Herpes virus oncolytic therapy reverses tumor immune dysfunction and facilitates tumor antigen presentation.

Abstract

We have previously shown that intratumor administration of HSV-1716 (an ICP34.5 null mutant) resulted in significant reduction of tumor growth and a significant survival advantage in a murine model of ovarian cancer. Herewith we report that oncolytic HSV-1716 generates vaccination effects in the same model. Upon HSV-1716 infection, mouse ovarian tumor cells showed high levels of expression viral glycoproteins B and D and were highly phagocyted by dendritic cells (DCs). Interestingly, increased phagocytosis of tumor-infected cells by DCs was impaired by heparin, and anti-HSV glycoproteins B and D, indicating that viral infection enhances adhesive interactions between DCs and tumor apoptotic bodies. Moreover, HSV-1716 infected cells expressed high levels of heat shock proteins 70 and GRP94, molecules that have been reported to induce maturation of DCs, increase cross-presentation of antigens and promote antitumor immune response. After phagocytosis of tumor-infected cells, DCs acquired a mature status in vitro and in vivo, upregulated the expression of costimulatory molecule and increased migration towards MIP-3beta. Furthermore, HSV-1716 oncolytic treatment markedly reduced vascular endothelial growth factor (VEGF) levels in tumor-bearing animals thus abrogating tumor immunosuppressive milieu. These mechanisms may account for the highly enhanced antitumoral immune responses observed in HSV-1716 treated animals. Oncolytic treatment induced a significantly higher frequency of tumor-reactive IFNgamma producing cells, and induced a robust tumor infiltration by T cells. These results indicate that oncolytic therapy with HSV-1716 facilitates antitumor immune responses.

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1194Cancer Biology & Therapy2008; Vol. 7 Issue 8
[Cancer Biology & Therapy 7:8, 1194-1205; August 2008]; ©2008 Landes Bioscience
We have previously shown that intratumor administration of
HSV-1716 (an ICP34.5 null mutant) resulted in significant reduc-
tion of tumor growth and a significant survival advantage in a
murine model of ovarian cancer. Herewith we report that oncolytic
HSV-1716 generates vaccination effects in the same model. Upon
HSV-1716 infection, mouse ovarian tumor cells showed high levels
of expression viral glycoproteins B and D and were highly phago-
cyted by dendritic cells (DCs). Interestingly, increased phagocytosis
of tumor-infected cells by DCs was impaired by heparin, and
anti-HSV glycoproteins B and D, indicating that viral infection
enhances adhesive interactions between DCs and tumor apoptotic
bodies. Moreover, HSV-1716 infected cells expressed high levels
of heat shock proteins 70 and GRP94, molecules that have been
reported to induce maturation of DCs, increase cross-presentation
of antigens and promote antitumor immune response. After phago-
cytosis of tumor-infected cells, DCs acquired a mature status in vitro
and in vivo, upregulated the expression of costimulatory molecule
and increased migration towards MIP-3β. Furthermore, HSV-1716
oncolytic treatment markedly reduced vascular endothelial growth
factor (VEGF) levels in tumor-bearing animals thus abrogating
tumor immunosuppressive milieu. These mechanisms may account
for the highly enhanced antitumoral immune responses observed in
HSV-1716 treated animals. Oncolytic treatment induced a signifi-
cantly higher frequency of tumor-reactive IFNγ producing cells, and
induced a robust tumor infiltration by T cells. These results indicate
that oncolytic therapy with HSV-1716 facilitates antitumor immune
responses.
Introduction
Replication-restricted oncolytic viruses carry specific genetic alter-
ations that prevent their replication in normal diploid cells, while
enabling them to replicate selectively in malignant cells by virtue
of compensatory overexpression of homologue eukaryotic genes or
loss of critical molecular checkpoints in the latter.1-6 Attenuated
replication-competent herpes simplex virus (HSV) strains have been
shown to selectively replicate in neuronal as well as epithelial tumors
and induce their death without exerting cytotoxicity on adjacent
differentiated cells.7-9 HSV-1 mutants have been generated that
harbor alterations in both copies of RL1 gene, a diploid fragment
of the HSV-1 genome.10 Its product, the infected cell protein (ICP)
34.5 protein, has been implicated in neurovirulence11 and is respon-
sible for preventing apoptosis related to premature shut-off of protein
synthesis in the infected host cells.12,13 A large amount of preclinical
data has demonstrated the efficacy of intracranial administration of
ICP34.5-deficient and other replication-selective HSV-1 mutants
against brain tumors,7,14-24 while the safety of this approach has
been demonstrated by two clinical trials in the human.25,26 We have
previously shown that oncolytic HSV is efficacious against chemo-
therapy-sensitive and chemotherapy-resistant ovarian carcinoma,27,28
while the efficacy of various strains has been reported in cervical,29
colorectal,30 breast and other common epithelial tumors.31-33
In the process of establishing a productive infection propagating
within the tumor, oncolytic HSV induces massive tumor cell death
and triggers a potent inflammatory response.33-37 Animal experi-
ments in syngeneic tumor models indicate that tumor inoculation
with replication-restricted HSV may result in activation of adaptive
antitumor immune response. For example, intratumoral injection of
multi-attenuated HSV-G207 was followed by the immune-mediated
tumor regression of distant uninfected tumor nodules.36 T cell clones
against an immunodominant tumor-associated, major histocompat-
ibility complex (MHC) class I-restricted epitope were identified in
mice treated with oncolytic HSV.37 In addition, recent evidence
indicates that antitumor immune response is required for oncolytic
HSV to achieve its therapeutic effect in vivo in the immunocompe-
tent host.33
The mechanisms by which oncolytic HSV triggers tumor vaccina-
tion are not known. Tumors express specific antigens, but successfully
evade immune recognition. The main cell population controlling
adaptive immune responses including immune response against
Research Paper
Herpes virus oncolytic therapy reverses tumor immune dysfunction and
facilitates tumor antigen presentation
Fabian Benencia,1,2,†,‡ Maria C. Courrèges,1,† Nigel W. Fraser3 and George Coukos1,2,*
1Center for Research on Ovarian Cancer Early Detection and Cure; 2Abramson Family Cancer Research Institute; and Division of Gynecologic Oncology; Department of Obstetrics
and Gynecology; 3Department of Microbiology; University of Pennsylvania; Philadelphia, Pennsylvania USA.
†These authors contributed equally to this work.
