Immunotherapy Can Reject Intracranial
Tumor Cells without Damaging the Brain
despite Sharing the Target Antigen
This information is current as
of June 16, 2017.
Byram W. Bridle, Jian Li, Shucui Jiang, Ruby Chang, Brian
D. Lichty, Jonathan L. Bramson and Yonghong Wan
J Immunol 2010; 184:4269-4275; Prepublished online 17
March 2010;
doi: 10.4049/jimmunol.0901447
http://www.jimmunol.org/content/184/8/4269
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References
The Journal of Immunology
Immunotherapy Can Reject Intracranial Tumor Cells without
Damaging the Brain despite Sharing the Target Antigen
Byram W. Bridle,* Jian Li,* Shucui Jiang,† Ruby Chang,† Brian D. Lichty,*
Jonathan L. Bramson,* and Yonghong Wan*
A
ntigen-specific, active immunotherapy offers a promising
approach to target both primary and metastatic cancers of
various types including intracranial (i.c.) tumors (1, 2). In this
regard, finding Ags exclusively expressed by tumor cells and incorporating them into therapeutic vaccines will not only increase the
likelihood of induction of anti-tumor immunity but also reduce the
possibility of autoimmune pathology (3). However, development of
vaccines directed at tumor-specific Ags is complicated by the fact that
each vaccine will likely need to be patient specific (4). As an alternate
strategy, efforts have been invested into developing vaccines to target
tumor-associated Ags (TAAs) that are typically nonmutated proteins
overexpressed in specific classes of tumors (5). Indeed, despite the lack
of mutation in these proteins, many cancer patients often display
spontaneous reactivity to TAAs. This realization has led to the development of different strategies that can overcome self-tolerance and
elicit an immune response against self-Ags.
Whether pathological damage to normal tissues that express TAAs
is a requisite outcome of successful anti-tumor immunotherapy remains
controversial. Evidence from animal studies has shown that vaccination
against melanoma-associated Ags (MAAs), such as tyrosinase-related
protein 1 or dopachrome tautomerase (DCT; also known as tyrosinaserelated protein 2), is often associated with damage to normal melanocytes, leading to autoimmune vitiligo (6–9). Coupling of anti-tumor
immunity and autoimmunity has also been observed in other tumor
*Department of Pathology and Molecular Medicine and †Neurosurgery Division,
Department of Surgery, McMaster University, Hamilton, Ontario, Canada
Received for publication May 7, 2009. Accepted for publication February 15, 2010.
This work was supported by grants to Y.W. from the Canadian Institutes of Health
Research (MOP-67066) and the Ontario Cancer Research Network.
Address correspondence and reprint requests to Dr. Yonghong Wan, Department of
Pathology and Molecular Medicine, McMaster University, Room MDCL-5024, 1200
Main Street West, Hamilton, Ontario L8N 3Z5, Canada. E-mail address: wanyong@
mcmaster.ca
Abbreviations used in this paper: Ad, recombinant adenovirus; DCT, dopachrome
tautomerase; DNFB, 2,4-dinitro-1-fluorobenzene; hDCT, human dopachrome tautomerase; i.c., intracranial; MAA, melanoma-associated Ag; TAA, tumor-associated
Ag.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901447
models, and furthermore, autoimmune symptoms appear to be a positive sign of clinical efficacy in cancer immunotherapy trials (10–13).
These results led to the hypothesis that autoimmune destruction is an
unavoidable or necessary consequence of effective anti-tumor immunity. However, although the damage to nonessential organs may be
acceptable, this hypothesis imposes a conceptual challenge to cancer
vaccines targeting Ags that are also expressed by cells in vital organs.
We and others have shown that tumor protection can be achieved in the
absence of autoimmune disease (14–17). Specifically, we demonstrated
in a murine melanoma model that a high-magnitude CTL response induced by recombinant adenovirus (Ad) expressing human DCT (hDCT)
did not result in vitiligo unless there was damage (i.e., trauma or inflammation) to the normal tissue (15). A similar observation was made
by Lang et al. (18) that large numbers of CTL specific for pancreatic Ag
did not necessarily result in autoimmune diabetes, and overt autoimmune disease occurred only after an inflammatory stimulus that upregulated MHC class I expression in the pancreas. These data identify
a concurrent inflammatory event in the target organ as a key factor determining the development of an autoimmune disorder following vaccination against self-tumor Ags.
