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IMMUNOBIOLOGY
Small molecule Toll-like receptor 7 agonists localize to the MHC class II
loading compartment of human plasmacytoid dendritic cells
Carla Russo,1 Ivan Cornella-Taracido,2 Luisa Galli-Stampino,1 Rishi Jain,2 Edmund Harrington,2 Yuko Isome,2
Simona Tavarini,1 Chiara Sammicheli,1 Sandra Nuti,1 M. Lamine Mbow,3 Nicholas M. Valiante,3 John Tallarico,2
Ennio De Gregorio,1 and Elisabetta Soldaini1
1Novartis Vaccines & Diagnostics, Siena, Italy; 2 Novartis Institutes for Biomedical Research, Cambridge, MA; and 3Novartis Vaccines & Diagnostics,
Cambridge, MA
TLR7 and TLR8 are intracellular sensors
activated by single-stranded RNA species generated during viral infections.
Various synthetic small molecules can
also activate TLR7 or TLR8 or both through
an unknown mechanism. Notably, direct
interaction between small molecules and
TLR7 or TLR8 has never been shown.
To shed light on how small molecule
agonists target TLRs, we labeled 2 imidazoquinolines, resiquimod and imiquimod, and
one adenine-based compound, SM360320,
with 2 different fluorophores [5(6) carboxytetramethylrhodamine and Alexa Fluor 488]
and monitored their intracellular localization in human plasmacytoid dendritic cells
(pDCs). All fluorescent compounds induced
the production of IFN-␣, TNF-␣, and IL-6
and the up-regulation of CD80 and CD86
by pDCs showing they retained TLR7stimulating activity. Confocal imaging of
pDCs showed that, similar to CpG-B, all
compounds concentrated in the MHC
class II loading compartment (MIIC), iden-
tified as lysosome-associated membrane
protein 1ⴙ, CD63, and HLA-DRⴙ endosomes. Treatment of pDCs with bafilomycin A, an antagonist of the vacuolar-type
proton ATPase controlling endosomal
acidification, prevented the accumulation
of small molecule TLR7 agonists, but not
of CpG-B, in the MIIC. These results indicate that a pH-driven concentration of
small molecule TLR7 agonists in the MIIC
is required for pDC activation. (Blood.
2011;117(21):5683-5691)
Introduction
Since the discovery of type I IFN (IFN-I) in 1957 and its potential
for treatment of viral infections and cancer,1 several small molecule
inducers of this factor have been identified. Many of these
compounds are now known to be TLR7 or TLR8 or both agonists.
Among them are imidazoquinolines,2,3 such as resiquimod (R848)
that activates both TLR7 and TLR8 in humans, and imiquimod
(R837) that activates TLR7 only.4,5 Imidazoquinolines have been
largely tested in humans, and R837, formulated as a cream
(Aldara), is licensed for topical treatment of genital warts, basal
cell carcinoma, and actinic keratosis.6 Similar to R837, purinelike molecules, such as 9-benzyl-8-hydroxy-2-(2-methoxyethoxy)
adenine (SM360320 or 1V136), are TLR7 agonists.7 TLR7 and
TLR8 are phylogenetically related, located on the X chromosome
in most mammals, and probably derive from a gene duplication
event. TLR7 and TLR8 are both activated by various singlestranded RNAs (ssRNAs) from viruses,8 synthetic guanosine- or
uridine-rich ssRNAs,9 and synthetic small molecules. TLR9, together
with TLR7 and TLR8, form a subfamily of TLRs that is based
on genomic structure and sequence homology.10 The natural
agonist of TLR9 is unmethylated, CpG-containing DNA of bacterial or viral origin that can be mimicked by synthetic ssCpG
oligonucleotides that can be classified as A-type (CpG-A), B-type
(CpG-B), or C-type (CpG-C) on the basis of their different
sequence motifs and biologic activities.11 Several reports have
suggested that TLR7, TLR8, and TLR9 can interact directly with
nucleic acids12-15; however, structural evidence confirming this
hypothesis is not available yet. No convincing evidence of direct
binding of imidazoquinolines or purine-like molecules to TLR7 or
TLR8 exists either.
TLR7 and TLR9 are the only TLRs expressed by plasmacytoid
dendritic cells (pDCs), which are a rare subset of circulating DCs
that are considered as a frontline defense against viral infections.
pDCs produce most of the systemic IFN-I (ie, IFN-␣ and -␤) after
viral infection and rapidly activate T cells with the use of presynthesized MHC class I and II molecules stored in their early and late
endosomal compartments, respectively.16,17
Although nucleic acid sensing provides protection from intracellular infection, nucleic acids are not exclusively from pathogens; therefore, a balance between self versus foreign responsiveness is necessary. Indeed, recognition of self nucleic acids by
TLR7 and TLR9 and the resultant IFN-I production have been
linked to autoimmune disorders such as lupus.18 To avoid autoimmunity driven by recognition of self nucleic acids, TLR3, which
binds double-stranded RNA,19 TLR7, TLR8, and TLR9 are sequestered in intracellular compartments. In particular, TLR7 and
TLR9 localize to the endoplasmic reticulum and the endolysosomal compartment, which is a still incompletely understood
vesiculotubular network of sorting and degradative organelles
that accept, redirect, and process molecules from the exterior or
interior of the cell.20 TLR7 and TLR9 translocation from the
endoplasmic reticulum to the endolysosomal compartment depends
on the chaperone protein UNC93B and is required for TLR7 and
Submitted December 29, 2010; accepted March 28, 2011. Prepublished online as
Blood First Edition paper, April 12, 2011; DOI 10.1182/blood-2010-12-328138.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
Presented in abstract form at the DC2010: Forum on Vaccine Science, Lugano,
Switzerland, September 26-30, 2010.
BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
© 2011 by The American Society of Hematology
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BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
RUSSO et al
TLR9 signaling.21,22 Recent evidence shows that TLR9 becomes
competent for signaling on proteolytic cleavage of its ectodomain
in the endolysosomal compartment.23-26 The need for proteases in
intracellular TLR activation may be a common phenomenon
because cleaved TLR7 has been found in RAW264.7 cells,23 and
Sepulveda et al25 claimed that human TLR7 is cleaved in DCs on
R837 stimulation, possibly by asparagine endopeptidase, such as
TLR9. Furthermore, several reports have shown that CpG concentrates in the endolysosomal compartment,13,27,28 supporting the idea
that the productive encounter between TLR9 and its ligand occurs
there. In particular, localization of CpG-A to transferrin receptor–
positive (TfR⫹) endosomes was associated with IFN-␣ production,
whereas localization of CpG-B in lysosome-associated membrane
protein 1–positive (LAMP-1⫹) endosomes was associated with
costimulatory molecule (eg, CD80 and CD86) up-regulation in
human pDCs.28
Unlike CpG, the intracellular localization of small molecule
TLR7/8 agonists has not been characterized. Thus, we labeled
R848, R837, and SM360320 with fluorophores to track them
in human pDCs. All 3 fluorescently labeled compounds were
TLR7 agonists and induced IFN-␣, TNF-␣, and IL-6 production as
well as CD80 and CD86 up-regulation by human pDCs, like their
unlabeled parent compounds and CpG-B, as described.28-30 Fluorescently labeled compounds colocalized with CpG-B in LAMP-1⫹,
CD63⫹, and HLA-DR⫹ endosomes, which belong to the MHC
class II loading compartment (MIIC) of pDCs, independently of
their chemotype and of the fluorophore used to label them.
Bafilomycin A (BafA), an antagonist of the vacuolar type proton
ATPase responsible for endosomal acidification,31 strongly inhibited the localization of the TLR7 agonist small molecules while
leaving unaltered the localization of CpG-B. We conclude that the
acidic pH present in the MIIC is the driving force for small
molecule TLR7 agonist localization and a key requirement for their
immunostimulatory activity.
Methods
Fluorescently labeled TLR7 agonist small molecules and CpGs
Synthesis of R848, R837, and SM360320, fluorescently labeled or not, is
described in supplemental Methods (available on the Blood Web site; see
the Supplemental Materials link at the top of the online article). CpG-B
(2006): 5⬘-tcg tcg ttt tgt cgt ttt gtc gtt-3⬘ and CpG-A (D19):
5⬘-ggTGCATCGATGCAGggggg-3⬘, labeled or not at the 3⬘-end with 5(6)
carboxytetramethylrhodamine (TAMRA), were from Primm. CpG-B–FITC
was from Invivogen. Bases shown in capital letters are phosphodiester and
those in lowercase letters are phosphorothioate.
Antibodies and chemicals
The following mouse mAbs were from BD PharMingen: FITCconjugated anti–LAMP-1, FITC-conjugated anti-CD63, allophycocyanin
(APC)–conjugated anti–HLA-DR, BD–Horizon V450–conjugated antiCD80, APC–conjugated anti-CD86, APC-conjugated anti–BDCA-2, FITCconjugated anti-CD123 (IL-3R), biotin-conjugated anti–HLA-DR, and
purified anti-TfR. Purified rabbit mAb anti-EEA1 was from Cell Signaling
Technology. Purified rabbit polyclonal anti–LAMP-1 Ab was from Abcam.
Alexa Fluor 488 (AF488)–, AF568-, and AF647-conjugated goat anti–
rabbit F(ab⬘)2 IgG, AF488-conjugated goat anti–mouse F(ab⬘)2 IgG, and
APC-conjugated streptavidin were from Molecular Probes (Invitrogen).
Live-Dead Fixable Aqua Dead Cell Stain kit was from Invitrogen. BafA
was from Sigma-Aldrich.
Luciferase assay
HEK293T cells stably transfected with the firefly luciferase gene under the
transcriptional control of NF-␬B together with human TLR7, TLR8, or
TLR9 (TLR-DST) were made in our laboratory. TLR-DST cells were
cultured overnight at 5 ⫻ 104 cells/well in microclear 96-well plates
(Greiner Bio-One). Cells were stimulated with serial dilutions of compounds in duplicate for 6 hours and then lysed with cell culture lysis reagent
(Promega), according to the manufacturer’s instructions. Luciferase assay
substrate (Promega) was added, and luciferase activity was measured by
SpectraMax L microplate reader (Molecular Devices). Luciferase activity
was expressed as fold induction over nonstimulated cells.
Donors
Buffy coats from healthy HIV-, hepatitis B virus–, and hepatitis C virus–
negative donors were obtained from the Blood Transfusion Section,
Alta Val D’Elsa Hospital, Poggibonsi, Italy. Informed consent was obtained before all blood donations. The study protocol was approved by the
Novartis Research Center Ethical Committee and conformed to the ethical
guidelines of the 1975 Declaration of Helsinki.
Purification and stimulation of pDCs
PBMCs were isolated from fresh buffy coats by Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation, following standard
procedures. Highly purified pDCs (ⱖ 98% BDCA-2⫹CD123⫹) were obtained with Diamond Plasmacytoid Dendritic Cell Isolation Kit (Miltenyi
Biotec), which consists of magnetic depletion of non-pDCs followed by
positive selection of pDCs with anti–BDCA-4 Ab. pDCs were cultured at
5 ⫻ 104 per well in 96-roundbottom plates in RPMI 1640 medium (Gibco)
supplemented with 10% FCS (HyClone), glutamine, penicillin, and streptomycin. Human rIL-3 (100 ng/mL; R&D Systems) was added to the cultures
for 16-24 hours before stimulation with TLR7 agonist small molecules or
CpGs to keep pDCs alive.32
Cytokine production, CD80 and CD86 up-regulation,
and compound uptake
pDCs were stimulated with different concentrations of TLR7 agonist small
molecules or CpGs overnight. IFN-␣ was quantified with human IFN-␣
multiple subtype ELISA kit (PBL Biomedical Laboratories). TNF-␣
and IL-6 were measured with the human proinflammatory 7-plex (TNF-␣,
IL-6, IFN-␥, IL-10, IL-12p70, IL-1␤, IL-8) tissue culture kit (MSD
Technology). Other cytokines were produced at low concentrations, except
IL-8 that was produced constitutively and poorly induced on stimulation
(data not shown). pDCs were washed twice with ice-cold PBS, stained with
anti-CD80 HorizonV450 and anti–CD86-APC, and analyzed for CD80 and
CD86 expression as well as for the fluorescence compound resulting from
uptake by FACS LSRII instrument (Becton Dickinson) with the use of the
BD FACSDiva v6.1.3 software (Becton Dickinson). Data were analyzed
with FlowJo software (TreeStar Inc).
