Report IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Responses Graphical Abstract Highlights d The high-affinity IgE receptor FcεRI facilitates antigen cross- presentation by DCs d IgE and FcεRI efficiently prime CTLs in response to free low- dose soluble antigen d MyD88 or IL-12 induction is not required for cross- presentation via IgE d IgE-mediated cross-presentation by DCs improves anti- tumor responses in vivo Authors Barbara Platzer, Kutlu G. Elpek, ..., Shannon J. Turley, Edda Fiebiger Correspondence edda.fi[email protected]In Brief Platzer et al. demonstrate a mechanism of cross-presentation executed by dendritic cells via IgE and the high-affinity IgE receptor FcεRI. IgE/FcεRI-mediated cross-presentation efficiently induces cytotoxic T cell responses, which are crucial for anti-tumor responses. This pathway provides a mechanistic explanation for epidemiologic data that show an inverse correlation between IgE- mediated allergies and cancer. Platzer et al., 2015, Cell Reports 10, 1–9 March 10, 2015 ª2015 The Authors http://dx.doi.org/10.1016/j.celrep.2015.02.015
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IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Responses
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Report
IgE/FcεRI-Mediated Antige
n Cross-Presentation byDendritic Cells Enhances Anti-Tumor ImmuneResponses
Graphical Abstract
Highlights
d The high-affinity IgE receptor FcεRI facilitates antigen cross-
presentation by DCs
d IgE and FcεRI efficiently prime CTLs in response to free low-
dose soluble antigen
d MyD88 or IL-12 induction is not required for cross-
presentation via IgE
d IgE-mediated cross-presentation by DCs improves anti-
tumor responses in vivo
Platzer et al., 2015, Cell Reports 10, 1–9March 10, 2015 ª2015 The Authorshttp://dx.doi.org/10.1016/j.celrep.2015.02.015
Eleonora Dehlink,1,5 Kai-Ting C. Shade,4 Robert M. Anthony,4 Richard S. Blumberg,3 Shannon J. Turley,2,6
and Edda Fiebiger1,*1Division of Gastroenterology and Nutrition, Boston Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA
02115, USA2Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA3Division of Gastroenterology, Brigham and Women’s Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02115,
USA4Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital,Harvard Medical School, Boston, MA 02129, USA5Present address: Department of Pediatrics and Adolescent Medicine, Division of Pediatric Pulmonology, Allergology and Endocrinology,
Medical University of Vienna, 1090 Vienna, Austria6Present address: Department of Cancer Immunology, Genentech, One DNA Way, South San Francisco, CA 94080, USA*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.celrep.2015.02.015
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
SUMMARY
Epidemiologic studies discovered an inverse as-sociation between immunoglobulin E (IgE)-mediatedallergies and cancer, implying tumor-protectiveproperties of IgE. However, the underlying immuno-logic mechanisms remain poorly understood. Anti-gen cross-presentation by dendritic cells (DCs) is ofkey importance for anti-tumor immunity because itinduces the generation of cytotoxic CD8+ T lympho-cytes (CTLs) with specificity for tumor antigens. Wedemonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and prim-ing of CTLs in response to free soluble antigen at lowdoses. Importantly, IgE/FcεRI-mediated cross-pre-sentation is a distinct receptor-mediated pathwaybecause it does not require MyD88 signals or IL-12induction in DCs. Using passive immunization withtumor antigen-specific IgE and DC-based vaccina-tion experiments, we demonstrate that IgE-mediatedcross-presentation significantly improves anti-tumorimmunity and induces memory responses in vivo.Our findings suggest a cellular mechanism for the tu-mor-protective features of IgE and expand the knownphysiological functions of this immunoglobulin.
INTRODUCTION
It is well established that immunoglobulin E (IgE) plays a key role
in allergies and mounts protective immune responses against
helminthes (Galli and Tsai, 2012). Additionally, evidence for a
role of IgE in tumor immunity has accumulated over the last
decade. Epidemiological studies discovered an inverse correla-
tion between elevated serum IgE levels (as seen in allergic pa-
tients) and the risk of developing childhood leukemia, pancreatic
cancer, brain cancers, and ovarian cancer, indicating a possible
function of IgE in anti-tumor immunity (Jensen-Jarolim et al.,
2008; Josephs et al., 2013). Furthermore, IgE with specificity
for tumor-associated antigens was found in humans (Fu et al.,
2008; Staff et al., 2012), and the existence of tumor-protective
features of IgE was supported by studies that employed murine
models (Daniels-Wells et al., 2013; Daniels et al., 2012; Fu et al.,
2008; Karagiannis et al., 2012; Nigro et al., 2009; Staff et al.,
2012). However, the underlying cellular and molecular mecha-
nisms remain largely unknown.
