Regulation of tumor immunity by tumor/dendritic cell fusions.

Abstract

The goal of cancer vaccines is to induce antitumor immunity that ultimately will reduce tumor burden in tumor environment. Several strategies involving dendritic cells- (DCs)- based vaccine incorporating different tumor-associated antigens to induce antitumor immune responses against tumors have been tested in clinical trials worldwide. Although DCs-based vaccine such as fusions of whole tumor cells and DCs has been proven to be clinically safe and is efficient to enhance antitumor immune responses for inducing effective immune response and for breaking T-cell tolerance to tumor-associated antigens (TAAs), only a limited success has occurred in clinical trials. This paper reviews tumor immune escape and current strategies employed in the field of tumor/DC fusions vaccine aimed at enhancing activation of TAAs-specific cytotoxic T cells in tumor microenvironment.

Full text

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Hindawi Publishing Corporation
Clinical and Developmental Immunology
Volume 2010, Article ID 516768, 14 pages
doi:10.1155/2010/516768
Review Article
Regulationof TumorImmunity by Tumor/DendriticCellFusions
Shigeo Koido,1,2,3SadamuHomma,3EiichiHara,4YoshihisaNamiki,2
AkitakaTakahara,1Hideo Komita,1EijiroNagasaki,2Masaki Ito,2
Toshifumi Ohkusa,1JianlinGong,5andHisaoTajiri1
1Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine,
Tokyo 277-8567, Japan
2Institute of Clinical Medicine and Research, The Jikei University School of Medicine, Tokyo 277-8567, Japan
3Department of Oncology, Institute of DNA Medicine, The Jikei University School of Medicine, Tokyo 105-8461, Japan
4Saitama Cancer Center Research Institute for Clinical Oncology, Saitama 362-0806, Japan
5Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA
Correspondence should be addressed to Shigeo Koido, shigeo koido@jikei.ac.jp
Received 29 May 2010; Accepted 22 September 2010
Academic Editor: E. Shevach
Copyright © 2010 Shigeo Koido et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The goal of cancer vaccines is to induce antitumor immunity that ultimately will reduce tumor burden in tumor environment.
Several strategies involving dendritic cells- (DCs)- based vaccine incorporating different tumor-associated antigens to induce
antitumor immune responses against tumors have been tested in clinical trials worldwide. Although DCs-based vaccine such as
fusions of whole tumor cells and DCs has been proven to be clinically safe and is efficient to enhance antitumor immune responses
for inducing effective immune response and for breaking T-cell tolerance to tumor-associated antigens (TAAs), only a limited
success has occurred in clinical trials. This paper reviews tumor immune escape and current strategies employed in the field of
tumor/DC fusions vaccine aimed at enhancing activation of TAAs-specific cytotoxic T cells in tumor microenvironment.
1.T Lymphocytesand Tumor Immunity
The T-cell receptor (TCR) interaction with complex of
peptides and major histocompatibility complex (MHC)
molecules is a critical event in T-cell-mediated responses.
