J Neurooncol (2015) 123:425–432
DOI 10.1007/s11060-015-1830-1
EDITORS’ INVITED MANUSCRIPT
Dendritic cell immunotherapy for brain tumors
Joseph P. Antonios1 • Richard G. Everson1 • Linda M. Liau1,2,3,4
Received: 2 April 2015 / Accepted: 25 May 2015 / Published online: 3 June 2015
Ó Springer Science+Business Media New York 2015
Abstract Glioblastomas are characterized by immuno-
suppression, rapid proliferation, angiogenesis, and invasion
into the surrounding brain parenchyma. Limitations in
current therapeutic approaches have spurred the develop-
ment of personalized, patient-specific treatments. Among
these, active immunotherapy has emerged as a viable
option for glioma treatment. The ability to generate an
immune response utilizing patient-derived dendritic cells
(DCs) (professional antigen-presenting cells) is especially
attractive. This approach to glioma treatment allows for the
immunologic targeting and destruction of malignant cells.
Data acquired in multiple pre-clinical models and clinical
trials have shown significant responses and prolonged
survival. Here we provide an overview of the current status
of DC vaccination for the treatment of gliomas.
Keywords Glioma
Glioblastoma
Immunotherapy

Dendritic cell vaccine
& Linda M. Liau
lliau@mednet.ucla.edu
1
Department of Neurosurgery, David Geffen School of
Medicine at UCLA, University of California Los Angeles,
Los Angeles, CA 90095, USA
2
Brain Research Institute, David Geffen School of Medicine at
UCLA, University of California Los Angeles, Los Angeles,
CA 90095, USA
3
Jonsson Comprehensive Cancer Center, David Geffen School
of Medicine at UCLA, University of California Los Angeles,
Los Angeles, CA 90095, USA
4
Department of Neurosurgery, David Geffen School of
Medicine at UCLA, University of California Los Angeles,
300 Stein Plaza, Suite 562, Box 956901, Los Angeles,
CA 90095-6901, USA
Introduction
Malignant gliomas are the most frequent and aggressive
intrinsic brain tumors of the central nervous system (CNS).
Characterized by diffuse infiltration into brain parenchyma,
gliomas often do not respond well to conventional treatments
[1, 2]. There is an urgent need for more effective and specific
therapies, as current treatment methods of surgery,
chemotherapy and radiation have failed to substantially alter the
poor prognosis of this disease [1, 3]. Immunotherapeutic
approaches to glioma treatment are especially attractive, as they
allow for specific targeting and destruction of malignant cells.
Indeed, while the CNS was once considered an immune-priv-
ileged site largely protected by the blood–brain barrier, it has
been shown that, instead, the CNS supports a highly-specialized
immune surveillance system that can recruit cells associated
with both innate and adaptive immunity [4]. Specifically, den-
dritic cell (DC) immunotherapy is an attractive approach for the
treatment of brain tumors, as immune cells theoretically can
seek out and selectively destroy invading malignant cells while
sparing normal brain tissues [3]. As professional antigen pre-
senting cells (APCs), DCs are the most potent stimulators of the
cell-mediated immune system. Its specialized ability to
orchestrate antigen-specific responses through CD4? helper T
cells and CD8? cytotoxic T cells makes DCs prime candidates
to induce anti-tumor immunity. This review discusses the pro-
mise these DC-based immunotherapies have shown for treating
malignant brain tumors.
Dendritic cells and cancer immunotherapy
DCs are the foremost coordinators of immune responses,
bridging non-antigen specific innate immunity with the
antigen-specific arm of adaptive immunity [5]. Though
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DCs are most often known as professional APCs, their
immature state is characterized by high levels of phago-
cytic activity and not antigen presentation [6]. They are
localized in most organs and tissues, especially where
pathogen entry is most likely, and act as local sentinels and
primary immune responders. By constantly sampling and
processing antigens, DCs make the crucial decision to
induce tolerance or begin the recruitment of effector cells.
