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All content in this area was uploaded by Christine Bangert on Apr 20, 2015
Content may be subject to copyright.
Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
The Journal of Experimental Medicine
ARTICLE
JEM © The Rockefeller University Press $15.00
Vol. 204, No. 6, June 11, 2007 1441–1451 www.jem.org/cgi/doi/10.1084/jem.20070021
1441
Interest in imiquimod (IMQ) rst came from the
observation that this imidazoquinoline exerts a
profound activity against viral acanthomas that
was originally explained by its IFN- inducing
capacity (1). When used topically for a prolonged
period of time, it can lead to the regression of
certain virus-induced (e.g., genital warts [2] and
molluscum contagiosum [3]) and other (e.g.,
basal cell carcinoma [BCC] [4, 5] and lentigo
maligna [6]) skin neoplasms. IMQ exerts its
biologic activity primarily by ligation of Toll-
like receptor (TLR) 7 (7) and, to a lesser extent,
TLR8, both of which have been identi ed as
natural receptors for single-stranded RNA
(8, 9). Cell stimulation via TLR7 and TLR8 leads
to downstream activation of NF-B and other
transcription factors (10, 11). Consequently,
several genes encoding mediators and e ector
molecules of the innate as well as the adap-
tive immune response are transcribed (12–14).
Because of their prominent expression of TLR7
and TLR8, plasmacytoid DCs (pDCs) and my-
eloid DCs (mDCs) (15, 16), respectively, are
therefore likely candidates for the initiation of
the IMQ-induced host defense reaction. Other
mechanisms explaining the antitumor activity
of IMQ are also under discussion. These include
(a) the reversal of CD4
regulatory T cell function
(17), (b) a TLR-independent immunostimu-
latory action of IMQ via adenosine receptor
signaling (18), (c) direct (19) and indirect, via
IFN- (20), IMQ-induced proapoptotic e ects
on tumor cells, and (d) an antiangiogenic ac-
tivity of IMQ, as shown in a mouse model of
angiogenesis (21, 22).
In a recent study, our group investigated
IMQ-induced tumor regression in mice and
found not only a good clinical response of the
tumors to the topically applied compound but
also a direct correlation between IMQ-induced
tumor regression and the density of DCs in the
peritumoral tissue (23). Not infrequently, cancer
cells in death were found in close contact with
DCs, which was compatible with a tumoricidal
property of the latter (24, 25). In this study, we
sought to determine whether similar phenome-
nona also occur during IMQ treatment of human
skin cancers and, if so, to unravel the mechanisms
responsible for IMQ-induced tumor regression.
Tumoricidal activity of TLR7/8-activated
in ammatory dendritic cells
Georg Stary,
1
Christine Bangert,
1
Martina Tauber,
2
Robert Strohal,
3
Tamara Kopp,
1
and Georg Stingl
1
1
Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University of Vienna,
1090 Vienna, Austria
2
Department of Pathology and
3
Department of Dermatology, Federal Academic Hospital Feldkirch, 6800 Feldkirch, Austria
Imiquimod (IMQ), a synthetic agonist to Toll-like receptor (TLR) 7, is being successfully used
for the treatment of certain skin neoplasms, but the exact mechanisms by which this com-
pound induces tumor regression are not yet understood. While treating basal cell carcinoma
(BCC) patients with topical IMQ, we detected, by immunohistochemistry, sizable numbers of
both myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) within the in ammatory
in ltrate. Surprisingly, peritumoral mDCs stained positive for perforin and granzyme B,
whereas in ltrating pDCs expressed tumor necrosis factor–related apoptosis-inducing ligand
(TRAIL). The biological relevance of this observation can be deduced from our further nd-
ings that peripheral blood–derived CD11c
mDCs acquired antiperforin and anti–granzyme
B reactivity upon TLR7/8 stimulation and could use these molecules to effectively lyse major
histocompatibility complex (MHC) class I
lo
cancer cell lines. The same activation protocol led
pDCs to kill MHC class I–bearing Jurkat cells in a TRAIL-dependent fashion. While suggesting
that mDCs and pDCs are directly involved in the IMQ-induced destruction of BCC lesions,
our data also add a new facet to the functional spectrum of DCs, ascribing to them a major
role not only in the initiation but also in the effector phase of the immune response.
CORRESPONDENCE
Georg Stingl:
georg.stingl@meduniwien.ac.at
Abbreviations used: APC, allo-
phycocyanin; BCC, basal cell
carcinoma; IMQ, imiquimod;
iNOS, inducible NO synthase;
mDC, myeloid DC; pDC,
plasmacytoid DC; TLR, Toll-
like receptor; TRAIL, TNF-
related apoptosis-inducing
ligand; TUNEL, Tdt-mediated
dUTP-biotin nick-end labeling.
T. Kopp and G. Stingl contributed equally to this study.
The online version of this article contains supplemental material.
1442 TUMORICIDAL FUNCTION OF ACTIVATED DENDRITIC CELLS | Stary et al.
RESULTS
Regression of BCC upon IMQ treatment
Seven patients with histopathologically con rmed super cial
BCC were treated with IMQ. After a treatment period of
6 wk, we observed a complete clinical (Fig. 1) and histopatho-
logical response in all patients. No signs of recurrence were
noted in any of the patients followed for at least 10 mo.
In accordance with a previous report (4), all patients de-
veloped, before tumor clearance, an in ammatory tissue re-
action at the site of IMQ application. It began as erythema
after 2–3 d of treatment; became erosive during the second
week (Fig. 1); appeared as crusting and, later, scaling plaques
after 3–4 wk; and resolved completely after cessation of IMQ
treatment. Systemic side e ects such as u-like symptoms,
lymphadenopathy, myalgia, or changes in laboratory values
never occurred in any of our patients.