The proteasomes in tumor cells degrade tumor-associated
antigens (TAAs) into short peptides (usually 8–10 amino
acids), mostly derived from endogenously synthesized pro-
teins as well as exogenous antigens in the endoplasmic
reticulum, and present them to cytotoxic T lymphocytes
(CTLs) that express the CD8 coreceptor. Therefore, CD8+
CTLs can directly lyse tumor cells [1, 2]. On the other
hand, CD4+ T cells recognize antigenic peptides (10–30
amino acids) associated with MHC class II molecules and
mediate their helper functions to induce antigen-specific
CTLs through secretion of cytokines such as interferon
(IFN)-γ. There are increasing evidences that CD4+ T cells
play a more direct role beyond delivery of assistance in the
generation of efficient stimulatory immunity [3]. CD4+ T-
cell responses can also elicit not only stimulatory but also
suppressive immunity. Now, it is becoming clear that there
is an enormous diversity in CD4+ T-helper (Th) cell polar-
ization patterns including Th1, Th2, Th17, and regulatory
T cells (Tregs). Th1 cells secrete type I cytokines such as
IFN-γ, resulting in the activation of antigen presenting cells
(APCs), which can stimulate CTLs [1, 2]. Tumor-specific
CD4+ T cells regulate the survival and persistence of CTLs
as memory cells [3]. Both CD8+ and Th1 cells secrete
IFN-γ, which can further sensitize tumor cells to CTLs by
upregulating MHC class I molecules and antigen-processing
machinery of APCs. Th2 cells secrete type II cytokines,
such as interleukin 4 (IL-4) and IL-10 [1, 2]. Th2 cells can
enhance the generation of a humoral immunity, antibody-
based antitumor response. The newly identified Th17 cells
secrete IL-17, eliciting tissue inflammation implicated in
autoimmunity.Finally,TregsinhibitthedevelopmentofCTL
responses [4]. Tregs are mainly derived from two origins,
whicharenaturallyoccurringthymus-derivedTregs(nTregs)
and adaptive or inducible Tregs (iTregs) [5]. Foxp3 has been
considered to be a master regulatory transcription factor

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2Clinical and Developmental Immunology
for Tregs [6]. It is becoming clear that Tregs play a pivotal
role in the tumor progression and the suppression of tumor
immunity [7] (Figure 1).
2.DendriticCells(DCs) andTumor Immunity
Dendritic cells (DCs) are professional APCs and key regu-
lators of T- and B-cell immunity, owing to their superior
ability to take up, process, and present TAAs [1, 2, 8].
DCs derive their potency from constitutive and inducible
expression of essential costimulatory ligands on the cell
surface including B7, ICAM-1, LFA-1, LFA-3, and CD40
[9, 10]. These proteins function in concert to generate
a network of secondary signals essential for reinforcing
the primary antigen-specific signal in T-cell activation [11,
12]. Therefore, DCs play a pivotal role on the initiation,
programming, and regulation of tumor-specific immune
responses. Various strategies to deliver TAAs into DCs have
been developed to generate potent CTL responses against
tumor cells. DCs have been pulsed with synthetic peptides
derived from the known TAAs, tumor cell lysates, apoptotic
tumor cells, and tumor RNA [13–17]. Another strategy is the
use of fusion cells generated by fusing DCs and whole tumor
cells [18]. The fusion process facilitates the entry of TAAs,
includingbothknownandunidentified,intotheendogenous
antigen-processing pathway and presents antigenic peptides
through MHC class I and II pathways in the context of the
potent immune-stimulatory machineries in the DCs [19–
22]. These antigen-loaded DCs have already been used as
vaccines to improve antitumor immunity [8].
3.Fusionsof Tumor Cell andDC
The fusions with whole tumor cell and DC (tumor/DC) by
polyethylene glycol (PEG) known as a chemical membrane
destabilizing agent [18, 23–25], physical [26–31], or biolog-
ical means [32, 33] create heterokaryons that express both
TAAs and DC-derived costimulatory molecules. Therefore,
the fused cells inherit the properties of their parental
cells (tumor cell and DC) (Figure 2). For example, the
membranes of fused cells are integrated into a single cell
whereas the nuclei are remained to be separate, at least in
the primary fusions [34]. Such a characteristic structure may
make it possible to maintain the functions of both original
cells, at least in part, including synthesis of antigens and
costimulatory molecules [34].
4.AntigenProcessingand Presentationby
Tumor/DC Fusions
It has been shown that antigens are processed and presented
through two major pathways by DCs. Endogenously syn-
thesized proteins, such as those expressed in viral infections
and certain exogenous antigens are processed and presented
through the MHC class I-restricted pathway to CD8+ T
cells [35, 36]. In contrast, exogenous antigens from the
extracellular environment are captured and delivered to the
compartments of the endosome/lysosome, where they are
degraded to antigenic peptides by proteases and peptidases,
which are complexed with MHC class II molecules and
recognized by CD4+ T cells [35, 36]. Importantly, DCs are
alsocapableofprocessingandpresentingexogenousantigens
on MHC class I molecules through an endogenous pathway,
a phenomenon called antigen cross-presentation [37, 38].