To directly address the question of whether an effective vaccine imposes a risk to a vital tissue, we extended our melanoma vaccine study
from the skin to the brain. Because both melanocytes and CNS tissue
differentiate from the neural crest during embryonic development, they
share MAAs, including DCT (19–21). This provides an ideal experimental setting to examine the relationship between anti-tumor immunity
and autoimmune pathology upon i.c. inoculation with melanoma cells.
Furthermore, melanoma commonly metastasizes to the brain in patients
(22), and many MAAs are expressed on human malignant gliomas (19,
20, 23); thus, a better understanding of melanoma Ag-based vaccines
may have a specific implication in the treatment of brain cancers.
Materials and Methods
Mice and tumor cells
Age-matched (8–10 wk old at study initiation) female C57BL/6 (H-2b) mice
(Charles River Laboratories, Wilmington, MA) were housed in specific pathogen-free conditions. Animal studies complied with Canadian Council on
Animal Care guidelines and were approved by McMaster University’s Animal
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Although vaccines targeting tissue differentiation Ags represent a promising strategy for cancer immunotherapy, the risk of triggering autoimmune damage to normal tissues remains to be determined. Immunizing against a melanoma-associated Ag, dopachrome tautomerase (DCT), which normal melanocytes and glial cells also express, allowed concurrent analysis of autoimmune
consequences in multiple tissues. We show that vaccination with recombinant adenovirus expressing DCT elicited a strong CTL
response in C57BL/6 mice, leading to protection against intracranial challenge with B16-F10 melanoma cells. Both histological
analysis and behavioral testing indicated that there was no evidence of neuropathology in vaccinated animals and long-term survivors. Although vitiligo or demyelination could be induced by additional stimuli (i.e., surgery or inflammation) in DCT-vaccinated
mice, it did not extend beyond the inflammatory area, suggesting that there is self-regulatory negative feedback in normal tissues.
These results demonstrate that it is possible to vaccinate against a tumor embedded in a vital organ that shares the target Ag. The
Journal of Immunology, 2010, 184: 4269–4275.
4270
SEPARATION OF ANTI-TUMOR ACTIVITY AND AUTOIMMUNE TOXICITY
Research Ethics Board. Murine melanoma (B16-F10) cells were grown in F11MEM containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate and
vitamin solution, 0.01 mM nonessential amino acids, 50 mM 2-ME, 100 U/ml
penicillin, and 100 mg/ml streptomycin (all from Invitrogen, Grand Island, NY).
Recombinant adenovirus. Adenoviral vector construction has been described
previously (24). Ad-hDCT expresses the full-length hDCT, and Ad-BHG contains no transgene. Viruses were propagated in 293 cells and purified on a cesium
chloride gradient.
Peptides
Immunodominant peptides from DCT that bind to H-2Kb [DCT180–188,
SVYDFFVWL; shared by hDCT and mDCT (25)] and I-Ab [hDCT88–102,
KFFHRTCKCTGNFA (26)] were synthesized by Biomer Technologies
(Hayward, CA).
Vaccination protocol
Anesthetized mice were immunized by i.m. injection of 1 3 108 PFU of
Ad-hDCT in 100 ml PBS (50 ml/hamstring). Control mice received AdBHG or PBS.
Stereotactic surgery
Abs
The following mAbs were used in flow cytometry assays: anti-CD16/CD32
(clone 2.4G2) to block FcRs, anti-CD3 (clone 145-2C11), anti-CD4 (clone RM45), anti-CD8 (clone 53-6.7) for detecting cell surface markers, and anti–IFN-g
(clone XMG1.2) (all from BD Biosciences, San Diego, CA) for intracellular
staining. Immunodepletion studies were conducted with the mAbs GK1.5 (antiCD4) and/or 2.43 (anti-CD8) from American Type Culture Collection (Manassas, VA). Purified mAbs (250 mg in 500 ml saline) were injected i.p. 2 d apart
and then twice a week thereafter at a maintenance dose of 200 mg/treatment. The
efficiency of specific depletion of lymphocyte subsets was .98% as measured
by flow cytometry. For phenotyping skin-infiltrating T cells, the following additional reagents were used: PE-conjugated Kb-SVYDFFVWL tetramer (The
Protein Core, Baylor College of Medicine, Houston, TX), recombinant mouse
E-selectin/CD62E Fc chimera (R&D Systems, Minneapolis, MN), biotinylated
polyclonal Ab to human IgG (Alexis Biochemicals, Plymouth Meeting, PA),
PE-Cy7–conjugated streptavidin (BD Biosciences), and anti-CD194/CCR4
(clone 2G12; BioLegend, San Diego, CA).