Confocal immunofluorescence microscopy
pDCs were incubated with fluorescent compounds (TLR7 agonist small
molecules or CpG) for 90 minutes. Cells were washed, fixed with 1% paraformaldehyde in PBS at room temperature for 15 minutes, and permeabilized with saponin buffer (0.5% saponin, 1% BSA in PBS). Samples
were blocked for 30 minutes with Image-iT FX signal enhancer (Invitrogen)
and stained with the indicated antibodies in saponin buffer. Cells were
cytocentrifuged on glass slides and mounted with SlowFade Gold antifade
reagent with 4,6 diamidino-2-phenylindole (DAPI; Invitrogen), as described.28 Images were acquired sequentially with separate laser excitations
to avoid cross talks between different fluorophores with the use of either
a Nikon Eclipse TE2000, Radiance 2100 Confocal System (Bio-Rad)
with the acquisition software Lasersharp 2000 (Zeiss) and processed with
Volocity 3.6 (Improvision Inc) or with a LSM 710 confocal microscope
System (Zeiss) and processed with Zen2008 software (Zeiss). An oilimmersion objective (63⫻/1.4 numerical aperture [NA]), with the pinhole
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BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
SMALL MOLECULE TLR7 AGONISTS LOCALIZE TO THE MIIC
5685
Figure 1. Fluorescently labeled R848, R837, and
SM360320 are TLR7 agonists. R848 (A), R837 (B), and
SM (C) were conjugated with TAMRA (in red) or with
Alexa Fluor 488 (AF488; in green) fluorophores at the
indicated tolerant linker site. A spacer was introduced
together with TAMRA (in orange). Both fluorophores
were a mixture of 5-6 regioisomers. Structures depicted
here represent the 5-regioisomer. The biologic activity
of these fluorescently labeled molecules was assessed
on HEK293T cells stably transfected with TLR7 and the
luciferase gene under the transcriptional control of NF-␬B
and (TLR7-DST) in parallel with their nonfluorescent
parent molecules. Luciferase activity was expressed as
fold induction (FI) over nonstimulated cells. EC50 values
are reported.
set for a section thickness of 0.8 ␮m (pinhole set to 1 airy unit in each
channel) was used. Unless otherwise stated, the images show one representative cell, as checked by DAPI staining of the nucleus, of ⱖ 30 cells
analyzed for each sample.
Results
Fluorescently labeled R848, R837, and SM360320 are
TLR7 agonists
Structure-activity relationship analysis of R848 led us to the
identification of a “tolerant” linker site. We used this site to
conjugate R848, R837, and SM360320 (SM) with TAMRA or
AF488 fluorophores (Figure 1). We then assessed the biologic
activity of these fluorescent molecules on HEK293T cells stably
transfected with the luciferase gene under the transcriptional
control of NF-␬B and TLR7 (TLR7-DST) and compared them
with their unlabeled parent molecules. R848 (Figure 1A) activity
on TLR7-DST was strongly decreased on conjugation with
TAMRA (EC50, 50 ␮m for R848-TAMRA vs 0.5 ␮m for R848),
R837 (Figure 1B) activity was slightly decreased on TAMRA
conjugation (EC50, 65.8 ␮m vs 23.1 ␮m for R837) and unaltered on
AF488 conjugation (EC50, 22.7 ␮m vs 23.1 ␮m for R837), whereas SM (Figure 1C) activity was not affected by conjugation with
either fluorophore (EC50, 0.9 ␮m for SM-TAMRA, 0.4 ␮m for
SM-AF488 vs 0.7 ␮m for SM). All fluorescent compounds were
inactive on TLR8-DST (supplemental Figure 1).
The activity of the fluorescently labeled TLR7 agonists was
tested on human pDCs that express TLR7 and TLR9. For practical
Figure 2. Fluorescently labeled R837 activates human pDCs. Human pDCs, purified from fresh buffy coats
and precultured with IL-3 for 24 hours, were stimulated
with different concentrations of R837, labeled or not
with TAMRA or AF488. After overnight incubation, cytokine concentrations in the culture supernatant fluids
were assayed, and cells were analyzed by flow cytometry
for CD80 and CD86 expression and for compound
uptake (TAMRA or AF488 fluorescence). Data shown are
from one donor and are representative of ⱖ 3 independent donors.
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5686
RUSSO et al
reasons in this study pDCs were cultured with IL-3 for 18-24 hours
before stimulation with TLR7 agonist small molecules or CpG to
prevent them from dying.32 pDCs were stimulated overnight with
labeled or unlabeled R848, R837, or SM360320. Cytokine concentrations in the supernatants were measured while cells were stained
for CD80 and CD86 and analyzed by flow cytometry for costimulatory molecule expression and for compound uptake (TAMRA or
AF488 fluorescence). R837-TAMRA and R837-AF488 induced
the production of IFN-␣, TNF-␣, and IL-6 and the up-regulation of
CD80 and CD86 in a dose-dependent manner (Figure 2). Internalization of R837-TAMRA and R837-AF488 was readily detected by
flow cytometry. Similar experiments were performed with fluorescent R848 and SM (supplemental Figure 2A-B). All the fluorescent
compounds tested induced mainly IFN-␣, although production of
TNF-␣ and IL-6 and up-regulation of CD80 and CD86 were also
observed, similar to the parent compounds and CpG-B (supplemental Figure 2C). The ability of each fluorescent compound to activate
human pDCs with respect to its parent compound was in good
agreement with the EC50 measured on TLR7-DST (Figure 1).