Tumor eradication depends heavily on the host’s ability to suc-
cessfully induce cytotoxic T cell (CTL) responses. Dendritic cells
(DCs) contribute to tumor defense via major histocompatibility
complex (MHC) class I-restricted cross-presentation, a pathway
that efficiently generates CTLs in response to exogenous anti-
gens such as those derived from tumors (Joffre et al., 2012;
Mende and Engleman, 2005). Importantly, the low amount of
circulating tumor antigens is considered a limiting factor for effi-
cient responses via cross-presentation in vivo. For increased ef-
ficiency in monitoring the antigenic environment, DCs can use
endocytic receptors that facilitate antigen uptake. Targeting of
such receptors (e.g., DEC205) is currently being tested in cancer
immunotherapy trials (Chatterjee et al., 2012; Dhodapkar et al.,
2014; Tacken et al., 2007).
Fc gamma receptors (FcgRs) were among the first receptors
identified to sample antigens for cross-presentation (reviewed
in Platzer et al., 2014b). FcgRs allow DCs to detect antigen in
the form of immunoglobulin G immune complexes (IgG-ICs),
but not in its soluble free form. They belong to the immune
recognition receptor family, with their ligand-binding a chains
Cell Reports 10, 1–9, March 10, 2015 ª2015 The Authors 1
Figure 1. IgE/FcεRI-Mediated Antigen Uptake Allows for Cross-Priming of CTLs in Response to Free Soluble Antigen at Low Dose
(A) Binding and uptake of fluorescently labeled OVA (OVAAF647) by DCs that were pre-loaded with OVA-specific IgE (histogram: red line). Representative
histogram overlay. See also Figure S1.
(B–G) CD8+ T cell priming via IgE.
(B) Schematic overview of IgE/FcεRI-independent (no IgE) and IgE/FcεRI-dependent (plus IgE) antigen sampling.
(C) In vivo T cell proliferation assay. Splenic DCs from IgER-TG and WT mice (without or with IgE) were pulsed in vitro with NP-OVA (0.05 mg/ml) and injected
into WT recipients. Prior to DC injection, recipients received CFSE-labeled CD8+ OT-I T cells. Representative FACS plots and quantification (mean ± SEM of
3 independent experiments, R2 mice per experiment).
(D) In vivo killing assay; dots represent individual mice (n = 2, mean ± SEM).
(E) In vitro T cell proliferation assay. Triplicates ± SEM of a representative experiment (n R 5). Granzyme B production was determined by ELISA. bd, below the
detection limit. Triplicates ± SEM, representative experiment (n = 3).
(legend continued on next page)
2 Cell Reports 10, 1–9, March 10, 2015 ª2015 The Authors
Please cite this article in press as: Platzer et al., IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Re-sponses, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.02.015
Please cite this article in press as: Platzer et al., IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Re-sponses, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.02.015
containing immunoglobulin-like domains and their FcRg-chain
dimer containing ITAM signaling modules. Interestingly, the
high-affinity IgE receptor FcεRI has close structural similarities
to FcgRs (Kinet, 1999). Therefore, we hypothesized that FcεRI
might also contribute to cross-presentation.
Human, but not mouse, DCs constitutively express a trimeric
isoform of FcεRI. Trimeric FcεRI contains the IgE-binding a
chain and the common FcRg-chain dimer, but lacks the
FcεRI-b chain, which is a component of the tetrameric isoform
as expressed on mast cells and basophils in humans and
mice. Comparably to tetrameric FcεRI, trimeric FcεRI serves
to coat the cell surface with monomeric IgE, creating the DC-
bound IgE pool in humans (Galli and Tsai, 2012; Platzer et al.,
2011). Since the DC-specific pool is absent in mice, we used
animals that were humanized for their FcεRI expression on
DCs (IgER-TG animals; Platzer et al., 2014a). Using this
approach, we described an IgE/FcεRI-mediated cross-presen-
tation pathway that allows for the generation of CTLs. We further
demonstrated the contribution of this pathway to anti-tumor im-
mune responses in vivo.