Emergence kinetics of leukocytic populations
in the peritumoral in ltrate upon IMQ treatment
Biopsies from BCCs were obtained before, during, and after
IMQ treatment and subjected to immuno uorescence analy-
sis using a broad panel of antibodies (Table I) to analyze, both
quantitatively and qualitatively, the composition and kinet-
ics of the IMQ-induced in ammatory in ltrate. In untreated
BCCs we found a sparse in ltrate, mainly consisting of T cells
of the helper phenotype (Table II). Upon 2 wk of IMQ treat-
ment, a dramatic increase of CD8
T cells and, to a much
lesser extent, of CD4
T cells was seen around tumor cell is-
lets (Table II). Fig. 2 (A and B) shows that, after 2 wk of topi-
cal IMQ treatment, BCC islets were surrounded and partly
in ltrated by dendritically shaped cells exhibiting either the
CD11c
/HLA-DR
mDC (Fig. 2 A) or the CD123
/HLA-
DR
pDC (Fig. 2 B) phenotype. This increase in in am-
matory-type mDCs and pDCs occurs at the expense of the
resident DC populations of normal human skin (Langerhans
cells and CD1c
dermal DCs; Table II). Other constituents
of the peritumoral in ltrate included (a) CD56
/CD94
NK
cells, (b) CD14
mononuclear phagocytes, and (c) CD15
/
HLA-DR
neutrophils (Table II). In contrast, B cell, eosinophil,
basophil, and mast cell counts displayed no major alterations
upon IMQ treatment. At the end of treatment, when tumor
clearance was achieved, the density of the various in ltrating
leukocyte subpopulations was comparable to the values before
IMQ application (Table II).
Detection of lytic molecules in the peritumoral tissue
upon IMQ treatment
Analyzing the mechanisms of IMQ-induced tumor regres-
sion, we searched, by Tdt-mediated dUTP-biotin nick-end
labeling (TUNEL) staining, for signs of apoptosis in IMQ-
treated BCC lesions. Although only a few TUNEL
tumor
cells were detected in untreated BCCs (Fig. 2 C), the decrease
Figure 1. Complete regression of super cial BCC after a 6-wk treat-
ment period with IMQ. IMQ topically applied ve times a week for a period
of 6 wk led to a local in ammatory response, which resulted in a complete
clinical and histopathological tumor clearance in all patients treated. The
clinical pictures are representative for all patients (n 7) treated with IMQ.
Table I. List of mAbs used in this study
Antibody speci city Clone (source of antibodies) Isotype
Staining experiments
BDCA-2 (FITC) AC144 (Miltenyi Biotec) mIgG1
CD1a (FITC) HI149 (BD Biosciences) mIgG1
CD1c (pur., biot.) M241 (Ancell) mIgG1
CD3 (FITC, PerCP) SK-7 (BD Biosciences) mIgG1
CD4 (pur.) SK-3 (BD Biosciences) mIgG1
CD8 (pur.) C8/144B (DakoCytomation) mIgG1
CD8 (allophycocyanin [APC]) B9.11 (Immunotech Coulter) mIgG1
CD11c (FITC) BU15 (Serotec) mIgG1
CD11c (PE, PE-Cy5) B-ly6 (BD Biosciences) mIgG1
CD14 (pur., FITC, PerCP) MP9 (BD Biosciences) mIgG2b
CD15 (FITC) 80H5 (Immunotech Coulter) mIgM
CD19 (pur., PerCP) SJ25C1 (BD Biosciences) mIgG1
CD45RA (pur.) L48 (BD Biosciences) mIgG1
CD56 (biot., PE-Cy5) B159 (BD Biosciences) mIgG1
CD83 (FITC) H15a (Immunotech Coulter) mIgG2b
CD94 (pur.) HP-3D9 (BD Biosciences) mIgG1
CD117 (c-kit, pur.) 95C3 (An Der Grub) mIgG1
CD123 (biot.) 9F5 (BD Biosciences) mIgG1
CD203c (PE) 97A6 (Immunotech Coulter) mIgG1
CD207 (pur.) DCGM4 (Immunotech Coulter) mIgG1
Fas ligand (pur.) NOK-1 (BD Biosciences) mIgG1
Granzyme B (pur.) GrB-7 (Monosan) mIgG2a
Granzyme B (PE) GB-11 (PeliCluster) mIgG1
HLA-DR (pur., FITC, APC) L243 (BD Biosciences) mIgG2a
IFN- 225.2C (Chromaprobe) mIgG2b
iNOS (FITC) 6 (BD Biosciences) mIgG2a
MBP (pur.) BMK13 (Chemicon) mIgG1
Pancytokeratin (pur.) AE1 and AE3 (BioGenex) mIgG1
Perforin (pur.) dG9 (Abcam) mIgG2b
Perforin (FITC) G9 (BD Biosciences) mIgG2b
TNF- (FITC) MAb11 (BD Biosciences) mIgG1
TRAIL (pur.) 75402 (R&D Systems) mIgG1
TRAIL (neutralizing; pur.) 75411 (R&D Systems) mIgG1
TRAIL R1 (pur.) 69036 (R&D Systems) mIgG1
Cell-sorting experiments
CD3 (pur.) UCHT1 (Immunotech Coulter) mIgG1
CD16 (pur.) 3G8 (Immunotech Coulter) mIgG1
CD19 (pur.) J4.119 (Immunotech Coulter) mIgG1
CD34 (pur.) 581 (Immunotech Coulter) mIgG1
CD41 (pur.) SZ22 (Immunotech Coulter) mIgG1
CD56 (pur.) C218 (Immunotech Coulter) mIgG1
CD235a (pur.) 11E4B-7-6 (Immunotech Coulter) mIgG1
biot., biotinylated; MBP, eosinophil major basic protein; pur., puri ed.
JEM VOL. 204, June 11, 2007 1443
ARTICLE
in pancytokeratin
BCC cells after 2 wk of therapy was ac-
companied by a concomitant increase of TUNEL
tumor
cells (Fig. 2 D). One may therefore conclude that BCC re-
gression upon IMQ treatment is at least partly accomplished
by apoptosis of tumor cells.
Based on the observation of IFN-–induced tumor re-
gression (26) and tumor cell apoptosis (27), we searched for
the presence of IFN-–producing cells in IMQ-treated BCC
biopsies and found 30% of all pDCs, but no other leuko-
cytes, to exhibit anti–IFN- staining (Fig. 2 E). It is therefore
not unreasonable to assume that IMQ-activated pDCs con-
tribute to BCC apoptosis by IFN- production.