Once they have trafficked into draining lymph nodes,
mature DCs can quickly and efficiently seek out rare
antigen-specific T cells to begin a proliferative burst
(Fig. 1) [7]. Their specialized ability to orchestrate antigen-
specific responses through CD4? helper T cells and CD8?
cytotoxic T cells makes DCs prime candidates to induce
anti-tumor immunity.
With respect to cancer, in the process of malignant
transformation and mutation, many neoplastic cells give
rise to proteins that are not normally expressed in most
tissues of the body. The presence of unique, identifying
antigens renders tumor cells susceptible to immune
recognition. When immune regulation remains competent,
tumor cells express these unique antigens, allowing circu-
lating DCs to recognize them as foreign. Consequently, DC
vaccination has proven to be promising in treating various
types of cancers, including human gliomas.
DCs originate in the bone marrow from precursor stem
cells. The most common in vitro method used to generate
Fig. 1 Dendritic cells originate in the bone marrow. Once they enter
tissues and organs, the immature dendritic cells sample and process
antigens from the environment. A dendritic cell that has processed
antigen will partially mature and migrate to a lymph node, where it
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J Neurooncol (2015) 123:425–432
DCs involves culturing peripheral monocytes with the
cytokines interleukin-4 (IL-4) and granulocyte–macro-
phage colony-stimulating factor [8]. The resulting pheno-
type of these cells most closely resembles conventional
DCs, which are characterized by high levels of CD11c and
MHC Class II expression and can stimulate T cell prolif-
eration. The resulting immature DCs have proven to be
fully effective in antigen uptake and subsequent T cell
activation once mature. Generation of immature DCs is
necessary for most methods of vaccine preparation. This
stems from the enhanced phagocytic abilities of immature
DCs compared to their mature counterparts, which allows
for the uptake of antigen sources [9]. Immature DCs can be
pulsed with autologous tumor-eluted peptides [10], tumor
lysate [11], or tumor-derived RNA [12]. These must then
be matured and activated—a necessary step for upregula-
tion of co-stimulatory molecules, antigen presentation, and
chemokine receptors associated with migration. DCs can
be administered intradermally for maturation in vivo; or
they can be matured ex vivo with the addition of adjuvants
(e.g., a-galactosylceramide, CD40-specific antibody,
inflammatory cytokines, or specific TLR ligands such as
polyI:C) and then pulsed with defined T cell epitopes
[13].More recently, Kalinski et al. developed mature, non-
exhausted ‘type-1-polarized’ DCs (DC1s) that produce
high levels of IL-12 by exposing immature DCs to types I
and II interferons in culture [14]. Okada et al. showed that
will mature and seek out an antigen-specific T cell to begin a
proliferative burst. HSC hematopoietic stem cell, CMP common
myeloid progenitor, MDP macrophage dendritic cell progenitor, CDP
common dendritic cell progenitor
J Neurooncol (2015) 123:425–432 427

these novel DC1-type DCs induced long-lived T-cell
responses against targeted antigens more efficiently than
mature DCs [15].
DC-based immunotherapy for brain tumors
Developing clinically relevant DC vaccine therapies has
proven challenging, with multiple approaches of preparing
DCs for vaccination tested in clinical trials for patients with
gliomas (Table 1).
Acid-eluted peptide DC vaccine
Liau et al. described the first case report of a glioblastoma
patient treated with DC-based immunotherapy, which
consisted of three intradermal injections of autologous DCs
pulsed with allogeneic acid-eluted peptides [16]. Due to the
marked heterogeneity of malignant gliomas, this was an
attractive approach to DC therapy [17]. The acid-elution
technique extracted tumor antigens bound to MHC Class I
molecules, allowing for the preparation of a unique tumor-
antigen specific vaccine. While clinical benefit was diffi-
cult to assess in this single case report, an increased CD3?