Key words: oncolytic, herpes, ovarian tumor, mouse, therapy, dendritic cells
*Correspondence to: George Coukos; Center for Research on Reproduction and
Women’s Health; University of Pennsylvania; 1355 BRB II/III; 421 Curie Boulevard;
Philadelphia, Pennsylvania 19104 USA; Tel.: 215.746.5136; Fax: 215.573.7627;
Email: gcks@mail.med.upenn.edu
Submitted: 04/27/08; Accepted: 05/01/08
Previously published online as a Cancer Biology & Therapy E-publication:
http://www.landesbioscience.com/journals/cbt/article/6216
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Oncolytic HSV facilitates tumor antigen presentation
www.landesbioscience.comCancer Biology & Therapy1195
tumors is dendritic cells (DCs).38 DCs take up, process and present
antigens to naïve T cells.39 Fully mature DCs can elicit potent T cell
stimulation and DCs have been utilized to expand tumor-specific
T cells ex vivo or for therapeutic tumor vaccination in vivo.40-44
Immature or partly mature DCs can instead induce tolerance.40,43,45
It follows that DC maturation is a critical prerequisite for oncolytic
HSV to induce tumor vaccination in situ. Immature myeloid DCs
express the herpes receptors herpes virus entry (HVE)-A and HVE-C
and are susceptible to infection by HSV.46 Importantly, HSV infec-
tion has been reported to suppress myeloid DC maturation and its
ability to stimulate naïve T cells.46,47 Furthermore, tumor-infiltrating
DCs may already be functionally impaired owing to tumor-derived
factors.14-22 For example, tumor-derived vascular endothelial growth
factor (VEGF) suppresses DC differentiation and maturation,48,49
mediating tumor immune evasion.50,51
Here we investigated the effects of oncolytic HSV on mechanisms
of tumor antigen presentation. We used a syngeneic mouse model of
ovarian carcinoma engineered in our laboratory to overexpress the
VEGF164 isoform. We have previously reported that these tumors
express MHC-I and are antigenic, but successfully evade immune
recognition.52 Moreover, we have shown that HSV-1716 is capable of
exerting oncolytic activity in vivo and in vitro in this model, inducing
intratumor recruitment of immune effector cells.53 Here we demon-
strate that inoculation of ICP34.5-deficient HSV-1716 induced in
situ tumor vaccination. This was achieved through increased antigen
uptake as well as enhanced DC function. Increased uptake of tumor
apoptotic bodies by DCs was mediated through adhesive interactions
between HSV glycoproteins on infected tumor cells and HSV recep-
tors on DCs. Tumor infection with oncolytic HSV-1716 sharply
decreased VEGF levels in the tumor microenvironment and induced
DC maturation. Stress response heat shock protein 70 (hsp70) and
glucose-related protein 94 (GRP94) were upregulated in infected
tumor cells thus increasing the antigenicity of these cells. Similar
mechanisms were observed with human monocyte-derived DCs,
providing important rationale for human tumor immunotherapy.
Results
HSV-1716 oncolytic effects on murine ovarian carcinoma.
We have previously reported that ID8 cells overexpressing VEGF
(ID8-VEGF) are sensitive to infection and killing by oncolytic
HSV-1716. Herewith we show that intratumoral injections of
HSV-1716 induced significant reduction in tumor growth (Fig. 1A).
Interestingly, a significant reduction in tumor growth was observed
in contralateral tumors of animals that received HSV-1716 only
to tumors located in the opposite flank compared to tumors of
animals that received UV-inactivated HSV (Fig. 1A). These results
are in agreement with evidence reported previously that oncolytic
HSV therapy exerts distant antitumor effects.37 To assess whether
HSV-1716 induces activation of adaptive immune response, we
examined treated and non-treated tumors for CD8+ cells. Tumors
receiving direct injection of HSV-1716 exhibited a significant
increase of tumor-infiltrating CD8+ cells compared to tumors of
animals that had not received the virus (Fig. 1B and C). Importantly,
contralateral tumors from animals that received HSV-1716 only to
the tumor in the opposite flank also exhibited markedly increased
infiltration by T cells (Fig. 1B and C).
HSV-1716 induces in situ tumor vaccination. The above results
suggest that HSV oncolysis induces activation of adaptive antitumor
immune response. To test whether oncolytic HSV-1716 induces
in situ tumor vaccination in the ID8-VEGF model, we analyzed
the frequency of peripheral tumor-reactive T cell precursors in
mice intratumorally treated with live or UV-inactivated HSV-1716
(mock). Splenocytes from these animals were incubated with synge-
neic bone marrow-derived DCs that were previously pulsed with
non-infected ID8-VEGF cells killed by UVB radiation. A signifi-
cantly higher proliferation rate was observed in splenocytes from
HSV-1716-treated mice compared to splenocytes from mock-treated
animals (Fig. 2A). No significant proliferation was observed when
splenocytes from either group were incubated with control DCs
pulsed with TC-1 cells or unpulsed mature syngeneic DCs. (Fig. 2A).
When incubated with DCs pulsed with dead, non-infected tumor
cells, splenocytes from HSV-1716-treated mice produced a higher
amount of IFNγ and IL-12 but not IL-4, as determined by ELISA
analysis of supernatants, compared to splenocytes obtained from
mock-treated animals (Fig. 2B).
Next, freshly isolated splenocytes from HSV-1716 or mock
treated animals were incubated with ID8-VEGF target cells. A
significantly higher frequency of IFNγ producing cells, as assessed
by ELISPOT, was observed among splenocytes obtained from mice
treated with HSV-1716 compared to mock-treated mice (Fig. 2C).
By flow cytometry, we measured a significantly higher frequency of
CD8+ lymphocytes positive for intracellular IFNγ among spleno-
cytes from HSV-treated mice compared to mock-treated mice (not
shown). Finally, splenocytes isolated from mice bearing i.p. tumors
treated with HSV-1716 killed non-infected ID8-VEGF cells more
efficiently compared to splenocytes from mock-treated mice, as
assessed by cytotoxicity assays (Fig. 2D). No killing of ID8-VEGF
cells was recorded when naïve lymphocytes were used (Fig. 2D). No
cytotoxicity was observed when splenocytes from the HSV-1716
treated group were incubated with control TC-1 cells (Fig. 2D).
Thus, HSV oncolytic therapy induces in situ tumor vaccination.
Viral infection increases the uptake of apoptotic cells. Because
the efficiency of antigen presentation may in part depend on the
amount of antigen available to DCs,54 we investigated the ability
of DCs to engulf apoptotic bodies of ID8-VEGF cells killed by
HSV-1716 or by UVB radiation. We quantified the uptake of
apoptotic bodies through flow cytometry. Tumor cells were labeled
with CFSE prior to pulsing of DCs. Cells killed by HSV-1716
were more efficiently engulfed by immature DCs than cells killed
by UVB (Fig. 3A).