In addition to IFN-–induced apoptotic events, perforin/
granzyme-based and death receptor ligand–mediated cancer
cell lysis are the immune system’s major tumoricidal e ector
mechanisms. When we analyzed BCC biopsies before, during,
and after IMQ treatment for the expression of lytic mole-
cules, we could hardly detect any expression of Fas ligand
(Fig. 2 F). In contrast, we observed a considerable up-regulation
of perforin, granzyme B, and TNF-related apoptosis-inducing
ligand (TRAIL) on in ltrating cells after 2 wk of IMQ treat-
ment when compared with the low baseline levels of un-
treated BCCs (Fig. 2 F).
The majority of anti-TRAIL reactivity was observed on
T cells (Fig. 3 A), but CD11c
mDCs, CD123
pDCs, and
CD14
mononuclear phagocytes also expressed this molecule
(Fig. 3, A and B). The biologic importance of this nding is
underscored by the further observation that tumor cells of
untreated (Fig. 3 C) and IMQ-treated (Fig. 3 D) BCC lesions
were positive for TRAIL receptor 1 (TRAIL R1) and sur-
rounded by a dense rim of CD123
cells (Fig. 3 D). Anti-
TRAIL R2, R3, and R4 stainings yielded negative results
(not depicted).
Somewhat to our surprise, we found that only a few T cells
and NK cells displayed antiperforin and anti–granzyme B
staining and that CD3
CD56
HLA-DR
cells were the main
source of these lytic molecules (Fig. 4, A and C). Additional
phenotyping revealed that these cells were CD11c
(Fig.
4, B and D) and lacked lineage markers such as CD3, CD56,
CD14, CD15, or CD19, as well as CD1a and CD207, thus
corresponding to mDCs. Similar to what has been observed
in untreated psoriatic skin lesions (28), CD11c
/HLA-DR
DCs in IMQ-treated BCC lesions were also found to re-
act with antibodies against inducible NO synthase (iNOS)
and TNF- (Fig. 4 E). At least phenotypically, these cells
therefore corresponded to TNF-– and iNOS- producing
DCs, an mDC population possibly involved in innate immune
defense against bacteria (29). In contrast to in ltrating pDCs
(Fig. S1 A, available at http://www.jem.org/cgi/content/full/
jem.20070021/DC1), the vast majority of CD11c
/HLA-
DR
DCs displayed the maturation marker CD83 (Fig. S1 B).
As it was quite unexpected not to nd cytotoxic T cells
Table II. Number of leukocytes occurring in BCCs before, after 2 wk, and at the end of IMQ treatment
Cell type Antigens Epidermis/dermis Untreated BCCs 2 wk IMQ 6 wk IMQ
T helper cells
CD4
/CD3
epidermis (cells/mm)
dermis (cells/mm
2
)
1.8 2
98.2 64.4
3.3 3.3
164 60.6
1.4 1.8
99 56.8
Cytotoxic T cells
CD8
/CD3
epidermis (cells/mm)
dermis (cells/mm
2
)
1.4 0.9
55.7 31.1
5.5 2.6
236.6 61.4
3.73.4
63.135.3
NK cells
CD56
/CD94
epidermis (cells/mm)
dermis (cells/mm
2
)
0
3 2.2
0.7 1.3
46.8 19
0
10.1 10.7
Langerhans cells
CD207
/CD1a
epidermis (cells/mm)
dermis (cells/mm
2
)
9 4.2
6.2 3.6
3.6 1.7
6.7 4.9
8.3 5.2
5.1 4.7
CD11c
mDCs CD11c
/HLA-DR
epidermis (cells/mm)
dermis (cells/mm
2
)
0
25.5 4.2
0
58.4 14.6
0
22.9 4.8
CD1c
mDCs CD1c
/HLA-DR
epidermis (cells/mm)
dermis (cells/mm
2
)
0.7 1.3
55.2 29.7
0.3 0.5
16.3 7.4
0.3 0.4
24.1 12.7
Phagocytes
CD14
epidermis (cells/mm)
dermis (cells/mm
2
)
1.4 3.8
21.2 15.2
2.4 3.6
47.6 6.1
1.3 3
35.1 12.7
pDCs
CD123
/ CD45RA
epidermis (cells/mm)
dermis (cells/mm
2
)
0.2 0.4
9.9 12.1
0
54.7 6.5
0
17.1 8.8
Neutrophils
CD15
/HLA-DR
epidermis (cells/mm)
dermis (cells/mm
2
)
0
7.8 8.9
0
62.1 14.3
0
15.4 13
Eosinophils
MBP
epidermis (cells/mm)
dermis (cells/mm
2
)
0
0
0
2.1 3.3
0
0.5 0.9
Basophils
CD203c
epidermis (cells/mm)
dermis (cells/mm
2
)
0
0
0
0
0
0
Mast cells
cKit
epidermis (cells/mm)
dermis (cells/mm
2
)
0
14.3 13.5
0
13.3 2.9
0
10.4 11.7
B cells
CD19
epidermis (cells/mm)
dermis (cells/mm
2
)
0
3.2 4.6
0
2.8 5.6
0
1.1 1.5
Cells were visualized using the indicated markers and were evaluated by immuno uorescence analysis. Numbers in bold indicate means SE of the mean of cells counted in
BCC lesions before, after 2 wk, and after 6 wk of IMQ treatment. MBP, eosinophil major basic protein.
1444 TUMORICIDAL FUNCTION OF ACTIVATED DENDRITIC CELLS | Stary et al.
and/or NK cells, but instead to find DCs containing per-
forin and granzyme B, we compared immuno uorescence
triple stainings of IMQ-treated BCCs with those of allergic
contact dermatitis, a disease in which perforin- and gran-
zyme B–containing T cells have been previously described
(30). In contrast to IMQ-treated BCC lesions in which most
perforin- and granzyme B–positive cells were CD3
as
well as CD8
, antiperforin and anti–granzyme B reactivity
in allergic contact dermatitis was mainly restricted to T cells
(Fig. S1, C–F).
