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IMMUNOBIOLOGY
Commonly used prophylactic vaccines as an alternative for synthetically produced
TLR ligands to mature monocyte-derived dendritic cells
Gerty Schreibelt,1 Daniel Benitez-Ribas,1 Danita Schuurhuis,1 Annechien J. A. Lambeck,1 Maaike van Hout-Kuijer,1
Niels Schaft,2 Cornelis J. A. Punt,3 Carl G. Figdor,1 Gosse J. Adema,1 and I. Jolanda M. de Vries1,3,4
1Department
of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;
of Dermatology, University Hospital Erlangen, Erlangen, Germany; 3Department of Medical Oncology, Radboud University Nijmegen Medical
Centre, Nijmegen, The Netherlands; and 4Department of Paedriatric Hemato-Oncology, Radboud University Nijmegen Medical Centre, Nijmegen, The
Netherlands
2Department
Currently dendritic cell (DC)–based vaccines are explored in clinical trials, predominantly in cancer patients. Murine
studies showed that only maturation with
Toll-like receptor (TLR) ligands generates
mature DCs that produce interleukin-12
and promote optimal T-cell help. Unfortunately, the limited availability of clinicalgrade TLR ligands significantly hampers
the translation of these findings into DCbased vaccines. Therefore, we explored
15 commonly used preventive vaccines
as a possible source of TLR ligands. We
have identified a cocktail of the vaccines
BCG-SSI, Influvac, and Typhim that contains TLR ligands and is capable of optimally maturing DCs. These DCs (vaccine
DCs) showed high expression of CD80,
CD86, and CD83 and secreted interleukin12. Although vaccine DCs exhibited an
impaired migratory capacity, this could
be restored by addition of prostaglandin
E2 (PGE2; vaccine PGE2 DCs). Vaccine
PGE2 DCs are potent inducers of T-cell
proliferation and induce Th1 polarization.
In addition, vaccine PGE2 DCs are potent
inducers of tumor antigen-specific CD8ⴙ
effector T cells. Finally, vaccine PGE2–
induced DC maturation is compatible with
different antigen-loading strategies, including RNA electroporation. These data
thus identify a new clinical application for
a mixture of commonly used preventive
vaccines in the generation of Th1-inducing clinical-grade mature DCs. (Blood.
2010;116(4):564-574)
Introduction
Dendritic cells (DCs) are the most potent professional antigenpresenting cells of the immune system. On infection or inflammation, immature DCs (imDCs) are activated and differentiate
into mature DCs that instruct and activate B and T lymphocytes,
the mediators of adaptive immunity.1 Currently, DC-based
immunotherapy is explored in clinical trials, predominantly in
cancer patients,2,3 including hematologic malignancies.4 Antigenloaded autologous DCs are administered to patients with the
intention of inducing antigen-specific T- and B-cell responses.
Although DC-based immunotherapy induces immunologic responses, thus far, only a limited number of clinical responses
have been observed.3 It remains unclear why some patients
respond to DC-based immunotherapy and others do not, but it
has been suggested that the current protocols used to generate
mature DCs may not result in optimal Th1 responses. To date,
most clinical studies use tumor necrosis factor-␣ (TNF-␣),
interleukin-1␤ (IL-1␤), IL-6, and prostaglandin E2 (PGE2) for
DC maturation. However, murine studies have shown that
activation of DCs by solely proinflammatory cytokines yields
DCs that support CD4⫹ T-cell clonal expansion but fail to
efficiently direct helper T-cell differentiation. DCs polarize
immune responses via secretion of soluble factors, such as
cytokines.5 IL-12p70 favors the differentiation of interferon-␥
(IFN-␥)–producing T helper 1 cells.6,7 In addition, IL-12p70 is
involved in the activation of CD8⫹ effector T cells8,9 and is thus
relevant in enhancing in vivo antitumor responses.10,11 DCs
matured with proinflammatory cytokines produce very little or
no IL-12p70. In contrast, exposure of DCs to pathogenassociated molecular patterns (PAMPs), such as Toll-like receptor (TLR) ligands, induces DCs that produce high levels of
IL-12p70 and promote efficient T-cell help.12-15
PAMPs are recognized by pattern recognition receptors.
TLRs are part of this family of proteins and sense microbial and
viral products. TLR engagement on DCs induces maturation and
cytokine secretion, including IL-12p70.16 In humans, 11 TLRs
have been described for which many specific ligands have been
identified.17-19 The signaling pathways associated with ligation
to each of these TLRs are not identical; therefore, distinct
biologic responses are initiated on ligation.20 Human monocytederived DCs (moDCs) express TLR1, TLR2, TLR3, TLR4,
TLR5, TLR6, and TLR8.18 We recently demonstrated that a
maturation cocktail combining the TLR3 ligand polyinosinicpolycytidylic acid (poly(I:C)) and the TLR7/8 ligand R848
supplemented with PGE2 yields DCs with both high migratory
capacity and IL-12p70 production on T-cell encounter.12 TLRmediated maturation of ex vivo–generated human moDCs may
thus be used to improve immunologic and clinical responses in
DC vaccination of cancer patients.
One possible drawback of the use of purified TLR ligands
was recently reported. We showed that the presence of the TLR3
ligand poly(I:C) in the maturation cocktail activates innate
immune mechanisms that induce an antiviral state in DCs.21,22
Submitted October 30, 2009; accepted April 20, 2010. Prepublished online as
Blood First Edition paper, April 27, 2010; DOI 10.1182/blood-2009-11-251884.
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.
© 2010 by The American Society of Hematology
564
BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
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BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
VACCINES INDUCE DC MATURATION VIA TLRs
This may lead to RNA degradation or inhibition of protein
synthesis, thus hampering protein expression of tumor antigen–
mRNA after electroporation. Therefore, we searched for an
alternative maturation cocktail containing clinically applicable
TLR ligands that give rise to IL-12–producing mature DCs and
allow electroporation with mRNA encoding tumor antigens.
Whereas several TLR ligands have been shown to yield
mature Th1-directing DCs, limited availability in Good Manufacturing Practice conditions impedes the use of these TLR
ligands for the generation of DCs for immunotherapy. Vaccines
against infectious diseases frequently contain PAMPs derived
from bacteria or viruses. These PAMPs may be recognized by
pattern recognition receptors and may therefore be good candidates for stimulation of DCs via TLRs. Generally, vaccines
consist of either live attenuated or killed microorganisms,
subunits of microorganisms, or inactivated toxic compounds,
often supplemented with adjuvants that further stimulate an
immune response against the pathogens. The importance of TLR
ligation for efficacy of vaccines is now underscored by investigators working on vaccine development,23,24 but for the majority
of currently used vaccines the presence of TLR ligands has not
been studied.25 In this study, we searched for new sources of
clinical-grade TLR ligands that can be used for maturation of
clinically applicable DCs. To activate DCs that express TLR
receptors, we explored the use of commercially available
prophylactic vaccines. We have identified vaccines that contain
TLR ligands and are capable of inducing DC maturation. This
knowledge provides a new application for these clinically
applicable agents: clinical-grade DC stimulators.