Imidazoquinolines localize to the MIIC of human pDCs
The localization of fluorescent TLR7 agonists in pDCs was
determined with the protocol applied by Guiducci et al28 to assess
BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
the localization of CpG-A and CpG-B in these cells. pDCs were
incubated with fluorescent TLR7 agonists or CpGs and stained for
LAMP-1, CD63, or TfR to identify distinct classes of endosomes.33,34 As shown by confocal microscopy (Figure 3A-B),
both imidazoquinolines labeled with TAMRA colocalized with
CpG-B in LAMP-1⫹CD63⫹ endosomes but not with CpG-A. As
a control, the ethanol amine-capped TAMRA fluorophore
when used alone did not localize in any intracellular compartment
(data not shown). However, neither R837-TAMRA nor CpG-B–
TAMRA localized to TfR⫹ endosomes, whereas CpG-A–TAMRA
showed a partial colocalization with TfR and almost no colocalization with R837-AF488 (Figure 3C). The ability of CpGs to
induce IFN-␣ production by freshly isolated human pDCs has
been correlated with their localization in TfR⫹ endosomes (early/
recycling endosomes).28 However, we found that fluorescently
labeled TLR7 agonist small molecules, like their parent compounds
and CpG-B, induced pDCs precultured in IL-3 to produce high
amounts of IFN-␣, yet neither R837-TAMRA nor CpG-B–TAMRA
colocalized with TfR. To confirm the absence of localization in
early endosomes we analyzed fluorescent TLR7 agonist small
molecules and CpG-B for colocalization with EEA1. None of the
fluorescent TLR7 agonist small molecules and CpG-B localized to
EEA1⫹ endosomes (Figure 3D; data not shown).
Figure 3. Imidazoquinolines localize to LAMP1ⴙCD63ⴙ
endosomes but not to TfRⴙEEA-1ⴙ endosomes in
pDCs. Human pDCs, precultured overnight with IL-3,
were stimulated for 90 minutes with (A) R848-TAMRA (20
␮m), R837-TAMRA (50 ␮m), CpG-B–TAMRA (10 ␮m), or
CpG-A–TAMRA (10 ␮m); (B) R837-TAMRA (50 ␮m),
CpG-B–TAMRA (10 ␮m), CpG-A–TAMRA (10 ␮m), or
R837-TAMRA (50 ␮m) together with CpG-B–FITC (10
␮m); (C) R837-TAMRA (50 ␮m), CpG-B–TAMRA (10
␮m), CpG-A–TAMRA (10 ␮m) alone or together with
R837-AF488 (20 ␮m); (D) R837-AF488 (20 ␮m), CpG-B–
FITC (10 ␮m), R848-TAMRA (20 ␮m), or CpG-B–TAMRA
(10 ␮m). Cells were fixed, permeabilized, and stained
with anti–LAMP-1-FITC Ab (A), anti–CD63-FITC Ab (B),
purified mouse mAb anti-TfR followed by anti–mouse Ab
labeled with AF488 (C), or purified rabbit mAb anti-EEA1
followed by anti–rabbit Ab labeled with AF568 (D, top and
upper middle) or AF488 (D, lower middle and bottom).
Images were acquired with a Bio-Rad TE2000-U (A-B) or
a ZEISS LSM 710 (C-D) confocal microscope and an
oil-immersion objective (63⫻/1.4 NA), with the pinhole
set for a section thickness of 0.8 ␮m (pinhole set to 1 airy
unit in each channel). Images were acquired sequentially
with separate laser excitations to avoid cross talks between different fluorophores and processed with Zen2008
software. Images show single cells from 1 representative
donor of 3.
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BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
Collectively, these data show that imidazoquinolines do not
diffuse randomly inside pDCs but concentrate in LAMP1⫹CD63⫹
endosomes despite that they are cell permeable and thus able to
spread readily throughout the cell.
LAMP-1 and CD63 are markers of late endosomes and
lysosomes, which are characterized by an acidic pH.34 Because in
pDCs, like in other antigen-presenting cells, the endolysosomal
compartment is specialized in antigen presentation onto MHC
class II molecules (MHC II),16 we stained for HLA-DR, which is a
MHC II receptor, and LAMP-1 to check whether the endosomes
where imidazoquinolines concentrated belonged to the MIIC. As
shown in Figure 4, we found that R848-TAMRA, R837-TAMRA,
and R837-AF488 localized to endosomes that were positive not
only for LAMP-1 but also for HLA-DR. In addition, R837-TAMRA
and R837-AF488 colocalized with each other in HLA-DR⫹
endosomes. As a control, the ethanol amine-capped AF488 fluorophore alone did not localize in any intracellular compartment
(data not shown).
Purine-like TLR7 agonist SM colocalizes
with imidazoquinolines in the MIIC
To assess if localization in the MIIC is a general feature of
TLR7 agonists, we looked at fluorescent SM, a purine-like
compound that is structurally distinct from imidazoquinolines.
As shown in Figure 5, SM labeled with TAMRA colocalized
with HLA-DR and SM-AF488 colocalized with LAMP-1. Moreover,
SM-AF488 colocalized with SM-TAMRA and with R837-TAMRA.
Thus, TLR7 agonists concentrated in LAMP-1⫹CD63⫹HLA-DR⫹
endosomes of pDCs independently of the compound chemical
scaffold and the fluorophore used for labeling. While we were
writing this article, Shukla et al35 described the synthesis of
yet another imidazoquinoline conjugated with different fluoro-
Figure 4. Fluorescently labeled R848 and R837 localize to
LAMP1ⴙHLA-DRⴙ endosomes in pDCs. pDCs, precultured overnight
with IL-3, were stimulated with R848-TAMRA (20 ␮m), R837-TAMRA (50
␮m), R837-AF488 (20 ␮m) alone or together with R837-TAMRA (50 ␮m)
for 90 minutes. Cells were fixed, permeabilized, and stained with purified
rabbit polyclonal anti-LAMP1 Ab followed by anti–rabbit Ab labeled with
AF488 (top and upper middle) or AF568 (lower middle) and anti–HLA-DRAPC. Images were acquired with a ZEISS LSM 710 confocal microscope
and an oil-immersion objective (63⫻/1.4 NA), with the pinhole set for a
section thickness of 0.8 ␮m (pinhole set to 1 airy unit in each channel).
