Download Role of the Immune Response during Neuro

[CANCER RESEARCH 60, 5714 –5722, October 15, 2000]
Role of the Immune Response during Neuro-attenuated Herpes Simplex
Virus-mediated Tumor Destruction in a Murine Intracranial
Melanoma Model1
Cathie G. Miller and Nigel W. Fraser2
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
ABSTRACT
Neuro-attenuated herpes simplex virus-1 (HSV-1) ␥34.5 mutants can
slow progression of preformed tumors and lead to complete regression of
some tumors. However, the role of the immune response in this process is
poorly understood. Syngenic DBA/2 tumor-bearing mice treated with
HSV-1 1716 fourteen days after tumor implantation had significant prolongation in survival when compared with mice treated with viral growth
sera (mock; 38.9 ⴞ 2.3 versus 24.9 ⴞ 0.6, respectively; P < 0.0001).
Additionally, 60% of the animals treated on day 7 had complete regression
of the tumors. However, no difference was observed in the mean survival
rates of viral- or mock-treated tumor-bearing SCID mice (15 ⴞ 1.7 versus
14.8 ⴞ 2.2, respectively). When DBA/2 mice syngenic for the tumor were
depleted of leukocytes by cyclophosphamide administration (before and
during viral administration), there was again no significant difference
observed in the survival times (19.0 ⴞ 1.9 versus 19.5 ⴞ 2.7, respectively).
These data demonstrate that the immune response contributes to the
viral-mediated tumor destruction and the increase in survival. Immune
cell infiltration was up-regulated, specifically CD4ⴙ T cells and macrophages (which are found early after viral administration). Prior immunity
to HSV-1 increased survival times of treated mice over those of naive mice,
an important consideration because 50 –95% of the adult human population is sero-positive for HSV-1. Our results imply that the timing of viral
administration and the immune status of the animals will be an important
consideration in determining the effectiveness of viral therapies.
INTRODUCTION
Previous reports have demonstrated the ability of a replication
competent neuro-attenuated HSV-13 to slow the progression of preformed tumors in experimental animals and, in some cases, to lead to
complete regression of tumors (1–3). One concern regarding the use
of neuro-attenuated HSV treatment for tumors is the effect of prior
immunity on viral-mediated tumor destruction because 50 –90% of the
adult population is sero-positive for HSV-1 (4). If the immune response contributes to tumor destruction, prior immunity to HSV may
decrease the effectiveness of the treatment by eliminating the virus
before it is able to spread throughout the tumor and destroy it.
The immune response to HSV has been characterized in the context
of both the peripheral nervous system and the CNS (5–12). Both arms
of the immune response, humoral and cellular, have been shown to be
important in limiting the severity of acute HSV infections. The humoral response limits the manifestation of CNS disease, although
antibody alone cannot protect mice from reactivation (13). CD4⫹ T
cells and CD8⫹ T cells clear the virus and prevent it from spreading
to the CNS (11, 14, 15). T cells secrete antiviral cytokines in the CNS
and thus limit HSV infection by primarily noncytolytic mechanisms
within the CNS (16). The primary cytokines secreted are IFN-␥ and
tumor necrosis factor ␣, with interleukin 6 also having a proposed role
(17). Additionally, microglia cells are activated, and macrophages are
recruited during HSV infection of the nervous system (18).
The immune response to tumors has been studied in detail, and
much is dependent on the model and therapeutic modality used in the
study. It is generally accepted that many tumors are recognized by the
immune system but that tumor suppression factors, such as interleukin
10, are released and down-regulate MHC class I expression through
various mechanisms, allowing the tumor to escape immune monitoring by CTLs (19 –21). A useful property for cancer therapeutics is to
not only destroy local treated tumor masses but to also develop new
tumor-specific immune responses to destroy distal tumor metastases.
Neuro-attenuated HSV-1 may have the potential to do this. Whereas
specific lytic infection of tumor cells will destroy these cells at the site
of injection, this lysis may also expose new tumor cell antigens to
immune cells infiltrating the tumor mass due to viral infection, potentially including CD4⫹ and CD8⫹ T cells. However, the immune
response could also interfere with the viral therapy by preventing the
spread of the virus throughout the tumor mass, thus reducing the
ability of the virus to destroy the tumor.