565
Methods
Vaccines
Vaccines include the following: Act-HIB (Aventis Pasteur), BCG vaccin
SSI (Nederlands Vaccin Instituut [NVI]), BMR vaccin (Bof- Mazelen-,
Rubellavaccin, NVI), FSME-IMMUN (Baxter AG), Havrix (GlaxoSmithKline BV), HBVAXPRO (Sanofi Pasteur MSD), Infanrix-IPV ⫹ HIB
(GlaxoSmithKline BV), Influvac 2007/2008 (Solvay Pharmaceuticals),
Neisvac-C (Baxter AG), Pneumo-23 (Aventis Pasteur), Prevenar (Wyeth),
Geïnactiveerd Rabiesvaccin Mérieux HDCV (Sanofi Pasteur MSD), Stamaril (Sanofi Pasteur MSD), Tetanus (NVI), and Typhim Vi (Sanofi Pasteur
MSD). Table 1 gives detailed information about the contents of the
vaccines.
TLR ligand screening
The presence of TLR ligands was tested on recombinant human embryonic
kidney 293 (HEK293) cell lines functionally expressing a given TLR
protein and a reporter gene driven by a nuclear factor-␬B–inducible
promoter. A recombinant HEK293 cell line for the reporter gene only was
used as negative control. Positive control ligands are PAM2 (100 ng/mL)
for HEK293-hTLR2, lipopolysaccharide (LPS); K12 (100 ng/mL) for
HEK293-hTLR4, and Flagellin (1 ␮g/mL) for HEK293-hTLR5. Vaccines
(10⫻ diluted) were added to the reaction volume. TLR ligand screenings
were performed by InvivoGen Europe.
Generation of DCs from peripheral blood precursors
DCs were generated from peripheral blood mononuclear cells (PBMCs)
prepared from leukapheresis products or from buffy coats as described.26,27
Buffy coats were obtained from healthy volunteers according to institutional guidelines. Plastic-adherent monocytes from leukapheresis or buffy
Table 1. Vaccine descriptions
Vaccine
Infectious agent
Disease
Type of vaccine
Supplier
Adjuvant
Bacteria
PREVENAR
Streptococcus pneumoniae
Pneumonia, otitis media, meningitis
Conjugated subunit
Wyeth
AlPO4
PNEUMO 23
Streptococcus pneumoniae
Pneumonia, otitis media, meningitis
Subunit
Aventis Pasteur
None
Tetanus
Clostridium tetani
Tetanus
Subunit
NVI
AlPO4, thiomersal
TYPHIM Vi
Salmonella typhi
Typhoid fever
Subunit
Sanofi Pasteur
None
Act-HIB
Haemophilis Influenzae type b
Meningitis, epiglottitis, pneumonia type b
Conjugated subunit
Aventis Pasteur
Tetanus toxoid
NEISVAC-C
Neisseria meningitidis
Meningitis, sepsis
Conjugated subunit
Baxter
Al(OH)3/tetanus toxoid
BCG
Mycobacterium bovis
Tuberculosis
Live attenuated
NVI
None
Viruses
HAVRIX
Hepatitis A virus
Liver disease, cancer
Inactivated
GlaxoSmithKline AlO(OH)
HBVAXPRO
Hepatitis B virus
Liver disease, cancer
Recombinant
Sanofi Pasteur
AlPO4
Al(OH)3
subunit
FSME
Tick-borne encephalitis virus
Tick-borne encephalitis
Inactivated
Baxter
Rabies
Rabies virus
Rabies
Inactivated
Sanofi Pasteur
Neomycin
STAMARIL
Yellow fever virus
Jaundice, kidney and liver failure
Live attenuated
Sanofi Pasteur
None
BMR
Measles virus
German measles, respiratory tract infection,
Live attenuated
NVI
None
Subunit, inactivated,
GlaxoSmithKline AlPO4, AlO(OH), tetanus
Mumps virus
mumps, meningitis, orchitis
Rubella virus
INFANRIX-IPV
Corynebacterium diphtheriae
Diphtheria
⫹HIB
Clostridium tetani
Tetanus
conjugated
toxoid
Pertussis
Bortella pertussis
Poliomyelitis, paralysis
Poliovirus
Meningitis, epiglottitis, pneumonia type b
Haemophilis influenzae type b
INFLUVAC
2006-2007,
INFLUVAC
2007-2008
Influenza virus A
Influenza virus B
Flu, respiratory diseases
Inactivated subunit
Solvay Pharma
None
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566
SCHREIBELT et al
coats were cultured in X-VIVO 15 medium (Lonza Walkersville) supplemented with 2% pooled human serum (HS; Bloodbank Rivierenland), IL-4
(500 U/mL), and granulocyte-macrophage colony-stimulating factor (800 U/
mL; both CellGenix). On day 6 or 7, DCs were either kept immature or one
of the following maturation cocktails was added for 48 hours: 10 ng/mL
recombinant TNF-␣ (CellGenix), 5 ng/mL IL-1␤ (ImmunoTools), 15 ng/
mL IL-6 (CellGenix), and 10 ␮g/mL PGE2 (Pharmacia; conventional DCs
[cDCs]); 20 ␮g/mL poly(I:C) (Sigma-Aldrich), and 5 ␮g/mL R848 (Axxora)
without PGE2 (TLR DCs) or with 10 ␮g/mL PGE2 (TLR PGE2 DCs); BCG
(4%), Typhim (4%), and Influvac (4%) without PGE2 (vaccine DCs) or with
10 ␮g/mL PGE2 (vaccine PGE2 DCs). Single vaccines were added at a
concentration of 5%.
Flow cytometric analysis
The phenotype of DCs was determined by flow cytometry. The following
primary monoclonal antibodies (mAbs) or appropriate isotype controls
were used: anti–human leukocyte antigen (HLA)–ABC (W6/32), anti–HLADR/DP (Q5/13), and anti-CD80 (all BD Biosciences), anti-CD83 (Beckman
Coulter), anti-CD86 (BD Biosciences PharMingen), anti-CCR7 (R&D
Systems), followed by Alexa 488–conjugated goat anti–mouse IgG (Invitrogen). Intracellular staining of gp100 and tyrosinase was performed as
described22 with NKI/beteb (IgG2b) and T311 (IgG2a; Cell Marque). Flow
cytometry was performed with a FACSCalibur flow cytometer equipped
with CellQuest software (BD Biosciences).
In vitro migration assays
For random migration on fibronectin, flat-bottomed 96-well plates (Corning
Life Sciences) were coated with 20 ␮g/mL fibronectin (Roche Diagnostics)
for 60 minutes at 37°C and blocked with 0.01% gelatin (Sigma-Aldrich) for
30 minutes at 37°C. A total of 4000 DCs per well were seeded on
fibronectin-coated plates and recorded for 60 minutes at 37°C, after which
migration tracks of individual DCs were analyzed using an automated
cell-tracking system.28
For CCR7-mediated migration, transwell inserts with 5 ␮m pore size
polycarbonate membranes (Corning Life Sciences) were preincubated with
100 ␮L of X-VIVO 15/2% HS in 24-well plates, each well containing
600 ␮L of medium. A total of 1 ⫻ 105 DCs were seeded in the upper
compartment. CCL21 (10 or 100 ng/mL; R&D Systems) was added to the
lower wells. Spontaneous migration and kinesis were measured by incubation of the cells in a transwell without or with 100 ng/mL CCL21 in both the
upper and the lower well, respectively. DCs were allowed to migrate for
60 minutes in a 5% CO2, humidified incubator at 37°C. After 60 minutes,
DCs were harvested from the lower chamber and counted by flow
cytometry. All conditions were tested in duplicate.
Determination of chemokine and cytokine production
IL-12p70 production was measured in the supernatants 48 hours after
induction of maturation using a standard sandwich enzyme-linked immunosorbent assay (ELISA; Pierce Biotechnology).
MIP1␣, RANTES, and IP-10 production was measured using FlowCytomix Simplex kits (Bender MedSystems).