Images were acquired sequentially with separate laser excitations to avoid
cross talks between different fluorophores and processed with Zen2008
software. Images show single cells from 1 representative donor of 3.
SMALL MOLECULE TLR7 AGONISTS LOCALIZE TO THE MIIC
5687
phores. Those investigators observed an intracellular localization of these compounds in the murine J774 macrophage cell line
that, although not characterized further, was compatible with our
observations.
BafA prevents the concentration of
small molecule TLR7 agonists in the MIIC
The pH of the MIIC is acidic for optimal protease activity
necessary for the generation of peptides to be complexed with
MHC II molecules.36 The acidic luminal environment is maintained
by v-ATPase, which pumps protons into the MIIC lumen. To
investigate whether the acidic pH plays a role in the concentration
of TLR7 agonist small molecules in the MIIC we used BafA to
block v-ATPase and raise the MIIC pH.31 As shown in Figure 6,
pretreatment of pDCs with BafA resulted in the near complete inhibition of SM-AF488 (Figure 6A) and R837-TAMRA
(Figure 6B) localization in LAMP1⫹HLA-DR⫹ endosomes. By
contrast, CpG-B–TAMRA localization in the BafA-treated and untreated cells were indistinguishable (Figure 6C). Moreover, BafA
strongly inhibited R837-TAMRA uptake while leaving CpG-B–
TAMRA uptake unaltered (Figure 6D), confirming what we observed by confocal microscopy (Figure 6B-C). In line with the
fact that BafA is a well-known inhibitor of TLR7- and TLR9induced signaling,26 in our experiments BafA blocked the production of IFN-␣, TNF-␣, and IL-6 in response to SM-AF488,
R837-TAMRA, or CpG-B–TAMRA by pDCs (data not shown).
Blocking of TLR9 signaling by BafA was probably because of
inhibition of proteases responsible for TLR9 cleavage.23,25 Although this mechanism could contribute also to the blockade of
TLR7 signaling,23,25 our findings highlighted that the acidic pH of
the MIIC is required for the localization of TLR7 agonist small
molecules in there.
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BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
RUSSO et al
weak base, passively diffuses inside the cells (fast phase, v-ATPase
independent, insensitive to 4°C) and then starts to accumulate in
the acidic vesicles of the MIIC because it gets protonated and
therefore trapped (slow phase, v-ATPase dependent, blocked at
4°C; Figure 7C).37
Discussion
Figure 5. Purine-like TLR7 agonist SM360320 colocalizes with imiquimod in the
MIIC. pDCs, precultured overnight with IL-3, were stimulated with SM-TAMRA
(20 ␮m) or with SM-AF488 (3 ␮m), alone or together with SM-TAMRA (20 ␮m) or
R837-TAMRA (20 ␮m), for 90 minutes. Cells were fixed, permeabilized, and stained
with anti–HLA-DR-APC or purified rabbit polyclonal anti-LAMP1 Ab followed by anti–
rabbit Ab labeled with AF647. Images were acquired with a ZEISS LSM 710 confocal
microscope and an oil-immersion objective (63⫻/1.4 NA), with the pinhole set for a
section thickness of 0.8 ␮m (pinhole set to 1 airy unit in each channel). Images were
acquired sequentially with separate laser excitations to avoid cross talks between
different fluorophores and processed with Zen2008 software. Images show single
cells from 1 representative donor of 3.
To get further insights on the mechanism of entry and accumulation of small molecule TLR7 agonists we studied the effect of
BafA on R837-TAMRA uptake over time. As shown in Figure 7A,
although BafA had little effect on the R837-TAMRA uptake at
30 minutes, it blocked further uptake of the compound almost
completely at later time points. As expected, CpG-B–TAMRA
uptake was almost unaffected by BafA (Figure 7B). To understand whether the internalization of TLR7 agonist small molecules
was the result of passive diffusion or an active mechanism, we
compared R837-TAMRA uptake at 4°C with uptake at 37°C
(standard culture condition). Although the R837-TAMRA uptake
after 30 minutes of incubation at 4°C was only marginally reduced compared with 37°C, further accumulation of the compound was blocked at 4°C. These results showed that an early
passive diffusion of R837 is followed by an active mechanism
that dramatically increases the accumulation of the TLR7 agonist
in the MIIC (Figure 7A). No CpG-B–TAMRA internalization
occurred at 4°C (Figure 7B), in agreement with previous data
showing that CpG-B enters cells by an active mechanism involving rapid translocation to tubular lysosomal compartment
(LAMP-1⫹) from early endosomes (EEA1⫹TfR⫹).13 On the basis
of these results we propose that R837, which is a cell-permeable
To understand the mechanism of action of small molecule TLR7
agonists more completely, we labeled R848, R837, and SM36020
with TAMRA or AF488 fluorophore and studied their subcellular
localization. All of the fluorescent compounds retained TLR7
agonist activity and induced human pDCs to produce cytokines and
IFN-␣ and to increase expression of costimulatory molecules.
Examination of pDCs by confocal microscopy showed that all
compounds colocalized with CpG-B in LAMP-1⫹CD63⫹HLA-DR⫹
endosomes, independently of their chemotype or labeling fluorophore. Our observations are in line with an elegant study by Sadaka
et al38 showing that MHC II molecules converge with LAMP-1 and
CD63 (but not with TfR and EEA1 markers) in human pDCs
treated with IL-3. LAMP-1 and CD63 are markers of late endosomes and lysosomes, whereas HLA-DR identifies MHC II
molecules. Therefore, the vesicles where small molecule TLR7
agonists accumulate appear to belong to the MIIC.20,38,39 Indeed, in
pDCs, like in other antigen-presenting cells, the endolysosomal
compartment is specialized for MHC II antigen presentation and
is considered as a lysosome-related organelle.39 The function of
MHC II molecules is to access the endolysosomal network, bind
peptides derived from the antigens, and display them on the cell
surface to CD4⫹ T cells. Notably, the regulation of MHC II
expression and transport is completely different in pDCs compared
with conventional DCs (cDCs) from both humans38 and mice.40,41
In particular, cDCs halt the synthesis and degradation of MHC II
after activation, whereas pDCs maintain synthesis and turnover
of MHC II. As a result pDCs are less efficient at presenting antigens than cDCs, but their more dynamic mode of antigen presentation should be advantageous in counteracting viruses that
exhibit mutation rates.40,41 Peptide binding by MHC II molecules
requires the degradation of the invariant chain by endolysosomal proteases. These proteases have been implicated in the
activation of TLR7 and TLR9.23-25 Our data showing that small
molecule TLR7 agonists and CpG-B localize to MIIC suggest
that this compartment is the crossroad where antigens and
MHC II molecules are assembled and nucleic acid–sensing
TLRs signal on encounter with their ligands. The commonality
of proteolytic cleavage to form functionally relevant forms of
both MHC II and TLRs and their colocalization could affect the
way pDCs inform T cells as to the specific nature of distinct pathogenic threats.
