. pDC cytotoxicity was largely
TRAIL-dependent, as demonstrated by the reduction in killing to
baseline levels after addition of a neutralizing TRAIL Ab. We
should not forget that the IFN-a doses (25,000 IU/ml) used in
these experiments are supraoptimal and far exceed the blood/
tissue levels measured in IFN-a–treated patients. We therefore
asked whether clinically relevant doses of IFN-a (250 IU/ml) in
combination with (suboptimal) IMQ doses (0.05 mg/ml) would
lead not only to optimal TRAIL expression (Fig. 3D), but also to
optimal tumor cell lysis. Fig. 6G shows that this is indeed the case.
To the best of our knowledge, we show for the first time that IFN-
a–treated pDCs exhibit cytotoxic activity against melanoma cells
and that the combination of suboptimal stimulation conditions for
IFN-a and IMQ may result in effective tumor cell lysis.
The finding thatDCs canexpresscytotoxicmoleculesafter TLR7/8
and TLR9 stimulation (8, 10) and can use them to effectively lyse
cancer cells (9, 15, 23) as well as virus-infected targets (12) adds
a novel facet to our current concept of DC biology. In this study,
we sought to unravel the molecular events governing pDC cyto-
toxicity and, thus, gain a better understanding of the potential role
of cytotoxic DCs in health and disease.
We were able to extend our previous results that TLR7/8-
activated (IMQ) pDCs express TRAIL (15) by the finding that
in the absence of other costimulators (i.e., IFN-a), TLR-induced
TRAIL expression is exclusively associated with the activation of
intracellular TLRs. This was not entirely unexpected, as pDCs
express only low levels of TLR1 and TLR6 mRNA, react poorly to
ligation of these moieties, and lack mRNA for all other extra-
cellular TLRs (5–7). The selective induction of TRAIL through
TLRs that are involved in viral DNA and RNA recognition may, at
least partly, be responsible for the prominent role of pDCs during
viral infection (3). It is of interest, however, that pDCs costimu-
A, Measurement of IFN-a, TNF-a, and IFN-g in the supernatants of TLR7/
8- (IMQ, CL-075) and TLR9-activated (CpG2216) pDCs and PBMCs
activated with PMA/ionomycin. Data represent mean cytokine levels 6
SEM (n = 2–5). B, Flow cytometric analysis of IFN-a/b receptor ex-
pression (open histogram) on pDCs compared with an isotype Ab (filled
histogram) using fresh PBMCs and gating on HLA-DR+BDCA-2+cells. C,
Freshly isolated pDCs were preincubated with neutralizing anti–IFN-a,
anti–IFN-b (5 mg/ml each), or isotype-matched control Abs for 30 min
prior to TLR stimulation. After overnight culture, the level of surface
TRAIL was assessed by flow cytometry. Data represent mean percentages
of TRAIL+cells 6 SD (n = 3). D, Histograms depict MFI of surface
TRAIL expression on pDCs after overnight stimulation with different
concentrations of IFN-a (0–50,000 IU/ml) alone or in combination with
low-dose IMQ and are representative of three independent experiments
performed. E, Flow cytometric analysis of costimulatory molecule ex-
pression on TRAIL+pDCs generated by IFN-a activation (25,000 IU/ml).
One representative staining series is shown (n = 4). Numbers indicate the
percentage of positive cells. C–E, Cells were gated on BDCA-2 and HLA-
PDCs upregulate TRAIL in an IFN-a/b–dependent fashion.