We hypothesized that HSV glycoproteins expressed on the surface
of infected cells enhance adhesive interactions between DCs and
tumor apoptotic bodies. Viral glycoprotein B (gB) and gC mediate
attachment of virions to cell surface heparan sulfate and initiation
of infection, a process inhibited by heparin. Viral gD, B and
C mediate viral entry through binding to herpes virus entry
(HVE)-A, B and C cell surface receptors.55,56 ID8-VEGF cells
infected with HSV-1716 were found to express gB and high levels
of gD on their surface (Fig. 3B). Incubation of HSV-1716 infected
cells with anti-gB or anti-gD decreased their phagocytosis by
CD11c cells (Fig. 3C). This effect was not observed when non-
infected cells were incubated with anti-HSV glycoprotein antibodies
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Oncolytic HSV facilitates tumor antigen presentation
1196Cancer Biology & Therapy2008; Vol. 7 Issue 8
(Fig. 3D). Furthermore, similar to what happens with
viral attachment to target cells,57 the adhesion of infected ID8-
VEGF cells to DCs was blocked by heparin (Fig. 3E). Thus,
HSV-1716 infection enhances up-take of tumor cells by DCs
through expression of herpes glycoproteins on apoptotic bodies.
Viral infection increases the immunogenicity of tumor cells. We
have previously reported that HSV-1716 kills human ovarian cancer
cells by necrosis or p53-independent apoptosis.28 Because apoptosis
but not necrosis may promote efficient antigen presentation by DCs
and tumor immunization,58 we examined the pattern of cell death
Figure 1. HSV-1716 oncolytic effect on ID8-VEGF syngeneic tumor model. (A) Tumor growth in animals bearing two tumors treated with live or UV-inactivated
HSV-1716. Animals received four intratumor doses (5 x 106 p.f.u.) of the virus (days 7, 14, 21 and 28 post-implantation of the tumor). Day 45, HSV-1716
vs. contralateral p < 0.05, HSV-1716 vs. mock p < 0.01, Contralateral vs. mock p < 0.01, Mann-Whitney. The experiment was repeated two times with
similar results. (B) By immunohistochemical analysis of mouse ovarian tumors, we detected a higher frequency of CD8+ leukocytes in HSV-1716 treated
specimens with respect to contralateral non-treated or mock treated tumors. (Magnification: x200). Nuclei were counterstained with hematoxylin. (C) Higher
numbers of CD8+ infiltrating cells were counted in tumors of live HSV-1716 treated animals respect to contralateral or mock treated animals. HSV-1716 vs.
contralateral p < 0.05, HSV-1716 vs. mock p < 0.05, Contralateral vs. mock p < 0.05, Mann-Whitney.
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www.landesbioscience.comCancer Biology & Therapy1197
Oncolytic HSV facilitates tumor antigen presentation
induced by HSV-1716 in murine ovarian cancer cells. We observed
that HSV-1716 infection induced apoptosis in ID8-VEGF cells, as
assessed by TUNEL assay (Fig. 4A), DNA ladder assay (Fig. 4B) and
annexin V staining (Fig. 4C)
Because DC function is potently enhanced by the presence of
“danger” signals that become abundant during pathogen infection,59
we asked whether HSV-1716 could induce such “danger” signals
upon infection of ID8 tumor cells. We thus examined the expression
of stress response proteins Hsp70 and GRP94, which reportedly can
induce maturation of DCs, increase cross-presentation of antigens
and promote antitumor immune responses.60-64 We observed that
ID8-VEGF cells infected by HSV-1716 strongly upregulated Hsp70
(Fig. 4D and E) and GRP94 (Fig. 4F and G) as determined by flow
cytometry analysis and immunohistochemistry respectively. In addi-
tion, the levels of both proteins were higher in HSV-infected cells
than in cells undergoing apoptosis induced by UVB radiation (Fig.
4D and F).
HSV oncolysis decreases tumor VEGF levels. VEGF has been
shown to suppress DC maturation and mediate tumor immune
evasion.48,49,65,66 We reasoned that a robust suppression of VEGF
levels in the tumor microenvironment might trigger efficient tumor
vaccination by HSV-1716. As shown in Fig. 5A, HSV-1716 infec-
tion did not induce VEGF expression within the peritoneal cavity
of normal mice. Furthermore, no increase in VEGF expression
was observed in mixed PEC obtained from the peritoneal cavity of
normal mice five days after i.p. HSV-1716 inoculation, and in DCs
or macrophages infected in vitro with oncolytic HSV (1 MOI) (Fig.
5B). On the contrary, VEGF protein levels were significantly lower in
ascites of animals bearing i.p. ID8-VEGF tumors five days following
i.p. treatment with live HSV-1716 compared to animals treated
with UV-inactivated HSV-1716 (mock) (Fig. 5C). Similarly, 72 h
after virus infection ex-vivo, a decrease in VEGF protein level was
observed in supernatants of primary cultures generated with mixed
single cell populations derived from ascites or from solid ID8-VEGF
tumors (Fig. 5D).
Tumor cells infected by HSV-1716 induce DC matu-
ration. Next, we examined the phenotype of DCs following
engulfment of ID8-VEGF cells killed by HSV-1716. To assess DC
Figure 2. Antitumoral immune response in treated animals. (A) Proliferation of splenocytes from treated animals and control animals upon stimulation with
DCs loaded with ID8-VEGF apoptotic cells. Data are representative of three experiments. (B) ELISA analysis of cytokines in supernatants of antigen-stimulated
splenocytes from treated animals with respect to controls. Data represent mean ± SD. n = 5. The experiment was repeated two times with similar results.
(C) ELISPOT analysis of IFNγ producing cells in activated splenocytes from the same animals. Data represent mean ± SD. n = 5. HSV-1716 vs. mock p <
0.001, HSV-1716 vs. näive p < 0.01, HSV-1716 vs. non-loaded DCs p < 0.001, mock vs. näive p < 0.05, mock vs. non-loaded DCs p < 0.05, näive vs.
non-loaded DCs non-significant, Anova. (D) Cytotoxic assay using ID8-VEGF cells as targets. Cytotoxic values were obtained from non-activated lymphocytes
purified from pooled spleens of animals treated with live or UV-inactivated HSV-1716 (mock) at different effector:target ratios. Naïve lymphocytes were used
as controls. TC-1 cells were used as targets for HSV-1716 splenocytes in order to determine specificity of the cytotoxic reaction. 30:1 ratio, HSV-1716 vs.
mock p < 0.05, HSV-1716 vs. näive p < 0.01, HSV-1716 vs. TC-1 target p < 0.001, mock vs. näive p < 0.05, mock vs. TC-1 target p < 0.05, näive vs.
TC-1 target: non-significant, Anova.
