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Contents lists available at ScienceDirect
European Journal of Cell Biology
journal homepage: www.elsevier.de/ejcb
Review
Third generation dendritic cell vaccines for tumor immunotherapy
Bernhard Frankenberger a,∗ , Dolores J. Schendel a,b
a
b
Institute of Molecular Immunology, Helmholtz Zentrum München, German Research Center for Environmental Health, Marchioninistrasse 25, 81377 Munich, Germany
Clinical Cooperation Group “Immune Monitoring”, Helmholtz Zentrum München, German Research Center for Environmental Health, Marchioninistrasse 25, 81377 Munich, Germany
a r t i c l e
i n f o
Article history:
Received 29 October 2010
Received in revised form 26 January 2011
Accepted 26 January 2011
Keywords:
Dendritic cells
Young dendritic cells
Dendritic cell-based vaccines
Antitumor response
Memory effector cytotoxic T lymphocytes
Dendritic cell maturation
Th1-polarization
a b s t r a c t
This review summarizes our studies of the past several years on the development of third generation
dendritic cell (DC) vaccines. These developments have implemented two major innovations in DC preparation: first, young DCs are prepared within 3 days and, second, the DCs are matured with the help
of Toll-like receptor agonists, imbuing them with the capacity to produce bioactive IL-12 (p70). Based
on phenotype, chemokine-directed migration, facility to process and present antigens, and stimulatory
capacity to polarize Th1 responses in CD4+ T cells, induce antigen-specific CD8+ CTL and activate natural
killer cells, these young mDCs display all the important properties needed for initiating good antitumor
responses in a vaccine setting.
© 2011 Elsevier GmbH. All rights reserved.
Introduction
Among the various types of antigen presenting cells (APCs),
dendritic cells (DCs) are considered to be the most potent
because they can efficiently prime naïve T cells during development of T cell-mediated immunity and stimulate adaptive
immune responses. Particularly, immature DCs have an exceptional ability to internalize different forms of antigen that are
subsequently processed and presented within major histocompatibility complex (MHC) class I and class II molecules at the
DC cell surface. However, concerns have been raised regarding
the use of immature DCs (iDCs) in clinical trials since antigen presentation by iDCs, in particular antigens representing
self-proteins, is known to tolerize T cells rather than immunize hosts (Schuler et al., 2003). Exposure of iDCs to danger
signals in the periphery leads to their differentiation to a
mature state characterized by high expression of MHC and
costimulatory molecules. Therefore, upon reaching the T-cell
zones of secondary lymphoid organs, mature DCs (mDCs) are
well equipped to activate antigen-specific T cells. Because of
these properties, mDCs generated and transfected with tumorassociated antigens (TAAs) in vitro, in a environment free of
tumor-associated inhibitory factors, have evolved as a powerful
vaccination tool for tumor immunotherapy. Several clinical studies using mDCs as tumor vaccines have been implemented and
∗ Corresponding author. Tel.: +49 897099344; fax: +49 897099300.
E-mail address: [email protected] (B. Frankenberger).
T cell responses were elicited against TAA epitopes derived from
self-proteins (Palucka et al., 2010).
Improved understanding of the biology of DCs has revealed that
three interactive signals impinge on lymphocyte responses and
are important for consideration in vaccine development in order
to achieve optimal activation of both innate and adaptive antitumor immunity: (i) adequate DC presentation of MHC-peptide
complexes for induction of antigen-specific T cells (signal 1), with
simultaneous expression of activation ligands for stimulation of
innate natural killer cells; (ii) dominant positive costimulation via
molecules such as CD40, CD80, and CD86 (signal 2) and (iii) secretion of cytokines that polarize immune responses in a Th1/Tc1
direction to create optimal antitumor responses (signal 3) (Fig. 1).
Signal 1: reproducible and efficient expression of TAAs in
mDCs after transfer of in vitro transcribed RNA
One of the most commonly used strategies of antigen delivery
to DCs is exogenous loading with synthetic peptides that represent
defined epitopes from known TAAs (Cerundolo et al., 2004; Schuler
et al., 2003; Jager et al., 2002). The short half-life of peptide-MHC
complexes (pMHC) and the MHC restriction of T cell recognition
that limits this approach to patients with specific MHC allotypes
are major disadvantages of providing DCs with this form of antigen. Furthermore, responses directed against single peptides often
allow immune selection of tumor variants that no longer express
the TAA or MHC-restricting molecules (Jager et al., 2002). Therefore,
activation of T cells recognizing multiple pMHC ligands is important
to avoid selection of tumor antigen-loss variants.