We have determined that the immune response can contribute to
tumor lysis and that the response is characterized by an early influx of
mainly CD4⫹ T cells, NK cells, and macrophages (although most
types of immune cells are found in the tumor mass after viral therapy).
Interestingly, prior immunity to HSV seems to aid in tumor lysis. This
novel result underscores the importance of patient selection in clinical
trials for viral therapies of tumors because it predicts that an immunecompromised patient with a large tumor mass will have inferior
results compared with a non-immune-compromised patient who has a
smaller tumor mass.
MATERIALS AND METHODS
Animals. Female DBA/2 mice (4 – 6 weeks old; body weight, approximately 20 grams) were obtained from Taconic (Germantown, NY). Immunodeficient female SCID (CB-17 scid/scid) mice, originally obtained from M.
Bosma (Fox Chase Cancer Center, Philadelphia, PA), were bred and maintained in a pathogen-free environment at the Wistar Institute animal facility.
Serum IgM titers of the mice were routinely tested by a direct ELISA when the
mice were 6 –7 weeks old, and only non-IgM-producing mice were used in our
studies. All animal work was approved by the University of Pennsylvania’s
and the Wistar Institute’s Institutional Animal Care and Use Committee
Received 2/4/00; accepted 8/17/00.
(IACUC).
The costs of publication of this article were defrayed in part by the payment of page
Tumor Cells. S91 Cloudman M3 melanoma cells (22) were obtained from
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
the American Type Culture Collection (Manassas, VA). Cells were grown
1
Funded by NIH Grant NS39546. C. G. M. was supported by NIH post-doctoral
using DMEM containing penicillin, streptomycin, and 15% fetal bovine serum.
training grant CA77903.
2
When originally obtained, cells were first implanted s.c. in the flanks of
To whom requests for reprints should be addressed, at Department of Microbiology,
syngenic DBA/2 mice. Tumors that arose in these mice were removed asepUniversity of Pennsylvania School of Medicine, 319 Johnson Pavilion, 3610 Hamilton
Walk, Philadelphia, PA 19104-6076. Phone: (215) 898-3847; Fax: (215) 898-3849;
tically, grown, and then frozen in 95% culture media/5% DMSO so that all
E-mail: [email protected].
experiments could be initiated with cells of a similar passage number. On the
3
The abbreviations used are: HSV-1, herpes simplex virus-1; CNS, central nervous
day of i.c. injection, cells in subconfluent monolayer culture were passaged
system; NK, natural killer; pfu, plaque-forming unit; BBB, blood-brain barrier; i.c.,
with 0.25% trypsin solution in EDTA, washed once in cell culture medium,
intracranial.
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IMMUNE RESPONSE TO HSV-1 1716-MEDIATED TUMOR DESTRUCTION
counted using trypan blue, resuspended at the appropriate concentration in
medium without serum, and kept on ice.
i.c. Tumor Production. Mice were anesthetized with ketamine/xylazine
(87 mg/kg ketamine/13 mg/kg xylazine; i.p.). The head was cleansed with 70%
ethanol. A small midline incision was made in the scalp, exposing the skull.
Stereotactic injection of tumor cell suspensions was performed using a small
animal stereotactic apparatus (Kopf Instruments, Tujunga, CA). Injections
were done with a Hamilton syringe through a 30-gauge needle. The needle was
positioned at a point 2 mm caudal of the bregma and 1 mm left of midline.
Using a separate 27-gauge needle, the skull was punctured at these coordinates.
The injection needle was advanced through the hole in the skull to a depth of
2 mm from the skull surface and then extracted 0.5 mm to create a potential
space.
Cells (5 ⫻ 104, DBA/2; 5 ⫻ 102, SCID) in a total volume of 10 ␮l were
injected over 2 min. After the injection, the needle was left in place for 2 min
and then slowly withdrawn. The skin was sutured closed.