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1198Cancer Biology & Therapy2008; Vol. 7 Issue 8
Oncolytic HSV facilitates tumor antigen presentation
Figure 3. (A) Flow cytometry analysis of CD11c cells 12 h after phagocytosis
of 1716-APO or UV-APO cells stained with CFSE. (B) Glycoprotein expres-
sion in ID8-VEGF cells 18 h after infection with UV-inactivated HSV-1716
(mock) or live HSV-1716 (—). (C and D) Flow cytometry analysis of CD11c
cells 12 h after phagocytosis of 1716-APO (C) or UV-APO cells (D) stained
with CFSE in the presence of anti-glycoprotein B or anti-glycoprotein D anti-
bodies. (E) Phase contrast microphotograph of DC culture after overnight
incubation with ID8 cells infected with HSV-1716. ID8 infected cells were
incubated with heparin (1000 μg/ml) (right) or media alone (left) before
administration to DCs. Magnification: 100x.
maturation, we tested their ability to induce non-specific prolifera-
tion of allogeneic lymphocytes and to migrate towards MIP-3α versus
MIP-3β. We also quantified the expression of MHC-II and costimu-
latory molecules CD86 and CD80. DCs loaded with cells killed
with HSV-1716 induced strong proliferation of allogeneic lympho-
cytes (Fig. 6A). A switch in chemotactic behavior was observed in DCs
loaded with tumor cells killed by HSV-1716; DCs lost the ability to
migrate towards MIP-3α and acquired the ability to migrate towards
MIP-3β, indicating maturation (Fig. 6B). Control unloaded DCs
matured with TNFα and LPS exhibited similar migration towards
MIP-3β than DCs loaded with ID8 cells killed by HSV-1716, while
immature DCs were chemoattracted mostly by MIP-3α. Finally,
increased maturation was seen when DCs were loaded with virally
killed tumor cells compared to UVB-killed tumor cells, as assessed
by expression of MHC-II, CD86 and CD80 (Fig. 6C). These data
collectively show that apoptosis induced by HSV-1716 enhances DC
antigen uptake and maturation.
Tumor-infiltrating DCs are loaded with antigen and undergo
maturation following HSV-1716 infection. The above results
collectively show that DCs avidly uptake apoptotic infected tumor
cells and undergo maturation in the context of HSV-1716 oncolysis.
Taking into account that controversy exists whether direct DC infec-
tion with wild-type HSV is able to suppress maturation and antigen
presentation,46,47,67,68 we further investigated the state of maturation
of DCs in tumors following inoculation of HSV-1716. Flow cytom-
etry analysis of ascites cells revealed that in non-treated animals (not
shown) or animals treated with UV-inactivated HSV-1716 animals
(mock) a large proportion of CD45+ cells were GFP+, likely repre-
senting leukocytes engulfing tumor cells (Fig. 7A). Furthermore,
virtually all CD11c cells within the tumor milieu uptake tumor
antigen as determined by their high GFP expression (Fig. 7B).
Importantly, in non-treated (not shown) or mock treated animals
virtually all DCs were CD86 negative, indicating an immature
state (Fig. 7C). Similar results were obtained with cells recovered
from solid tumors subjected to enzymatic digestion (not shown).
Five days following i.p. administration of HSV-1716, a dramatic
decrease in CD45-GFP+ cells (tumor cells) was noted as a result of
the oncolytic effect of the virus (Fig. 7A). Furthermore, a significant
influx of CD45+ cells was also seen, with increased frequency of
CD45+GFP+ cells (Fig. 7A). A significant increase in the frequency
of CD11c+CD86+ cells (within the GFP gated population) was
observed; indicating that DCs underwent maturation as a result of
phagocytosis of HSV-1716 killed tumor cells (Fig. 7C). To further
test whether DCs engulfing infected apoptotic bodies of tumor cells
undergo maturation, primary mixed cell cultures harvested from
ascites or solid tumor were infected ex vivo with HSV-1716 at 1
MOI. Gated CD11c+GFP+ cells were found to exhibit an increase in
MHC-II as well as CD80 and CD86 expression 72 h after addition
of the virus (Fig. 7D).
Human DCs mature upon phagocytosis of tumor cells killed by
oncolytic HSV-1716. The above results have important implications
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www.landesbioscience.comCancer Biology & Therapy1199
Oncolytic HSV facilitates tumor antigen presentation
for clinical applications of oncolytic HSV in
the human. To test whether the observed effects
are recapitulated by human DCs, we used the
ovarian human ovarian cancer cell line A2780
and monocyte-derived DCs. We have previously
shown, using TUNEL and flow cytometry DNA
content analysis, that HSV-1716 efficiently
infects and kills A2780 cells by apoptosis.69
These results were confirmed by annexin V
staining and DNA ladder assay (not shown).
Next, we examined the state of maturation of
DCs engulfing tumor cells infected with onco-
lytic HSV-1716. A2780 cells were infected with
HSV-1716 at 1 MOI and one day later were
pulsed onto immature DCs at a 1:1 ratio (tumor
cells:DCs) for another 24 hrs. Control DCs
were matured with rhTNFα for 48 h. Similar to
murine DCs, upon phagocytosis of HSV-1716-
infected, A2780 cells human DCs upregulated
the expression of HLA-DR, CD80, CD86,
CD83 and CD40 and downregulated CD32
(Table 1), thus exhibiting features of mature
antigen presenting cells. In multiple experi-
ments, upregulation of costimulatory molecules
was stronger in DCs pulsed with HSV-infected
tumor cells than in DCs exposed to rhTNFα
for 48 h.
Discussion
Different reports have shown the oncolytic
efficacy of replication-competent HSV lacking
the ICP34.5 gene.4,14,21,22,70,71 Similarly, we
observed that HSV-1716 exerted in vitro and
in vivo oncolytic activities in a syngeneic model
of ovarian carcinoma developed in C57BL/6
mice. We have recently shown that intratumor
injection of HSV-1716 induced expression
of IFNγ, MIG and IP-10 in the tumor. This
was accompanied by a significant increase
in the number of tumor-associated NK and
CD8+ T cells expressing CXCR3 and CD25.53
We have also demonstrated that ascites from HSV-1716-treated
animals efficiently induced in vitro migration of NK and CD8+
T cells, which was dependent on the presence of MIG and IP-10.