RESULTS
IgE/FcεRI-Mediated Cross-Presentation EfficientlyInduces Proliferation of CTLs in Response to Low Dosesof Free Soluble AntigenWe hypothesized that IgE/FcεRI-mediated uptake of tumor anti-
gens by DCs and consequent cross-presentation allow IgE to
contribute to cancer immunosurveillance. An examination of
DCs from IgER-TG mice that were loaded with ovalbumin
(OVA)-specific IgE and incubated with fluorescently labeled
OVA showed that IgE-bearing DCs were far superior in capturing
soluble free antigen compared with DCs lacking IgE (Figure 1A).
Using the uptake of fluorescently labeled transferrin as a control,
we showed that crosslinking of IgE/FcεRI does not change the
overall endocytic capacity (Figure S1A). By following the intracel-
lular route of IgE/FcεRI in a human cell line commonly used as
model for antigen trafficking (Zwart et al., 2005), we found that
the crosslinked receptor slowly entered Rab5+ early endosomes,
where it remained detectable over a prolonged period of time
(�45 min) and then appeared in Rab7+ and LAMP1+ late
endo-/lysosomal vesicles (Figures S1B and S1C). This slow re-
ceptor trafficking pattern suggests that IgE/FcεRI can indeed
target antigens to compartments that favor MHC class I-specific
presentation by protecting antigenic epitopes against rapid
degradation in the more acidic environment of late endo-/lyso-
somes (Kreer et al., 2011). Using human DCs and human IgE
with specificity for OVA, we next confirmed that IgE/FcεRI-me-
diated uptake shuttles free antigen into Rab14+ endosomes
(Figure S1D), which have been described as cross-presentation
vesicles (Weimershaus et al., 2012). These data suggested that
(F) Signaling induced by NP-BSA through IgE/FcεRI crosslinking does not augmen
loaded with NP-specific IgE (+) or not (�) prior to simultaneous stimulation w
Representative experiment (n = 2).
(G) Comparison of IgE- and IgG-mediated antigen cross-presentation after incub
overview: (I) DCs were pre-incubated with monomeric IgE or IgG1, washed, and t
to form ICs and then added to DCs. NT, not treated with antigen. Triplicates of a
antigen that enters DCs via IgE/FcεRI is targeted for cross-
presentation.
Cross-presentation of antigens by DCs primes naive CD8+
T cells to proliferate and differentiate into CTLs. Thus, we first as-
(B) Vaccination with DCs loaded with tumor-specific antigen via IgE/FcεRI increases tumor-free survival. Control (CTRL) mice did not receive DCs. Unprimed DCs
were loaded with IgE but not incubated with antigen. Pooled data from two experiments; 20 mice per group, CTRL and unprimed, n = 10 mice. Tumor cells were
injected subcutaneously (s.c.).
(C) Tumor-free mice from the experiments shown in (B) were re-challenged with OVA-expressing B16 tumor cells and monitored for tumor growth.
(D and E) Tumor-specific IgE mediates tumor protection in vivo.
(D) Overview of the tumor experiment after passive immunization with IgE. Mice in which expression of FcεRI was restricted to DCs were treated with OVA-
specific IgE or DNP-specific IgE, and OVA-expressing B16 cells were injected i.v.
(E) Tumor count in lungs. Quantification (symbols are representative of n = 5 mice per group, mean ± SEM, **p < 0.01) and representative images are shown.
Please cite this article in press as: Platzer et al., IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Re-sponses, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.02.015
(Figures 3E and S3). This set of data indicates that during Th2-
type inflammation, IL-4 prevents an overshooting CTL response
induced by IgE, and explains why allergen-specific CD8+ T cells
are highly uncommon.