Detection of perforin and granzyme B in mDCs
upon TLR7 and TLR8 stimulation and of TRAIL
on pDCs upon TLR7 stimulation
To determine the biological signi cance of anti-TRAIL and
antiperforin/anti–granzyme B staining of DCs in IMQ-
treated BCC lesions, we decided, for quantitative reasons, to
work with DCs isolated from peripheral blood rather than
from these skin lesions. Using the protocol described in
Materials and methods, we routinely obtained mDC and
pDC populations of 93–99% (mean 97%) purity. By FACS
analysis, these cells were HLA-DR
, expressed either high
levels of CD11c or BDCA-2, and lacked the lineage markers
CD3, CD56, CD14, and CD19 (Fig. 5 A). None of the lytic
molecules searched for were detected on freshly isolated
mDCs. In contrast, stimulation of puri ed peripheral blood–
derived mDCs with TLR7 and TLR8 ligands, but not with
the vehicle control, led to the intracellular expression of per-
forin and granzyme B in 10–15% of these cells (Fig. 5, B
and C). Expression of TRAIL was not detectable on mDCs
after stimulation with TLR7/8 ligands (Fig. 5 D).
Unstimulated pDCs abundantly contained granzyme B
within their cytoplasm (Fig. 5 E). After 12 h of stimulation
with ligands to TLR7 and TLR7/8, but not with the vehicle
control or the TLR8 ligand alone, the intracellular expres-
sion of granzyme B in pDCs had decreased. (Fig. 5, E and F).
This is consistent with the previous nding of pDCs express-
ing TLR7 but not TLR8 (15). In contrast to mDCs, TLR7/8
Figure 2. Induction of tumor cell apoptosis is accompanied by
peritumoral accumulation of DCs and up-regulation of lytic
molecules on in ammatory cells. (A and B) Immuno uorescence triple
labeling of cryostat sections of IMQ-treated BCC lesions with antipan-
cytokeratin (TRITC), anti–HLA-DR (FITC), and either (A) anti-CD11c (APC) or
(B) anti-CD123 (Cy5) revealed a close proximity of both DC populations
with tumor cells. Arrows indicate double-positive DCs. (C) Untreated
BCCs and (D) BCCs after 2 wk of IMQ treatment were stained with an
antipancytokeratin antibody (TRITC) and TUNEL staining (FITC). Note
the double-positive tumor cells occurring after 2 wk of IMQ treatment
(arrows). The pictures are representative for all evaluated biopsies. The
dotted lines point out the margins of BCCs. (E) Identi cation of IFN-–
producing pDCs in 2-wk–treated BCCs by triple stainings with anti–
IFN- (TRITC), anti–HLA-DR (FITC), and anti-CD123 (Cy5) mAbs. The
arrow indicates a triple-positive cell. (F) Quantitative analysis of lytic
molecules expressed by in ammatory cells of the peritumoral in ltrate.
Immuno uorescence single stainings were performed using antiperforin,
anti–granzyme B, anti-Fas ligand, and anti-TRAIL antibodies (all TRITC).
Data are given as absolute numbers of positive cells SEM for the indi-
cated markers.
Figure 3. Upon IMQ treatment, TRAIL R1
BCC islets are sur-
rounded by TRAIL-expressing pDCs. (A) Detailed quantitative in situ
analysis of the expression pattern of TRAIL on leukocytes before, during,
and after IMQ treatment was performed by immuno uorescence double
stainings with an anti-TRAIL mAb and lineage markers in all patients
treated. The data are given as absolute numbers of double-positive
cells SEM. (B) A representative view of anti-CD123 (FITC), anti–HLA-DR
(APC), and anti-TRAIL (TRITC) triple stainings of a BCC lesion after 2 wk of
IMQ treatment identi es pDCs as TRAIL-expressing cells. Immuno uores-
cence triple labeling of (C) untreated and (D) IMQ-treated BCCs with
antipancytokeratin (TRITC), anti-TRAIL R1 (A488), and anti-CD123
(Cy5) reveals TRAIL R1
BCC cells (C and D) surrounded by CD123
cells (arrows; D).
JEM VOL. 204, June 11, 2007 1445
ARTICLE
agonists induced up-regulation of TRAIL on the surface of
pDCs (Fig. 5 G). Similar to the pretreatment situation, the
cell populations stimulated with TLR7/8 agonists for 12 h
failed to express CD3 and CD56, strongly arguing against the
possibility of the involvement of T cells and/or NK cells.
These ndings indicate that, upon stimulation with TLR7/8
agonists, mDCs and pDCs from peripheral blood acquire the
phenotype of mDCs and pDCs found in the peritumoral tis-
sue of IMQ-treated BCC lesions.
mDCs release perforin and granzyme B upon
TLR7 and TLR8 stimulation
The cytotoxic e ect of the perforin–granzyme B pathway
is only functional if the cytotoxic proteins are degranulated
and come into contact with target cells. To reveal possible
cytotoxic functions of perforin- and granzyme B–expressing
TLR7/8-stimulated DCs, we analyzed perforin and gran-
zyme B levels in the supernatants of sorted DCs after TLR7
and TLR8 ligation by ELISA. The viability of DCs was
controlled by annexin V ( ow cytometry) and trypan blue
stainings (counting chamber) and exceeded 90% before and
after cultures. Supernatants of PMA/ionomycin-stimulated,
negatively selected T cells served as positive controls. We
observed a substantial release of perforin and granzyme B
Figure 4. IMQ treatment induces the up-regulation of perforin
and granzyme B on mDCs in the peritumoral in ltrate. A detailed
quantitative analysis of the expression pattern of (A) perforin and
(C) granzyme B on leukocytes before, during, and after IMQ treatment
was performed by immuno uorescence double stainings with antibodies
against the indicated lytic molecules and lineage markers in all seven
treated patients. The data are given as absolute numbers of double-positive
cells SEM. Representative pictures of immunofluorescence triple
stainings with anti-CD11c, anti–HLA-DR, and (B) antiperforin or (D) anti–
granzyme B mAbs of the peritumoral in ltrate after 2 wk of treatment
with IMQ. (E) Triple immuno uorescence labeling of BCCs upon IMQ
treatment with anti-CD11c (PE), anti–HLA-DR (APC), and anti-iNOS (FITC)
or anti–TNF- (FITC) mAbs identi es iNOS and TNF- to be produced by
CD11c
HLA-DR
cells (arrows).