0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ejcb.2011.01.012
Please cite this article in press as: Frankenberger, B., Schendel, D.J., Third generation dendritic cell vaccines for tumor immunotherapy. Eur. J.
Cell Biol. (2011), doi:10.1016/j.ejcb.2011.01.012
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Fig. 1. Three interactive signals impinge on lymphocyte responses. Three interactive signals that impinge on innate and adaptive lymphocyte responses need to be considered
to achieve optimal activation of antitumor immunity in the development of DC-based vaccines. Signal 1: the first signal to MHC-restricted T cells involves DC presentation of
MHC-peptide ligands specific for TAAs in order to induce MHC class I and class II-restricted T cells. Signal 2: positive costimulation of naïve or activated effector lymphocytes
via CD80 and CD86 can be counter-regulated by negative signals delivered by inhibitory molecules of the B7 family, such as B7-H1, B7-H2, B7-H3 and B7-H4; kinetics and
quantitative expression of these counter-regulating ligands can influence the efficiency of DC stimulation of effector lymphocytes. In addition, expression of these molecules
on tumor cells can regulate effector cell function upon contact with tumor cells. These negative signaling cascades can impact on both innate and adaptive antitumor
immunity. Signal 3: the cytokines secreted by DCs determine the types of effector cells that will be activated. For example, T helper 1 cell polarization can only occur if mature
DCs secrete IL-12(p70). Importantly, DCs that secrete IL-12 or IL-2 also have the capacity to initiate innate immune responses by supporting NK and non-MHC-restricted T
cell proliferation, respectively. Therefore, means are needed to obtain DCs that secrete the necessary cytokines in order to achieve optimal antitumor immunity through DC
vaccination.
RNA offers an attractive form of antigen provision that allows
DCs to generate multiple pMHC ligands from individual or multiple proteins. By this means, vaccines can be developed for all
patients independent of their genetic backgrounds. Successful de
novo priming of T cells by DCs pulsed with single-species RNA
encoding individual TAAs was first described by Gilboa and coworkers (Boczkowski et al., 1996). An early clinical trial using DCs
loaded with RNA encoding the prostate-specific antigen demonstrated the feasibility and safety of this approach. Furthermore, T
cell responses to PSA were induced in some patients (Heiser et al.,
2002). Results of more recent clinical trials further support the
potential of this vaccine concept (Van Tendeloo et al., 2010; Kyte
et al., 2007; Coulie and van der Bruggen, 2003). It has also been
speculated that the full repertoire of TAAs displayed by a tumor
could be transferred to recipient DCs via use of total RNA from a
patient’s individual tumor (Gilboa and Vieweg, 2004).
RNA delivery of individual TAAs imbues mDCs with excellent
capacity to reactivate effector-memory CTLs
Our goal was to develop a DC-based vaccine that not only could
prime T cell responses de novo but also could reactivate preexisting repertoires of effector-memory cytotoxic T lymphocytes
(CTLs) that may be prevalent in some tumor patients (Goff et al.,
2010; Geiger et al., 2009; Spiess et al., 1987). Previous studies of
others analyzed transfer of RNA-encoded TAAs into iDCs because
of their exceptional ability to spontaneously internalize materials
from their surroundings, including RNA. Meanwhile, however, concerns have been raised regarding the use of iDCs in clinical studies
because of their capacity to tolerize rather than immunize T cells
(Schuler et al., 2003).
Therefore, we focused on optimizing RNA transfer into mDCs
using the method of electroporation. We found that in vitro transcribed RNA (ivt-RNA) indeed provided a highly suitable source to
achieve efficient TAA expression in DCs, yielding excellent pMHC
display at the DC surface (Javorovic et al., 2005). By applying ivtRNA encoding enhanced green fluorescent protein (EGFP), we could
observe strong EGFP protein expression in 85–90% of mDCs around
12 h after electroporation and excellent expression of full protein
was detected for at least 48 h. Importantly, electroporation of ivtRNA did not significantly alter the expression of typical surface
markers of mDCs, such as CD40, CD80, CD83, CD86, and HLA-DR.