Small molecule TLR7 agonists and CpG-B did not localize to
early endosomes (TfR⫹ or EEA1⫹), yet they induced high amounts
of IFN-␣ production. These results are in contrast with the proposed model that in human pDCs TLR9 signaling from early
endosomes (TfR⫹) leads to IFN-␣ production, whereas signaling
from late endosomes (LAMP-1⫹) leads to up-regulation of costimulatory molecules.28 However, our observations are in line with a
recent report from Sasai et al42 which showed that in mouse
BM-derived macrophages TLR9 signals leading to IFN-I production are generated in acidic endosomes (LAMP-2⫹LysoTracker⫹).
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BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
SMALL MOLECULE TLR7 AGONISTS LOCALIZE TO THE MIIC
5689
Figure 6. BafA prevents the localization of small
molecule TLR7 agonists to the MIIC. pDCs, precultured overnight with IL-3, were treated with BafA
(100nM) for 2 hours before stimulation with SM-AF488 at
3 ␮m (A), R837-TAMRA at 50 ␮m (B), or CpG-B–TAMRA
at 10 ␮m (C). After 90 minutes of stimulation, cells were
fixed, permeabilized, and stained with anti–HLA-DR-APC
and purified rabbit polyclonal anti–LAMP-1 Ab followed by anti–rabbit Ab labeled with AF568 (A) or
with AF488 (B-C). Images were acquired with a ZEISS
LSM 710 confocal microscope and an oil-immersion
objective (63⫻/1.4 NA), with the pinhole set for a section
thickness of 0.8 ␮m (pinhole set to 1 airy unit in each
channel). Images were acquired sequentially with separate laser excitations to avoid cross talks between different fluorophores and processed with Zen2008 software. Images show several cells from 1 representative
donor of 3. Cells in squares are shown in higher magnification on the right. (D) After 18 hours of stimulation with
R837-TAMRA (50 ␮m) or CpG-B–TAMRA (10 ␮m), compound uptake by pDCs was evaluated by flow cytometry.
It is unclear why CpG-B is a poor inducer of IFN-␣ by freshly
isolated pDCs28 but a good inducer for IL-3 pretreated pDCs.43
In both pDC populations CpG-B localizes to LAMP-1⫹ endosomes, suggesting that IL-3 treatment leads to changes in the
constituents of these vesicles. Indeed, IL-3 treatment induced the
relocalization of MHC II to LAMP-1⫹ endosomes, and it is
tempting to speculate that key IFN-I–signaling components, such
as IFN regulatory factor 7, are recruited to the MIIC in response to
IL-3 treatment.
A key feature of the MIIC is low pH. Blockade of vesicle
acidification by BafA or NH4Cl (data not shown) abolished the
accumulation of R837-TAMRA in the MIIC, suggesting that
the electrochemical proton gradient is the driving force for the
accumulation of small molecule TLR7 agonists in this compartment. Interestingly, a similar mechanism has been shown
to cause the accumulation of neurotransmitters and hormones
into secretory vesicles.44 At the acidic pH found in the MIIC of
pDCs (pH 5-628), small molecule TLR7 agonists are predicted
to be positively charged and protonated and are expected to
reach concentrations in the acidic compartment ⬃ 100-fold
higher than the rest of the cell.37 This substantial accumulation of compound is consistent with what we observed in live
pDCs (data not shown) and could serve to drive low-affinity
interactions of small molecule TLR7 agonists with TLR7 or a
coreceptor molecule or both above the threshold necessary
for signaling.
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5690
BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
RUSSO et al
Figure 7. BafA prevents the concentration of imiquimod in the MIIC. pDCs, precultured with IL-3 for
24 hours, were treated or not with BafA (100nM) for
2 hours. Then, cells were stimulated with R837-TAMRA
at 50 ␮m (A) or CpG-B–TAMRA at 10 ␮m (B) for the
indicated times at 37°C or at 4°C. Cells were stained
with Live/Dead and analyzed by flow cytometry for compound uptake gating on live cells. The mean fluorescence of cells alone cultured under the same conditions
was subtracted (␦ mean fluorescence). Cell viability was
not affected by BafA treatment. Data were obtained with
pDCs purified from a single donor and were reproduced
with pDCs from 2 other donors. (C) Model for the concentration of TLR7 agonist small molecules in acidic
endosomes on the basis of the previous data. R837 is a
cell-permeable weak base that passively diffuses everywhere inside the cell (fast phase, v-ATPase-independent)
and then starts to accumulate in the class II loading
compartment where it gets protonated and therefore
trapped (slow phase, v-ATPase-dependent). In the presence of BafA, v-ATPase is blocked, and endosomal pH is
no longer acidic. Therefore, R837 does not get protonated in endosomes and does not accumulate there.
Acknowledgments
We thank Michela Brazzoli for her initial support in confocal microscopy analysis; Susanna Aprea, Sara Valentini, and Ugo D’Oro for
providing TLR-DST cell lines; Diego Piccioli for his advice on
pDCs; Giorgio Corsi for artwork; Alberto Visintin, Qian Huang,
Xinming Cai, Tom Wu, and Alex Cortez for discussion; and
Sylvie Bertholet for careful revision of the manuscript.