Vitiligo assessment
Mice were immunized with Ad-hDCT, and 14 d later, their skins were “painted”
with 120 ml 2,4-dinitro-1-fluorobenzene (DNFB, 0.2% diluted in acetone/olive
oil at a 4:1 ratio) (Sigma-Aldrich). Vitiligo development was monitored weekly
for 8 wk after the DNFB challenge.
Behavioral assessments
To further evaluate the long-term effect of vaccination therapy on brain functions,
a set of preliminary behavioral assessments were performed in mice that survived
.90 d posttherapy and age-matched unvaccinated mice, as described previously
(28). Briefly, each individual mouse was placed in an empty cage where object
avoidance, olfactory, auditory, and visual cliff tests were conducted. Neurological reflexes including balance, eye blink, ear twitch, whisker orientation, and
righting were assessed (29, 30). Upon completion of all tests for all animals
within the same cage, behavior, such as locomotion, interaction with littermate,
and grooming, were noted (31). A general health assessment consisting of body
weight, body temperature, and fur condition were also recorded at the end of the
test session.
Statistical analyses
GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA)
was used to graph data and for statistical analyses. If required, data were normalized by log transformation. T cell responses were analyzed by Student twotailed t test, one- or two-way ANOVA. Differences between means were considered significant at p # 0.05. Means plus SE bars are shown. Survival data were
analyzed using the Kaplan-Meier method and the log-rank test.
Results
Ad-hDCT immunization induced effective protection against
i.c. challenge with B16-F10 cells
Murine melanoma B16-F10 cells are known for their aggressiveness
and we have previously demonstrated that immunization with AdhDCT provides robust protection against s.c. and challenge with these
Detection of DCT-specific T cell responses
Ag-specific T cell responses were quantified by flow cytometric analysis 7 d
postvaccination. Blood wascollectedfrom the periorbitalsinus and RBCslysed.
To study skin-infiltrating T cells, postmortem damaged and normal shaved skin
samples measuring 1 3 5 cm were excised from each mouse. Damaged skin
had been injected with CFA 48 h prior to harvest or taken from a surgical lesion.
Skin was minced, suspended in HBSS containing 0.1% collagenase (Invitrogen), incubated in a shaker at 37˚C at 200 rpm for 45 min, and then pressed
through a nylon mesh filter with a 40-mm pore size to obtain a single-cell
suspension. Mononuclear cells were stimulated with peptides (1 mg/ml
DCT180–188 + 20 mg/ml hDCT88–102) in the presence of brefeldin A (GolgiPlug;
BD Pharmingen [San Diego, CA], 1 mg/ml added after 2 h of incubation). After
6 h, total incubation time cells were treated with anti-CD16/CD32 and surface
markers fluorescently labeled by addition of Abs. Cells were then permeabilized and fixed with Cytofix/Cytoperm (BD Pharmingen) and stained for
intracellular cytokines. Data were acquired using a FACSCanto flow cytometer
with FACSDiva 5.0.2 software (BD Pharmingen) and analyzed with FlowJo
Mac Version 6.3.4 software (Tree Star, Ashland, OR).
Histological analyses
Brains were fixed for 3 d in 10% formalin, transferred to 70% ethanol, paraffinembedded, sectioned at a thickness of 10 mm, and stained with H&E (SigmaAldrich Canada, Oakville, Ontario, Canada). To stain myelin, sections (10–25
mm thick) were dehydrated in graded alcohol and placed in 0.1% Luxol fast
blue at 37˚C overnight. Sections were then differentiated in 0.05% LiCO3 for
FIGURE 1. Immunization with Ad-hDCT provides protection against i.c.
tumor challenge. A, Gross pathological analysis of brains (dorsal view) from
C57BL/6 mice that were harvested 13 d after an i.c. challenge with 1 3 103
B16-F10 cells. Seven days prior to challenge, mice were injected i.m. with AdBHG (left panel) or Ad-hDCT (right panel). Scale bar, 1 mm. B, Summary of
animal survival data following vaccination with Ad-BHG (n = 13) or Ad-hDCT
(n = 22). Of 22 mice immunized with Ad-hDCT, 6 never grew tumors (27%).