A detailed kinetic analysis using real-time PCR to quantify EGFP
RNA after transfer into mDCs was performed to assess the stability of the transfected RNA. These quantitative analyses revealed
that the amount of ivt-RNA decreased rapidly after transfer, with
loss of more than 99% of specific RNA at 24 h as compared with
the amount present 30 min after electroporation of mDCs. In contrast, EGFP protein expression was found to be increased at the time
when most ivt-RNA had already been eliminated. This discrepancy
revealed that sufficient template was available in the mDC for protein expression despite the rapid loss of RNA. Because EGFP is a
stable protein with a long half-life of approximately 24 h, it is likely
that protein expression of an antigen with a shorter half-life will
peak sooner. However, once peptides have been processed and presented at the cell surface they are stable over several days, providing
mDCs with adequate time to present their new TAA-derived pMHC
ligands to responding T cells. Thus, rapid loss of TAA-encoding RNA
and corresponding full length protein is not a hindrance for provision of antigen in this form to mDCs.
After full characterization of the parameters of EGFP ivtRNA and protein expression in mDCs, we turned to analysis of
melanoma-associated antigens. Tyrosinase was selected as a model
antigen because epitopes seen by HLA-A2-restricted CTLs are well
defined. This allowed us to evaluate the impact of RNA transfer
on the capacity of mDCs to reactivate tyrosinase-specific effectormemory CTLs. We assessed the capacity of ivt-RNA-pulsed mDCs
to present tyrosinase-derived pMHC ligands that could activate
the TyrF8 CTL clone, which recognizes an HLA-A*0201-restricted
tyrosinase369–377 peptide (Visseren et al., 1995). These experiments
Please cite this article in press as: Frankenberger, B., Schendel, D.J., Third generation dendritic cell vaccines for tumor immunotherapy. Eur. J.
Cell Biol. (2011), doi:10.1016/j.ejcb.2011.01.012
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demonstrated that this CTL clone recognized RNA-pulsed mDCs just
as well as melanoma cells. In comparison with direct coculture
of CTLs with mDCs immediately after RNA delivery, we detected
better CTL stimulation when mDCs were given 6–25 h to process
transfected RNA before initiating cocultures with CTLs, indicating
that time was needed for optimal display of pMHC ligands. Of note,
this time varied with different TAAs, dependent upon their kinetics
of expression in mDCs. These experiments demonstrated that provision of ivt-RNA encoding a single TAA by electroporation allowed
highly reproducible transfer and subsequent pMHC presentation
by mDCs, providing them with an excellent capacity to specifically
reactivate effector-memory CTLs.
Failure of total tumor-derived ivt-RNA to reconstitute important
CTL ligands after transfer to mDCs
It was proposed that transfer of total RNA derived from a tumor
would be an optimal strategy to provide DCs with a multiplex of
TAAs, particularly when immunogenic antigens were unknown for
particular tumor types (Ashley et al., 1997; Boczkowski et al., 1996).
Furthermore, this would allow epitopes derived from mutated proteins that were unique to individual tumors to be presented to T
cells if they contained binding motives for patient MHC molecules.
Since tumor-derived RNA should contain message for many different TAAs, the selection of epitopes presented by different MHC
molecules is solely dependent on antigen processing and presentation by the DC, thus knowledge about MHC-binding peptide
sequences of a TAA is not required for vaccine development, as is the
case with peptide-based vaccines. However, patient tumor material is often limited, therefore a strategy was devised to amplify
tumor RNA through reverse transcription PCR with subsequent
in vitro transcription, providing an unlimited amount of ivt-RNA
for patient-individualized vaccine preparation (Boczkowski et al.,
2000). How this complex process impacted on display of different pMHC epitopes remained unknown, in particular for epitopes
derived from proteins that were not over-expressed in tumor cells.