Figure 5. Peripheral blood–derived DCs express lytic molecules
after TLR7/8 stimulation. (A) Characterization of isolated peripheral
blood–derived pDCs and mDCs as HLA-DR
BDCA-2
and HLA-
DR
CD11c
, respectively, and lineage-negative cell populations by ow
cytometry. (B and C) Analysis of intracellular expression levels of perforin
and granzyme B of isolated mDCs before and after culture with TLR7/8
agonists by ow cytometry. (B) Representative dot plots of mDCs show
double-positive cells for perforin (FITC) and granzyme B (PE) after stimu-
lation with TLR7, TLR8, and TLR7/8 agonists. (C) Quantitative analysis of
granzyme B
and perforin
mDCs. Data are given as mean percentages of
perforin- or granzyme B–positive mDCs SEM from three independent
experiments. (D) Freshly isolated and TLR7/8-stimulated mDCs do not
display membrane-bound TRAIL (A488). Representative histogram plots
from three independent experiments show the anti-TRAIL reactivity (bold
line) and the matching isotype control (dashed line). (E and F) Determina-
tion of intracellular expression levels of perforin and granzyme B on
isolated pDCs before and after stimulation with TLR agonists. (E) Repre-
sentative dot plots of pDCs show a decrease of granzyme B–positive cells
after stimulation with TLR7 and TLR7/8 agonists. (F) Quantitative analysis
of pDCs expressing perforin and granzyme B. Data are given as mean
percentages of perforin- and granzyme B–positive cells SEM from
three independent experiments. (G) TLR7/8-stimulated, but not freshly
isolated, pDCs display membrane-bound TRAIL (A488). Representative
histogram plots from three different experiments show the anti-TRAIL
reactivity (bold line) and the matching isotype control (dashed line).
Dead cells were excluded by gating out propidium iodide–positive cells in
all experiments.
1446 TUMORICIDAL FUNCTION OF ACTIVATED DENDRITIC CELLS | Stary et al.
after stimulation of mDCs with TLR7 and TLR8 ligands, in
contrast to the vehicle control (Fig. 6, A and B). We de-
tected granzyme B in the supernatants of pDCs after TLR7
and TLR7/8 ligation but not after TLR8 ligation alone or
vehicle control (Fig. 6 B), which was consistent with our
FACS data described in the previous section. Perforin could
not be detected at any setting in the supernatant of pDCs
(Fig. 6 A). These data demonstrate that mDCs and pDCs
can be activated to release lytic molecules in a biologically
relevant fashion.
TLR7/8-stimulated mDCs and pDCs become killer cells:
involvement of different cytotoxic pathways
To evaluate the functional signi cance of lytic molecules
expressed by DCs after TLR7 and TLR8 ligation, we per-
formed cytotoxicity assays with puri ed peripheral blood–de-
rived mDCs and pDCs as e ector cells, and perforin-sensitive
K562 (Fig. S2 A, available at http://www.jem.org/cgi/
content/full/jem.20070021/DC1) and TRAIL-sensitive Jurkat
cell lines (31) as target cells. Although freshly isolated mDCs
hardly displayed any cytotoxic activity against K562 and Jur-
kat cells, mDCs stimulated with TLR7/8 agonists e ectively
lysed K562 but not Jurkat cells (Fig. 7 A). We observed the
opposite e ect in pDCs, in that they exhibited substantial
cytotoxic activity against Jurkat and not K562 cells after
TLR7/8 stimulation, but not when unstimulated (Fig. 7 B).
This diverse killing behavior indicated distinct mechanisms
operative in DC-mediated cytotoxicity after stimulation with
TLR7/8 ligands. To investigate the di erent cytotoxic path-
ways in greater detail, we performed inhibition experiments
with (a) concanamycin A, a speci c inhibitor of the perforin-
based cytotoxic pathway (32), (b) a neutralizing anti-TRAIL
antibody to block TRAIL-mediated killing, and (c) a neutral-
izing anti-Fas ligand antibody to inhibit Fas-induced killing.
As shown in Fig. 7 C, coincubation of TLR7/8-stimulated
mDCs and K562 in the presence of concanamycin A led to
a complete disappearance of the speci c lysis, whereas anti-
TRAIL and anti-Fas ligand antibodies had no e ect on the
mDC-mediated cytotoxicity. On the other hand, preincuba-
tion of TLR7/8-stimulated pDCs with an inhibitory anti-
TRAIL antibody essentially abolished the pDC cytotoxicity
against Jurkat cells (Fig. 7 D); the speci city of this inhibition
is also documented by the failure of concanamycin A and
anti-Fas ligand antibodies to block the killing of Jurkat cells
by TLR7/8-activated pDCs. To exclude the theoretical pos-
sibility of contaminating NK/NKT cells exhibiting cytotoxic
Figure 6. Secretion of perforin and granzyme B by mDCs upon
TLR7 and TLR8 stimulation. (A) Perforin and (B) granzyme B protein
levels were determined by ELISA in media conditioned by puri ed blood–
derived mDCs and pDCs after 12 h of stimulation with TLR7 and TLR8
agonists. Media conditioned by 12-h PMA/ionomycin-stimulated T cells
were used as positive controls. Data are given as means SEM from two
independent experiments with 2 10
5
cells in each setting.
Figure 7. Tumoricidal activity of blood-derived mDCs and pDCs
after stimulation with TLR7/8 agonists. Unstimulated, TLR7/8-
stimulated, or vehicle-stimulated (A) mDCs and (B) pDCs were cultured
with K562 or Jurkat as target cells for a conventional 2-h europium-TDA
release assay at the indicated effector/target cell ratios. Data represent the
means of triplicate wells SEM from two independent experiments.
(C and D) For inhibition of different cytotoxicity-inducing pathways, 12-h
TLR7/8-stimulated mDCs and pDCs as effector cells were preincubated
with 100 nM concanamycin A for 120 min, 5 g/ml anti-TRAIL for
30 min, 5 g/ml anti-Fas ligand for 30 min, or 5 g/ml of an IgG1 isotype
for 30 min. Target cells were (C) K562 for TLR7/8-stimulated mDCs and
(D) Jurkat for TLR7/8-stimulated pDCs. Cytotoxicity was determined by a
2-h europium-TDA release assay at the indicated effector/target cell
ratios. Data represent the means of triplicate wells SEM from two
independent experiments.