However, the antigen cross-presentation is generally not
efficient to induce CTL responses in the absence of carrier
proteins or particles [39].
It is now well known how the fusion cells assemble
and present the MHC class I- and II-restricted peptide
complexes. One possibility is that antigenic peptides are
complexed with tumor-derived MHC class I molecules and
the complexes are simply transferred and presented by
tumor/DC fusions. Moreover, the fusions can efficiently
process TAAs from tumors through an endogenous antigen-
processing pathway [34]. Therefore, an advantage of the
fusions-strategy over DCs pulsed with tumor lysates is
that endogenously synthesized antigens have better access
to MHC class I pathway [40]. Indeed, tumor/DC fusion
vaccines are superior to those involving other methods of
DCs loaded with antigenic proteins, peptides, tumor cell
lysates, or irradiated tumor cells in animal studies [41].
Moreover, the important advantage of tumor/DC fusions
approach is that modifications of tumor cells and DCs are
independently possible, which their characters persist after
fusion process [22].
5.CTL InductionbyTumor/DC Fusions
Immature DCs take up tumor antigens, mature into IL-
12-producing cells, and stimulate Th1 cells in the draining
lymph node, resulting in IFN-γ production. These stimu-
lated Th1 cells help during the priming of CD8+ T cells
with the capacity for optimal secondary expansion upon re-
encounter with antigens. Even in the absence of CD4+ T
cells, these memory CD8+ T cells can be rapidly expanded in
response to secondary antigens exposure. Expanded CD8+
CTLs can destroy tumor cells through effector molecules
such as granzyme B and perforin [42]. Therefore, efficient
CTL induction requires the stimulation of both CD4+
and CD8+ T cells. Expression of MHC class I and II
molecules, costimulatory molecules (CD80 and CD86), and
adhesion molecules (ICAM-1 and LFA-3) on tumor/DC
fusions is essential for antigen processing, presentation, and
subsequent activation of both CD4+ and CD8+ T cells
[25, 43, 44]. In animal models, the fusion cells, like DCs, can
also migrate into regional lymph node as early as 18 hours
after s.c. injection. Then, the fusion cells localize to the T-cell
area in the lymph node and form clusters with CD4+ and
CD8+ T cells simultaneously [45].
To dissect the role of antigen-presentation through MHC
class I and II pathways by tumor/DC fusions, we created
four types of fusions by alternating fusion cell partners:
(1) wild-type fusions (WT-FCs), (2) MHC class I knockout
fusions (IKO-FCs), (3) MHC class II knockout fusions
(IIKO-FCs), and (4) MHC class I and II knockout fusions
(I/IIKO-FCs) [46]. Immunization of wild-type mice with

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Clinical and Developmental Immunology3
CD4Th
Th1
Th2
Dendritic cells
IL-10
IL-4
IL-17
IL-22
IL-23
iTreg
CD8+
B cell
Plasma cell
Perforin
granzyme B
Cellular immunity
Antibody
Humoral immunity
Suppressive
immunity
Th17
Autoimmunity
CTL
IL-10
IL-4
IL-12
IL-18
IFN-γ
TGF-β
IL-10
IFN-γ
IL-2
TGF-β
Figure 1: The role of helper T cells in tumor immunity. CD4+ T-helper cells play extensive roles and are able to interact with the tumor
cell and immune effectors. Th1 cells secrete type I cytokines such as interleukin 2 (IL-2) and IFN-γ, resulting in the activation of DCs,
which can stimulate CTLs. Tumor-specific Th1 cells regulate the survival and persistence of CD8+ effector T cells as memory cells. Th2 cells
secrete type II cytokines, such as IL-4 and IL-10. Th2 cells can enhance the generation of humoral, antibody-based antitumor responses.