To assess the potential of mDCs to display a variety of different
pMHC epitopes, including an epitope originating from a mutated
protein, we introduced single-species RNAs encoding tyrosinase
and mutated CDK4 proteins into mDCs and compared them with
mDCs pulsed with native or amplified total tumor RNA for their
capacity to activate effector-memory CTLs. By this means we could
directly test the contention that transfer of tumor-derived RNA
allowed mDCs to display the same repertoire of pMHC ligands as
tumor cells themselves. In this comparison, we were particularly
interested in the CTL response to the mutated epitope of CDK4, since
the levels of mRNA encoding this TAA were not necessarily high
in tumor cells, even though they were well recognized by a specific CTL clone. We prepared total cellular RNA from an established
melanoma cell line (SK29-MEL-1) that expressed both tyrosinase
and mutated CDK4 R24C, which encodes an HLA-A*0201-restricted
epitope spanning this mutation (Wolfel et al., 1995). Such mutated
epitopes are thought to be very immunogenic since they are recognized as foreign peptides by T cells (Mumberg et al., 1996). We
compared the responses of CTLs specific for epitopes derived from
these two TAAs after stimulation with mDCs that were provided
with either native or amplified total tumor RNA of SK29-MEL1 cells. In parallel, mDCs were loaded with single-species RNAs
encoding these two TAAs. CTLs specific for a tyrosinase epitope and
the CDK4 R24C epitope responded to mDCs loaded with singlespecies RNAs far better than to stimulation with mDCs loaded
with total tumor RNA. The T cell responses to mDCs expressing
individual RNAs were similar to those seen with peptide-pulsed
mDCs, whereas mDCs expressing total cellular melanoma RNA
were either not recognized or low but non-reproducible responses
were seen with the effector-memory CTLs. A clear correlation was
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found between the levels of TAA message in tumor-derived RNA
and pMHC ligand reconstitution on mDCs, as measured by CTL
recognition. Further studies led us also to conclude that individual
RNA species were likely to behave differently during the amplification procedure, influenced by their starting quantity, sequence
and size, resulting in differences in the efficiency of in vitro transcription. This leads to variations that were not seen with tumor
cells that can constantly produce new RNA through endogenous
gene transcription. Furthermore, these results revealed that TAAs
with highly immunogenic mutated epitopes were not effectively
transferred using tumor-derived ivt-RNA at a level sufficient to
allow mDCs to induce reactivation of some important pre-existing
effector-memory CTLs.
On this basis we concluded that transfer of defined pools of
selected individual RNAs would provide a more reliable means to
generate DC vaccines that would reproducibly display a multitude
of pMHC epitopes at sufficient levels for T cell stimulation.
Presentation of multiple antigens by mDCs using pools of ivt-RNA
Tumor cells that show down-regulation of some TAAs may
be able to escape immune-mediated elimination if vaccinationinduced T cell responses are limited to a single pMHC epitope. To
compensate for tumor immune escape, efficient vaccine strategies
need to target multiple TAAs simultaneously, enabling polyclonal
CTL responses to develop that are more efficient at eliminating
malignant cells, which are not hampered by loss of a single TAA
ligand (Schendel, 2007). Indeed, clinical trials with DCs expressing
complex mixtures of TAAs induced polyclonal CTL responses that
were superior at killing tumor cells compared to CTLs specific for
individual antigens (Milazzo et al., 2003; Muller et al., 2003; Su
et al., 2003; Heiser et al., 2001).
Since total tumor-derived RNA resulted in poor stimulation
of pre-sensitized CTLs, due to insufficient transfer of individual
mRNAs in the native RNA pool, we elected to apply a pool of defined
individual RNAs encoding several TAAs that could be delivered in
higher amounts to DCs. This approach should yield DCs presenting greater numbers of specific pMHC ligands while still achieving
greater complexity in TAA presentation. This generic approach
allows DC vaccines to be easily tailored to different tumor types
through selection of the TAAs for inclusion in the RNA pools.
To assess how RNA pool complexity impacted on presentation
of pMHC ligands by mDCs, we analyzed the stimulatory capacity of cells following introduction of pools composed of three or
six individual RNA species (Javorovic et al., 2008). The TAAs were
chosen based on the availability of CTL clones that recognized corresponding TAA-derived epitopes so that competition for epitope
presentation could be judged. As expected, the time frame and the
level of TAA expression in mDCs were dependent on the amount of
transferred RNA. When the pool of three RNAs was introduced, the
mDCs stimulated the corresponding CTLs at levels of approximately
80% of those seen when mDCs were loaded with the individual
RNAs. Others noted that pools of this complexity could also be used
to create composite vaccines (Schaft et al., 2005). However, simultaneous provision of six RNAs strongly reduced the ability of the
mDCs to activate the CTLs by more than 50%. Thus, diminished presentation of individual epitopes occurred when more complex RNA
pools were used. This effect was dependent upon the quantity of
transferred RNA, revealing a limit to the total amount of RNA that
could be introduced into mDCs without impairing their stimulatory
capacity.