Virus. To produce virus stocks, subconfluent monolayers of baby hamster
kidney 21 clone 13 cells were infected with HSV-1 strain 1716 (stock titer,
1 ⫻ 108 pfu/ml). The creation of HSV-1 strain 1716 has been described
previously (23). Briefly, HSV-1 1716 has a 759-bp deletion that deletes part of
the genes encoding ICP34.5, mLAT, and orfP (23). Virus was concentrated
from the culture and titrated by plaque assay as described previously (24). All
viral stocks were stored frozen in viral culture medium (serum-free DMEM
containing penicillin and streptomycin) at ⫺70°C and thawed rapidly just
before use. Serum-free medium was used for control (mock) inoculation
studies as a negative control.
i.c. Viral Inoculation. Mice were anesthetized i.p. with ketamine/xylazine
(as described above), and the head was cleansed with 70% ethanol. Using a
Hamilton syringe with a 30-gauge needle, the appropriate amount of virus was
injected in a total volume of 10 ␮l through a midline incision at the same
stereotactic coordinates used for tumor cell injection. The needle was passed
through the hole punctured previously for tumor implantation. The injection
was performed over a 2-min period, and after the injection, the needle was left
in place for 2 min and then slowly withdrawn. The amount of HSV-1 1716
used in all experiments was 5 ⫻ 104 pfu/mouse, the amount determined to give
the best survival at the lowest dosage.4
Interocular Viral Inoculation. For latent mice, DBA/2 mice were anesthetized i.p. with ketamine/xylazine as described above. Eyes were scarified in
a cross pattern 10 times, and HSV 17⫹ (5 ⫻ 103 pfu) was pipetted onto the
eyes in 10 ␮l of viral culture media. Mice were monitored daily for signs of
inflammation, and proparacaine hydrochloride was administered ocularly as
needed. Virus infection was allowed to become latent for 28 days before tumor
injections were done as described above.
Immunohistochemistry. Mice were sacrificed by cervical dislocation on
days 0, 1, 3, 5, 7, 10, 14, 15, 17, 19, and 21 after tumor implantation, and the
brains were dissected for histological and immunohistochemical analysis.
These time points were chosen to provide information involving the immediate
immune response after tumor cell and viral injection and to provide information on the specific immune response seen later after tumor cell and viral
injection. The methods for tissue processing and light microscopic immunohistochemical analysis were similar to those described elsewhere (25, 26).
Antibodies used were as follows: (a) anti-HSV-1, polyclonal rabbit antibody
(American Qualex, Santa Clemente, CA; 1:1000); (b) MHC II, rat monoclonal
antirat TRIBu (class II polymorphic) and mouse H-21-A (Biosource International, Camarillo, CA; 1:5 dilution); (c) MHC I, biotin-conjugated mouse
antimouse H-2Kb/H-2Db monoclonal antibody (PharMingen, San Diego, CA;
1:1000 dilution); (d) secondary antibody, biotin-conjugated goat antirat immunoglobulin (Biosource International; 1:1000 dilution); (e) CD8a, purified rat
antimouse CD8a (Ly-2) monoclonal antibody (PharMingen; 1:1000 dilution);
(f) CD4, rat monoclonal antimouse L3/T4 CD4 (Biosource International;
1:1000 dilution); (g) neutrophils, rat monoclonal antimouse neutrophil (polymorphic; Biosource International; 1:1000 dilution); (h) macrophage, rat monoclonal antimouse pan macrophage marker (Biosource International; 1:25 dilution); (i) B cells, purified rat antimouse CD19 monoclonal antibody