JEM VOL. 204, June 11, 2007 1447
ARTICLE
activity within isolated DC populations, we performed cyto-
toxicity assays with e ector cells consisting of irrelevant ller
cells (i.e., A431) containing titrated numbers of NK cells. In
a setting of 7% NK cells titrated into A431 as ller cells, we
observed a 10% cytotoxic activity against K562 and Jurkat
(Fig. S2 B), which represented less than half of the mDC-
mediated killing and less than one third of pDC-mediated
cytotoxicity. In addition, NK cells killed K562 and Jurkat
cells with a comparable intensity and did not show selective
killing like mDCs and pDCs. As NK cells and T cells never
accounted for >1% within puri ed DC populations (Fig.
5 A), these experiments strongly argue against contaminating
cells being even partly responsible for the cytotoxic activity
of the DC populations.
D I S C U S S I O N
IMQ is now an established therapeutic option in the treat-
ment of certain skin neoplasms such as genital warts (2), ac-
tinic keratoses (33), and BCCs (4, 5). In BCCs, cure rates are
similar to those achieved with other therapeutic procedures
(34, 35). Our study con rms these observations.
After binding to and signaling through TLR7 (7), IMQ
leads to up-regulation of cytokines with growth-inhibitory
(e.g., IFN-) and/or proin ammatory (e.g., TNF-, IL-1,
and IL-6) properties (1, 36). TLR-independent mechanisms,
operating directly by induction of apoptosis (19) and/or indi-
rectly by adenosine receptor signaling (18), could also con-
tribute to the clinically observed tumor regression. Even
though the IMQ-induced regression pattern of skin cancers
shows no signs of T cell memory, evidence exists that CD8
T cells are abundantly present in the in ammatory in ltrate
of IMQ-treated skin cancers (12, 14). A role for NK cells has
also been claimed (37). To address the involvement of cellu-
lar cytotoxicity in IMQ-induced tumor regression, in this
study we searched for the expression of lytic molecules on/in
di erent cell populations forming this in ltrate.
As opposed to the pretreatment situation, we detected
sizable numbers of perforin/granzyme B–positive cells, as
well as TRAIL-expressing cells, after 2 wk of IMQ treat-
ment. Interestingly, we observed that the majority of CD8
T cells and CD56
NK cells, which together formed a major
portion of the IMQ-induced in ltrate, were largely devoid
of antiperforin and anti–granzyme B reactivity. The reasons
for this nding are not entirely clear. One could argue that
CD8
T cells and NK cells are simply bystander cells within
the in ammatory in ltrate and are not concerned with cyto-
toxic e ector functions. It is also conceivable that CD8
T cells discharge their cytotoxic granules directly after migra-
tion into the peritumoral tissue; after 2 wk of IMQ treat-
ment, the time point we had chosen for taking the biopsies,
these granules would therefore no longer be detectable
within their cytoplasm. We cannot de nitively exclude this
possibility. The occurrence of staining artifacts is unlikely, as
we detected perforin- and granzyme B–positive T cells in
skin biopsies of allergic contact eczema (Fig. S1, C and E).
To our surprise, double and triple labeling of IMQ treated
BCC sections revealed perforin and granzyme B to be ex-
pressed predominantly by lineage-negative CD11c
/HLA-
DR
mDCs. On the other hand, TRAIL, a molecule directly
involved in tumor cell killing by binding to its corresponding
death receptors (38–40), was expressed on several cell types,
including pDCs. We sought to better characterize the mDC
subset expressing cytotoxic molecules in situ and found that
they coexpress TNF- and iNOS, two proin ammatory
mediators with antitumor activity (41, 42). Thus, these cells
corresponded to TNF-– and iNOS-producing DCs, which
were shown to originate from blood-derived monocytes (43)
and exhibit e ector functions in bacterial infections in mice
(29) and to correlate with disease activity in psoriasis in
humans (28).
We were not able to isolate DCs from IMQ-treated BCC
lesions in numbers that would have su ced to conduct
meaningful functional experiments. We therefore decided
to test the functional relevance of our immunostaining re-
sults in studies with peripheral blood–derived mDCs and
pDCs stimulated with TLR7 and TLR8 agonists. When
activated in vitro with such compounds, peripheral blood–
derived mDCs not only expressed and released perforin
and granzyme B but also exhibited substantial cytotoxicity
against MHC class I–negative (K562) but not MHC class
I–positive (Jurkat) tumor cell lines. This lytic activity was
abolished by pretreatment of e ector cells with concana-
mycin A, an inhibitor of perforin-mediated cytotoxicity.
To rule out the possibility that T cells and/or NK/NKT
cells would contaminate the mDC e ector cell population,
we analyzed puri ed DCs of the peripheral blood by FACS
stainings and by real-time PCR. Results obtained showed
that these cells lacked all lineage markers at the protein level
(Fig. 5 A) and were devoid of CD3 transcripts (unpub-
lished data). We also titrated CD56
cells into a population
of nonfunctional ller cells and found that the lytic capacity
of this cell mixture was considerably lower than that of
puri ed TLR7/8-activated DCs, even when numbers of
CD56
NK cells were greater than the peak levels of all
lineage
CD11c
cells ever encountered in our mDC prep-
arations (Fig. S2 B). Evidence for a direct cytotoxic poten-
tial of mDCs has also been presented by other investigators,
but their ndings di er from ours with regard to the acti-
vation status of the DCs and the type of lytic molecules
involved (24, 44–48).
The emergence of pDCs in the peritumoral tissue of
IMQ-treated skin cancers has been demonstrated in mice
(23) and in humans (49). We con rmed the occurrence of
IFN-–producing pDCs in the peritumoral in ltrate during
IMQ treatment. As IFN- induces cytotoxic molecules on
NK cells (50) and T cells (51), pDCs may indirectly contrib-
ute to the elimination of tumor cells. In this study, we asked
whether pDCs could directly acquire e ector functions via
up-regulation of cytotoxic molecules and, thus, play an active
role in tumor clearance. In contrast to mDCs, pDCs occur-
ring within the peritumoral in ltrate expressed neither perfo-
rin nor granzyme B. At rst glance, this was rather surprising,
1448 TUMORICIDAL FUNCTION OF ACTIVATED DENDRITIC CELLS | Stary et al.
because unstimulated pDCs from peripheral blood clearly
displayed intracellular anti–granzyme B (Fig. 5 E) (52, 53).