Thus, excellent T-cell activation was achieved by selecting individual RNAs for a composite pool of TAAs, endowing mDCs with a
multiplicity of pMHC ligands. While there was a limitation on the
total amount of RNA and thereby the complexity of TAAs that could
be introduced simultaneously into one mDC population, it would
Please cite this article in press as: Frankenberger, B., Schendel, D.J., Third generation dendritic cell vaccines for tumor immunotherapy. Eur. J.
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Fig. 2. Preparation of young mDCs within only three days. Our DC vaccine strategy
utilizes young mDCs that are prepared within only three days, in contrast to standard
procedures that utilize a 7-day culture period.
be possible to prepare more complex vaccines by introducing different TAA pools into subfractions of mDCs that are finally mixed
to form the final composite vaccine.
Signal 2: optimal costimulatory profiles are displayed by
young mDCs generated in 3 days in vitro
To date, many different protocols have been described for the
preparation of mDCs, which vary both with respect to the signals
used for maturation and the time periods used for induction of mDC
in vitro. The most common methods require around 7 days of cell
culture and frequently use a maturation cocktail that was developed by Jonuleit et al. (1997), which includes TNF-␣, IL-1␤, IL-6
and PGE2 . Dauer and colleagues demonstrated that “fast DCs” could
be rapidly generated from peripheral blood monocytes within 48 h
(Dauer et al., 2003; Obermaier et al., 2003). These mDCs could prime
naïve T cells and also reactivate effector-memory CTLs, but showed
some impairment in migratory capacity (Jarnjak-Jankovic et al.,
2007; Dauer et al., 2003, 2005). We originally prepared 6d iDCs from
peripheral blood monocytes using leukaphresis and elutriation, followed by culture with GM-CSF and IL-4 and subsequent maturation
(Zobywalski et al., 2007). We altered this protocol to prepare young
mDCs by modifying the method for generation of “fast DCs” through
extending the time period for development of iDCs to 48 h, followed
by addition of the maturation signals for another 24 h, giving a total
culture time of 72 h (Fig. 2). This time frame reliably yielded a stable population of mDCs. It has the advantage that it saves time and
costs for GMP-compliant vaccine development as compared to a
7d-production protocol. Young mDCs may also better reflect the
function of mDCs in vivo (Randolph et al., 1998).
To demonstrate that young mDCs (3d mDCs) would display
comparable functions and antigen processing and presentation
capacities as standard mDCs (7d mDCs), we assessed cells prepared in both time frames from the same donors for phenotype,
migration capacity, and antigen processing and presentation of long
peptides and ivt-RNA. They were also compared for their capacity
to stimulate effector-memory CTLs (Bürdek et al., 2010).
Young mDCs were more robust cells, reflected in a smaller morphology and lower granularity. This led to a higher yield of cells
with greater viability; these differences were particularly apparent
after electroporation and cryopreservation. Both 3d and 7d mDCs
displayed high levels of CD83 and no CD14, demonstrating the hallmark signature of mDCs. Young mDCs were equivalent to 7d mDCs
in their capacity to activate effector-memory CTLs after exogenous
loading of short peptides or following introduction of a long peptide
into the mDCs from which a short pMHC epitope was produced by
intracellular processing and presentation. In contrast, initial studies
of young mDCs showed inferior CTL stimulation after electroporation of ivt-RNA. This was traced to poor uptake of RNA, requiring
an alteration in electroporation parameters in order to achieve efficient transfer of long ivt-RNAs into the compact and robust young
mDCs. With this change, 3d mDCs were comparable, if not better,
in their capacity to process and present TAA-derived epitopes from
ivt-RNA for CTL stimulation.
Interesting differences were seen when 3d and 7d mDCs were
assessed for expression of various costimulatory molecules, such as
CD40, CD80, and CD86, as well as other functionally relevant surface
molecules, including CD209 (DC-SIGN), HLA-DR and CCR7. Despite
a shortened production time, young mDCs expressed higher levels
of CD40, CD209 and HLA-DR than 7d mDCs. In contrast, expression
of the inhibitory molecule CD274 (B7-H1, PD-1L) was consistently
lower on young mDCs. The relevance of this difference was particularly striking when the expression of the positive costimulatory
molecule CD80 was compared with the level of CD274, since it was
found that 3d mDC had higher levels of CD80 relative to CD274,
whereas 7d mDCs showed a reciprocal pattern. This is likely to be
functionally important because CD274 transmits negative signals
through interaction with CD279 (PD-1) on T cells (Selenko-Gebauer
et al., 2003; Freeman et al., 2000).