(PharMingen; 1:1000 dilution); (j) NK cells, purified rat antimouse pan-NK
cell monoclonal antibody (PharMingen; 1:1000 dilution); (k) IgG2a k isotype,
4
B. Randazzo, personal communication.
purified mouse IgG2a monoclonal immunoglobulin isotype standard
(PharMingen; 1:1,000 dilution); (l) IgG2b k isotype, purified mouse IgG2b
monoclonal immunoglobulin isotype standard (PharMingen; 1:1000 dilution);
and (m) IgG2c k isotype, purified mouse IgG2c monoclonal immunoglobulin
isotype standard (PharMingen; 1:1000 dilution). Cells were detected by an
indirect avidin-biotin immunoperoxidase method (Vectastain ABC kit; Vector
Laboratories, Burlingame, CA) as specified by the manufacturer, with a slight
modification. Briefly, tissue sections were rehydrated, quenched in peroxide
(H2O2), and blocked in 3.5% goat serum (Sigma Chemical Co., St. Louis,
MO). Tissue sections were incubated overnight at 4°C with the primary
antibody, used at concentrations stated above. Next, the tissues were incubated
at room temperature with biotinylated goat antirabbit IgG or goat antirat IgG,
the avidin-biotin horseradish peroxidase complex, and 3,3⬘-diaminobenzidine
as the chromagen. Sections were counterstained with hematoxylin and examined under the light microscope. Sections were washed twice with 0.01 M
Tris-HCl (pH 7.9) and then washed twice with 0.01 M Tris-HCl containing 5%
goat serum between every step except addition of chromagen, in which
sections were washed twice with 0.01 M Tris-HCl only. As an additional
control for the specificity of immunostaining, tissues were processed as described above, except that 5% nonimmune goat serum alone was substituted
for the primary antibody for the overnight incubation and was then followed
with secondary antibody. Quantification of positively stained cells was done
using the Phase 3 Imaging program (Phase 3 Imaging, Glen Mills, PA).
Briefly, the positively stained cells and tumor cells were counted, and the
percentage of positively stained cells per number of total tumor cells was
determined. Two fields per tumor were counted for each antibody. Each
treatment includes three mice, and two slides were counted for each mouse, for
a total of 12 fields per antibody.
Apoptosis Detection. We used the Dead End Colorimetric Apoptosis Detection Assay from Promega (Madison, WI) per the manufacturer’s directions.
Briefly, slides were deparaffinized and rehydrated with sequential ethanol
washes. Slides were then incubated in 0.85% NaCl and washed with PBS, and
tissue was fixed with 4% paraformaldehyde. The slides were washed again and
then incubated with proteinase K. After an additional wash, slides were
covered with equilibration buffer, and the biotinylated nucleotide mixture was
made. Terminal deoxynucleotidyltransferase reaction mixture was added to
slides, and slides were incubated overnight at 4°C. The reaction was stopped
by incubating slides in SSC, and the slides were washed and colorized using
3,3⬘-diaminobenzidine as described above. Negative controls were prepared by
treating a sample with DNase I, and these slides were treated as described
above but in separate jars to prevent contamination of sample slides with
DNase I. Positive cells were counted as described above.
Cyclophosphamide Administration. Mice for cyclophosphamide experiments were implanted with tumors as described above. On days 6 and 8 after
tumor implantation, 75 mg/kg cyclophosphamide (Sigma Chemical Co.) was
injected i.p., followed every 2 days by 50 mg/kg cyclophosphamide. Mice
were bled to insure leukocyte depletion, which was determined to be ⬎97% in
sampled mice throughout the study period. Viral or mock therapy was administered as described above on day 10, and mice were followed for signs of
morbidity or neurological symptoms, at which point they were sacrificed.
Statistics. Data analysis, including calculations of means, SDs, repeated
measures ANOVA, Fisher’s PLSD for survival, and unpaired t test, was
performed using StatView statistical software (Abacus Concepts, Berkley, CA)
on an Apple Macintosh computer (Cupertino, CA).
RESULTS
HSV-1 1716 Prolongs Survival of i.c. Tumor-bearing Immunocompetent Mice. We have previously shown that neuro-attenuated
HSV-1 1716 is able to significantly prolong survival of i.c. HardingPassey tumor-bearing C57/Bl6 mice when the virus is administered at
the midpoint of survival (1). However, the Harding-Passey tumor line
is not a syngenic tumor line because it is derived from an outbred
mouse strain. Because we are interested in the role of the immune
response in viral-mediated tumor destruction, we have used the M3
Cloudman S91 melanoma (S91) model in syngenic DBA/2 mice. Here
we demonstrate that HSV-1 1716 is also able to significantly prolong
survival of i.c. S91 tumor-bearing immunocompetent DBA/2 mice.
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Specifically, 6-week-old DBA/2 mice were injected i.c. with S91
cells as described in “Materials and Methods.” Fourteen days after this
implantation, the animals were split into two groups, with half of the
animals receiving HSV-1 1716 treatment, and the other half of the
animals receiving mock treatment. Animals were followed for signs of
severe illness and sacrificed according to IACUC guidelines. A second group of mice was treated similarly to the above-mentioned
group, but virus or mock treatment was administered 7 days after
tumor cell implantation.