1588 MECHANISMS OF IMQ- AND IFN-a–INDUCED pDC CYTOTOXICITY
lated with low-dose IFN-a acquire the ability to upregulate
TRAIL in response to TLR2/1 and TLR2/6 agonists, suggesting
that IFN-a and TLR agonists may exert combined and additive
effects. Notably, we also found modest TRAIL expression on
pDCs that had been cultured with IL-3 (Fig. 1F). Because IL-3
lacks IFN-a–inducing properties (24), the reason for this phe-
nomenon is yet unknown. TNF-a and CD40L apparently have
a bimodal effect on pDCs. They both induce maturational events
(e.g., upregulation of CD83) but, in contrast, fail to trigger TRAIL
positivity. The latter effect is probably due to downregulation of
IFN-a (25). This demonstrates that TRAIL expression on pDCs is
not simply the result of promiscuous cell activation but prefer-
entially occurs after stimulation with IFN-a–inducing agents, such
as viruses and antimicrobial peptide-complexed autologous RNA
Different from the situation with TRAIL, we found the lytic
molecule granzyme B to be abundantly expressed on freshly
isolated/IL-3–activated pDCs but considerably less so after stim-
ulation with TLR7/8 and TLR9 agonists. The functional role of
this lytic molecule on pDCs has yet to be clarified. Several
observations from us and others (15, 27) argue against its in-
volvement in pDC-induced killing. First, unstimulated granzyme
B+pDCs fail to lyse tumor targets (Fig. 4B). Second, the reported
dependence of granzyme B on other lytic molecules (e.g., per-
forin, granulysin) for CTL and NK cell effector functions (28) is
in agreement with our findings of pDCs being consistently devoid
of perforin, granulysin, FasL, and lysozyme. Other investigators
reported that, after activation, pDC-derived granzyme B limits
T cell expansion (29) and effectively kills tumor cells (27), and
they also detected lysozyme (17). Methodological differences may
well account for these discrepancies, but one may also entertain
the possibility that pDC-associated granzyme B alone may exert
its lytic function only after prolonged contact with its target (27).
An important observation of our study was that TLR-activated,
TRAIL-expressing pDCs exhibit a mature phenotype and are ef-
ficient inducers of T cell proliferation in an allogeneic MLR (Fig.
2A–C). The latter result was not necessarily expected, given that
IFN-a has known antiproliferative properties and that both HIV-
infected pDCs (12) and CMV-infected monocyte-derived DCs
a TRAIL-dependent fashion. A, Expression of TRAIL-R1 and -R2 on
Jurkat T cells was assessed by flow cytometry. B–E, Cytotoxic activity of
IMQ- (5 mg/ml), CpG2216- (2.5 mM), and IFN-a–activated (25,000 IU/
ml) pDCs or pDCs that were left unstimulated overnight against Jurkat
T cells was determined after 2 h. In B–D, activated pDCs were pre-
incubated with neutralizing anti-TRAIL, anti–IFN-a, anti–IFN-b (10 mg/
ml each), or isotype-matched control Abs for 30 min prior to addition of
target cells. In E, freshly isolated pDCs were preincubated with neutral-
izing anti–IFN-a/b or isotype-matched control Abs for 30 min prior to
induction of TRAIL with IMQ. Data in B, D, and E represent mean of
duplicates of specific lysis 6 SD, and one representative example of three
TLR- and IFN-a–activated pDCs kill Jurkat cells in
independent experiments yielding similar results is shown. Data in C show
mean of specific lysis 6 SD of results from two independent experiments.
concentration of sTRAIL in the supernatants of pDCs (5 3 105cells/ml)
cultured with TLR7/8 (IMQ; n = 5) or TLR9 (CpG2216; n = 4) ligands
overnight and in the serum of five healthy donors was determined by
ELISA; limit of detection 32 pg/ml. Each dot denotes one individual
measurement. B, Cytotoxic activity of IMQ-stimulated pDCs (E:T ratio
20:1) or their supernatants (from cultures of 5 3 105cells/ml) against
Jurkat target cells was determined after 2 h. Data represent mean per-
centage of specific lysis 6 SD (n = 3).
Jurkat cell lysis by pDCs requires cell contact. A, The
The Journal of Immunology1589
(30) can delete activated T lymphocytes. The reasons why IFN-a
and TRAIL do not influence T cell proliferation could be that the
immunostimulatory effect of a mature DC outweighs its cytotoxic
capacity or, most likely, that once T cells become sensitive to
TRAIL by upregulating their TRAIL-Rs (after 3 d), pDCs have
already lost their TRAIL expression (usually after 48 h) and/or
have ceased to produce IFN-a.