MLR
The ability of DCs to induce Th1 cells was studied in a mixed lymphocyte
reaction (MLR). DCs were added to 1 ⫻ 105 freshly isolated allogeneic
nonadherent PBLs from a healthy donor in the presence or absence of
neutralizing anti–human IL-12 antibody (R&D Systems). Cytokine production was measured in MLR supernatants after 48 hours by cytometric bead
array (Th1/Th2 Cytokine CBA 1; BD Biosciences PharMingen).
KLH-specific proliferation assay
Cellular responses against the protein keyhole limpet hemocyanin (KLH)
were measured in a proliferation assay. In our vaccination studies, KLH is
added to immature DC cultures as an immunomonitoring tool. Peripheral
BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
blood lymphocytes (PBLs) were isolated from blood samples from
3 patients taken after 4 biweekly vaccinations with mature DCs. The
PBMCs were plated in a 96-well tissue-culture microplate with autologous
cDCs, vaccine DCs or vaccinePGE2 DCs that were cultured with or without
KLH. After 4 days of culture, 1 ␮Ci/well of tritiated thymidine was added
for 8 hours, and incorporation of tritiated thymidine was measured in a
␤-counter.
Generation of CD45RAⴙCD8ⴙ gp100-specific T cells
The vectors pGEM4Z-TCR␣296 and pGEM4Z-TCR␤296 are encoding
TCR-␣ and -␤ chains originating from a gp100:280-288/HLA-A2-specific
CTL clone. Gp100-specific T cells were generated by transferring the TCR
␣ and ␤ chain to T cells by electroporation of RNA, resulting in transient
expression of the TCR chains as described previously.29 In vitro transcription of gp100 TCR RNA was performed as described.29
CD8⫹CD45RA⫹ T cells were isolated from PBMCs of an HLA-A2.1–
positive donor. Monocytes were removed via adherence, and CD8⫹ T cells
were isolated by positive isolation using fluorescein isothiocyanate (FITC)–
conjugated anti–human CD8 (BD Biosciences) and anti-FITC microbeads
(Anti-FITC Multisort kit; Miltenyi Biotec) according to the manufacturer’s
instructions. Subsequently, CD45RA⫹ T cells were isolated from the CD8⫹
T-cell fraction by negative selection using CD45RO microbeads (Miltenyi
Biotec). Purity of CD8⫹CD45RA⫹ T cells was 90% to 95%, as assessed by
double staining using FITC-conjugated anti–human CD8 and phycoerythrin (PE)–conjugated anti–human CD45RA mAbs (BD Biosciences).
For RNA electroporation, CD45RA⫹CD8⫹ T cells or CD8⫹ T cells
were washed once with phosphate-buffered saline and once with OptiMEM
without phenol red (Invitrogen). A total of 10 to 12 ⫻ 106 cells were
incubated for 3 minutes with 15 to 20 ␮g of RNA in 200 ␮L OptiMEM in a
4-mm cuvette (Bio-Rad). Subsequently, cells were pulsed in a Genepulser
Xcell (Bio-Rad). Pulse conditions were square-wave pulse, 500 V, 5 ms.
Immediately after electroporation, the cells were transferred to X-VIVO 15
medium without phenol red (Cambrex) supplemented with 6% HS. After
4 hours of incubation at 37°C, cells were frozen in liquid nitrogen.
Expression of the gp100 TCR was verified by flow cytometry using
PE-conjugated anti-TCRV␤14 mAb from Coulter Immunotech (supplemental Figure 1A, available on the Blood Web site; see the Supplemental
Materials link at the top of the online article). Functionality of the
gp100-specific T cells was shown by up-regulation of the early activation
marker CD69 after overnight stimulation with gp100:280-288 peptideloaded HLA-A2⫹ imDCs (supplemental Figure 1B).
Electroporation of DCs
Mature DCs were washed twice in phosphate-buffered saline and once in
OptiMEM without phenol red (Invitrogen). A total of 20 ␮g of RNA (gp100
or tyrosine RNA, Curevac) was transferred to a 4-mm cuvette (Bio-Rad),
and 10 ⫻ 106 DCs were added in 200 ␮L of OptiMEM and incubated for
3 minutes before being pulsed with an exponential decay pulse at 300 V,
150 ␮F in a Genepulser Xcell (Bio-Rad) as described previously.22
Immediately after electroporation, the cells were transferred to warm
(37°C) X-VIVO 15 without phenol red (Cambrex) supplemented with 6%
HS, and left for at least 2 hours at 37°C, before further manipulations were
performed.
gp100-specific activation of CD45RAⴙCD8ⴙ T cells and
CD8ⴙ T cells
moDCs from an HLA-A2.1⫹ donor were matured with different maturation
cocktails for 48 hours and loaded with either specific peptide (gp100:280288) or control peptide (gp100:154-167 or tyrosinase:369-376) for 1 hour
(1 ␮g/7 ⫻ 103 DCs). DCs (7 ⫻ 103 per well) were washed and coincubated
with CD45RA⫹CD8⫹ gp100:280-288-specific T cells derived from the
same donor (5 ⫻ 104 per well) in round-bottom 96-well plates. T-cell
stimulatory capacity of electroporated DCs was tested by coculturing
7 ⫻ 103 DCs 4 hours after electroporation with gp100-encoding or control
(tyrosinase) mRNA with 5 ⫻ 104 CD8⫹ gp100:280-288-specific T cells.
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BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
VACCINES INDUCE DC MATURATION VIA TLRs
After overnight incubation, CD69 expression was measured by flow
cytometry using FITC-conjugated mouse anti–human CD69 (BD Biosciences PharMingen), and IFN-␥ production was measured using a
standard sandwich ELISA (Pierce Chemical). After 4 days of culture,
1 ␮Ci/well of tritiated thymidine was added for 8 hours, and incorporation
of tritiated thymidine was measured in a ␤-counter. After 5 days, granzyme
B expression was measured by flow cytometry using PE-conjugated mouse
antihuman granzyme B antibodies (Sanquin). Antigen-specific degranulation was measured in gp100;280 TCR-expressing CD8⫹ T cells cocultured
with differently matured DCs for 5 hours in the presence of Golgi-stop
(monensin; BD Biosciences) and PE-Cy5–labeled anti-CD107a (BD Biosciences PharMingen) as described previously.30 CD107a expression was
measured by flow cytometry.
RNA isolation
Total RNA was isolated from DC cultures using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions, with minor modifications. RNA integrity was determined by analyzing the ribosomal 28S and
18S bands on 1% agarose gel. The reverse transcription reaction was
performed using Moloney murine leukemia virus reverse transcriptase
(Invitrogen) according to the manufacturer’s instructions. For each sample,
an “-RT” control was included in which the reverse transcriptase was
replaced by diethyl pyrocarbonate–treated Milli-Q. cDNA was stored at
⫺80°C until further use.
Quantitative PCR
Quantitative analysis of gene expression in DCs was performed using
SYBR Green-based quantitative polymerase chain reaction (PCR). The
quantitative PCR reactions were performed in a 25-␮L volume containing
12.5 ␮L of SYBR Green mix (Applied Biosystems), 1.5 ␮L of forward/
reverse primer (300nM end concentration), 4.5 ␮L of Milli-Q, and 5 ␮L of
cDNA dilution. Reactions were performed on an ABI 7900HT Sequence
Detection System (Applied Biosystems). Analysis was performed using
Sequence Detection software (Version 2.0, Applied Biosystems). Primer
sequences are available on request and were designed using the freely
accessible Primer Bank program.31
Statistics
Data were analyzed using the Student t test and 1-way analysis of variance.