T-cell infiltrate into the tumor was noted. In a Phase I dose-
escalation trial, 12 malignant glioma patients were treated
with DCs loaded with acid-eluted peptides from autologous
tumor. This DC immunotherapy was found to be safe and
well-tolerated, as no cases of either autoimmunity or dose-
limiting toxicity were reported with up to 107 DCs injected
per dose [10]. Patients in this study exhibited increased
progression-free survival and median overall survival
compared to historical controls, with five long-term sur-
vivors ([36 months). Of the eight patients who developed
new areas of contrast enhancement on follow-up MRI
scans, four demonstrated prolonged survival following
surgical biopsy and resection. Specimens obtained from
these patients demonstrated a significant CD3? T-cell
infiltrate, predominantly composed of memory T cells
(CD8?/CD45RO?). Data from these initial trials also
suggested that DC-based immunotherapy works best in the
absence of actively progressive tumor and with minimal
residual disease.
Subsequently, Yu et al. also reported on glioblastoma
patients treated with intradermal DCs loaded with acid-
eluted tumor peptides with similar findings [18]. Never-
theless, this approach specifically utilizing DCs prepared
with acid-eluted peptides was somewhat limited. This was
largely due to technical difficulties related to the require-
ment for large numbers of primary tumor cells needed for

Table 1 Clinical trials assessing dendritic cell immunotherapy

Therapy/protocol Center/principal investigator Phase- ND, P, R WHO ClinicalTrials.gov References
enrollment (newly grades identifier
diagnosed,
persistent,
recurrent)

Acid-eluted tumor peptide UCLA, Los Angeles, CA/Liau I-28 ND III or IV NCT00068510 [14]
dendritic cell vaccine
Autologous tumor lysate UCLA, Los Angeles, CA/Liau II-23 ND, R III or IV NCT01204684 [11, 15]
dendritic cell vaccine
Autologous tumor lysate UCLA, Los Angeles, CA/Liau III-348 ND IV NCT00045968
dendritic cell vaccine
Autologous tumor lysate Cedars-Sinai Medical Center, Los II-50 R III or IV NCT00576537 [17, 19]
dendritic cell vaccine Angeles, CA/Yu
Autologous tumor lysate University Hospital Gasthuisberg, 56 R IV [20]
dendritic cell vaccine Leuven, Belgium/van Gool
Autologous tumor lysate University Hospital Gasthuisberg, 45 ND III or IV [21]
dendritic cell vaccine Leuven, Belgium/van Gool
Tumor-specific EGFRvIII Duke University, Durham, NC and II-40 ND IV NCT00643097 [23]
vaccine MD Anderson Cancer Center at
UT Houston, Houston, TX/
Vlahovic and Sampson
Autologous DC ? brain tumor Duke University, Durham, NC/ I-50 R IV NCT00890032
stem cell-mRNA Sampson
Autologous DCs cancer stem Huashan Hospital, Shanghai, II ND, R III or IV NCT01567202
cell vaccine China/Zhou
Table categorized based on therapy and protocol, with associated information regarding phase, grade, clinical trial identifier, and any associated
references

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acid-elution and the inability to consistently grow out
sufficient tumor cells for this purpose.
Tumor-lysate pulsed DC vaccine
More recently, tumor lysate prepared from autologous
tumor cells obtained from surgical resection have been
utilized to pulse DCs. In this technique, patient tumor cells
are lysed using freeze/thaw cycles to generate cellular
fragments; and exposure of DCs to these tumor fragments
allows for phagocytosis and subsequent peptide antigen
presentation on both MHC Classes I and II. Although there
is a theoretical disadvantage of the increased potential for
selection of auto-reactive T cells due to the lack of antigen
specificity, no severe auto-immunity has ever been reported
with the use of tumor lysate-pulsed DC vaccination for
CNS tumors.