IFN-a exhibits known TRAIL-inducing properties on PBMCs
(31–33). In this study, we show that TLR-induced TRAIL expres-
sion on pDCs (8, 9) and also, as a consequence, TRAIL-mediated
pDC cytotoxicity is IFN-a dependent. More importantly, we dem-
onstrate that tumor targets lysed by IFN-a–stimulated pDCs also
include melanoma cells (Fig. 6E, 6F). In this situation, IFN-a may
even exert a dual effect, as it also sensitizes tumor cells to TRAIL-
dependent apoptosis (9, 34). This mechanism could be, at least
partly, responsible for the reported efficacy of IFN-a as adjuvant
treatment in tumor patients (e.g., high-risk primary melanoma)
Given our findings, one may ask whether there is any potential
advantagein usingIMQ aloneor incombinationwith IFN-a.There
exist several reasons why we believe that this is the case. Firstly,
IMQ when applied topically or injected into the tissue stimulates
the release of proinflammatory cytokines and chemoattractants
(Fig. 3A) (13). DCs represent a major portion of such an IMQ-
induced infiltrate (14, 15, 21). Secondly, IMQ may have additional
benefits in a therapeutic setting when compared with IFN-a alone.
As we show in this study, the TRAIL-inducing properties of
IFN-a at physiological concentrations are rather limited, but can
be greatly enhanced even by suboptimal doses of IMQ (Figs. 3D,
6G). Lastly, what was said above should be re-emphasized, that
IMQ, in contrast to IFN-a, causes phenotypic and functional pDC
maturation, providing these cells with the capacity to induce
productive primary immune responses. The combination of their
tumoricidal and immunostimulatory properties thus makes TLR-
stimulated DCs a double-edged sword in its true and best sense.
Following this reasoning, cancer cell lysis effectuated by these
and -R2 on melanoma target cells (SKMel2, WM793) was assessed by flow cytometry. B, C, E, and F, Cytotoxic activity of IMQ- (5 mg/ml) and IFN-a–
activated (25,000 IU/ml) pDCs against the melanoma cell lines SKMel2 (B, E) and WM793 (C, F). D, The maximum level of TRAIL-induced lysis of
SKMel2, WM793, and Jurkat cells was evaluated using recombinant skTRAIL (100 ng/ml). G, Evaluation of TRAIL expression on pDCs (left panel) and
cytotoxic activity of the same cells (right panel) against WM793 targets after stimulation with IMQ (5 mg/ml; low-dose 0.05 mg/ml), IFN-a (25,000 IU/ml;
low-dose 250 IU/ml), and combinations thereof. In all cytotoxicity assays (B, C, E–G), pDCs were preincubated with neutralizing anti-TRAIL (10 mg/ml)
or isotype-matched control Abs for 30 min prior to performing the assay at E:T target ratios of 20:1. Data represent mean of duplicates of specific lysis 6
SD. One representative example of three experiments performed and yielding similar results is shown for all assays.
Melanoma cells are sensitive to TRAIL-induced apoptosis mediated by IMQ- and/or IFN-a–activated pDCs. A, Expression of TRAIL-R1
1590 MECHANISMS OF IMQ- AND IFN-a–INDUCED pDC CYTOTOXICITY
cells would result in the release of tumor-associated Ags which, Download full-text
when taken up and presented by the very same cell population,
would hopefully result in protective cancer immunity. Data ob-
tained in a humanized mouse model support this notion (35).
Clinical trials are needed and currently being devised to test both
safety and efficacy of this approach.
We thank Dr. Patrick Brunner for assistance with multiplex cytokine
analysis and Ba ¨rbel Reininger for assistance with cell isolation.
The authors have no financial conflicts of interest.
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