P values less than .05 were considered to be statistically significant.
Results
Vaccines contain TLR ligands
TLR ligands, such as LPS or poly(I:C), are frequently used for in
vitro DC maturation. However, the availability of synthetic TLR
ligands for clinical usage is limited. To search for alternative
Act-HIB
Vaccines induce DC maturation and IL-12 production
Because the TLR ligand screening revealed that several vaccines
contain TLR ligands, we studied whether these vaccines can be
used for DC maturation in vitro to obtain clinically applicable
TLR-matured DCs. Given that, in addition to TLR, other pattern
recognition receptors such as dectin-1 are involved in antigenpresenting cell maturation,32 we also included vaccines that did not
activate TLRs tested in TLR ligand screening. The vaccines were
added at 5% (vol/vol) concentration to the culture medium of
imDCs at day 6 for 48 hours. The majority of the vaccines were
nontoxic, and the viability was comparable with that of cDCs in the
concentrations used (Figure 2A). Only Infanrix and tetanus strongly
affected cell viability (25% viable cells), even when added at 1%
(vol/vol; data not shown).
To study the effect of all 15 vaccines on DC maturation, the
expression of major histocompatibility molecule HLA-DR/DP,
costimulatory molecules CD80 and CD86, and the DC-maturation
marker CD83 at the surface of DC was measured by flow
cytometry. Act-HIB, BCG, and Typhim significantly increased the
expression of CD80 on moDCs compared with imDCs (Figure 2B).
BCG
BMR
0
1
0
TLR-
TLR2
TLR4
TLR5
2
OD
1
Infanrix
2
OD
OD
OD
sources of clinical-grade TLR ligands for maturation of clinically
applicable DCs, we tested 15 readily available vaccines (Table 1)
for their capacity to interact with extracellular TLRs known to be
expressed by moDCs (TLR2, TLR4, and TLR5). HEK293 cells
stably transfected with plasmids encoding human TLR genes were
used to investigate the 15 vaccines. The HEK293 cell line was
selected for its null or low basal expression of endogenous TLR
genes. Four of 15 vaccines were able to activate TLR-expressing
HEK293 transfectants (Figure 1). TLR2-mediated activation was
observed with BCG (tuberculosis vaccine containing live attenuated Mycobacterium bovis) and Infanrix (containing antigens of
diphtheria, Clostridium tetani, pertussis, poliovirus, and Haemophilus influenzae type b), whereas Act-Hib (vaccine against H influenzae
type b) and Infanrix were able to activate TLR4. BCG activated the
TLR2-expressing HEK293 cell line as efficient as the TLR2 ligand
PAM2. BMR, a vaccine composed for vaccination against measles,
mumps, and rubella, was able to activate TLR5. The remaining
11 vaccines did not activate one of the TLRs tested (data not shown).
To exclude that vaccine adjuvants play a major role in the
observed DC activation, vaccine preparations were compared that
all share the same adjuvant (Table 1). Because we observed that
neither all vaccines containing aluminum hydroxide nor all vaccines with aluminum phosphate induced TLR activation, we can
exclude a prominent role for the adjuvants present in the vaccine
formulations in activating TLRs.
2
2
1
0
TLR-
TLR2
TLR4
TLR5
Positive control
567
1
0
TLR-
TLR2
TLR4
TLR5
TLR-
TLR2
TLR4
TLR5
Vaccine
Figure 1. Vaccines contain TLR ligands. Vaccines were added (10⫻ diluted) to recombinant HEK293 cell lines, functionally expressing a given TLR protein and a reporter
gene driven by an nuclear factor-␬B–inducible promoter. TLR stimulation was measured as activation of the reporter gene. The negative control value for each clone is the
background signal. Positive control ligands used are: PAM2 for the HEK293-hTLR2 cell line, LPS K12 for the HEK293-hTLR4 cell line, and Flagellin for the HEK293-hTLR5 cell
line. Data are expressed as optical density (OD) values.
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BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
SCHREIBELT et al
**
2
*
1
0
imDC
cDC
TLR-DC
Act-HIB
BCG
BMR
FSME
Havrix
HBVaxPro
Infanrix
Influvac
Neisvac-C
Pneumo23
Prevenar
Rabies
Stamaril
Tetanus
Typhim
0
E
HLA-DR/DP
CD80
C
6
D
CD83
**
5
4
*
3
2.5
Fold increase MFI
25
CD80
**
2
1
0
CD83
CD86
CD86
2.0
1.5
1.0
0.5
0.0
cDC
Act-HIB
BCG
BMR
FMSE
Havrix
HBVaxPro
Infanrix
Influvac
Neisvac-C
Pneumo23
Prevenar
Rabies
Stamaril
Tetanus
Typhim
*
**
50
**
Fold increase MFI
75
3
cDC
Act-HIB
BCG
BMR
FMSE
Havrix
HBVaxPro
Infanrix
Influvac
Neisvac-C
Pneumo23
Prevenar
Rabies
Stamaril
Tetanus
Typhim
B
100
Fold increase MFI
F
**
600
400
300
200
Typhim
Tetanus
Rabies
Stamaril
Prevenar
Pneumo23
Infanrix
Influvac
Neisvac-C
HBVaxPro
FSME
Havrix
BCG
BMR
0
cDC
100
Act-HIB
BCG
500
imDC
cDC
IL-12p70 (pg/mL)
viability (%)
A
cDC
Act-HIB
BCG
BMR
FMSE
Havrix
HBVaxPro
Infanrix
Influvac
Neisvac-C
Pneumo23
Prevenar
Rabies
Stamaril
Tetanus
Typhim
568
Figure 2. Vaccines induce DC maturation. imDCs were incubated with the conventional cytokine cocktail (TNF-␣, IL-6, IL-1␤, and PGE2) or with different preventive vaccines
for 48 hours. (A) Viability was analyzed by Trypan blue exclusion. Data are mean ⫾ SD of 3 independent experiments performed with DCs from different donors. **P ⬍ .01
compared with cDCs. (B-D) The expression of maturation markers HLA-DR/DP, CD80 (B), CD83 (C), and CD86 (D) was measured by flow cytometry. Results are shown as fold
increase of mean fluorescence intensity relative to imDCs. Data are mean ⫾ SEM of 3 experiments with different donors. *P ⬍ .05. **P ⬍ .01. The dotted line indicates fold
increase 1.0 (unchanged fluorescence intensity compared with imDCs). (E) Example of expression of HLA-DR/DP, CD80, CD83, and CD86 (bold line) on cDCs and DCs
treated with BCG. The thin line represents the isotype control. (F) At 48 hours after addition of the vaccines, IL-12p70 secretion was measured in the supernatant by ELISA. Per
condition, each symbol represents 1 donor. Mean values are shown for each vaccine. **P ⬍ .01 compared with cDCs.
Influvac and rabies caused a moderate up-regulation of CD80. In
addition, Typhim significantly increased CD83 expression on
moDCs (Figure 2C). Act-HIB, BCG, and Typhim slightly increased
the expression of CD86 (Figure 2D). Figure 2E shows the
expression of maturation markers on DCs matured with the
conventional cytokine cocktail or with BCG. Although BMR
contains a TLR5 ligand, it did not enhance the expression of
maturation markers on moDCs.
On TLR triggering, DCs rapidly produce high levels of
IL-12p70.12,33 We analyzed the IL-12p70 concentration in the
culture medium of DCs 48 hours after addition of the 15 vaccines.