We also found that antigen-presenting cells were responsible for
the intratumor production of MIG and IP-10 upon HSV-1716
treatment. Moreover, human ovarian carcinomas showed high
numbers of monocytes and DCs and upon HSV-1716 infec-
tion, human monocyte-derived DCs produced large amounts of
IFNγ and upregulated MIG and IP-10 expression. These results
indicate that HSV-1716 induces an inflammatory response that
may facilitate antitumor immune response upon oncolytic ther-
apy.53 Similar studies in syngeneic tumor models indicate that
tumor inoculation of replication-restricted HSV results in tumor
vaccination.37,72 Consistently, herewith we showed that HSV-1716
treatment notably improved the antitumor immune response in
the ID8-VEGF model. We showed that splenocytes obtained from
HSV-1716 treated animals highly proliferate when activated with
non-infected ID8 cells, producing higher levels of IFNγ and IL-12
when compared from splenocytes obtained from UV-inactivated
HSV-1716 treated animals. Moreover, splenocytes recovered from
HSV-1716 treated, tumor-bearing animals killed non-infected
ID8 cells more efficiently than splenocytes obtained from mock
treated animals. Thus, oncolytic treatment of ovarian tumors with
HSV-1716 increased specific antitumor immune responses in our
mouse model.
We hypothesized that one mechanism underlying tumor vacci-
nation triggered by HSV-1716 could be the upregulation of the
immunogenic properties of infected cells. First, we observed that
HSV-1716 infection significantly increased tumor antigen up-take
by DCs. We showed that this mechanism was mediated through
receptor-mediated interactions between HSV receptors present in
the surface of DCs and HSV glycoproteins expressed in the surface
Figure 4. (A) TUNEL assay of ID8 cells infected with UV-inactivated HSV-1716 (mock) or live
HSV-1716 (1 MOI) 48 h post-infection. (B) DNA ladder assay of ID8-VEGF cells 72 h after infec-
tion with live or UV-inactivated HSV-1716. (C) Flow cytometry analysis of annexin V in cells 36
h after infection with live (—) or UV-inactivated HSV-1716 (10 MOI) (■). (D–G) Upregulation of
danger signals in HSV-1716 infected cells. (D) Flow cytometry analysis of HSP70 expression in
ID8 cells killed by HSV-1716, UVB irradiation (1500 μW/cm2 for 10 min) (■) or treated with
UV-inactivated HSV-1716. (E) Immunohistochemistry of HSP70 antigen in ID8 cells infected with
live or VV-inactivated (mock) HSV-1716, 36 h p.i., 1 MOI. (F) Flow cytometry analysis of GRP94
expression in ID8 cells killed by HSV-1716, UVB irradiation (1500 μW/cm2 for 10 min) (■) or
treated with UV-inactivated HSV-1716. (G) Immunohistochemistry of GRP94 antigen in ID8 cells
infected with live or VV-inactivated (mock) HSV-1716, 36 h p.i., 1 MOI.
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1200Cancer Biology & Therapy2008; Vol. 7 Issue 8
Oncolytic HSV facilitates tumor antigen presentation
of tumor-infected cells. Moreover, we detected overexpression of
danger signals such as Hsp-70 and GRP-94 in HSV-infected tumor
cells. These heat shock proteins play a critical role in promoting
antigen cross-presentation in the MHC-I class I pathway and
eliciting tumor-specific protective immunity tumor. As well as
other heat shock proteins they activate NFκB mediated signal and
cytokine production in APC, promoting maturation of DCs.60-64
Consistently, phagocytosis of ID8 cells killed by HSV-1716 infection
induced phenotypical and functional maturation of bone marrow
derived DCs. This effect on DC maturation was observed in vitro,
and in vivo when DCs loaded with tumor antigen were studied
after HSV-1716 treatment. Similar results on DC maturation were
observed when human DCs phagocytosed HSV-1716 infected
A2780 human ovarian carcinoma cells.
Epithelial ovarian carcinoma is characterized for maintaining
a strong immunosuppressive milieu.48-50,65,66,73-76 Tumor associ-
ated VEGF has been reported playing a suppressive role in DC
maturation.48-50,66,74 We observed that upon HSV-1716 treatment,
VEGF levels were downregulated in vivo. This effect, which may be
related to viral killing of tumor cells, disrupts the tumor immunosup-
pressive milieu. Thus, tumor associated DCs may be more able to
stimulate antigen-specific T cells upon phagocytosis of highly immu-
nogenic HSV-1716 infected cells, triggering antitumor responses.
In summary, oncolytic HSV-1716 induced strong antitumor
immune response in a model of murine tumor overexpressing VEGF.
We observed that viral infection increased the immunogenicity of
tumor cells which result in highly activated DCs upon phagocy-
tosis. These antitumor DCs may account for the marked antitumor
cellular response observed in our model. Besides affording direct
tumor killing, oncolytic HSV is therefore able to promote tumor
immune recognition and attack.
Materials and Methods
Animals and virus. Six- to 8-week-old female C57BL6 mice
(Charles River Laboratories, Wilmington, MA) were used in protocols
approved by the Institutional Animal Care and Use Committee and
the University of Pennsylvania.
Replication-competent HSV-1716 was originally provided by Dr.
S.M. Brown (Glasgow University, UK)77 as reported elsewhere.27,28,69
The genome of this virus carries a 759-bp deletion located within
each copy of the BamHI fragment of the long repeat region removing
most of the RL1 gene. Thus, cells infected with the HSV-1716
mutant fail to make ICP34.5 protein. The virus used in this study
was passed in our laboratory in baby hamster kidney cells (BHK),
purified by means of sucrose gradients and stored at -70°C. Plaque
assays for virus titration were performed on BHK cells as described
previously.27 In most experiments controls were performed by using
virus inactivated by UV-irradiation (1500 μW/cm2, 20 min, 4°C).
No live virus was detected after treatment, as determined by plaque
assays.
Figure 5. Oncolytic treatment abrogates VEGF levels in tumors. (A) ELISA of peritoneal washings obtained from naïve animals treated i.p. with live or
UV-inactivated HSV-1716. No significant differences were observed. (B) ELISA analysis of supernatants from PEC obtained from peritoneal cavity of naïve
animals treated i.p. with HSV-1716, or macrophages and DCs infected in vitro with live or UV-inactivated HSV-1716 (1 MOI). No significant difference was
observed between groups treated with live or UV-inactivated HSV-1716. (C) ELISA analysis shows a decrease in the levels of VEGF protein (n = 6) values
5 days after HSV-1716 treatment (5x106 p.f.u./ 500 μl serum free RPMI i.p.) in the intraperitoneal model of murine ovarian carcinoma. (D) ELISA analysis
of supernatants shows a decrease in the levels of VEGF protein (n = 3) values 72 h after infection of primary tumor cultures with live or UV-inactivated HSV-
1716 (mock) (1 MOI).