Tumor-bearing mice treated with HSV-1 1716 fourteen days after
tumor implantation had a mean survival of 38.9 ⫾ 2.3 days, whereas
tumor-bearing mice mock-treated with viral growth media had a mean
survival of 24.9 ⫾ 0.6 days (P ⬍ 0.0001; Fig. 1A). To further
characterize this model, we determined whether the previously seen
increase in survival with earlier (day 7) administration of virus (1) was
also seen. When HSV-1 1716 or mock treatment was administered on
day 7 after tumor implantation, 60% of the HSV-1 1716-treated mice
showed complete regression of tumors. There was also a significant
prolongation of mean survival in the remaining mice (54 ⫾ 9.1 days),
whereas mock-treated mice had a mean survival of 27.8 ⫾ 1.1 days
(P ⬍ 0.0003; Fig. 1B). Although a more significant prolongation in
Fig. 1. Survival of DBA/2 mice after i.c. injection of 5 ⫻ 104 S91 melanoma cells
followed by treatment injection. A, viral treatment on day 14. There is a significant
increase in survival of mice receiving 5 ⫻ 104 pfu HSV-1 1716 (E) compared with those
receiving mock treatment (䡺). n ⫽ 10 for HSV-1 1716-treated mice; n ⫽ 10 for
mock-treated mice. P ⬍ 0.0001. B, viral treatment on day 7. There is a significant increase
in survival of mice receiving 5 ⫻ 104 pfu HSV-1 1716 (E) compared with those receiving
mock treatment (䡺). n ⫽ 10 for HSV-1 1716-treated mice; n ⫽ 10 for mock-treated mice.
P ⬍ 0.0003.
survival of mice after viral treatment than mock treatment is seen
when virus is administered on day 7 after tumor implantation, the size
of the tumor was small at this time, and it was difficult to insure that
virus was administered within the tumor mass. Therefore, the results
were not as consistent as those of mice with treatment administered at
day 14, and we have used this model for further studies. These data
demonstrate the feasibility of using this model to study the role of the
immune response in neuro-attenuated HSV-1 therapy of murine i.c.
melanoma.
HSV-1 1716 Does Not Prolong Survival of i.c. Tumor-bearing
Immunodeficient Mice. We have previously shown that HSV-1
1716 is able to decrease human tumor cell growth in SCID and nude
mice (27–29). The ability of HSV-1 1716 to destroy tumors in
immunodeficient mice implies that the immune system does not
contribute substantially to viral-mediated tumor destruction. However, nonspecific immune responses to the xenographic tumor cells
may be triggered by viral administration, and these responses, along
with unchecked viral replication throughout the tumor mass, may be
sufficient to mediate tumor destruction. Additionally, it has been
shown that HSV-1 replicates more efficiently in human tissue than in
rodent tissue (30). This fact may make the viral-mediated destruction
of tumors more effective in the human tumor/mouse host models than
in mouse tumor/mouse host models.
We have evaluated whether a specific immune response contributes
to viral-mediated tumor cell destruction using an allogenic immunodeficient model. Six-week-old SCID mice were implanted with S91
cells as described in “Materials and Methods.” Due to the accelerated
growth of S91 melanoma cells in SCID mice as compared with
syngenic DBA/2 mice, we injected 2-fold less tumor cells and administered the virus on day 7. Fig. 2A shows the mean survival for HSV-1
1716-treated and mock-treated S91 i.c. tumor-bearing SCID mice
treated on day 7. No significant difference in survival was seen
between mock- and HSV-1 1716-treated mice (14.8 ⫾ 2.2 and
15.0 ⫾ 1.7 days, respectively; P ⫽ 0.95). This may be due to the
accelerated growth kinetics of the S91 melanoma cells in SCID mice
because the tumor may have grown too large for the virus to kill a
significant portion of the tumor and thus have an effect on survival.
We have shown previously that administration of the virus at a later
period during tumor growth decreases its effectiveness in tumor
destruction and decreases survival times (30).