Cytokine-matured DCs (IL-1␤, TNF-␣, IL-6, PGE2; cDCs) hardly
produced any IL-12p70, whereas BCG and Typhim induced
secretion of IL-12p70 (Figure 2F), suggesting that these vaccines
activate TLRs on DCs.
Combined vaccines have a synergistic effect on DC maturation
Previous studies described that different TLRs act in synergism and
induce a stronger immune response when activated simultaneously
or successively.33,34 Therefore, we combined vaccines that contain
different TLR ligands and/or induced DC maturation. We compared different combinations of vaccines (data not shown) and the
cocktail of BCG, Influvac, and Typhim (all 4% vol/vol) induced
optimal expression of maturation markers and IL-12p70 production. This vaccine cocktail was chosen for further analysis. The
morphology of DCs matured with BCG, Influvac, and Typhim
(vaccine DCs) was comparable with that of DCs matured with the
TLR ligands R848 or poly(I:C)12 (and data not shown). Vaccine
DCs had an elongated phenotype and were more adherent to plastic
than cDCs, which have a semiround appearance with multiple
dendrites (data not shown). Vaccine DCs have a fully mature
phenotype, and expression of maturation markers is comparable
with the expression on cDCs (Figure 3A). The phenotype of
vaccine DCs was stable for at least 24 hours after removal of
vaccines and cytokines from the culture medium (data not shown).
Compared with DCs treated with single vaccines, such as BCG or
Typhim, IL-12p70 production of vaccine DCs was strongly enhanced, indicating a synergistic effect of the separate vaccines
(Figure 3B).
PGE2 is essential for migration of vaccine DC
For clinical studies, it is important that DCs are able to migrate
from the injection site to the T-cell areas of lymph nodes to present
tumor antigens to the targeted T cells, even when injected
intranodally. Migration to the lymph nodes is mediated by the
chemokines CCL19 and CCL21 and their receptor CCR7.35 The
migratory capacity was tested 48 hours after vaccine-induced
maturation in a random migration assay on the extracellular matrix
protein fibronectin and in a transwell migration assay toward the
lymph node chemokine CCL21. In the random migration assay,
single cells were tracked with an automated cell-tracking system
and their traversed paths were analyzed.28 Compared with cDCs,
random migration of vaccine DCs was strongly decreased (Figure
4A). In addition, vaccine DCs showed reduced CCR7-mediated
migration toward CCL21 compared with cDCs (Figure 4C).
However, the addition of PGE2 to the vaccine cocktail, which
increased expression of the chemokine receptor CCR7 (Figure 4B),
restored the migratory capacity of vaccine DCs, both on fibronectin
(Figure 4A) and toward CCL21 (Figure 4C). Addition of PGE2 did
not affect the expression of maturation markers (Figure 3A).
Although addition of PGE2 to the vaccines reduced IL-12p70
production, vaccine PGE2 DCs still produce 100-fold more IL12p70 than cDCs (Figure 3B).
Vaccine PGE2 DCs have high stimulatory capacity
For effective immunotherapy, DCs need to have a high T-cell
stimulatory capacity and induce Th1 cells. Vaccine DCs produce
high levels of the inflammatory and Th1-attracting chemokines
MIP1␣, RANTES, and IP-10 (supplemental Figure 2). Although
addition of PGE2 reduced the production of these chemokines,
vaccine PGE2 DCs produce more MIP1␣, RANTES, and IP-10
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BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
A
HLA-DR/DP
CD80
VACCINES INDUCE DC MATURATION VIA TLRs
CD83
B
CD86
**
10000
*
cDC
IL-12p70 (pg/mL)
cDC
569
vaccine-DC
vaccinePGE2-DC
1000
100
10
C
D
C
2-
E
G
eP
va
c
ci
n
va
c
ci
ne
-D
C
cD
im
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at
ur
e
D
C
1
Figure 3. Combined vaccines have a synergistic effect on DC maturation. DCs were matured for 48 hours with the conventional cytokine cocktail (TNF-␣, IL-6, IL-1␤, and
PGE2), preventive vaccines (BCG, Typhim, and Influvac), or vaccines with PGE2, and the expression of maturation markers and IL-12p70 production was evaluated. (A) The
expression of maturation markers HLA-DR/DP, CD80, CD83, and CD86 (bold line) was measured by flow cytometry. The thin line represents the isotype control. (B) IL-12p70
production was measured by ELISA in the supernatant of DC cultures 48 hours after maturation. Per condition, each symbol represents 1 donor. Mean values are shown for
each maturation cocktail. *P ⬍ .05, **P ⬍ .01 compared with cDCs.
than imDCs and cDCs. To analyze whether vaccine DCs induce a
Th1 type of immune response, we measured the secretion of
cytokines by PBLs in supernatants of primary MLRs (Figure 5A).
cDCs induced very low levels of the Th1 cytokines IFN-␥ and
TNF-␣, whereas cocultures of vaccine DCs and vaccine PGE2 DCs
showed considerably higher production of these cytokines. Neutralization of IL-12 reduced the production of IFN-␥ in primary MLRs
with vaccine DCs or vaccine PGE2 DCs, suggesting that IL-12 is
involved in the induction of Th1 responses by vaccine DCs and
vaccine PGE2 DCs (Figure 5B). The secretion of IL-10 was slightly
increased in cocultures with both vaccine DCs and vaccine PGE2
DCs, but the secreted levels were less than 70 pg/mL. There were
no differences in secretion of Th2 cytokines IL-4 and IL-5 between
cDCs, vaccine DCs, and vaccine PGE2 DCs.
We also investigated whether vaccine DCs are capable of
stimulating antigen-specific T cells, which was demonstrated by
KLH-specific proliferation of PBLs isolated from patients who had
been previously vaccinated with KLH-loaded DCs. Figure 5C demon-
strates that vaccine DCs and vaccine PGE2 DCs are able to induce
KLH-specific T-cell proliferation. Interestingly, PBLs from these patients showed already a higher proliferation in cocultures with vaccine
DCs and vaccine PGE2 DCs cultured without KLH, which may be
directed against one of the vaccines used for DC maturation.
Besides skewing CD4 T cells toward a Th1 cytokine profile,
IL-12p70 is involved in the activation and induction of CD8⫹
T cells with CTL activity.8,9 To study whether vaccine PGE2 DCs
are capable of inducing effector T cells, differently matured,
peptide-loaded DCs were coincubated with autologous naive
CD8⫹CD45RA⫹ T cells expressing gp100:280-288–specific
T-cell receptor-␣ and -␤. All differently matured DCs induced
peptide-specific T-cell activation, as demonstrated by CD69
expression and IFN-␥ production after 16 hours of coincubation
of DCs and CD8⫹CD45RA⫹ T cells (Figure 6A and B,
respectively). Interestingly, naive CD8⫹ T cells activated by
DCs matured in the presence of PGE2 (PGE2 DCs: cDCs, TLR
PGE2 DCs, vaccine PGE2 DCs) produced higher levels of IFN-␥
**
100
CCR7
B
*
75
cDC
50
25
0
cDC
vaccines
vaccines + PGE
2
vaccine-DC
C
migrated cells (% of cytokine DC)
Figure 4. PGE2 is essential for migration of vaccine DCs.
(A) Random migration on fibronectin. cDCs, vaccine DCs, and
vaccine PGE2 DCs were added to a fibronectin-coated plate, and
migration of individual cells was monitored for 60 minutes. Data
represent the percentage of migrating cells (mean ⫾ SEM) of
3 experiments with cells from different donors. For each experiment, migration of 50 cells per condition was monitored. *P ⬍ .05.