Thus, young mDCs displayed a clear advantage in their profile
of costimulatory molecules which favors positive costimulation
(signal 2). This may imbue them with a better capacity to prime
naïve T cells, without activating a negative regulatory loop involving CD274–CD279 interactions. Also 3d mDCs may have a greater
capacity to reactivate effector-memory T cells in vivo, particularly if
T cells express high levels of CD279 (PD-1) due to chronic exposure
to tumor-antigens in the patient.
Signal 3: TLR-activated mDCs show an optimized capacity to
polarize Th1/Tc1 immune responses
Analyses of Toll-like receptor (TLR)-signaling in DCs demonstrated activation of the NF-kB pathway, with resultant modulation
of their cytokine secretion (reviewed in Kaisho and Tanaka, 2008).
For this reason, synthetic TLR agonists are of interest for inclusion in
maturation mixtures for mDCs to enable them to optimally induce
antitumor responses. Many different TLRs are expressed by human
monocyte-derived DCs (Ito et al., 2002). Several immunomodulatory genes are induced following ligation of TLRs (Napolitani et al.,
2005; Gautier et al., 2005; Roelofs et al., 2005), leading to cytokine
and chemokine synthesis, including production of TNF-␣, IFN-␥, IL6, IL-10, IL-12, and MIP-1␣, in various cell types (Philbin and Levy,
2007; Gorden et al., 2005). To date, a variety of TLR ligands, such as
imiquimod (R837), resiquimod (R848), S-27609, CL097, CL075 (3M002), CL087, and loxoribone, have been studied for their potential
to activate corresponding receptors in various human cell types
(Hemmi et al., 2002; Jurk et al., 2002).
We investigated the use of TLR agonists to regulate specific
cytokine production in young mDCs, allowing them to provide
appropriate signals for polarization of CD4+ T cell responses in
a Th1-direction, to efficiently stimulate CD8+ CTL responses and
to activate natural killer cells. Kaliniski and coworkers generated human Th1-polarizing DCs (alpha DC) using TLR3 agonists to
produce cells that secreted bioactive IL-12(p70) (Mailliard et al.,
2004). Recently, 2d mDCs were reported that secreted bioactive IL12(p70) after stimulation with TLR4 and TLR7/8 agonists (Dauer
et al., 2008). Earlier, we reported use of R848 and poly(I:C) as
corresponding agonists for TLR7/8 and TLR3, respectively, in combination with several cytokines for maturation of DCs in a 7d-protocol
(Zobywalski et al., 2007). More recently, we have demonstrated
that CL075, a synthetic thiazoloquinoline compound that activates
Please cite this article in press as: Frankenberger, B., Schendel, D.J., Third generation dendritic cell vaccines for tumor immunotherapy. Eur. J.
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TLR8, can also polarize young mDCs to produce bioactive IL-12(p70)
(Spranger et al., 2010). We compared 3d mDCs matured without TLR agonists to those induced with a TLR3 agonist [poly(I:C)]
alone or in combination with a TLR7/8 agonist (R848 or CL075).
Comprehensive studies demonstrated that all populations of mDCs
displayed the expected array of costimulatory molecules but with
some differences. Most notably, inclusion of the TLR8 agonist CL075
yielded mDCs with somewhat higher levels of HLA-DR and CCR7.
The good expression of CCR7 was paralleled by spontaneous migratory capacity as well as positive chemotactic responses of mDCs to
CCL19 chemokine signals.
TLR ligation changed the cytokine secretion pattern of the mDCs
towards high production of bioactive IL-12(p70), which occurred
when only a TLR3 signal or the combination of TLR3 and TLR7/8 agonists were used. However higher levels of cytokine were achieved
when the combined signals were applied (Spranger et al., 2010).
These findings supported an earlier report that showed that TLR8signaling resulted in high production of IL12(p70) by 7d mDCs
(Larangé et al., 2009).
Furthermore, use of these maturation mixtures had a strong
functional impact on innate immune responses since TLR-matured
mDCs were able to (i) upregulate CD69 on both CD56dim and
CD56bright NK cell subpopulations, (ii) induce secretion of high levels of IFN-␥ as well as (iii) activate strong killing capacity by NK
cells.