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To establish brain tumors or local inflammation, mice received i.c. injections of
1 3 103–106 B16-F10 cells or 10 mg LPS in 2 ml saline. Mice were placed in
a stereotaxis (Xymotech Biosystems, Mt. Royal, Quebec, Canada), and under
both general and local anesthesia, an incision was made in the scalp to expose
the skull. A needle mounted on a 10-ml Hamilton syringe (Hamilton, Reno,
NV) was positioned over the right hemisphere of the brain, 2.25 mm lateral to
Bregma. A small burr hole was drilled through the skull, and the bevel of the
needle inserted into the brain parenchyma to a depth of 3 mm. Cells or LPS
were injected over a period of 1 min. The needle was left in place for 2 min prior
to withdrawal to minimize reflux along the injection tract. The scalp incision
was closed with stainless steel clips that were removed 7–10 d later.
4 min and then dehydrated in graded alcohol and xylene (all reagents from
Sigma-Aldrich, St. Louis, MO). For CD8 detection, brains frozen in optimal
cutting temperature medium were sectioned at a thickness of 10 mm, fixed with
acetone, treated with peroxidase, stained with rat anti-mouse CD8 (clone 536.7; BD Biosciences) rat on mouse HRP polymer kit (Biocare Medical, Concord, CA), and resolved using an 3-amino-9-ethylcarbazole substrate kit (Invitrogen). Myelin was quantified by measuring integrated density of Luxol fast
blue in stained sections taken through the injection site using ImageJ software
(version 1.42q, http://rsb.info.nih.gov.ij; W. Rasband, National Institutes of
Health, Bethesda, MD) (27)
The Journal of Immunology
4271
FIGURE 2. Tumor protection is mediated by CD8+
T cells. To evaluate effector mechanisms, mice received
i.m. injections of Ad-BHG or Ad-hDCT. The DCT180–188specific CD8+ (A) and DCT88–102-specific CD4+ (B) T cell
responses were measured by flow cytometric detection of
intracellular IFN-g following in vitro peptide restimulation. Each bar represents five mice. C, Fourteen days
post-Ad injection, mice received i.c. injections of 1 3 103
B16-F10 cells. Immunized mice were depleted of CD4+
and/or CD8+ T cells by administration of Abs 2 d prior to
and the day of tumor implantation and twice per week
thereafter. Survival data are shown (n = 5/group). D, Inflammatory infiltrates (upper panels, original magnification 3100) and CD8+ T cells (lower panels, original
magnification 340) were examined by H&E and immunohistochemical staining, respectively, in sham-treated
(left panels) and Ad-hDCT–immunized (right panels)
mice subsequently challenged i.c. with 1 3 104 B16-F10
cells. Scale bars, 100 mm.
4 d (Fig. 3A, left panel). However, immunization with Ad-hDCT 7 d
prior to challenge prevented tumor dissemination (Fig. 3A, right
panel), confirming the potency of Ad-hDCT. Four days after the high-
Protection against i.c. B16-F10 tumors depended on CD8+
T cells
To evaluate the induction of Ag-specific T cells by Ad-hDCT, we
determined the frequency of both CD8+ and CD4+ T cells that were
capable of producing IFN-g in response to restimulation with their
corresponding peptides. At 8 d postvaccination, .3% of bloodderived CD8+ T cells were producing IFN-g in response to the
immunodominant epitope DCT180–188, shared between mice and
humans (Fig. 2A). A significant level of CD4+ T cells (0.4%) specific
for hDCT88–102, a recently identified helper peptide (26), was also
evident by IFN-g production (Fig. 2B). To determine their relative
contribution to tumor protection, immunized mice were depleted of
CD4+ and/or CD8+ T cells 2 d before i.c. challenge. Protection was
completely abrogated by depletion of CD8+ but not CD4+ T cells,
indicating that CD8+ T cells were the primary effectors for i.c. tumor
rejection (Fig. 2C). This notion is further supported by histological
staining that revealed more immune infiltrates at the tumor challenge site in DCT-vaccinated animals compared with controls (Fig.
2D, upper panels), and many of these infiltrates were CD8+ T cells
(Fig. 2D, lower panels).
No evidence of acute neuropathology despite autoimmune
targeting of DCT
For evaluation of potential acute autoimmune or bystander damage,
micewere immunized with Ad-hDCTand challenged i.c. 7 d later with
a high dose of 1 3 106 B16-F10 cells to ensure survival of tumor cells
so malignant lesions could be identified. Rapid dissemination of the
melanoma to the ventral surface and brain stem was observed in only
FIGURE 3. Protection against high-dose tumor challenge in the absence of
overt neuropathology. A, Ventral view of brains harvested 4 d after i.c. challenge with 1 3 106 B16-F10 cells. Seven days before implantation, mice were
injected i.m. with Ad-BHG (left panel) or Ad-hDCT (right panel). Scale bar,
1 mm. B, These brains were sectioned and stained with H&E (left panels,
original magnification 3100) and Luxol fast blue (right panels, original
magnification 3100; to detect myelin) in adjacent serial sections to assess
potential damage to normal neural tissue. Upper panels, A naive mouse that
never received an i.c. challenge. The sections were taken at the depth at which
tumors were implanted in other mice. Middle and lower panels, Ad-BHG and
Ad-hDCT-treated mice, respectively. These sections represent those in which
the main tumor mass was largest. Circles indicate the location of the tumor at
the implantation site (or the equivalent area in the naive control). Squares show
a neural fiber-rich area suitable for evaluating myelination. Areas encompassed
by the circles and squares are shown at high magnification. Scale bars, 100 mm.