Prins et al. reported on a Phase I/II clinical trial that
involved patients with both newly diagnosed and recurrent
glioblastoma receiving immunotherapy with autologous
tumor lysate-pulsed DCs, as well as adjuvant imiquimod or
poly-ICLC during booster vaccinations. Safety of the vac-
cines and the adjuvants were demonstrated, with no sig-
nificant adverse events reported. Patients in this trial had
substantially better progression-free and overall survival
rates compared to historical controls, with a median sur-
vival of 31.4 months for newly diagnosed glioblastoma
patients. Improved outcome appeared also to stratify by
tumor molecular subtype as measured by gene expression
profiling, with increased survival shown for patients within
the mesenchymal subgroup of glioblastoma [11]. Currently,
a Phase III multi-center trial of DC vaccination for newly
diagnosed glioblastoma is underway, with approximately
348 patients to be enrolled (NCT00045968) [19].
Yu et al. also reported a Phase II trial with DC
immunotherapy in patients with recurrent glioblastoma that
utilized DCs loaded with autologous tumor lysate [20]. They
found evidence of systemic antitumor CTL activity, as well
as intra-tumoral lymphocyte infiltration in half of patients
undergoing re-operation. Median survival in the DC vacci-
nated patients was markedly increased to 133 versus
30 weeks for matched historical control patients treated at
the same institution. Wheeler et al. reported that after vac-
cination, PBMCs restimulated by tumor lysate were found to
increase production of IFN-c by greater than 1.5-fold in a
significant percentage of patients on the trial [21]. These
patients, who were characterized as ‘‘vaccine responders,’’
demonstrated an increased survival compared to ‘‘non-re-
sponders’’. Furthermore, vaccine response appeared to
potentiate the effects of subsequent chemotherapy.
De Vleeschouwer et al. reported on the treatment 56
patients with recurrent glioblastoma with matured DCs in
varied vaccination schedules [22]. They also included
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J Neurooncol (2015) 123:425–432
booster vaccines of tumor lysate alone. Patients on the trial
had a median survival of 9.6 months, with some patients
surviving more than 3 years from diagnosis of recurrent
glioblastoma. Patients who underwent complete resection
of tumor demonstrated a statistically significant improve-
ment in survival versus those who did not. A small per-
centage of patients in this trial displayed transient
worsening of focal neurologic deficits that had typically not
been reported in other trials. Whether or not these patients
had residual tumor was not reported. Nine of 21 patients on
this trial demonstrated a positive delayed-type hypersen-
sitivity (DTH) response. However, DTH responses did not
correlate with overall survival. Ardon et al. expanded their
study to evaluate the efficacy of DC vaccination in pedi-
atric patients who presented with a variety of high-grade
malignant brain tumors [23]. Autologous tumor lysate was
used as the antigen source for the autologous monocyte-
derived DC vaccine. Of those in the study, patients with
high-grade glioma and atypical teratoid–rhabdoid tumors
seemed to respond favorably to treatment, whereas patients
with ependymoma or medulloblastoma/PNETs did not.
Similar to their adult trials, this group observed a subset of
patients diagnosed with high-grade gliomas exhibited long-
term survival.
Glioma-associated antigen pulsed DC vaccine
Several glioma-associated peptides have also been used to
pulse DCs for clinical trials in malignant glioma patients.
These tumor-associated antigens (TAAs) have included the
mutated form of the epidermal growth factor receptor
(EGFRvIII), as well as the IL-13 receptor a2 (IL-13a2) and
gp100 [15, 24]. These peptides were able to induce specific
responses in patients, as measured by in vitro proliferation
assays and measurement of tetramer positive CD8? T cells.
Sampson et al. targeted EGFRvIII with DCs pulsed with a
peptide spanning the fusion junction of EGFRvIII conju-
gated to KLH [24, 25]. EGFRvIII expression in the tumor
was not an eligibility criterion for entry into the trial which
was a Phase I toxicity study. They intended to treat 15
patients, 3 of which progressed prior to receiving the
vaccine. Of the vaccinated patients, many displayed
EGFRvIII-specific immune responses with 10 out of 12
demonstrating antigen-specific T-cell proliferation and 5
out of 9 showing DTH responses to EGFRvIII.