IgE/FcεRI-Mediated Cross-Presentation Affects theEfficiency of Anti-Tumor Responses In VivoThus far, we have shown that IgE/FcεRI-mediated antigen uptake
can be extremely efficient in priming CTL responses. Since in-
duction of tumor-specific CTLs is a major goal in cancer im-
munotherapy, we next performed a classical tumor vaccination
experiment. Our approach involved ex vivo antigen loading of
DCs because IgE/FcεRI crosslinking on mast cells and basophils
can result in systemic activation of the allergic effector cascade,
including increased IL-4 production, which we found to inhibit
CTL priming (Figure 3E). DCs were isolated from IgER-TG mice,
loaded with antigen-specific IgE, pulsed with soluble free OVA,
and injected into recipient mice. Next, mice were injected subcu-
taneously with OVA-expressing B16 melanoma cells (Figure 4A).
6 Cell Reports 10, 1–9, March 10, 2015 ª2015 The Authors
We found significant protection from tumor development, as evi-
denced by prolonged tumor-free survival, in animals that could
use IgE/FcεRI-mediated cross-presentation compared with ani-
mals that could not (Figure 4B). Importantly, 40% of the mice
did not display any signs of tumor until day 50. To test whether
the tumor-free survivors had developed memory T cell re-
sponses, we re-injected the animals with B16 melanoma cells.
Indeed, mice that had been vaccinated with DCs capable of
IgE/FcεRI-mediated cross-presentation remained protected
upon re-challenge (Figure 4C).
We next asked whether in vivo recognition of tumor-specific
antigen via tumor-specific IgE conveys anti-tumor protection
(Figures 4D and 4E). Thus, we used passive immunization with
tumor-specific IgE (i.e., OVA-specific IgE) and tumor-irrelevant
IgE (i.e., dinitrophenyl [DNP]-specific IgE). To avoid activation
of mast cells and basophils, we used an IgER-TG strain in which
the FcεRI expressionwas restricted to DCs (Platzer et al., 2014a).
Whenwemonitored the capacity of OVA-expressing B16 cells to
metastasize to the lung after intravenous injection (Baker et al.,
Barbara Platzer, Kutlu G. Elpek, Viviana Cremasco, Kristi Baker, Madeleine M. Stout,
Cornelia Schultz, Eleonora Dehlink, Kai-Ting C. Shade, Robert M. Anthony, Richard S.
Blumberg, Shannon J. Turley, and Edda Fiebiger
SUPPLEMENTAL FIGURES
Figure S1. Antigen uptake and endo/lysosomal trafficking of activated IgE/FcεRI, related to Figure 1. (A) Antigen uptake and quantification of mean fluorescence intensity (MFI) of DC after incubation with OVAAF647 for 30 min and 60 min. Uptake of transferrin (Trf) is comparable whether DCs are loaded with IgE (gray bars) or not (white bars). (B-C) Trafficking of cross-linked FcεRI into endo/lysosomal compartments in MelJuso cells. (B) Representative images of internalized FcεRI in Rab5+ early endosomes at indicated time points. Co-localization of FcεRI with Rab5 was quantified after 5 min and 20 min and compared to transferrin uptake. (C) Co-localization of FcεRI with Rab5+ endosomes, Rab7+ endosomes, and LAMP-1+ lysosomes was quantified at indicated time points. (D) Human DCs use IgE/FcεRI to shuttle free soluble antigen into Rab14+ cross-presentation compartments. Co-localization of OVAAF647 and human IgE in Rab14+ endosomes. Monocyte-derived DCs were preloaded with OVA-specific human IgE and incubated with OVAAF647 for 15 min (blue), fixed, and stained for IgE (red) and Rab14 (green). Representative images are shown.
Figure S2. Functional characterization of Myd88-deficient IgER-TG animals on the C57BL/6 background, related to Figure 2. (A) FcεRI expression on DCs from MyD88-/- x IgER-TG mice is similar to DCs from IgER-TG mice that express MyD88. Splenic DCs purified by magnetic selection from indicated mouse strains were cultured overnight in the presence or absence of IgE and analyzed for FcεRI expression. Binding of IgE increases FcεRI surface expression as described. (B) IgE-independent antigen cross-presentation requires the presence MyD88 in DCs. In vitro T cell proliferation induced by DCs following IgE/FcεRI-independent antigen uptake. DCs isolated from the indicated mouse strains were incubated with 5 µg/ml OVA. LPS was added where indicated (+) and co-cultured with OT-I T cells.