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www.landesbioscience.comCancer Biology & Therapy1201
Oncolytic HSV facilitates tumor antigen presentation
Tissues and cells. ID8 mouse ovarian carcinoma cells expressing
both green fluorescent protein (GFP) and the VEGF 164 isoform
(ID8-VEGF) were engineered in our laboratory through retroviral
transfection as previously described52 and maintained in DMEM
(Invitrogen, Carlsbad, CA) supplemented with 2 mM L-glutamine,
100 μg/ml penicillin, 100 U/ml streptomycin, and 10% heat-inacti-
vated fetal bovine serum (FBS) (all Invitrogen).
TC-1, a C57BL/6 mouse lung adenocarcinoma cell line trans-
formed with HPV-16 E6 and E7 was a generous gift from Dr. Yvonne
Paterson, University of Pennsylvania.78 TC-1 cells were maintained
in RPMI (Invitrogen) supplemented with 2 mM l-glutamine; 100 U/
ml penicillin; 100 μg/ml streptomycin; 10% FBS and geneticin (1
mg/ml) (Invitrogen) in a 5% CO2 atmosphere at 37°C.
BKH cells were maintained in DMEM supplemented with 5%
FBS; 100 U/ml penicillin; and 100 μg/ml streptomycin, in a 5%
CO2 atmosphere at 37°C.
Murine DCs were generated from bone marrow precursors by
the method of Lutz et al.,79 from femurs and tibiae of 4–8 week old
female C57BL/6 mice, as previously described.52 Briefly, marrow
cells were dispersed by vigorous pipetting and cultured in RPMI-
1640 supplemented with penicillin (100 μg/ml), streptomycin
(100 U/ml), L-glutamine (2 mM), 2-mercaptoethanol (50 μM;
Sigma-Aldrich, St. Louis, MO) and 10% heat-inactivated FBS
in the presence of 20 ng/ml of recombinant mouse granulocyte-
macrophage colony-stimulating factor (GM-CSF, 315-03, Peprotech
Inc., Rocky Hill, NJ) for 8 days. GM-CSF was replenished on
days 3 and 6. In some experiments, maturation was induced by
culturing the cells for 2 days in the presence of 10 ng/ml GM-CSF,
20 ng/ml mouse tumor necrosis factor alpha (TNFα, 315-01A,
Peprotech) and 100 ng/ml bacterial lipopolysaccharide (LPS from E.
coli, serotype 0111:B4, L2630, Sigma).
Human adherent monocytes were obtained through
leukapheresis and cultured for 7 days in AIM-V media (Invitrogen)
supplemented with 800 IU/ml human GM-CSF (Immunex, Seattle,
WA) and 500 IU/ml human interleukin-4 (IL-4; R&D Systems,
Minneapolis, MN) to generate immature DCs.80 On day 7, cells
were collected, counted by trypan blue exclusion, and cultured for an
additional 4 days at 5 x 105 cells/ml in AIM-V media supplemented
with 3% heat-inactivated filtered human AB serum (BioWhittaker/
Cambrex, East Rutherford, NJ), 1000 IU/ml GM-CSF, and 1000
IU/ml IL-4. DC maturation was induced by adding 20 ng/ml TNFα
(R&D Systems) on day 7 and an additional 10 ng/ml TNFα on
Figure 6. Maturation of dendritic cells upon phagocytosis of HSV-1716 infected cells. (A) Proliferation of allogeneic of BALB/c splenocytes induced by
C57BL/6 DCs that had phagocyted ID8 cells killed by UVB irradiation or HSV-1716. (B) Chemotaxis analysis of DCs 48 h after phagocytosis of ID8 cells
killed by UVB irradiation or HSV-1716. (C) Flow cytometry analysis of maturation markers in CD11c+ cells 48 h after ingestion of ID8 cells killed by UVB
irradiation (- - - -) or HSV-1716 (—). Isotype control (■).
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1202 Cancer Biology & Therapy 2008; Vol. 7 Issue 8
Oncolytic HSV facilitates tumor antigen presentation
day 9. Cells were harvested for phenotypic and functional analysis
on day 11.
Tumors. Syngeneic flank or intraperitoneal (i.p.) tumors mice
were generated in C57BL/6 as previously described.52 In brief, for
flank injections, a single-cell suspension of ID8-VEGF cells was
prepared in PBS mixed with an equal volume of cold MatrigelTM
(BD Biosciences) at 10 mg/ml. A total volume of 0.5 ml containing
7 x 106 cells was subcutaneously injected into one flank of C57BL/6
mice. Tumors were detectable two weeks later and were measured
weekly using a Vernier caliper. Tumor volumes were calculated by
the formula V= ½ L x W2, where L is length (longest dimension)
and W is width (shortest dimension). For i.p. injections, 1 x 107
ID8-VEGF cells were suspended in a total volume of 0.7 ml PBS
and inoculated i.p.
In vivo inoculation of HSV-1716. Animals carrying solid flank
tumors, received an intratumoral dose of 5 x 106 plaque forming
units (p.f.u.) of HSV-1716 suspended in 50 μl of serum-free RPMI
at days 7, 14, 21 and 28 after tumor inoculation. Control animals
received a similar volume of UV-inactivated HSV-1716. Animals
were euthanized 45 days after tumor inoculation, and tumors were
recovered for immunostaining. For some studies, animals bearing
intraperitoneal tumors were inoculated i.p. with 5 x 106 p.f.u. of live
or UV-inactivated HSV-1716, and ascites was recovered 5 days later
for different studies.
Tumor cell apoptosis. Subconfluent cultures of ID8-VEGF cells
were rinsed twice in phosphate buffer saline (PBS) and exposed
to different amount of ultraviolet-B (UVB) radiation for 10 min.
Alternatively; cells were incubated with HSV-1716 at 1 multiplicity
of infection (MOI). Apoptosis was by flow cytometry detection of
annexin-V staining using the TACSTM Annexin-Biotin Apoptosis
detection kit (R&D Systems, Minneapolis, MN) and confirmed with
the ApopTag peroxidase in situ detection kit (Intergen, Purchase,
NY) and the Apoptotic DNA-Ladder Kit (Roche), according to the
manufacturers’ instructions.
Figure 7. Dendritic cell maturation upon in vivo phagocytosis of HSV-1716 infected cells. (A) Flow cytometry analysis demonstrated an increase in the pro-
portion of peritoneal CD45+ GFP+ cells in ascites of HSV-1716 (5 x 106 p.f.u.) (right) treated animals with respects to mock (left) at day five p.i. (B) Most
DCs present in the ascites are loaded with tumor antigen. (C) Tumor-associated DCs (GFP-gated) increase levels of maturation markers upon oncolytic treat-
ment. (D) Flow cytometry analysis of maturation markers in CD11c+ GFP+ cells 72 h after infection of primary tumor cultures with live (—) or UV-inactivated
HSV-1716 (■) (1 MOI).