DCs loaded with tumor-derived mRNA or cancer stem
cell antigens have also been studied [26]. An ongoing
Phase I trial is currently examining the applicability of a
vaccine utilizing mRNA-loaded DCs (NCT00890032) for
patients with gliomas. A Phase II trial is alternatively
examining DCs loaded with glioma stem-like cell antigens
(NCT01567202). Although these studies have not demon-
strated significantly increased overall survival, their ability
J Neurooncol (2015) 123:425–432
to induce antigen specific responses may warrant further
investigation.
Vaccine adjuvants
The ultimate goal of DC vaccine immunotherapy is to
induce a long-term immune response in glioma patients.
Gliomas express cytokines [such as transforming growth
factor-b2 (TGF-b2) and IL-10] that are known to sup-
press immune responses both directly and indirectly
(through T regulatory cells; Fig. 2). To promote a suc-
cessful long-term anti-tumor response and counter
immunosuppressive factors, the use of adjuvant treatments
alongside DC vaccine to counter tumor immunosuppres-
sion has been extensively studied [27].
Initially hampered by unfavorable side effects, pre-
clinical work has revived the use of immunotherapeutic
adjuvants, with the advent of engineered local delivery
systems with lower associated toxicities [28, 29].
Interferons, which activate and mediate immune
response, are divided into two classes: types II and I. Type
Fig. 2 Glioma tumor cells release cytokines that promote a suppres-
sive environment by both directly inhibiting the anti-tumor response
and promoting further release of inhibitory factors from cells such as
T regulatory cells
429
I interferons, IFN-a, IFN-b and IFN-x, are produced in the
setting of viral antigens and immune-modulatory signaling.
IFN-a induces tumor cell apoptosis and stimulates DCs. At
this point, however, clinical studies have not shown sig-
nificant benefit with the adjuvant use of IFN-a [30]. Type II
interferons, such as IFN-c, are released from activated T
cells to promote anti-tumor activity [31]. In pre-clinical
studies, administration of IFN-c alongside glioma-lysate
vaccination promoted anti-tumor activity with a significant
increase in long-term survival [32].
Additionally, ILs—another family of cytokines—regu-
late immune response. IL-2 promotes T-cell proliferation
and function. Therapies using IL-2 alone in pre-clinical
tumor-bearing animal models prolonged survival, with
some promise shown in patients when used as an adjuvant
therapy [33].Systemic IL-4, produced by T cells, promotes
a Th2 response [34]. It has been shown, however, that local
delivery of IL-4 at the site of immunization induces IL-12
production [35]. IL-12 promotes the Th1 response, as well
as T-cell activation and IFN-c production. Similarly, pre-
clinical models have shown a survival benefit with
administration of this cytokine both alone and as an adju-
vant to other therapeutic approaches [36].
Recently, Mitchell et al. demonstrated that using a recall
antigen to pre-condition the vaccine site allowed for
improved vaccination efficiency. They demonstrated
improved clinical responses in glioblastoma patients via this
technique; specifically, skin that was preconditioned with
tetanus/diphtheria toxoid stimulated increased CCL3 pro-
duction, in turn allowing DCs to better traffic into the local
lymph nodes and promote better T cell responses [37].
DC immunotherapy and checkpoint inhibitors
The immune system is intrinsically primed to respond to
infectious agents and harmful substances. From recogniz-
ing, containing, and eliminating pathogens to developing
immunologic memory and responding to re-exposure, the
immune system is able to react to most biologic insults. In
the case of tumor, however, several mechanisms come into
play. The first line of anti-tumor defense, innate immunity,
relies on phagocytic cells, granulocytes, and lymphoid
progenitor-derived natural killer cells to mediate an initial
immune response. DC activation and migration to lymphoid
tissue upon phagocytosis of malignant, dying, or stressed
cells then leads to activation of secondary immune response.