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cells (15, 32). We found that i.c. injection of 1 3 103 B16-F10 cells
resulted in formation of lethal tumors within only 13 d of injection
(Fig. 1A, left panel). However, immunization with Ad-hDCT 7 d prior
to challenge effectively prevented tumor growth (Fig. 1A, right
panel), demonstrating the potency of Ad-hDCT as a potential vaccine
for brain cancers that express DCT. This efficacy observed at early
time points translated into a significant extension of survival (Fig. 1B).
Mice treated with PBS (data not shown) or a control Ad vector (AdBHG) had a median survival of 15 d posttumor challenge, whereas
immunization with Ad-hDCT resulted in a significant survival benefit
in all treated mice (median = 64 d; p , 0.0001). More importantly,
27% (6 of 22) of vaccinated mice completely rejected implanted tumor cells, offering a unique model for the subsequent analysis of
the acute and chronic impact of vaccine-induced autoimmunity in
a vital organ.
4272
SEPARATION OF ANTI-TUMOR ACTIVITY AND AUTOIMMUNE TOXICITY
dose challenge, mice were euthanized, their brains harvested, and
frozen sections were stained (Fig. 3B) with H&E (left panels) or Luxol
fast blue to detect myelin (right panels). A normal brain from an agematched mouse was included. Again, the potency of Ad-hDCT immunization on inhibition of tumor growth within the brain parenchyma was evident relative to sham-treated controls (Fig. 3B, left
middle and left lower panels; see circles). This acute phase of DCTspecific cytotoxicity in the brain did not trigger any obvious damage to
normal tissues. Although swelling of tumor-adjacent brain tissues
could be observed in some untreated mice (example in Fig. 3A, left
middle panel with high-magnification inset), their neurons and myelin
sheaths remained intact (Fig. 3A, right middle panel with highmagnification inset).
Immunization with Ad-hDCT caused autoimmune pathology in
inflamed normal tissues
FIGURE 4. Autoimmune vitiligo is associated with
damaged skin, but neural tissue remains intact in longterm survivors. A, Mice receiving Ad-BHG and i.c.
challenged with B16-F10 cells never developed vitiligo
(top panel). Mice immunized with Ad-hDCT and
challenged with B16-F10 cells always developed severe vitiligo limited to the scalp incision area (bottom
panel). B, Mice vaccinated with Ad-hDCT had CD8+
(left panel) or CD4+ (right panel) T cells depleted right
before i.c. melanoma challenge. C, Skin dissected from
Ad-hDCT–immunized mice 48 h after injection of
CFA was used to assess the phenotype of infiltrating,
DCT-specific T cells by flow cytometry following tetramer and Ab staining. A typical contour plot of
E-selectin ligand and CCR4 expression on skininfiltrating Kb-DCT180–188+ T cells is shown (left panel)
along with a paired sample stained with isotype control
Abs (right panel). D, Mice treated with Ad-BHG (upper panel) or Ad-hDCT (lower panel) had their backs
shaved and the hapten DNFB painted on their skin 7 d
later. Only Ad-hDCT–immunized mice developed
vitiligo that was limited to the area of inflammation.