Th17 cells secrete IL-17 elicit tissue inflammation implicated in autoimmunity. Inducible CD4+ regulatory T cells (iTreg) exhibit a strong
immunosuppressive activity for antitumor immunity.
Proteosome
TAP
Tumor
nucleus
nucleus
Peptides
DC
Endosome
MHC
class II
vesicles
Lysosome
Tumor/DC fusions
CD8
CD4
TAAs
αβ-TCR
MHC class I
MHC class II
CD28
B7
Invariant chain
β2m
Golgi complex
Rough endoplasmic reticulum
Figure2:Characterizationoftumor/DCfusions.Tumor/DCfusionsexpressMHCclassI,II,costimulatorymoleculesandtumor-associated
antigens (TAAs). The fusions are able to process tumor-derived peptides and MHC class I peptides derived from DCs. They form MHC class
I-peptide complexes, in the endoplasmic reticulum, which are transported to the cell surface and presented to CD8+ T cells. Similarly, the
fusions can synthesize MHC class II peptides derived from DC in the endoplasmic reticulum, which are transported to the cytoplasm where
MHC class II-peptide complexes are assembled with tumor-derived peptides and presented to CD4+ T cells.

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4Clinical and Developmental Immunology
WT-FCs, IKO-FCs, IIKO-FCs, or I/IIKO-FCs provided 100,
91.7, 61.5, and 15.4% protection, respectively, against tumor
challenge with MHC class I positive tumor cells. Moreover,
IKO-FCs induced slightly decreased tumor prevention and
treatment. Importantly, IIKO-FCs abolished IFN-γ produc-
tion of CD4+ and CD8+ T cells and CTLs induction.
Therefore, antigen presentation through MHC class II is
essential for the activation of antigen-specific CD4+ T cells
and the induction of potent CD8+ CTL responses against
tumor. Although development of vaccine has been directed
toward activation and amplification of CD8+ T cells, there
is increasing evidence that CD4+ T cells play a broader
role in antitumor immunity [3]. CD4+ T cells contribute to
antitumor immunity through diverse mechanisms, in which
they are required not only for the maintenance of CD8+
CTLs but also for the infiltration of CD8+ CTLs at the tumor
site [3]. Indeed, adoptive transfer of antigen-specific CD4+
T cells controlled tumor growth [46]. Although maximal
antitumor immune responses require both MHC class I and
II antigen-presentation, MHC class II plays more important
roles on the antitumor immunity in cancer vaccines [3, 46].
Therefore, for the design of cancer vaccines, it is essential for
activating robust and long-lasting CD4+ and CD8+ T cell
responses in patients with cancer.
6.Tumor/DC FusionsVaccine
Tumor/DC fusions have been strongly effective in animal
studies using melanoma [26, 31, 31, 47–52], colorectal [18,
30, 45, 50, 51, 53–59], breast [60–65], esophageal [66],
pancreatic [67, 68], hepatocellular [69–73], lung [74–78],
renal cell carcinoma [79], sarcoma [80–85], myeloma [86–
93], mastocytoma [94], lymphoma [95], and neuroblastoma
[96]. More importantly, in preclinical studies the fusions
were also effective to induce CTL responses in vitro using
colorectal [25, 97–102], gastric [103, 104], pancreatic [105],
breast [43, 106–110], laryngeal [111], ovarian [34, 44, 112],
lung [113], prostate [114, 115], renal [116, 117], and
hepatocellular [118–120] carcinoma, leukemia [121–126],
myeloma [127, 128] sarcoma [129, 130], melanoma [29,
131–133], glioma [124], and plasmacytoma [134].