In addition, mDCs induced with TLR7/8 ligands could trigger
CD4+ and CD8+ T cells to produce IFN-␥ yielding higher numbers
of Th1-polarized T cells. Furthermore, young mDCs could generate higher numbers of CTLs with specific killing capacity for tumor
cells. Lastly, we observed that the surface expression of CD80 was
significantly increased on mDC induced with TLR8 signals compared to maturation mixtures lacking a TLR agonist. In contrast,
levels of CD274 were not significantly different in both DC types.
Therefore, the positive ratio of CD80 to CD274 that was found to
be associated with young mDCs was further fostered by use of TLRagonists for mDC maturation (Spranger et al., 2010). The phenotypic
and functional differences noted in mDCs matured with or without TLR-signaling was associated with strong differences in STAT1
activation, that account for altered expression of multiple STAT1dependent genes, including cytokines and costimulatory molecules
(Larangé et al., 2009; Liu et al., 2007).
in which CD80 expression was no longer dominant over CD274
expression. Thus by utilizing young mDCs, we were able to improve
the balance of B7 molecules in favor of positive costimulation (signal 2). Moreover, we found that TLR-signaling could enhance the
levels of CD80 expression on 3d mDCs, providing a further improvement in the positive costimulatory profile of young mDCs.
Additionally, the generation of young mDCs resulted in a high
yield of robust cells that better withstood the stress of electroporation needed for efficient RNA loading and subsequent
cryopreservation for later application. We demonstrated that
young mDCs could process and present epitopes of TAAs with the
same efficiency as 7d mDCs. Thus, this new procedure allows 3d
mDCs to replace 7d mDCs for use in DC-based vaccines for clinical trials that utilize long peptides, proteins or ivt-RNA as sources
of specific antigen (signal 1). Furthermore, it reduces the costs of
vaccine generation by reducing the amounts of cytokine needed for
mDC cultivation and the occupancy time needed in a clean room
facility for large scale vaccine production.
Recently, we have utilized our DC technologies to open up
new avenues for immunotherapy using adoptive T cell therapy
by imbuing recipient lymphocytes with high affinity T cell receptors (TCRs) obtained from high avidity T cells that are generated
by in vitro priming of naïve lymphocytes using ivt-RNA-pulsed
mDCs (Leisegang et al., 2010; Wilde et al., 2009). In the future, we
envision that adoptive T cell therapy using TCR-transgenic lymphocytes (Sommermeyer et al., 2006) will be combined with vaccine
strategies to retain the potency of T cell-mediated responses
in vivo over longer periods of time. Thus, our DC vaccine strategies will hopefully find additional application in future combination
immunotherapies.
Conclusions
References
In our DC vaccine strategy, we utilize young mDCs that are prepared within only three days in contrast to standard procedures
that utilize a 7-day culture period. Other groups are exploring the
properties of fast DCs (Jarnjak-Jankovic et al., 2007; Dauer et al.,
2003, 2005) but we are not aware of any ongoing clinical trials at
this time employing these cells. Furthermore, we developed new
maturation mixtures that include synthetic TLR agonists for TLR3
and TLR7/8. We could clearly demonstrate that 3d mDC, matured
with TLR agonists, displayed a much higher stimulatory capacity
for naïve T cells than 3d mDC matured with TNF␣, IL-1␤, IL-6 and
PGE2 alone (Spranger et al., 2010). These maturation mixtures stimulated young mDCs to secrete high levels of bioactive IL-12(p70)
upon encounter with T cells, while causing only minimal secretion of IL-10 (signal 3). Young mDCs producing bioactive IL-12(p70)
induced Th1-polarized CD4+ T cells and effectively activated CD8+
T cells as well as natural killer cells, with potent antitumor cytolytic
activity. We made the important observation that young mDCs
showed an improved expression of CD80 (B7.1) positive costimulatory molecules compared with expression of negative CD274
(B7-H1) coregulatory molecules. This ratio shifted as the mature
DCs aged over 7 days, with a resultant phenotype in aged mDCs
Acknowledgments
The authors thank the German Research Foundation (SFB-455,
SFB-TR36), the European Union under the 6th Framework Programme “Allostem”, the Helmholtz Society Alliance “Immunotherapy of Cancer” and the BayImmuNet Program for financial support
and the members of the laboratory for many valuable contributions to the development of DC vaccines and helpful suggestions on
this manuscript. We especially thank S. Spranger for preparation of
figures.
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