Our further observation that pDC expression levels of gran-
zyme B are continuously decreasing upon TLR7 ligation may
be an explanation for the results of our in situ stainings. In any
event, granzyme B does not seem to contribute to the antitumor
activity of pDCs, as inhibition of the perforin/granzyme B–
mediated cytotoxic pathway of TLR7/8-stimulated pDCs
had no e ect on the cytotoxic activity of these cells. The
physiological role of granzyme B in pDCs therefore remains
to be established.
On the basis of our ndings that pDCs occurring in the
IMQ-induced in ltrate expressed TRAIL and that TLR7-
stimulated peripheral blood–derived pDCs displayed TRAIL
on their surface and used this molecule for cytotoxic pur-
poses, we would like to propose that pDCs have the potential
to act as anticancer e ector cells. Our results con rm and
extend observations by others demonstrating the TRAIL-
mediated cytotoxicity of pDCs after stimulation by in uenza
virus or TLR agonists (25).
The biological importance of our ndings was underlined
by the observation that IMQ-treated BCC cells, surrounded
by CD123
pDCs, are TRAIL 1 (DR4)–positive (Fig. 3 D)
and express only negligible amounts of MHC class I mole-
cules in situ (unpublished data). Even though we are aware of
the fact that TLR7/8-stimulated DCs from peripheral blood
are not necessarily equivalent to DCs that accumulate in the
skin in response to TLR activation, our data suggest that per-
forin/granzyme B–bearing cytotoxic mDCs and TRAIL-
expressing pDCs participate in IMQ-induced tumor regression
by MHC class I–independent and death receptor–dependent
killing of BCC cells, respectively. This does not exclude the
possibility that other e ector molecules (e.g., TNF-) are also
involved in this process.
Many questions remain to be resolved. To begin with, it
has to be determined whether all DCs or only subpopulations
thereof can be transformed into e ector cells upon appropri-
ate stimulation. Although we did not nd NKG2D, CD94,
CD16, or CD56 to be expressed on cytotoxic mDCs or pDCs
before or after TLR7/8 stimulation (unpublished data), the
relationship of human DCs described in our study to the
newly described IFN-producing killer DCs in mice (54, 55)
has to be de ned. It also needs to be clari ed whether nat-
ural TLR7/8 ligands (viral single-stranded RNA) or stimuli
other than TLR7/8 agonists can endow DCs with lytic prop-
erties, which signaling cascades are involved in this process,
and whether tumor cells, by an as of yet unknown recog-
nition process, are triggering the release of cytotoxic gran-
ules by activated DCs. All of this information is required to
devise strategies for the use of cytotoxic DCs as tools in anti-
cancer immunotherapy.
Finally, it will be of great interest to determine whether DC
cytotoxicity ever occurs in the course of pathophysiological
DC-driven immune responses. If so, the attractive possibility
exists that DCs may use this e ector mechanism to down-
regulate the immune reactions that they initiate themselves.
MATERIALS AND METHODS
Patients and tissue material. Seven patients with histologically con rmed
super cial BCC (three females and four males; mean age 78.3 8.2) were
enrolled in the study after giving their written informed consent. BCCs were
located on the head (n 2), the neck (n 2), and the back (n 3). None
of the patients were immunosuppressed or had any dermatological disease
that could exacerbate upon IMQ (Aldara; 3M Pharmaceuticals) treatment.
BCCs included in this study were therapy naive and did not receive any
BCC-speci c therapy (e.g., photodynamic therapy, 5- uorouracil, or IFN-)
before this study.
Patients received a once daily, ve-times-a-week dosing regimen of
IMQ 5% cream for 6 wk, according to the o cial treatment guidelines
approved for BCC. The cream was applied as a thin layer without occlusion
overnight exactly on the BCC sites. Safety and e cacy were clinically
monitored weekly and by blood examinations at weeks 3 and 6. After 6 wk
of treatment, response rates were determined by clinical examination
and histological analysis of a punch biopsy taken from formerly a ected
BCC sites.
For immuno uorescence analysis, 4-mm punch biopsies were taken un-
der local anesthesia (1% lidocaine) from untreated BCCs after 2 wk of IMQ
treatment, when the clinical signs of in ammation were strongest, and after
6 wk of IMQ therapy at the treatment endpoint. The biopsies were embedded
in optimum cutting tissue compound (Tissue-Tek; Sakura Finetek), snap-
frozen in liquid nitrogen, and stored at –80C until further processing.
Studies involving patient material were performed according to the
Declaration of Helsinki. Biopsies were routinely taken from every patient for
diagnostic purposes, followed by an informed consent about a possible scien-
ti c use of the data obtained as recommended by the local ethics committee
(Federal Academic Hospital Feldkirch).
Immuno uorescence staining. Single and multicolor immuno uores-
cence staining procedures were performed as previously described (56). The
mAbs used in this study and their sources are shown in Table I.
Apoptotic cells were detected using a kit from Roche Molecular Bio-
chemicals to visualize DNA breaks as TUNEL
, according to the manufac-
turer’s guidelines. After blocking with normal mouse serum for 20 min to
diminish background staining, sections were counterstained with a puri ed
antipancytokeratin mAb overnight at 4C. Puri ed antibody binding was
visualized by incubating the sections with rhodamine (TRITC)-conjugated
A niPure F(ab)2 fragment goat anti–mouse IgG (HL). The evaluation of
immuno uorescence results was performed as previously described (56). In
brief, biopsy specimens were read in a blinded fashion by two independent
investigators with a mean observer coe cient <10%. Labeled cells were
enumerated per visual eld and expressed as the number of cells per millimeter/
basement membrane epidermis and millimeter
2
dermis SEM.