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We previously reported that i.m. immunization with Ad-hDCT did not
cause melanocyte damage (vitiligo) unless there was trauma/inflammation within the skin (15). Consistent with this earlier observation, all Ad-hDCT–immunized mice in the current model developed
severe vitiligo along the scalp incision site, whereas this lesion was
absent in all sham-treated mice (Fig. 4A). Depletion of CD8+ T cells
before the surgery completely abrogated vitiligo induction, whereas
CD4 depletion had a significant but incomplete effect, suggesting that
CD8+ T cells are the primary effectors in this autoimmune destruction
(Fig. 4B). Tetramer staining indicated that a substantial number of
DCT-specific CD8+ T cells were indeed recruited into damaged skin
and 50.4% (6 SE of 3.5; n = 3) of these expressed E-selectin ligand
and/or CCR4 (Fig. 4C), a surface phenotype consistent with skinhoming T cells (33). Interestingly, in all cases, the vitiligo was limited
to the area immediately surrounding the incision. To further delineate
the relationship between skin damage and vaccine-induced vitiligo,
a hapten DNFB was used to induce skin sensitization in a controlled
fashion. As shown in Fig. 4D, DNFB alone did not damage melanocytes (top panel), whereas the size of the vitiligo lesion directly
correlated with the extent of the DNFB-induced skin inflammation in
Ad-hDCT–immunized mice (bottom panel). The results reinforce the
idea that the number of autoreactive T cells did not limit the extent of
vitiligo, but their access to normal skin was apparently controlled by
the degree of local inflammation.
To determine whether additional inflammatory stimulation causes
autoimmune pathology in the brain, LPS was injected i.c. 14 d after
treatment with Ad-hDCT or PBS. Compared with sham injection, LPS
elicited more obvious inflammation, although it was limited to the injection site (Fig. 5, upper panels) and resolved in 3 d (data not shown).
Considerably more CD8+ T cells were evident in Ad-hDCT–vaccinated
mice (Fig. 5, middle panels), which correlated with localized demyelination (Fig. 5, lower panels; mean integrated density of Luxol fast
blue stain = 2.7 6 1.4 SE, n = 5 for Ad-hDCT + LPS versus 10.2 6 2.6,
n = 5 for PBS + LPS; p = 0.027). Sham injection or LPS challenge alone
was insufficient to induce tissue damage (Fig. 5, lower panels). These
observations confirm the concomitant requirement of both Ag-specific
autoimmunity and local tissue inflammation for the induction of autoimmune sequelae.
The Journal of Immunology
4273
these survivors, despite immunization with Ad-hDCT and tumor destruction in the brain (data not shown). Because DCT-immunized
mice challenged with LPS had evidence of acute demyelination at
the injection site, we repeated the study to determine whether there
were any signs of chronic damage. At 90 d post-LPS challenge,
these mice passed all behavioral tests (Table I) and did not have any
demyelination (mean integrated density of Luxol fast blue stain =
241.1 6 1.0 SE, n = 5 for naive controls versus 240.2 6 1.9, n = 5 for
LPS challenge).
Ad-hDCT vaccination provided a potent therapeutic effect
Long-term survivors did not have altered behaviors or chronic
neuropathology
Long-term survivors generated in our study provided an opportunity to
analyze potential neural damage beyond static pathology. Twenty-one
mice that survived i.c. challenge with B16-F10 cells and 22 agematched naive mice were enrolled in a behavior study to assess
multiple sensory motor skills. This analysis included tests that evaluated olfactory, visual, and auditory functions, all of which reflect the
integrity of CNS functionality. As summarized in Table I, mice that
survived their i.c. tumor challenge did not show any defects in the
tested aspects, compared with controls. Furthermore, brains harvested
from these mice did not show any evidence of tumor cells, suggesting
these were completely eradicated instead of their growth simply being
suppressed. Notably, there was no evidence of any neuropathology in
Discussion
Strategies to eliminate cancer cells by breaking tolerance to self-Ags
have raised an issue regarding the potential risk for collateral autoimmune damage to normal tissues (34–36). Using a murine melanoma
model, we and others have demonstrated that the development of effective anti-tumor immunity indeed results in autoimmune vitiligo,
especially when there is a concurrent inflammatory event in healthy
tissues that express Ags carried by the vaccine (8, 15, 37, 38). Results from several clinical trials also indicate that development of
Table I. Mice protected from i.c. melanoma exhibit normal behavior
Physical Evaluation
Mice
Rejected tumors
Naive
Ad-hDCT + LPS
Object
Avoidance
21/21
27/27
5/5
Neurological Reflex
Auditory
Test
Visual Cliff Test
(Head Dip/min)a
Olfactory Test Time
Sniffing (s)a
21/21
27/27
5/5
11.88 6 1.30
11.02 6 0.82
10.40 6 2.48
5.53 6 0.99
5.73 6 2.50
4.59 6 0.79
Balance
b
20/21
27/27
5/5
Whisker
Orientation
c
19/21
27/27
5/5
Righting
b
20/21
27/27
5/5
Eye Blink
Ear Twitch
21/21
27/27
5/5
21/21
27/27
5/5
Mice vaccinated with Ad-hDCT were challenged i.c. with B16-F10 cells or LPS 7–14 d later. Some mice completely rejected the implanted tumor cells. Twenty-one longterm survivors of the tumor challenge (.90-d survival), 5 mice at 90 d post-LPS challenge, and 27 age-matched naive controls were enrolled in a series of behavior studies
designed to assess general health and neurophysiological functions. Absence of brain tumors in these mice was confirmed by postmortem gross and histological examination
following testing.
a
Means (6 SEM) were not significantly different.
b
The mouse that failed these tests was the longest-term survivor and severely overweight.
c
The whiskers on two of these mice were absent because of barbering.