To readdress whether a specific immune response is important to
viral-mediated tumor cell destruction, we used a second murine tumor
model using i.p. cyclophosphamide injections to deplete leukocytes in
syngenic DBA/2 mice (31–34). After implantation of 5 ⫻ 104 S91
melanoma cells, cyclophosphamide administration was initiated on
day 6 and continued until the mice were moribund, at which point they
were sacrificed. On day 10 after tumor implantation, viral therapy was
administered. After HSV-1 1716 or mock treatment, leukocyte-depleted mice showed no significant difference in mean survival
(19.0 ⫾ 1.9 and 19.5 ⫾ 2.7 days, respectively; P ⫽ 0.6702; Fig. 2B).
Mice were bled before viral treatment and throughout the posttreatment monitoring period to determine the degree of leukocyte depletion (⬎97%; data not shown). Because there was some variance in
average mean survival times between experiments and to assess the
effect of cyclophosphamide treatment on tumor growth, a third group
of mice was implanted with tumor cells concurrently with the other
groups but was not treated with cyclophosphamide or given the virus.
These mice had a mean survival of 25 ⫾ 0.8 days. Therefore, the
leukocyte depletion due to the cyclophosphamide therapy may contribute to a slightly accelerated tumor growth rate. Furthermore, we
saw no difference in the growth rates of s.c. S91 flank tumors between
cyclophosphamide-treated, HSV-1-treated, and mock-treated mice
(data not shown). Taken together, these data suggest that an immune
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Fig. 2. Survival of immunodeficient mice after i.c. injection of tumor cells. A, survival
of CB17 (scid/scid) mice after i.c. injection of 5 ⫻ 102 S91 melanoma cells followed 7
days later by treatment injection. There was no significant difference in survival of mice
receiving mock treatment (viral culture media; 䡺) compared with those receiving 5 ⫻ 104
pfu HSV-1 1716 (E). n ⫽ 11 for HSV-1 1716-treated mice; n ⫽ 10 for mock-treated mice.
P ⫽ 0.9484. B, survival of cyclophosphamide-treated DBA/2 mice after i.c. injection of
5 ⫻ 104 S91 melanoma cells followed 14 days later by treatment injection. There was no
difference in survival of mice receiving mock treatment (viral culture media; 䡺) compared to those receiving 5 ⫻ 104 pfu HSV-1 1716 (E). n ⫽ 8 for HSV-1 1716-treated
mice; n ⫽ 8 for mock-treated mice. P ⫽ 0.6702.
response develops after viral administration that aids in tumor cell
destruction.
Immune Cells Infiltrate the Tumor but not the Surrounding
Brain Mass. The S91 melanoma cell line used in these studies is
immunogenic (35). Thus we investigated the immune response to the
i.c. implantation of tumor cells in DBA/2 mice to better understand
the response to the tumor and virus administration. The major class of
cells infiltrating into tumors after implantation was CD4⫹ T lymphocytes (10.6%; Fig. 4A). Significant numbers of CD8⫹ T lymphocytes,
NK cells, and microglia cells (7.2%, 9.1%, and 6.8% respectively)
were also seen (Fig. 4A). The immune cells were often located in edge
regions of the tumor mass, although positively stained cells were
found throughout the tumor masses. This response could be due to the
breach of the BBB during tumor implantation, although the recruitment of leukocytes into the CNS is now considered common in
response to virus, bacteria, and so forth. However, no significant
immune cell infiltration was seen when mock-implanted mice were
examined (Fig. 3E). Therefore, breaching the BBB is not enough to
cause recruitment of immune cells into the CNS; stimulation is
required. The infiltration into tumor-bearing mouse tissue disappeared
after several days; however, on day 12, a slight increase in infiltrating
cells was again seen for a short period of time. Specifically, CD4⫹ and
CD8⫹ T cells (4.1% and 3.7%, respectively), NK cells (3.5%), and B
cells (3.0%) were detected (Fig. 4A). At the time of virus administration,
little or no immune cells were present (data not shown).