Figure S3. CTL priming via IgE- and IgG- immune complexes (ICs) is regulated by IL-4, related to Figure 3. DCs were pulsed with preformed IgE-IC or IgG-ICs and co-cultured with CD8+T cells in the presence of recombinant IL-4 for 3 days.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Flow cytometry
Cell suspensions were pretreated with mouse Fc-BlockTM (BD Biosciences/ Pharmingen) and
incubated with fluorophore-labeled antibodies or appropriate isotype controls. Data acquisition
was performed with a BD FACSCanto II (BD Bioscience). Data analysis was done using BD
FACSDiva and FlowJo (Treestar INC.) software. DC subsets were sorted on a Dako-
Cytomation MoFlo Legacy (Dako North America Inc.) equipped with Summit for Windows
Version 4.3. The following antibodies were used in this study: Phycoerythrin-(PE) and
allophycocyanin-(APC) anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or
BioLegend), Pacific blue- and APC-anti-CD11c, Alexa Fluor® (AF)-647 anti-mouse CD8α
(Biolegend), and APC-Cy7 anti-mouse CD3 (eBiosciences).
Antibodies and Reagents
Hapten-specific chimeric human IgE anti-4-hydroxy-3-nitrophenylacetyl (NP) was derived from
Jw 8/5/13 cells (clone JW8/1, AbD Serotech). NP(7)-PE, NP-BSA, and NP-OVA (~19 NP-
moieties per OVA) were purchased from Biosearch Technologies Inc. NP-OVA was size
purified (10kDa > NP-OVA < 100 kDa) to remove OVA peptide and higher MW OVA
aggregates in the soluble antigen solution with Amicon Ultra Ultracel 10k and 100k centrifugal
filters (EMD Millipore). To generate human OVA-specific IgE, the OVA-specific variable
chains were cloned from the OVA-specific IgE hybridoma, TOε (Sawada et al., 1997), and fused
to the constant chain cDNAs of human IgE. Next, OVA-specific human IgE was generated by
transient transfection of light and heavy chains into HEK293T cells, as described (Nimmerjahn
and Ravetch, 2005).
Microscopy
Human monocyte-derived DCs were attached to poly-lysine D coated coverslips and preloaded
with 300ng/ml human OVA-specific IgE over night at 37°C. Cells were incubated for 15 min
with 1µg/ml AF-647-labeled OVA at 37°C. After fixation, cells were stained with rabbit α-
Rab14 (Sigma Aldrich, 1:250d, 5µg/ml) and goat α-rabbit AF-488 (Life Technologies). IgE was
detected with mouse PE labeled-anti-human IgE (Invitrogen). All cells were fixed with 4%
paraformaldehyde (Electron Microscopy Sciences) for 20 min before mounted using Prolong
Antifade reagent (Invitrogen). Confocal images were acquired on a Nikon TE2000 inverted
microscope coupled to a Yokogawa spinning disk confocal unit (Perkin-Elmer Inc.) and an Orca
AG scientific-grade cooled CCD camera (Hamamatsu Photonics K.K.). Slidebook software
(Intelligent Imaging Innovations Inc.) was used for image capture, and processing. All
microscopy was performed at the Harvard Digestive Disease Center Core Facility.
Measurement of cytokine production
IL-2, granzyme B, and IL-12p40 production of the DC/T cell cultures was measured using
commercially available Ready SET Go ELISAs!® (eBioscience).
Quantification of soluble free antigen uptake through IgE
DCs were isolated from spleens of IgER-TG animals and pre-loaded overnight at 37°C with
OVA-specific IgE (clone E-C1, Chondrex Inc.). Cells were washed to remove unbound IgE
before Alexa Fluor®(AF)-647-labeled OVA (0.05µg/ml) and AF-568-labeled transferrin
(Molecular Probes) were added. DCs were incubated at 37°C for indicated times, washed with
ice-cold medium, and analyzed by flow cytometry.
IgE-loading of DCs, IgE-dependent uptake of free antigen and uptake of immune
complexes
DCs were isolated from spleens using CD11c MicroBeads (Miltenyi Biotec). Purity (>90%) was
assessed by flow cytometry. For IgE-mediated uptake of soluble antigen (i.e. NP-OVA) DCs
were cultured over night at 37°C with NP-specific IgE (500 ng/ml). DCs were cultured in RPMI-