Based on these unique features of tumor/DC fusions
with antitumor immunity in murine and preclinical studies,
initial Phase I/II clinical trials have been conducted in a
variety of tumors (Table 1). Tumor/DC fusions vaccine was
first reported in patients with melanoma. Allogeneic DCs
were fused with autologous melanoma cells by electrofusion
and vaccinated in 16 patients with disseminated melanoma
refractory to standard therapy [135, 136]. There were no
serious side effects associated with the administration of the
vaccine. Seven of the 16 patients responded to the vaccina-
tion, one with complete response, one with partial response,
and five with stable disease, following to previous rapid
progression. Similar results in patients with melanoma were
reported from another group using autologous melanoma
cells fused to DCs either from healthy donors [137] or
the patients [138]. Although Tumor/DC fusions vaccine
was also coadministrated with rIL-2, efficient antitumor
immunity was not observed in patients with melanoma
[139]. Moreover, vaccination with fusions of HLA class
I-mismatched DCs from healthy donor and autologous
melanoma cells failed to find unequivocal beneficial effects
[139]. In addition, in malignant glioma, autologous fusions
vaccine produced partial clinical responses in two of six
patients [140]. In a similar trial by the same group, a combi-
nation of autologous fusions and rIL-12 was administered to
patients with malignant glioma, melanoma, breast, gastric,
colorectal, and ovarian cancer [23, 24, 141]. Three of 12
patients with malignant glioma achieved a partial response
and one patient a minor response [24] but the response to
other types of malignant tumors was muted [23]. Another
group tested fusions vaccine in 23 patients with metastatic
breast and renal cancer [142]. Immunologic and clinical
responses were observed in a subset of patients. Two patients
with breast cancer exhibited disease regression, including a
nearly complete response of a large chest-wall mass. Five
patientswithrenalcellcarcinomaandonepatientwithbreast
cancer showed stable disease. In a subsequent trial from
same group, autologous renal cell carcinoma cells were fused
with allogeneic DCs [143]. Although antitumor immune
responseswereobservedin10/21evaluablepatients,apartial
clinical response was demonstrated in two patients and
stable disease in eight patients. In patients with renal cell
carcinoma, fusions vaccine generated with allogeneic DCs
and autologous tumor cells showed immunologic, but not
effective clinical responses [138, 144, 145]. Together, only
limited therapeutic results were obtained in all these clinical
trials.
7.ImmunosuppressioninTumor
Microenvironment
Tumor/DC fusions aimed for inducing efficient antitumor
immunity have provided important proofs of principle in
both murine models and preclinical human models. How-
ever, immunological responses by DC/tumor fusions vaccine
havenotbeenassociatedwithsignificantclinicalresponses.A
majorreasonofthediversityisimmunosuppressivemicroen-
vironmentwithinthetumor.Themicroenvironmentinsolid
tumors is consisted of tumor cells and stroma cells such
as cancer-associated fibroblasts (CAFs), tolerogenic DCs,
myeloid-derived suppressor cells (MDSCs), immunosup-
pressive tumor-associated macrophages (TAMs), and Tregs
[66, 146–149] (Figure 3). Tumor cells and CAFs produce
immunosuppressive substances such as vascular endothelial
growth factor (VEGF) [150], IL-6 [151], IL-10 [151], trans-
forming growth factor-β (TGF-β) [152], soluble Fas ligand
(Fas-L)[153],andindolamine-2,3-dioxygenase(IDO)[154].
Tolerogenic DCs express low levels of MHC class I, II, and
costimulatory molecules and produce increased levels of
TGF-β, all of which are associated with generation of Tregs
[155–157]. MDSCs suppress the activation of CD4+ and
CD8+ T cells [158, 159] and also facilitate the generation
of tumor-specific Tregs [160, 161]. TAMs promote tumor
progression by generation of Tregs [162] and abolish tumor-
specific CTLs [163]. As the results, generation of Tregs

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Clinical and Developmental Immunology5
Table 1: Asessment of clinical trials by tumor/DC fusions-based vaccine.