This work was supported in part by the Transformational
Medical Technologies program (contract HDTRA1-07-9-0001)
from the Department of Defense Chemical and Biologic Defense
program through the Defense Threat Reduction Agency (DTRA).
This work is part of the PhD thesis for C.R.
DTRA was founded in 1998 to integrate and focus the
capabilities of the Department of Defense (DOD) that address the
threat by weapons of mass destruction (WMDSs). DTRA’s mission
is to safeguard the United States and its allies from chemical,
biologic, radiologic, nuclear, and high-yield explosive WMDSs
by providing capabilities to reduce, eliminate, and counter the
threat and mitigate its effect. DTRA combines DOD resources,
expertise, and capabilities to ensure the United States remains
ready and able to address present and future WMDS threats. For
more information on DTRA, visit www.dtra.mil. Transformational
Medical Technologies (TMT) was created by DOD to protect the
warfighter from emerging and genetically engineered biologic
threats by discovering and developing a wide range of medical
countermeasures through enhanced medical research, develop-
ment, and test and evaluation programs. The TMT program office
is matrixed from the joint science and technology office—DTRA
and joint program executive office—chemical and biologic defense
with oversight from the office of the secretary of defense. For more
information on TMT, visit www.tmti-cbdefense.org.
Authorship
Contribution: C.R. performed experiments, analyzed data, and
edited the manuscript; I.C.-T. and R.J. supervised the synthesis
of small molecule TLR7 agonists; L.G.-S. performed experiments; E.H. and Y.I. synthesized small molecule TLR7 agonists;
S.T., C.S., and S.N. advised on flow cytometric experiments and
analysis; M.L.M. advised on study design; J.T. designed and supervised the synthesis of small molecule TLR7 agonists; N.M.V. and
E.D.G. advised on study design and wrote the paper; and E.S.
designed, supervised and performed experiments, and wrote the
manuscript.
Conflict-of-interest disclosure: All authors are employees of
Novartis.
The current affiliation for I.C.-T. is Sanofi-Aventis Oncology,
Cambridge, MA.
Correspondence: Elisabetta Soldaini, Novartis Vaccines &
Diagnostics, via Fiorentina, 1, 53 100 Siena, Italy; e-mail:
[email protected]; and Ennio De Gregorio,
Novartis Vaccines & Diagnostics, via Fiorentina, 1, 53 100 Siena,
Italy; e-mail: [email protected].
References
1. Isaacs A, Lindenmann J. Virus interference, I:
the interferon. Proc R Soc Lond B Biol Sci. 1957;
147(927):258-267.
2. Miller RL, Gerster JF, Owens ML, Slade HB,
Tomai MA. Imiquimod applied topically: a novel
immune response modifier and new class of
drug. Int J Immunopharmacol. 1999;21(1):1-14.
3. Gester S, Wuest F, Pawelke B, Bergmann R,
Pietzsch J. Synthesis and biodistribution of an
18F-labelled resveratrol derivative for small animal positron emission tomography. Amino Acids.
2005;29(4):415-428.
4. Hemmi H, Kaisho T, Takeuchi O, et al. Small antiviral compounds activate immune cells via the
TLR7 MyD88-dependent signaling pathway.
Nat Immunol. 2002;3(2):196-200.
5. Jurk M, Heil F, Vollmer J, et al. Human TLR7 or
TLR8 independently confer responsiveness to
the antiviral compound R-848 [letter]. Nat Immunol. 2002;3(6):499.
6. Stanley MA. Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential. Clin Exp Dermatol. 2002;27(7):571-577.
7. Kurimoto A, Ogino T, Ichii S, et al. Synthesis
and structure-activity relationships of 2-amino8-hydroxyadenines as orally active interferon inducing agents. Bioorg Med Chem. 2003;11(24):
5501-5508.
8. Diebold SS, Kaisho T, Hemmi H, Akira S,
Reis e Sousa C. Innate antiviral responses by
means of TLR7-mediated recognition of singlestranded RNA. Science. 2004;303(5663):
1529-1531.
9. Forsbach A, Nemorin JG, Montino C, et al.
Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
BLOOD, 26 MAY 2011 䡠 VOLUME 117, NUMBER 21
immune responses. J Immunol. 2008;180(6):
3729-3738.
10. Chuang TH, Ulevitch RJ. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine
Netw. 2000;11(3):372-378.
11. Vollmer J, Weeratna R, Payette P, et al. Characterization of three CpG oligodeoxynucleotide
classes with distinct immunostimulatory activities.
Eur J Immunol. 2004;34(1):251-262.
12. Rutz M, Metzger J, Gellert T, et al. Toll-like receptor 9 binds single-stranded CpG-DNA in a
sequence- and pH-dependent manner. Eur J
Immunol. 2004;34(9):2541-2550.
13. Latz E, Schoenemeyer A, Visintin A, et al. TLR9
signals after translocating from the ER to CpG
DNA in the lysosome. Nat Immunol. 2004;5(2):
190-198.
14. Latz E, Verma A, Visintin A, et al. Ligand-induced
conformational changes allosterically activate
Toll-like receptor 9. Nat Immunol. 2007;8(7):
772-779.
15. Haas T, Metzger J, Schmitz F, et al. The DNA
sugar backbone 2⬘ deoxyribose determines tolllike receptor 9 activation. Immunity. 2008;28(3):
315-323.
16. Burgdorf S, Kautz A, Bohnert V, Knolle PA,
Kurts C. Distinct pathways of antigen uptake
and intracellular routing in CD4 and CD8 T cell
activation. Science. 2007;316(5824):612-616.
17. Palucka K, Banchereau J, Mellman I. Designing
vaccines based on biology of human dendritic cell
subsets. Immunity. 2010;33(4):464-478.
18. Krieg AM, Vollmer J. Toll-like receptors 7, 8, and
9: linking innate immunity to autoimmunity.
Immunol Rev. 2007;220:251-269.
19. Liu L, Botos I, Wang Y, et al. Structural basis of
toll-like receptor 3 signaling with double-stranded
RNA. Science. 2008;320(5874):379-381.