Cell preparations from peripheral blood. Peripheral blood samples
were purchased as bu y coats from the Vienna Red Cross Center. DCs were
isolated as follows: PBMCs were obtained by Ficoll-Plaque density gradient
centrifugation (Histopaque-1077; Sigma-Aldrich) and depleted of T cells,
B cells, NK cells, hemopoietic stem cells, monocytes, platelets, and erythro-
cytes by anti-CD3/CD19/CD56/CD34/CD14/CD16/CD41/CD235a
(3 g/ml each) immunolabeling and anti–mouse IgG immunomagnetic deple-
tion (MACS; Miltenyi Biotec). The remaining cell fraction was then divided
into mDCs and pDCs by positive selection using anti-CD1c and, thereafter,
anti-CD304 (BDCA-4) microbeads (MACS). The purity of isolated DC popu-
lations was 93–99%, as determined by ow cytometric analysis (FACScan; BD
Biosciences) with either anti-CD11c/HLA-DR or anti-CD303 (BDCA-2)/
HLA-DR stainings. None of the other leukocyte subpopulations tested
individually (T cells, B cells, NK cells, and monocytes) accounted for > 1%
of the sorted cell population. T cells were prepared as a positive control
for the analysis of the perforin and granzyme B release by negative selec-
tion from PBMCs using anti-CD11c/CD14/CD16/CD19/CD34/CD41/
CD56/CD123/CD235a (3 g/ml each) immunolabeling and anti–mouse
IgG immunomagnetic depletion (MACS). NK cells used as positive controls
JEM VOL. 204, June 11, 2007 1449
ARTICLE
for cytotoxicity assays were separated by anti-CD56 (3 g/ml each) immu-
nolabeling and anti–mouse IgG immunomagnetic selection after T cell de-
pletion (MACS). The purity of T cells and NK cells was >99%, as determined
by FACS analysis.
DC stimulation with TLR agonists. Synthetic agonists to TLR7 (3M-
001), TLR8 (3M-002), TLR7/8 (3M-003), and an inactive small molecule
TLR7/8 analogue that served as a negative control (3M-006) were provided
by R.L. Miller (3M Pharmaceuticals, St. Paul, MN). Isolated mDCs and
pDCs were washed and resuspended in RPMI 1640 medium (Invitrogen)
supplemented with heat-inactivated 10% FCS (Invitrogen) and 1% penicillin/
streptomycin (Invitrogen) and cultured with 4 M 3M-001, 3M-002,
3M-003, or 3M-006 for 12 h at 2 10
6
cells/ml in 0.25 ml in 96-well plates
or 0.5 ml in 48-well plates. Viability of the cells was measured with trypan
blue and annexin V (BD Biosciences) before and after cultures, according to
the manufacturer’s instructions. Cells were analyzed by FACS stainings for
detection of lytic molecules or used as e ector cells in cytotoxicity assays.
Media conditioned by DC subsets were collected and stored at 20C for
further analysis.
Flow cytometry. To analyze lytic molecules on blood-derived DCs, we
performed intra- and extracellular FACS stainings of isolated DC popu-
lations before and after culture with TLR agonists. After staining of DC
populations with anti–BDCA-2 (Miltenyi Biotec) or anti-CD11c (BD Bio-
sciences) mAbs, cells were xed and permeabilized with a cell permeabili-
zation kit (An Der Grub), according to the manufacturer’s instructions for
intracellular protein detection. Cells were incubated with antiperforin FITC
(BD Biosciences) and anti–granzyme B PE (PeliCluster; Sanquin) antibodies.
Surface TRAIL expression was visualized by staining with an anti-TRAIL
mAb (R&D Systems) after labeling the puri ed antibody with a protein
labeling kit (FluoReporter Oregon Green A488; Invitrogen), according to
the manufacturer’s instructions. Purity controls of DC populations before
and after culture were performed by triple stainings with antibodies against
HLA-DR FITC (BD Biosciences), BDCA-2 PE (Miltenyi Biotec), or
CD11c PE (BD Biosciences) and CD3 PerCp, CD14 PerCp, CD19 PerCp,
or CD56 PECy5.
After incubation with these mAbs, DCs were washed twice with ice-
cold PBS and subjected to ow cytometric analysis using a ow cytometer
(FACScan).
Measurement of perforin and granzyme B in the supernatants. Super-
natants were harvested after stimulation of isolated mDCs and pDCs with
TLR7, TLR8, or TLR7/8 agonists or vehicle control for 12 h and stored at
20C. Perforin and granzyme B levels were quanti ed by ELISA kits (Dia-
clone Research), according to the manufacturer’s instructions. Supernatants
of T cells stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 1 g/ml
ionomycin (Sigma-Aldrich) for 12 h served as positive controls.
Cytotoxicity assays. The ability of DCs to exert cytotoxicity was assessed
in a conventional 2-h europium-TDA release assay (PerkinElmer), as previ-
ously described (57), according to the manufacture’s instructions. Data were
expressed as the percentage of cytotoxicity calculated by the following
formula: cytotoxicity (%) (experimental release – spontaneous release)/
(maximum release – spontaneous release) 100.
We used the chronic myelogenous leukemia cell line K562 (provided
by C. Wagner, Medical University of Vienna, Vienna, Austria) and the
acute T cell leukemia cell line Jurkat (provided by P. Meraner, Medical
University of Vienna, Vienna, Austria) as target cells. In a 96-well plate,
5 10
3
target cells per well were incubated with mDCs and pDCs in dif-
ferent e ector/target ratios (starting at 25:1) in triplicates. For inhibition of
TRAIL- and Fas ligand–dependent lysis, 5 g/ml of azide-free neutralizing
anti-TRAIL (clone 75411; R&D Systems) and 5 g/ml of anti–Fas ligand
(clone NOK-1; BD Biosciences), respectively, were added to e ector cells
30 min before the addition of target cells. A 30-min preculture of e ector
cells with 5 g/ml of an IgG1 isotype (Sigma-Aldrich) served as negative
control. For inhibition of the perforin-based cytotoxicity, e ector cells were
treated for 120 min with 100 nM concanamycin A (Sigma-Aldrich) before tar-
get cells were added.
Online supplemental material. Fig. S1 shows that mDCs, but not
pDCs, display CD83 in IMQ-treated BCC and that perforin/granzyme
B–containing T cells occur in lesions of allergic contact dermatitis, but not
IMQ-treated BCC. Fig. S2 depicts NK cell–mediated cytotoxicity. Online
supplemental material is available at http://www.jem.org/cgi/content/full/
jem.20070021/DC1.
We thank Richard L. Miller (3M Pharmaceuticals) for providing agonists to TLR7
and TLR8 and Drs. Dieter Maurer and Ethan Shevach for critically reading the
manuscript. We would also like to thank Sabine Altrichter for technical advice and
Andreas Ebner for preparing the gures.
The authors have no con icting nancial interests.
Submitted: 2 January 2007
Accepted: 9 May 2007
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