**P ⬍ .01. (B) The expression of CCR7 (bold line) on cDCs,
vaccine DCs, and vaccine PGE2 DCs was measured by flow
cytometry. The thin line represents the isotype control.
(C) CCR7-mediated chemotaxis of cDCs, vaccine DCs, and
vaccine PGE2 DCs was determined by the number of cells that
had migrated into the lower compartment of a transwell system
containing 10 or 100 ng/mL CCL21, counted by flow cytometry. To
measure spontaneous migration, cells were incubated in a
transwell without CCL21 in the upper and lower compartment
(medium) or with 100 ng/mL CCL21 in both compartments (kinesis). Migration of cDCs in the presence of 100 ng/mL CCL21 was
regarded as 100% (100% corresponds to 26 190 ⫾ 10 636 migrated cells). The graph represents means ⫾ SEM from 3 experiments (with cells from different donors) performed in duplicate.
*Significant difference (P ⬍ .05) compared with medium. #Significant difference (P ⬍ .05) compared with vaccine DCs.
migrating cells (% of total)
A
125
*
*
100
75
50
cDC
vaccine-DC
vaccine-PGE2-DC
*#
*#
25
0
CCL21
CCL21
vaccinePGE2-DC
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BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
SCHREIBELT et al
vaccine-PGE 2 -DC
30
20
10
0
IFN γ
TNF α IL-10
IL-5
IL-4
IL-2
139
cDC
139
9
5
2
46
vaccine-DC
vaccinePGE2-DC
1444
285
67
3
92
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2236
117
56
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43
205
*
100
≠
≠
+KLH
-KLH
300
#
200
***
#
100
0
0
vaccine-DC
vaccine-PGE2-DC
C
40
***
400
D
vaccine-DC
control
anti-IL-12
no
50
200
cD
C
cDC
va
cc
in
eD
va
C
cc
in
ePG
E
2D
C
60
C
*
proliferation (% of cDC)
B
cytokine production (fold increase)
A
IFNγ production (% of vaccine-DC)
570
Figure 5. Vaccine PGE2 DCs have a high stimulatory capacity. (A) The profile of cytokines secreted by PBLs on contact with allogeneic cDCs, vaccine DCs, and vaccine
PGE2 DCs was measured by cytokine bead array. The graph represents the fold change in cytokine production of vaccine DCs and vaccine PGE2 DCs relative to cDCs of 3
different donors. The table presents the mean concentration (picograms per milliliter) of each cytokine in absolute numbers for all conditions. (B) IFN-␥ production by PBLs
) or presence (
) of neutralizing anti–IL-12 antibody. The graph represents the mean ⫾ SD from 2
cocultured with vaccine DCs or vaccine PGE2 DCs in the absence (
experiments (with cells from different donors) of relative IFN-␥ production compared with control vaccine DCs (100% corresponds to 48 ⫾ 5 pg/mL). *P ⬍ .05. (C) KLH-specific
proliferation of PBLs from a patient vaccinated with KLH-loaded DCs. PBLs were cocultured with autologous DCs matured with the cytokine cocktail, vaccines, or vaccines with
PGE2 with or without KLH. Proliferation was measured by incorporation of tritiated thymidine. The graph represents mean ⫾ SEM counts per minute relative to cDCs ⫹ KLH
represents DCs loaded with KLH; and
represents DCs without
(100% corresponds to 1201 ⫾ 818 cpm) of 3 experiments with different donors, performed in triplicate.
KLH. *P ⬍ .05. ***P ⬍ .001. ⫽Significant difference (P ⬍ .05) with cDCs with KLH. #Significant difference (P ⬍ .05) with cDCs without KLH.
PGE2 DCs induce CTL with the capacity to kill target cells, we
have transfected effector CTLs with mRNA encoding the
gp100:280 T-cell receptor and measured CD107a surface expression after stimulation with differently matured gp100:280loaded DCs. CD107a surface expression is a measure for
degranulation of cytotoxic granules and correlates with target
killing by CTLs.30 Like granzyme B expression, antigen-specific
CD107a surface expression on CD8⫹ T cells was induced by all
***
100
***
***
75
50
25
*
50
cD
C
0
**
***
50000
40000
30000
20000
***
**
10000
C
C
D
*
*
TL
*
100
*
50
25
0
gp100280-288
10
irrelevant peptide
C
C
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*
20
cD
E
CD107a surface expression (%)
TL
TL
cD
C
0
D
C
proliferation (cpm)
60000
***
**
***
100
cD
**
70000
granzyme B expression (% of cDC)
C
***
***
TL
cD
R
-D
TL
C
R
-P
G
E
2D
C
va
cc
in
va
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2D
C
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150
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-D
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cc
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ne
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C
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C
***
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C
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cc
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ne
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cc
C
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E
2D
C
B
125
C
CD69 expression (% of cDC)
A
IFNγ production (% of cDC)
than T cells activated by DCs matured in the absence of PGE2
(Figure 6B). Similarly, PGE2 DCs induced stronger antigenspecific CD8⫹ T-cell proliferation compared with DCs matured
in the absence of PGE2 (Figure 6C). Moreover, only T cells
incubated for 5 days with peptide-loaded PGE2 DCs express the
effector T-cell marker granzyme B (Figure 6D), suggesting that
naive CD8⫹ T cells incubated with PGE2 DCs, including vaccine
PGE2 DCs, acquire CTL function. To study whether vaccine
Figure 6. Vaccine PGE2 DCs induce antigen-specific effector
CD8ⴙ T cells. Naive CD45RA⫹CD8⫹ T cells specific for gp100:
280-288 (50 000 per well) were cocultured with autologous cDCs,
TLR DCs, TLR PGE2 DCs, vaccine DCs, or vaccine PGE2 DCs
(7000 per well) that were loaded with either gp100:280-288 or an
irrelevant peptide. After 16 hours, antigen-specific activation of
CD8⫹ T cells was analyzed by measurement of CD69 surface
expression (A) and secretion of IFN-␥ in the supernatant (B).
T-cell proliferation was analyzed by 3H-thymidine incorporation
after 4 days (C). Granzyme B expression was measured by
intracellular FACS staining after 5 days (D). Antigen-specific
degranulation was measured by CD107a surface expression on
gp100;280 TCR-expressing PBLs cocultured for 5 hours with
differently matured DCs in the presence of Golgi-stop and
represents DCs loaded
PE-Cy5–labeled anti-CD107a (E).
represents DCs
with specific peptide (gp100:280-288); and
loaded with irrelevant peptide. (A-B,D) Mean ⫾ SEM values of
the relative mean fluorescence intensity (A,D) or relative IFN-␥
production (B) compared with cDCs of 3 (B) or 4 (A,D) independent experiments performed with cells from different donors;
100% corresponds to 40% ⫾ 27% CD69-positive cells (A),
834 ⫾ 697 pg/mL IFN-␥ (B), and a mean fluorescence intensity of
238 ⫾ 363 (D). Panel C shows mean ⫾ SEM of 1 representative
experiment of 4 performed. (E) Mean ⫾ SEM values of the
percentage of cells expressing CD107a. *P ⬍ .05. **P ⬍ .01.
***P ⬍ .001.
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BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
VACCINES INDUCE DC MATURATION VIA TLRs
differently matured DCs; however, PGE2 DCs induced stronger
CD107a surface expression than DCs matured in the absence of
PGE2 (Figure 6E).