20. Bird PI, Trapani JA, Villadangos JA. Endolysosomal proteases and their inhibitors in immunity.
Nat Rev Immunol. 2009;9(12):871-882.
21. Brinkmann MM, Spooner E, Hoebe K, Beutler B,
Ploegh HL, Kim YM. The interaction between the
ER membrane protein UNC93B and TLR3, 7, and
9 is crucial for TLR signaling. J Cell Biol. 2007;
177(2):265-275.
SMALL MOLECULE TLR7 AGONISTS LOCALIZE TO THE MIIC
22. Kim YM, Brinkmann MM, Paquet ME, Ploegh HL.
UNC93B1 delivers nucleotide-sensing toll-like
receptors to endolysosomes. Nature. 2008;
452(7184):234-238.
23. Ewald SE, Lee BL, Lau L, et al. The ectodomain
of Toll-like receptor 9 is cleaved to generate a
functional receptor. Nature. 2008;456(7222):
658-662.
24. Park B, Brinkmann MM, Spooner E, Lee CC,
Kim YM, Ploegh HL. Proteolytic cleavage in an
endolysosomal compartment is required for activation of Toll-like receptor 9. Nat Immunol. 2008;
9(12):1407-1414.
25. Sepulveda FE, Maschalidi S, Colisson R, et al.
Critical role for asparagine endopeptidase in endocytic Toll-like receptor signaling in dendritic
cells. Immunity. 2009;31(5):737-748.
26. Barton GM, Kagan JC. A cell biological view of
Toll-like receptor function: regulation through
compartmentalization. Nat Rev Immunol. 2009;
9(8):535-542.
5691
by a screening procedure for intracellular antigens in eukaryotic cells. J Biol Chem. 1991;
266(5):3239-3245.
34. Eskelinen EL, Tanaka Y, Saftig P. At the acidic
edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 2003;13(3):
137-145.
35. Shukla NM, Malladi SS, Mutz CA, Balakrishna R,
David SA. Structure-activity relationships in
human toll-like receptor 7-active imidazoquinoline analogues. J Med Chem. 2010;53(11):
4450-4465.
36. Trombetta ES, Ebersold M, Garrett W, Pypaert M,
Mellman I. Activation of lysosomal function during
dendritic cell maturation. Science. 2003;299(5611):
1400-1403.
37. Siebert GA, Hung DY, Chang P, Roberts MS. Iontrapping, microsomal binding, and unbound drug
distribution in the hepatic retention of basic drugs.
J Pharmacol Exp Ther. 2004;308(1):228-235.
27. Honda K, Ohba Y, Yanai H, et al. Spatiotemporal
regulation of MyD88-IRF-7 signalling for robust
type-I interferon induction. Nature. 2005;
434(7036):1035-1040.
38. Sadaka C, Marloie-Provost MA, Soumelis V,
Benaroch P. Developmental regulation of MHC II
expression and transport in human plasmacytoidderived dendritic cells. Blood. 2009;113(10):
2127-2135.
28. Guiducci C, Ott G, Chan JH, et al. Properties
regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation. J Exp Med. 2006;203(8):1999-2008.
39. Raposo G, Fevrier B, Stoorvogel W, Marks MS.
Lysosome-related organelles: a view from immunity and pigmentation. Cell Struct Funct. 2002;
27(6):443-456.
29. Gibson SJ, Lindh JM, Riter TR, et al. Plasmacytoid dendritic cells produce cytokines and mature
in response to the TLR7 agonists, imiquimod and
resiquimod. Cell Immunol. 2002;218(1-2):74-86.
40. Young LJ, Wilson NS, Schnorrer P, et al. Differential MHC class II synthesis and ubiquitination
confers distinct antigen-presenting properties on
conventional and plasmacytoid dendritic cells.
Nat Immunol. 2008;9(11):1244-1252.
30. Colonna M, Trinchieri G, Liu YJ. Plasmacytoid
dendritic cells in immunity. Nat Immunol. 2004;
5(12):1219-1226.
31. Yoshimori T, Yamamoto A, Moriyama Y, Futai M,
Tashiro Y. Bafilomycin A1, a specific inhibitor of
vacuolar-type H(⫹)-ATPase, inhibits acidification
and protein degradation in lysosomes of cultured
cells. J Biol Chem. 1991;266(26):17707-17712.
41. van den Hoorn T, Neefjes J. Activated pDCs:
open to new antigen-presentation possibilities.
Nat Immunol. 2008;9(11):1208-1210.
42. Sasai M, Linehan MM, Iwasaki A. Bifurcation of
Toll-like receptor 9 signaling by adaptor protein 3.
Science. 2010;329(5998):1530-1534.
32. Kadowaki N, Antonenko S, Lau JY, Liu YJ. Natural interferon alpha/beta-producing cells link innate and adaptive immunity. J Exp Med. 2000;
192(2):219-226.
43. Jaehn PS, Zaenker KS, Schmitz J, Dzionek A.
Functional dichotomy of plasmacytoid dendritic
cells: antigen-specific activation of T cells versus
production of type I interferon. Eur J Immunol.
2008;38(7):1822-1832.
33. Metzelaar MJ, Wijngaard PL, Peters PJ, Sixma JJ,
Nieuwenhuis HK, Clevers HC. CD63 antigen. A
novel lysosomal membrane glycoprotein, cloned
44. Marshansky V, Futai M. The V-type H⫹-ATPase
in vesicular trafficking: targeting, regulation and
function. Curr Opin Cell Biol. 2008;20(4):415-426.
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2011 117: 5683-5691
doi:10.1182/blood-2010-12-328138 originally published
online April 12, 2011
Small molecule Toll-like receptor 7 agonists localize to the MHC class II
loading compartment of human plasmacytoid dendritic cells
Carla Russo, Ivan Cornella-Taracido, Luisa Galli-Stampino, Rishi Jain, Edmund Harrington, Yuko
Isome, Simona Tavarini, Chiara Sammicheli, Sandra Nuti, M. Lamine Mbow, Nicholas M. Valiante,
John Tallarico, Ennio De Gregorio and Elisabetta Soldaini
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