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FIGURE 5. Inflammation in the brain is only associated with damage in
Ad-hDCT-immunized mice. Mice were vaccinated with PBS (middle panels)
or Ad-hDCT (right panels) for 14 d and then challenged i.c. with 10 mg LPS.
Naive mice with sham injection (left panels) were included as controls. Inflammatory infiltrates (top panels), including CD8+ T cells (middle panels),
were evaluated at the injection site by H&E and immunohistochemical
staining, respectively. Myelination at the injection site and surrounding area
was examined by Luxol fast blue staining and quantified as mean integrated
density 6 SE (2.7 6 1.4, n = 4 for Ad-hDCT + LPS versus 10.2 6 2.6, n = 9
for PBS + LPS; p = 0.027; 4.3 6 2.2, n = 3 for sham injection). Scale bars,
100 mm (upper and middle panels, original magnification 340) or 500 mm
(lower panels, original magnification 3100).
We next extended the analysis to evaluate the therapeutic potential
of Ad-hDCT in mice bearing pre-existing tumors in the brain. In
this regard, mice were injected i.c. with 1 3 103 B16-F10 cells and
then treated with Ad-hDCT 5 d later. Eleven days after treatment,
the whole brains were harvested from both naive (tumor-free) and
treated (tumor-bearing) mice for quantitative analysis of efficacy.
Tumor burden was determined by subtracting the mean weight of
tumor-free brains from those in the tumor-bearing groups. Fig. 6A
shows that the mean tumor weight in Ad-hDCT–immunized mice
was 4.7-fold less than that in mice treated with a control Ad vector
(47.1 6 21.3 versus 9.9 6 2.8 mg; p , 0.05). Furthermore, mice
treated 5 d postengraftment had a significantly longer median
survival time following Ad-hDCT treatment, compared with
sham-treated controls (Fig. 6B). An additional study extended this
observation of Ad-hDCT–mediated therapeutic efficacy by demonstrating that immunization of mice with 7-d-old brain tumors
increased median survival to 27 d versus only 15 for sham-treated
controls (p = 0.0067) (Fig. 6C). As had been observed in the
prophylactic challenge model, vitiligo was evident in all mice
treated in the therapeutic setting (Fig. 6D). However, these lesions
were less severe than those observed in the prophylactic model
(Fig. 4A), presumably due to altered intervals between the peak of
the anti–self-immune response (10–14 d after immunization) and
surgery-induced skin damage (1–2 d after surgery). Specifically,
immunization was carried out several days after tumor inoculation, such that a significant amount of wound healing had
occurred by the time the CTL response was fully activated.
4274
SEPARATION OF ANTI-TUMOR ACTIVITY AND AUTOIMMUNE TOXICITY
autoimmune toxicities correlated with tumor regression (13, 39–41).
These observations point to the possibility that autoimmune pathology may be an unavoidable or even favorable outcome of cancer
immunotherapies.
However, because the potential destruction of normal brain tissue
could have severe consequences, the balance between anti-tumor immunity and autoimmune pathology in the case of cerebral malignancies needed to be investigated. The tumor model and vaccination
approach presented in this study provided several unique features to
address this issue. First, the potency of the Ad-hDCT vaccine allowed
us to achieve treatment efficacy against i.c. growth of B16-F10 melanomas and provided an opportunity to analyze pathological consequences both in early stages and in long-term survivors. Second, the
potential autoimmune destruction couldbe concomitantly evaluatedin
the skin and brain, both of which express the target Ag. Third, i.c.
challenge with B16-F10 cells did not only provide a brain tumor model
but also served as a local inflammatory stimulus within the brain.