Tumor/DC Fusions
Tumor Cells
Autologous
Patient
Number
16
Clinical
Responses
1 (CR)
1 (PR)
5 (SD)
9 (PD)
1 (PR)
1 (SD)
15 (PD)
8 (SD)
3 (SD)
2 (N)
4 (PD)
1 (SD)
10 (PD)
2 (PR)
1 (SD)
5 (PD)
3 (PR)
2 (MR)
4 (SD)
3 (PD)
14 (SD)
2 (PD)
3 (OR)
3 (N)
5 (SD)
8 (PD)
2 (PR)
8 (SD)
10 (PD)
3 (SD)
5 (PD)
1 (SD)
3 (PD)
1 (PR)
6 (SD)
3 (PD)
2 (PR)
1 (SD)
7 (PD)
1 (SD)
1 (PD)
1 (SD)
2 (PD)
1 (PD)
2 (SD)
1 (PD)
Tumor
Melanoma
Dendritic Cells
Allogeneic
CoadministrationRef.
[135]
[136]
Autologous Autologous 17[137]
Autologous Allogeneic 13[138]
Autologous
Autologous
Autologous
Allogeneic
rh IL-12
rh IL-2
4
11
[23]
[139]
Glioma AutologousAutologous8 [23]
AutologousAutologousrh IL-12 12[24]
Renal cell carcinomaAutologous Allogeneic 22[138]
Autologous Autologous13[142]
Autologous Allogeneic 20[143]
Allogeneic Allogeneic8 [144]
AutologousAllogeneic4[144]
AutologousAllogeneic 10[145]
Breast cancerAutologous Autologous 10[142]
AutologousAutologousrh IL-122[24]
Gastric/Colorectal cancer AutologousAutologousrh IL-123 [24]
Hepatocellular carcinoma
Ovarian cancer
Autologous
Autologous
Autologous
Autologous
1
3
[118]
[24] rh IL-12
CR: complete response; PR: partial response; MR: mixed response; SD: stable disease; PD: progressive disease.
OR: objective response; N: not evaluated.

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6Clinical and Developmental Immunology
CD8
CTL
Naive
T cell
Treg
Tumor
TAM
MDSC
Immature
DC
Mature DC
Treg
ROS
NO
IL-10
TGF-β
VEGF
IL-6
IL-10
TGF-β
Fas-L
IDO
Figure 3: Immunosuppression in tumor microenvironment.
Tumors secrete various factors such as VEGF, IL-6, IL-10, TGF-
β, Fas-L, and IDO, all of which promote the accumulation
of heterogeneous populations of tumor-associated macrophages
(TAMs), myeloid-derived suppressor cells (MDSCs), or immature
DCs. These immunosuppressive cells inhibit antitumor immunity
by various mechanisms, including depletion of arginine and elabo-
ration of reactive oxygen species (ROS) and nitrogen oxide (NO).
The tumor microenvironment also promotes the accumulation
of regulatory T cells (Tregs) that suppress CD8+ CTL function
through secretion of IL-10 or TGF-β from Tregs and tumors.
evades the antitumor immunity [164]. Indeed, an increase
of Tregs population has been observed in the peripheral
blood from patients with advanced cancer [165, 166] and
is inversely related to the outcome of several human cancer
treatments [167, 168]. Therefore, tumor/DC vaccines that
struggle against the tumors with CTLs as well as depletion
of Tregs may tip the balance in favor of immunostimulation.
8.Activationor Inactivationof Antitumor
Immunityby Tumor/DC Fusions
Progress in antitumor immunotherapy has been aided by
advances in the understanding of antigen presentation by
DCs and the rules for governing polarization of subsequent
immune responses toward CD4+ (Th1/Th2 phenotypes)
or CD8+ T cells [2]. Importantly, the immunosuppressive
microenvironment in tumors evades CTL responses during
their induction and effector phase [165, 166]. Indeed, in
cancer patients vaccinated with tumor/DC fusions, soluble
factors derived from tumor cells inhibited the induction of
CTL responses and promoted the generation of Tregs with
immunosuppressive capacities [118]. One way to improve
the CTL induction phase may be blockade of the negative
soluble factors from tumor/DC fusions. In murine model,
tumor-derived TGF-β reduced the efficacy of tumor/DC
fusions vaccine via an in vivo mechanism [55]. However,
the reduction of TGF-β derived from fusions inhibited
Tregs generation and enhanced antitumor immunity [66].