Overall, these data show that vaccine PGE2 DCs can stimulate
both CD4⫹ T helper cells and CD8⫹ CTLs in an antigen-specific
manner.
gp100- or tyrosinase-mRNA, and protein expression was analyzed
using flow cytometry 4 hours after electroporation. Electroporation of
both cDCs and vaccine PGE2 DCs was very effective, as indicated by
high expression of gp100 and tyrosinase (Figure 7B). To examine
whether DCs electroporated with gp100-encoding mRNA can present
gp100-derived epitopes to T cells, we incubated cDCs and vaccine
PGE2 DCs, 4 hours after electroporation with gp100 mRNA or control
mRNA (tyrosinase), with gp100:280-specific CD8⫹ T cells. CD69
expression, IFN-␥ production, and IP-10 production by gp100:280
specific CD8⫹ T cells were induced by gp100 mRNA–electroporated
cDCs and vaccine PGE2 DCs, but not by tyrosinase mRNA–
electroporated DCs (Figure 7C). Together, these data show that gp100
mRNA–electroporated vaccine PGE2 DCs efficiently process the gp100
protein and present the processed peptides in major histocompatibility
complex class I to specific T cells.
Vaccine PGE2 DCs can efficiently be electroporated with mRNA
encoding tumor antigens
A crucial aspect of DC-based immunotherapy is the method of
antigen delivery to DCs. We previously showed that maturation
with the TLR3 ligand poly(I:C) hampers protein expression of
tumor antigen-mRNA after electroporation because of increased
expression of the viral sensor RIG-I and the effector molecules
PKR and 2,5-OAS.22 Therefore, we examined whether vaccines
also provoke the expression of these proteins. We measured gene
expression of PKR, RIG-I, and 2,5-OAS in DCs matured with
poly(I:C), which has been shown to induce the expression of these
genes, the single vaccines BCG, Influvac, and Typhim, or with a
cocktail of these vaccines. poly(I:C) strongly increased the expression of PKR, RIG-I, and 2,5-OAS (Figure 7A). Influvac induced
low mRNA expression of these molecules, whereas BCG and
Typhim had no effect. The vaccine cocktail induced expression of
PKR, RIG-I, and 2,5-OAS, which was, however, very low.
Because vaccines did not induce strong expression of viral sensors
and effector molecules, we next investigated whether vaccine DCs can
be efficiently electroporated with mRNA encoding the tumor antigens
gp100 and tyrosinase, which we use for DC vaccination of melanoma
patients.36 cDCs and vaccine PGE2 DCs were electroporated with
Discussion
0.5
0.4
0.3
0.2
0.1
C
cc
in
e-
D
m
Ty
ph
i
va
va
In
flu
va
c
)
po
ly
B
(I:
C
G
0.0
D
m
e-
(I:
po
ly
C
0.0
2,5-OAS
0.6
C
0.1
in
C
0.2
D
m
ein
Ty
ph
i
va
cc
C
G
In
flu
va
c
C
(I:
po
ly
B
)
0.0
cc
0.1
0.3
Ty
ph
i
0.2
0.4
In
flu
va
c
0.3
0.5
)
0.4
0.6
C
G
0.5
0.7
B
0.7
0.6
RIG-I
0.8
C
mRNA expression (relative to GAPDH)
PKR
mRNA expression (relative to GAPDH)
In this study, we identified commonly used preventive vaccines as
an alternative source of clinical-grade TLR ligands. We demonstrate that these vaccines induce DC maturation, yielding clinicalgrade DCs that have high expression of maturation markers and
high production of the Th1-polarizing cytokine IL-12. Addition of
PGE2 to the vaccine cocktail gave rise to migratory DCs that are
potent inducers of an MLR and antigen-specific T-cell activation.
Importantly, vaccine DCs also express and present tumor antigens
after electroporation with mRNA encoding these antigens.
0.8
B
vaccinePGE2-DC
cDC
72%
82%
65%
71%
gp100
tyrosinase
CD69
35
*
30
*
25
20
15
10
5
0
cDC
IP10
IFNγ
tyrosinase
vaccine-PGE2-DC
gp100
IFNγ production (pg/mL)
gp100
15
tyrosinase
*
*
10
5
0
cDC
vaccine-PGE2-DC
gp100
IP-10 production (pg/mL)
C
CD69 expression (% positive cells)
Figure 7. Vaccine-PGE2 DCs do not express viral
sensors and effector molecules and express and
present tumor antigens after mRNA electroporation.
(A) DCs were matured for 48 hours with the conventional
cytokine cocktail (cDCs), combined vaccines (BCG, Typhim, and Influvac; vaccine DCs), poly(I:C), or with
separate preventive vaccines. mRNA levels of PKR,
RIG-I, and 2,5-OAS were determined using quantitative
PCR 48 hours after maturation. (B) DCs were matured
for 48 hours with the conventional cytokine cocktail or
combined vaccines (BCG, Typhim, and Influvac; vaccine
DCs) with PGE2. DCs were electroporated with mRNA
encoding gp100 or tyrosinase. After 4 hours, gp100 or
tyrosinase protein expression was determined by FACS
analysis. Filled curves represent staining with specific
antibody; and thin-lined curves represent the isotype
control. Numbers indicate percentage of cells expressing
the antigen. Data are a representative experiment of
3 performed. Average antigen expression (mean ⫾ SEM)
of 3 experiments was 73 ⫾ 7 for gp100 and 76 ⫾ 3 for
tyrosinase on vaccine PGE2 DCs, and 65 ⫾ 7 for
gp100 and 73 ⫾ 4 for tyrosinase on cDCs. (C) A total of
50 000 gp100:280-specific CD8⫹ T cells were coincubated with 7000 cDCs or vaccine PGE2 DCs 4 hours
after electroporation with gp100 mRNA or tyrosinase
mRNA as a control, and T-cell activation was analyzed
by measurement of CD69 surface expression (left),
IFN-␥ production (middle), and IP-10 production (right).
Data are mean ⫾ SEM of 1 representative experiment
of 3 performed in triplicate (CD69 and IFN-␥) or
1 representative experiment (IP-10). *P ⬍ .05.
mRNA expression (relative to GAPDH)
A
571
tyrosinase
350
300
250
200
150
100
50
0
cDC
vaccine-PGE2-DC
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
572
SCHREIBELT et al
Several PAMPs that are used as immunomodulators have been
shown to induce DC maturation.14,37,38 Our difficulties in obtaining these
products for clinical studies and for the production of DC vaccines
prompted us to search for alternatives. We here show that various
preventive vaccines contain different TLR ligands and stimulate DCs. In
line with previous studies in human blood DCs39,40 and murine DCs,41,42
we found that BCG activates human TLR2 and induces moDC
maturation and IL-12 production. Because mycobacterial antigens are
also recognized by other pathogen recognition receptors, including the
C-type lectins DC–specific intercellular adhesion molecule-3–grabbing
nonintegrin (SIGN) and dectin-1,32,43 it is probable that these pattern
recognition receptors are also involved in DC activation by BCG and
may act synergistically with TLR2 in our DC cultures. Act-HIB contains
TLR4 ligands and enhances CD80 expression on moDCs. Infanrix
activated both TLR2 and TLR4, thus making it a promising candidate
for DC maturation. However, Infanrix was too toxic for the DCs to be
used in our studies. Although BMR activated TLR5 on transfected
HEK293 cells, it did not induce a mature phenotype or IL-12 production
when added to our DC cultures. Previous studies describing TLR5
expression on human moDCs are inconsistent.18 Conceivably, TLR5
expression on our moDCs is either absent or too low to induce effective
DC maturation. For Influvac and Typhim, we did not find any ligands of
extracellular TLRs using the TLR ligand screening. However, Typhim
enhanced the expression of maturation markers on moDCs and induced
IL-12p70 production. Influvac induced some up-regulation of mRNA
expression of viral sensors and effector molecules, which is mediated by
TLR3 ligation,21 indicating that DCs are activated by Influvac as well.