Table 1 Human DCs were generated from peripheral
blood of healthy donors



HLA-DR
CD80
CD86
CD83
CD40
CD32
% of expression
with TNFα

96.33
64.35
94.75
47.76
63.16
23.56
without TNFα

40.42
25.34
35.49
27.80
26.43
72.01
pulsed with HSV-1716
infected A2780
67.22
83.68
95.86
86.83
78.30
51.27
Human DCs were generated from peripheral blood of healthy donors. Peripheral blood mononuclear cells
(PBMC) were separated by density gradient centrifugation of whole blood cells. The non adherent cells were
removed to start the differentiation to DCs. Adherent monocytes were cultured for seven days in AIM-V
supplemented with 800 IU/ml GM-CSF and 500 IU/ml interleukin 4. Differentiation into immature DCs was
assessed by flow cytometry detection of specific DCs markers. DCs maturation was induced by culturing at
5 x 105 cells/ml in AIM-V media supplemented with 3% human AB serum, 1000 IU/ml GM-CSF and 1000
IU/ml IL-4 with the addition of 20 ng/ml TNFα on day 7 and 9 or by co-incubation with HSV-1716 infected
A2780 cells at a 1:1 ratio (tumor cells:DCs) for 24 hrs. Cells were subjected to four-color flow cytometry on
a FACSCalibur flow cytometer using CellQuest 3.2.1f1 software (Becton Dickinson, San Jose, CA). For each
sample, we collected 10,000 events.
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www.landesbioscience.comCancer Biology & Therapy1203
Oncolytic HSV facilitates tumor antigen presentation
Loading of DCs. ID8-VEGF cells were infected with HSV-1716
or exposed to UVB as above to induce apoptosis; rinsed three times
with PBS; subjected to gamma-irradiation (100 Gy from a 137Cs
source) and co-incubated with immature murine DCs at a 1:1 ratio
(tumor cells, DCs). In some experiments, TNFα (20 ng/ml) and
LPS (100 ng/ml) were added for two additional days. DCs were
then harvested; rinsed and live cells were counted by the Trypan blue
exclusion method.
In some experiments, following induction of apoptosis, tumor
cells were labeled with 5-(and-6)-carboxyfluorescein diacetate, succin-
imidyl ester (5(6)-CFDA, SE (CFSE) (5 nM) (Molecular Probes,
Carlsbad, CA) for 5 min at room temperature. RPMI supplemented
with 10% FBS was added to stop the reaction and cells were rinsed
three times prior to using them for pulsing DCs.
Flow cytometry. Cells were subjected to four-color flow cytometry
on a FACSCalibur flow cytometer using CellQuest 3.2.1f1 software
(Becton Dickinson, San Jose, CA). We collected 20.000 events per
sample. Non-specific staining was blocked with anti-CD16/CD32
antibody (Fc block, 2.4G2; BD Pharmingen, San Diego, CA).
Fluorochrome-conjugated monoclonal antibodies against CD11c
(HL3), CD80 (16-10A1), CD86 (GL1), MHC-II (KH74; all BD
Pharmingen, San Diego, CA) were used at 1/100 dilution. For intra-
cellular cytokine staining of mouse cells, Brefeldin A (Sigma, 5 μg/
ml) was added to cells 12 h before harvesting. Cells were first stained
for cell surface antigens as above; washed; fixed; permeabilized with
PBS, 0.2% bovine serum albumin (BSA, Sigma), 0.5% saponin
for 5 min; and stained with monoclonal antibody against murine
interferon-gamma (IFNγ[XMG1.2], BD Pharmingen). Samples
were then washed with saponin-containing buffer and analyzed by
flow cytometry. For intracellular staining of heat shock proteins,
anti-mouse Hsp70 (453220; BD Transduction Lab, San Diego, CA)
as well as rabbit polyclonal antibody against GRP94 (gp96; (H-212)
Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1/200
dilution. For analysis of human cells, the following antibodies were
used: HLA-DR (G46-2.6), CD80 (BB1), CD86 (IT2.2), CD83
(HB15e), CD40 (5C3) and CD32 (3D3) (all BD Pharmingen)
(1:100 dilution).
Coculture experiments. Murine dendritic cells were cultured 12 h
with ID8 cells killed by UVB irradiation or by HSV-1716 infection
(1 MOI). In some experiments ID8 cells were stained with CFSE.
Cells were cocultured at 37°C in RPMI, 10% FBS. After incuba-
tion cells were washed and stained with anti-mouse CD11c (BD
Pharmingen). For blocking experiments, DCs were incubated on
ice for 30 min with 1/20 normal goat serum (Vector Laboratories,
Burlingame, CA) and 1/40 anti-mouse CD16/32 (BD Pharmingen).
ID8 cells killed by UVB irradiation or HSV-1716 infected were
incubated on ice for 30 min with 1/20 dilution of anti-glycoprotein
B (MCA2020) or anti-glycoprotein D (MCA2022) both Serotec,
Raleigh, NC. After incubation cells were cocultured for 12 h at 37°C
in RPMI 10% FBS.
In some experiments phagocytosis of HSV-1716 infected cells was
blocked by incubating tumor cells with 1 mg/ml of heparin (H4784,
Sigma) before coculture.
Allogeneic lymphocyte proliferation. Murine C57BL/6 DCs
were loaded with ID8-VEGF cells killed with HSV-1716 and
incubated for 48 h; harvested with EDTA; washed twice with
PBS; and subjected to gamma-irradiation (30 Gy). DCs were then
seeded in 96-well round-bottom plates at various dilutions in RPMI
containing 10% FBS. Spleens were resected from healthy BALB/c
mice and minced in a sterile fashion to yield a single cell suspen-
sion. Erythrocytes were eliminated by hypotonic shock. Splenocytes
were plated in culture dishes in RPMI media under standard condi-
tions for 120 min and the non-adherent fraction was subjected to
Percoll gradient centrifugation to recover a highly pure population
of lymphocytes. Constant numbers of BALB/c lymphocytes (1 x
105 cells/well) were incubated with irradiated C57BL/6 DCs at
increasing ratios (DC:lymphocytes) for five days at 37°C. [3H]
thymidine (NEN Life Science, Boston, MA, 1 μCi/well) was added
for 18 h at 37°C. Samples were recovered on glass fiber filters using
a Skatron cell harvester, and incorporation of radioactivity was
measured by liquid scintillation counting using a Microbeta Trilux
(Perkin Elmer Wallac, Inc., Gaithersburg, MD). Each experimental
value was determined three times.