Therefore, attention to these immunological bottlenecks
may prove critical to fully harness the therapeutic potential
of the fusions vaccine. Another approach for blocking
the suppressive soluble factors from fusions is the use of
adjuvants. The recognition of microbes by innate immune
cells initiates activation of the whole immune system [169].
Toll-like receptors (TLRs) recognize various components
of invading pathogens. It has been reported that DCs
maturation by microbial products through TLRs is essential
for abrogating the activity of Tregs in induction phase of T
cells [170]. Moreover, crosspriming by DCs is based on the
transfer of proteasome substrates that are transcriptionally
upregulated by heat treatment in human tumor cells [171].
Therefore, we have generated mature fusions by fusing
DCs stimulated with the TLR agonists and heat-treated
tumor cells [100, 101]. The mature fusions had potent APC
functions in induction phase of T cells, as demonstrated
by (1) upregulation of multiple heat-shock proteins (HSPs),
MHC class I and II, TAAs, CD80, CD86, CD83, and IL-12;
(2) activation of CD4+ and CD8+ T cells able to produce
IFN-γ at higher levels; (3) potent induction of cytotoxic
activity specific for TAAs (CEA and MUC1) against tumors.
Incorporating heat-treated tumor cells and TLR stimulated-
DCs may increase the immunogenicity of tumor/DC fusions
in induction of CTL responses. Similar results were also
obtained from fusions generated with gastric cancer patients
[172]. Immature fusions may stimulate a mixed T cell
response characterized by the expansion of both CTL and
Treg populations [109]. In addition, tumor/DC fusions
activated by TLR agonists, IL-12, and anti-CD3/CD28
preferentially limited the generation of Tregs and promoted
expansion of activated CTLs [109, 110]. Therefore, mature
fusions have more active to stimulate CTL responses in
the immunosuppressive environment in the growing tumor
burden (Figure 4). Indeed, in murine models, tumor/DC
fusionscoadministratedwithTLR9(syntheticoligodeoxynu-
cleotides (ODNs) containing specific bacterial unmethylated
CpG motifs (CpG ODNs)) and TLR3 agonists (Poly(I:C))
significantly reduced melanoma metastasis through IL-12
production,comparedwithfusionsalone[59,82].Moreover,
tumor/DC fusions transduced with IL-12 [30, 87, 91, 96],
IL-18 [90, 96], GM-CSF [47], IL-4 [88], CD40L [89] genes
induced potentially increased therapeutic efficacy.
Another approach designed to improve the efficacy of
cancer vaccine is HSP70-based vaccine using tumor/DC
fusion technology. The HSP70/peptide complexes (HSP70.
PC)derivedfromtumor/DCfusionswereespeciallydifferent
from those derived from tumor cells in enhanced association
with immunologic peptides in animal models [173] and
human models [172, 174]. HSP70.PC from human fusions
induced T cells that expressed higher levels of IFN-γ
and exhibited increased levels of killing of tumor cells,
compared with those induced by HSP70.PC derived from
tumor cells [172, 174]. Moreover, enhanced immunogenicity
of HSP70.PC from fusions was associated with improved
composition of the vaccine.
9.Combination of Treg Blockade and
Tumor/DC FusionsVaccine
Cancer vaccines must include some strategies to regulate
the immunosuppressive cell types and tumor byproducts.

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Clinical and Developmental Immunology7
Immature
Fusions
CD8 + CTL
Treg cell
immunity
Stimulatory
immunity
Suppressive
Perforin
granzyme B
Mature
Fusions
TGF-β
IL-10
TGF-β
IL-10
IDO
MHC class I
MHC class II
Peptide
Peptide
Costimulatory molecules
IL-12
HSP
Figure 4: Activation or inactivation of T cells by tumor/DC fusions. After acquired antigens in the periphery, tumor/DC fusions migrate
to the draining lymph nodes, where they encounter a cognate CD4+ or CD8+ T cells. The mature tumor/DC fusions produce stimulatory
factors, such as IL-12 and heat-shock proteins (HSPs), while the immature fusions produce suppressive factors (TGF-β, IL-10, or IDO,
etc.). High expression of costimulatory and MHC class I and II molecules by mature fusions is essential to promote survival and proliferative
capacityoftheactivatedCD8+CTLs.MaturefusionsinduceefficientCD8+T-cellactivationwithhighproductionofperforinandgranzyme
B. On the other hand, immature fusions may induce, at least in part, Tregs. In tumor microenvironment, the consequence of products from
tumor cells enhances local suppressive immunity.