This adaptive immunity is mediated by the presentation of
TAAs by APCs to induce TAA-dependent clonal expansion
of T cells (CD8?) cytotoxic T cells (CTLs), CD4? helper T
cells, and regulatory T lymphocytes (Tregs) [38].
Several factors may limit the ability of the immune
system to mount a successful adaptive response against
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gliomas (Fig. 3). Relevant to this discussion, cytotoxic
T-lymphocyte associated antigen 4 (CTLA-4) and the
programmed death 1 (PD-1) receptor have both been
identified as negative immune regulators [39]. CTLA-4 is a
co-inhibitory receptor expressed on immune cells that
competes with CD28? receptor binding of CD80 and CD86
to inhibit the initial activation of T cells [40]. Recent
studies similarly show that the inhibition of CTLA-4 by
antibody blockade results in objective tumor regression in
patients with metastatic melanoma [41, 42].
Glioma cells themselves reduce T-lymphocyte prolifer-
ative function, decrease Th1 cytokine synthesis, limit
interactions with normal cells, and alter HLA Class I
antigen expression [43]. Expression of TGF-b inhibits
T-cell proliferation and CTL development, as well pro-
motes tumor vascularization and glioma cell migration
[44]. PD-1, an immune regulatory receptor associated with
inhibition of T cells with prolonged antigen exposure (a
phenomenon noted in chronic disease manifestations, such
as viral infections and cancer), plays an important role in
regulating the effector phase of the T-cell response [45].
Activation of this receptor by PD-L1 and PD-L2, expressed
by glioma cells and regulatory cells in the tumor
microenvironment, promotes immune suppression [39, 41,
42]. Early studies have shown that inhibition of the PD-1
receptor is associated with a significantly increased
immune response to cancer [41, 46].
Fig. 3 Mechanisms of inhibition in the tumor environment exist to
restrict anti-glioma immune response
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J Neurooncol (2015) 123:425–432
These findings suggest that gliomas are able to success-
fully alter the tumor microenvironment such that effective
anti-tumor immune responses are suppressed. Indeed,
clinical studies inhibiting these suppressive mechanisms
have been pioneered and suggested therapeutic benefit in
melanoma [45]. Pre-clinical glioma models have corrobo-
rated this evidence, with Wainwright et al. demonstrating
that blockade of CTLA-4, PD-1, and IDO in established
intracranial glioma models resulted in decreased tumor-in-
filtrating T regulatory cells and increased long-term sur-
vival mediated by cytotoxic T lymphocytes [47]. Similarly,
Zeng et al. showed that PD-1 blockade in the setting of
radiation therapy led to long-term survival in mice
implanted with a murine glioma cell line [46]. These results
suggest promise for the inhibition of these immune-regu-
latory mechanisms for the potentiation of immune response
induced by DC-based vaccination strategies.
Conclusion
In conclusion, DC vaccines have shown efficacy in multi-
ple pre-clinical studies and Phase I/II clinical trials for
gliomas. While acid-eluted peptide and glioma-associated
peptide preparations have shown some clinical benefit, the
most widespread progress has been made using autologous
tumor lysate-pulsed DC vaccine approaches. Although it
was initially hypothesized that non-specific lysate prepa-
rations could induce severe autoimmune reactions in the
CNS, no major adverse autoimmune events have been
reported with now over 600 brain tumor patients treated to
date worldwide. Additionally, these vaccines have been
shown to induce antigen-specific responses, potentiate
immune cell infiltration into the tumor, and increase overall
survival in a subset of patients. Further developments in
adjuvant therapies will allow us to modulate and fine-tune
these anti-tumor immune responses. In the future, further
genomic sequencing and molecular characterizations of
brain tumors will undoubtedly allow for personalized DC
vaccines for glioblastoma patients that should generate
more potent and long-lasting anti-tumor immunity.
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