Chemotaxis assay. Migration of murine DCs towards macrophage
inflammatory protein (MIP)-3α or MIP-3β (R&D Systems,
Minneapolis, MN) was assessed in 96-well chemotaxis cham-
bers using an 8 μm-pore nitrocellulose membrane (Neuroprobe,
Gathersburg, MD). Pyrogen-free RPMI 1640 containing 1% BSA
was used as chemotactic media. Results are presented as chemotactic
index (CI), defined as fold increase in cell migration in the presence
of chemotactic factors compared to chemotactic media alone. Each
experiment was performed in triplicate.
Autologous lymphocyte proliferation. DCs loaded with
ID8-VEGF cells killed with UVB and incubated with TNFα and
LPS for 48 h were harvested with EDTA, washed twice with PBS and
subjected to gamma-irradiation (30 Gy). DCs were then seeded in
96-well round-bottom plates at various dilutions in RPMI containing
10% FBS. A constant amount of syngeneic C57BL/6 splenocytes
(1 x 105 per well) obtained from naïve animals, tumor bearing
animals treated with live HSV-1716 or treated with UV-inactivated
HSV-1716 (mock) was added to DCs. Splenocyte proliferation was
assessed by [3H]thymidine incorporation as above.
IFNγ ELISPOT. For ELISPOT assay, 107 autologous non-
adherent murine splenocytes were cultured with tumor-loaded or
control unloaded irradiated DCs at a 10:1 ratio (T cells: DCs)
in RPMI medium supplemented with 10% FBS. MultiScreen-IP
plates (Millipore) were coated overnight at 4°C with monoclonal
antibody against mouse IFNγ (R4-6A2), IL-4 (BVD4-1D11) and
IL-12 (all BD Pharmingen; 50 ng/well). Plates were blocked with
RPMI 10% FBS. T cells suspended in RPMI 10% FBS (4 x 105
T cells/ml) with irradiated DCs at a ratio of 10:1 (T cell:DCs) and
plated in triplicate at 100 μl/well. After 20 h of incubation in stan-
dard culture conditions, cells were rinsed with PBS-0.1% Tween-20.
Biotinylated monoclonal antibody against mouse IFNγ (XMG1.2),
IL-4 (BVD6-24G2) and IL-12 (all BD Pharmingen; 2 μg/ml) was
added to each well for 2 h in PBS containing 0.5% mouse serum and
0.1% Tween-20. The reaction was developed with streptavidin-HRP
(BD Pharmingen) and aminoethyl-carbamide (AEC; Sigma) reagent.
Spots were scanned and counted by computer-assisted ELISPOT
image analysis (Hitech Instruments, Edgemont, PA). Digitalized
images were analyzed for the presence of areas in which color density,
spot size and circularity exceeded background by a factor set on the
basis of the comparison of control wells.
Cytotoxic T lymphocyte (CTL) assays. We performed standard
lactate dehydrogenase-release assays to evaluate the development
of tumor-specific cytotoxic T lymphocytes as described in detail
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1204Cancer Biology & Therapy2008; Vol. 7 Issue 8
Oncolytic HSV facilitates tumor antigen presentation
elsewhere.81 We used T cells obtained from splenocytes of naïve or
tumor bearing animals, through a Percoll gradient, as effector cells
and ID8-VEGF or TC-1 cells as targets. The effector cells were not
stimulated in vitro with target cells prior to the CTL assay.
ELISA. Cytokine concentrations in culture supernatants, peri-
toneal washings and ascites were quantified by antigen capture
ELISA. We used the following purified antibodies for capture:
anti-mouse IFNγ (R4-6A2), IL-4 (BVD4-1D11), IL-12 (C17.8)
(all BD Pharmingen), and anti-mouse-VEGF (BAF493, R&D
Systems). For detection we used biotin anti-mouse IFNγ (XMG1.2),
IL-4 (BVD6-24G2), IL-12 (C17.8) (all BD Pharmingen), and
biotin anti-mouse VEGF (AF-493-NA, R&D Systems) at 1 μg/
well. Standard curves were constructed using recombinant murine
IL-4 (214-14), IP-10 (250-16), IFNγ (315-05), IL-12 (210-12)
and VEGF (450-32) (all Peprotech). Each dilution of recombinant
standard or sample was assayed in duplicate. The reaction was
revealed by using streptavidin-horseradish peroxidase (554066,
BD Pharmingen) and the 2,2'-azino-di-[3-ethylbenzthiazoline
sulfonate(6)] (ABTS) substrate system (Roche Diagnostics GmbH,
Mannheim, Germany). The blue-green color produced by enzymatic
activity was quantitated at 405 nm in an ELISA microplate reader
(Multiskan RC).
Immunohistochemistry. Solid tumor samples (n = 10 for
each experimental group) were snap-frozen in Optimal Cutting
Temperature (OCT) medium (Tissue Tek, Sakura, Torrance, CA).
Immunohistochemical staining was performed using the avidin-
biotin-peroxidase method. Sections were fixed in cold acetone for 10
minutes, pretreated with 3% H2O2 for 20 min to block endogenous
peroxidase activity and incubated in matched normal sera (Vector
Laboratories). Biotinylated rat anti-mouse CD8α (53-6.7) (BD
Pharmingen) was diluted at 1:100. The Vectastain ABC kit was applied
as described by the manufacturer (Vector Laboratories). Sections were
counterstained with Gill’s hematoxylin (Vector Laboratories). Images
were acquired through Cool SNAP Pro color digital camera (Media
Cybernetics, Carlsbad, CA). Ten different fields for each sample at
x400 magnification were evaluated for cell counting.
Tumor cell cultures were immunostained using the VECTASTAIN
ABC kit (Vector Laboratories), as recommended by the manufacturer.
The primary antibodies used were: mouse monoclonal antibody
against HSP70 (BD Transduction Lab) and rabbit polyclonal anti-
body against GRP94 (gp96, Santa Cruz Biotechnology). Anti-mouse
and anti-rabbit VECTASTAIN ABC kits (Vector Laboratories) were
used as recommended by the manufacturer. Slides were counter-
stained with hematoxylin.
Statistical analysis. A two-tailed Student’s t-test was applied to
determine differences between two groups. For multiple compari-
sons we performed ANOVA analysis with post-analysis comparisons
by the Tukey-Kramer multiple comparisons test. Non-parametric
studies were performed by using the Mann-Whitney U test. A value
of p < 0.05 was considered significant. Data are expressed as mean
± SD. Data were analyzed by using the Graph Pad Instat software
(GraphPad Software, Inc., San Diego, CA).
Acknowledgements
This work was supported by Ovarian SPORE CA83638. F.B. and
M.C.C. were supported by NIH Research Grant #D43 TW00671
funded by the Fogarty International Center.
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