Moreover, our data show that stimulation with a cocktail of BCG,
Influvac, and Typhim gives rise to fully mature DCs that produce large
amounts of IL-12p70, suggesting that these 3 vaccines have a synergistic effect on DC maturation. Possibly, Influvac and Typhim activate
intracellular TLRs, which could not be tested with the HEK293 system,
or Influvac and Typhim act on distinct receptors on DCs that operate in
synergism with TLRs, thus enhancing DC activation.33,34
For efficient antigen presentation to T cells, DCs used for vaccination of cancer patients need to migrate from the injection site to T-cell
areas of regional lymph nodes. Our data show that, compared with
cDCs, vaccine DCs have a strongly reduced migratory capacity, as we
previously demonstrated for TLR DCs matured with poly(I:C) and
R848.12 DCs homing to the lymph nodes are guided by chemokines
CCL19 and CCL21, ligands for the chemokine receptor CCR7. The
presence of CCR7 on the DC cell surface is induced by PGE2 and is
essential for DC migration to the lymph node.44-46 In line with our
previous studies,12 addition of PGE2 to the maturation cocktail enhanced
surface expression of CCR7 and partially restored the migratory
capacity of vaccine DCs. Although the migratory capacity of vaccine
PGE2 DCs was not as high as that of cDCs, vaccine PGE2 DCs migrate
efficiently compared with both imDCs and vaccine DCs that do not
migrate at all.47
Although PGE2 restores the motility of vaccine-matured DCs,
its presence during DC maturation reduced IL-12p70 production,
as shown before.45,46,48 However, in our studies, secreted levels are
still 100-fold higher than that of cDCs. And in line with previous
studies,12,49 our data show that the levels produced by vaccine
PGE2 DCs are sufficient to induce IFN-␥– and TNF-␣–secreting
Th1 cells, which produce only low levels of IL-4 and IL-5.
Together, our data show that, although they produce reduced levels
of IL-12p70 compared with vaccine DCs, vaccine PGE2 DCs have
the capacity to induce antigen-specific T-cell activation and Th1
responses. Based on IL-12 production during maturation and the
induction of Th1 responses, we expected that TLR DCs and
BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
vaccine DCs would induce stronger CTL responses than cDCs.
Unexpectedly, stimulating naive CD8⫹ T cells by cDCs, vaccine
PGE2 DCs and TLR PGE2 DCs resulted in equal levels of effector
markers. We hypothesize that a mature phenotype is more important in the direct activation of CD8⫹ T cells in the assays used here
than cytokines produced by the DCs. The elevated levels of IL-12
produced by vaccine PGE2 DCs and TLR PGE2 DCs may have a
more indirect effect on CD8⫹ T cells by Th1 induction. Indeed, the
neutralization of IL-12 in a CD4-dependent allogeneic response
significantly reduced IFN-␥ production.
An important aspect of DC vaccination is the method of antigen
loading of the DCs. To date, most clinical studies use DCs loaded
with tumor lysates or tumor peptides.3,50 Alternatively, DCs can be
loaded with RNA coding for tumor antigens, preferentially via
electroporation because this method is not considered gene therapy
and can therefore be used to generate clinical-grade DC vaccines.
We and others recently showed that electroporation of DCs
matured with the TLR3 ligand poly(I:C) is very ineffective. This is
caused by the induction of an antiviral state in the DCs by
poly(I:C). poly(I:C) is synthetic dsRNA that triggers the expression
of viral sensors and effector molecules,22,38 which may lead to RNA
degradation or inhibition of protein synthesis, thus hampering
effective protein expression after RNA electroporation. We here
demonstrate that, in contrast to poly(I:C), the vaccine cocktail does
not trigger the expression of viral sensors and effector molecules up
to a level that RNA electroporation is reduced. Interestingly,
Influvac enhanced the expression of RIG-I, PKR, and 2,5-OAS to
some extent, despite negative results in the TLR ligand screening.
However, the vaccine cocktail did not suppress protein expression
after mRNA electroporation, demonstrating that maturation with a
BCG, Typhim, and Influvac yields TLR-matured DCs that can be
electroporated very efficiently.
A potential disadvantage to the use of vaccines for DC
maturation may be that high amounts of antigens derived from the
vaccines can mask weakly immunogenic tumor antigens and thus
hamper antitumor responses. Using PBLs and DCs of patients who
had previously been vaccinated with KLH-loaded DCs, we showed
that vaccine DCs and vaccine PGE2 DCs also induce some T-cell
proliferation in the absence of KLH, which may be a specific
response to one of the vaccines present in the maturation cocktail.
However, we also show that vaccine DCs and vaccine PGE2 DCs
can induce KLH-specific immune responses. Moreover, vaccine
PGE2 DCs loaded with tumor peptides or mRNA are able to
activate gp100 specific CD8⫹ T cells, suggesting that vaccine DCs
can induce antitumor responses despite the presence of vaccinederived antigens. As with KLH, responses to the vaccines may even
provide T-cell help and thus facilitate antitumor responses.51
In conclusion, we present here a new source of clinical-grade
TLR ligands: commercially available vaccines. We introduce a new
clinical-grade maturation cocktail consisting of a mixture of
preventive vaccines and PGE2 that yields clinically applicable
Th1-inducing TLR ligand-matured DCs. Clinical studies are now
being performed to investigate whether vaccine-matured DCs
improve antitumor responses in vivo.
Acknowledgments
The authors thank Nicole Meeusen-Scharenborg, Annemiek de
Boer, and Mandy van de Rakt for technical support.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
VACCINES INDUCE DC MATURATION VIA TLRs
This work was supported by the Dutch Cancer Society (grants
KWF 2003-2917, KWF 2004-3126, and KWF 2004-3127), The
Netherlands Organization for Scientific Research (grant VIDI
91776363), the TIL Foundation, the NOTK Foundation, and the
European Union (Cancerimmunotherapy and DC-Thera).
Authorship
Contribution: G.S. designed and performed research, analyzed
data, and wrote the paper; D.B.-R. contributed to experimental
design and performed research; D.S., A.J.A.L., and M.v.H.-K.
573
performed research; N.S. contributed vital reagents; C.J.A.P.
contributed to experimental design; C.G.F. and G.J.A. contributed
to experimental design and writing of the paper; and I.J.M.d.V.
supervised the study and contributed to experimental design and
writing of the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: I. Jolanda M. de Vries, Department of Tumor
Immunology, Nijmegen Centre for Molecular Life Sciences, PO
Box 9101, 6500 HB Nijmegen, The Netherlands; e-mail:
[email protected].
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
2010 116: 564-574
doi:10.1182/blood-2009-11-251884 originally published
online April 27, 2010
Commonly used prophylactic vaccines as an alternative for synthetically
produced TLR ligands to mature monocyte-derived dendritic cells
Gerty Schreibelt, Daniel Benitez-Ribas, Danita Schuurhuis, Annechien J. A. Lambeck, Maaike van
Hout-Kuijer, Niels Schaft, Cornelis J. A. Punt, Carl G. Figdor, Gosse J. Adema and I. Jolanda M. de
Vries
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