Even if tumor/DC fusions were activated by TLRs, Tregs
were not a little induced [105, 109, 118]. As Tregs is one of
major obstacles for therapeutic cancer vaccines, depletion or
blockade of Tregs might enhance rejection of endogenous
immune-escaped tumor and improve tumor immunity. In
most patients with melanoma (90%), recombinant IL-2-
diphteria toxin fusion protein (ONTAK) treatment resulted
in depletion of Tregs and sufficient induction of melanoma-
specificCTLresponses[175,176].Moreover,CTL-associated
antigen-4 (CTLA-4) antagonistic antibodies also release a
key negative regulatory pathway on T cells and enhance
antitumor immunity [177–179]. Other antibodies, such
as CD137 (4-1BB) [180], CD40 [181], and programmed
death-1 (PD-1) [182] antagonists are currently investigated
in various stages of preclinical and clinical development.
In tumor/DC fusions approach, it has been reported that
the fusions coadministrated with Treg depletion by anti-
CD25 antibody enhanced the efficacy of immunotherapy in
murine pancreatic models [68]. Therefore, a combination
of control of Tregs and concomitant vaccination of mature
tumor/DCs fusions may be a more promising approach for
the induction of therapeutic antitumor immunity in patients
with advanced cancer.
Recently, to overcome negatively regulated pathway by
Tregs, a combination therapy of vaccine and chemotherapy
has been designed to counteract this immune suppression.
For example, when adoptive immunotherapy was combined
with nonmyeloablative lymphodepleting chemotherapy, 18
(51%) of 35 treated patients with refractory metastatic
melanomaexperiencedobjectiveclinicalresponsesincluding
three ongoing complete responses and 15 partial responses
[183]. This improvement of clinical responses is most likely
owing to the elimination of MDSCs and Tregs. Indeed,
cytotoxic chemotherapy not only affects the tumor but
also depletes MDSCs and Tregs [184]. Postchemotherapy
immune system reconstitution may provide a unique oppor-
tunity for therapeutic intervention by shaping the repertoire
towards responses to tumor antigens [147, 185, 186].
10.Summary
Although immunological responses have been observed in
patients with advanced stage of cancer after being vaccinated
with DC-based vaccines including tumor/DC fusions, the
clinicalresponsesarenotasvigorousasintheanimalmodels.
Several aspects of cancer vaccines require the reduction of
Tregs networks or suppressive tumor-microenvironments
that inhibit the function of antitumor immune responses. To
date, most of clinical trials have been enrolled patients who
are in the advanced stages of cancer, which may have limited
the clinical effectiveness because such individuals may not be
able to mount an effective immune response. As tumor/DC
fusions vaccine has been established as safe in phase I/II
trials, the fusions vaccine should be tested in patients with
early stage of cancer. Importantly, a combination therapy

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8Clinical and Developmental Immunology
of cancer vaccines and other therapies such as conventional
chemotherapy should be a more promising approach.
Conflict of Interests
The authors have no relevant affiliations or financial involve-
ment with any organization or entity with a financial interest
in or financial conflict with the subject matter or materials
discussed in the paper.
Acknowledgments
This work has been supported by Foundation for Promotion
of Cancer Research, Mitsui Life Social Welfare Foundation,
Grants-in-Aid for Scientific Research (B) from the Ministry
of Education, Cultures, Sports, Science and Technology
of Japan, Grant-in-Aid of the Japan Medical Association,
Takeda Science Foundation, and Pancreas Research Founda-
tion of Japan.
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