We previously reported that vitiligo lesions following melanoma
immunotherapyrequiredtwoevents:Ag-specificimmunity plusdamage
to the skin (15). The current study corroborates this. Specifically, vitiligo
was associated with the scalpel incision site in Ad-hDCT–immunized
mice. Moreover, vitiligo was severe in the prophylactic model where
immune responses were peaking at the time of injury to the skin. In
contrast, vitiligo severity was reduced in the therapeutic model where
.1 wk of wound healing occurred before significant DCT-specific
T cell responses were detectable. These observations reinforce our hypothesis that autoimmune pathology following effective cancer vaccination is the result of secondary trauma to normal tissues, and its severity
is affected by the degree and persistence of normal tissue damage. We
speculate that tissue inflammation/trauma is required to recruit autoreactive T cells, as demonstrated in Fig. 4C, and to modulate expression
Acknowledgments
We thank Duncan Chong, Xueya Feng, Ying Chuyan, Mary-Jo Smith, Mary
Bruni, Cai Jiang, and Jie Huang for technical assistance and Dr. Zhou Xing
for helping with histological examination.
Disclosures
The authors have no financial conflicts of interest.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 6. Therapeutic vaccination against established tumors. A, Mice
with 5-d-old brain tumors were treated with Ad-BHG (n = 4) or Ad-hDCT
(n = 5). Ten days later, Ad-treated mice and 10 age-matched, tumor-free
controls were euthanized, and their brains were weighed. The mean weight
of control brains was subtracted from those in the tumor-bearing groups to
quantify tumor burden. Mean tumor weights are shown with SE bars. Mice
carrying 5-d (B) or 7-d-old (C) tumors were treated with 1 3 108 PFU of
Ad-hDCT or Ad-BHG. Survival data are shown. Treatment of 5-d-old
tumors has been repeated 10 times with similar results. D, Vitiligo was
manifested in all Ad-hDCT–treated animals, which was limited to the
scalp incision area.
of MHC molecules and target Ags. Our results are consistent with the
finding by Lang et al. (18) that highly activated CD8+ T cells could
coexist with pancreatic b islet cells expressing the relevant autoantigen;
however, overt autoimmune disease occurred when an inflammatory
response was coupled.
Despite the presence of robust DCT-specific CD8+ T cells and
the evidence that DCT is expressed in normal brain (19–21, 23,
42), we never saw any neuropathology in Ad-hDCT–immunized
mice. This may be explained by the inherent immune-privileged
nature of the brain (43) or the lack of inflammatory signals to recruit autoreactive T cells into sites distal to the i.c. tumors. Our results confirm that Ag-specific vaccines are capable of inducing an
anti-tumor response within the immunologically privileged brain
and that the CNS is accessible to systemic immunotherapy (44).
More importantly, similar to what we observed in skin, immune
destruction appeared to only focus on the inflammatory area (i.e.,
tumor) and did not extend beyond the site of tumor growth. Furthermore, the lack of behavioral anomalies or demyelination in
normal brain tissue by long-term survivors provides further evidence that cerebral cancer can be eliminated by autoreactive
T cells while leaving the CNS unharmed. The linkage between
tissue inflammation and autoimmune pathology is further supported by deliberate i.c. injection of LPS where only Ad-hDCT–
immunized animals exhibit localized, acute demyelination, presumably due to the recruitment of CD8+ T cells that recognize
DCT expressed by normal glial cells. The fact that demyelination
was no longer evident at 90 d post-LPS challenge suggests the
cells that were acutely destroyed could be renewed following
cessation of inflammatory stimulation. Taken together, these results clearly demonstrate that the inflammation is tightly regulated
in normal tissues, which limits the initiation and progression of
autoimmune destruction in the vaccinated host.
It is possible that additional stringent mechanisms may also be
operational in the brain to regulate local immune responses to avoid
autoimmune pathology. For instance, although most tumors are inherently inflammatory and the integrity of the blood-brain barrier is
locally compromised allowing extravasation of activated T cells,
their trafficking may be more limited in normal parenchyma (45).
However, it remains to be determined whether the local environment
functionally influences brain-infiltrating lymphocytes. The fact that
immunotherapy is less efficacious in i.c. versus s.c. compartments
using the same vaccination strategy suggests anatomical location of
the tumor can influence the outcome of cancer vaccines, with the
brain being a particularly difficult site (46).
Overall, our findings provide proof-of-principle that self-Ags can
safely be targeted by cancer immunotherapy, even if the tumor is embedded within a vital tissue sharing the same Ag. Compelling evidence
supports the concept that anti-tumor immunity will only spill over into
autoimmune pathology if linked by inflammation. Given the demonstration of MAA expression by gliomas, our findings on vaccination
efficacy and the relationship between anti-tumor immunity and autoimmune pathology have significant implications for the design of
therapies directed not only against brain melanoma but also potentially
for primary brain cancers.
The Journal of Immunology
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