We next characterized the immune cell infiltration into the tumor
after viral administration. The main infiltrating cells early after viral
treatment were CD4⫹ T cells (11.7%) and macrophages (8.2%), but
PMN (7%), CD8⫹ T cells (2.1%), B cells (3.2%), NK cells (4.4%),
and microglia cells (3.6%) were also present (Fig. 4B). This immune
cell infiltration was sustained until day 21, when tumor size in most
sampled mice was negligible. These mice would have survived until
the maximum time (i.e., day 50 after tumor implantation) because a
small number of tumor cells escaped destruction and would have
resumed tumor growth. No infiltration or signs of inflammation occurred in the surrounding normal brain tissue on any day (data not
shown). Infiltration into the tumor proceeded from small, localized
sites on days 1 and 3 to more widespread sites in the tumors on days
7 and 12 (Fig. 3, F–N). Significant NK (16.3%) and polymorphonuclear leukocytes (16.5%) infiltration was seen on day 7, with significant CD4⫹ T cells (14.5%) again present on day 12. Staining for
HSV antigen showed a growth curve throughout sampling, with the
highest staining on day 7 (15.3%; Fig. 4). HSV-1 staining was found
throughout the tumor mass (Fig. 5, A–H). No staining was seen in
mock-treated tumor-bearing mice (Fig. 5H). MHC class I expression
was down-regulated 3 days after viral therapy in treated mice when
compared with mock-treated mice. This is in accordance with reports
on the ability of HSV-1 to down-regulate MHC class I expression
through ICP47 (36 –38). The down-regulation of MHC class I expression also corresponds with the concurrent shift from CD4⫹ and
CD8⫹ T cells to NK cell and PMN infiltration. This correlates with
the proposed escape from CTL recognition of tumors and the importance of NK cells in tumor clearance (39). This corresponds the
findings of Lewandowski et al. (40), who state that HSV-1 KOS is
able to block the surface expression of MHC class II after infection,
but that strain F is not able to do so. Because HSV strains KOS and
17⫹ are both more pathogenic than strain F, it is interesting to suggest
that more pathogenic strains are able to down-regulate MHC class II
expression through some as yet unknown mechanism.
Apoptosis Increases within the Tumor Mass after Viral Administration. An important factor in understanding the mechanism of
tumor destruction is whether tumor cells are being destroyed via
necrosis and/or apoptosis. Necrosis leads to potentially undesirable
inflammatory responses within the CNS. However, necrosis also
increases the potential development of a tumor-specific immune response because infiltrating immune responses clean up necrotic cell
debris and present tumor cell and viral antigens to lymphocytes. Death
through apoptosis, on the other hand, would limit the inflammation at
the site of tumor destruction but may block the development of a
tumor-specific immune response that would be useful in potentially
eliminating distant metastases. To determine whether tumor destruction occurs by apoptosis, we used the Dead-End Colorimetric Apoptosis Detection System (Promega) to localize apoptotic cells within
the tumor mass of viral-treated and mock-treated mice. Detection of
apoptotic cells increased throughout the monitoring period in viraltreated mice, beginning at 5% on day 0 and increasing to ⬎30% by
day 15 (Fig. 6). The apoptotic cells were found throughout the
viral-treated tumor mass (Fig. 5, I–N). In mock-treated mice, baseline
apoptotic cell detection was seen early in monitoring period (days
1– 6) but was not detected after day 7. These results suggest that
although apoptotic cell death is induced after viral administration,
necrotic cell death may also be occurring because some cells in
viral-treated tumor masses appear necrotic and do not stain for apoptosis (data not shown).
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Fig. 3. Detection of infiltrating inflammatory cells after i.c. implantation of S91 melanoma cells into DBA/2 mice. All pictures were taken within 1 h of tumor cell implantation.
A, CD4⫹ T cells; B, CD8⫹ T cells; C, NK cells; D, microglia cells; E, negative control. Note the lack of detection of infiltrating inflammatory cells after mock tumor implantation.
F–N, detection of infiltrating inflammatory cells after i.c. implantation of S91 melanoma cells into DBA/2 mice. Fourteen days later, they were inoculated with 5 ⫻ 104 pfu HSV-1
1716. F, CD4⫹ T cells, day 1; G, macrophages, day 1; H, PMN, day 7; I, CD8⫹ T cells, day 3; J, B cells, day 7; K, NK cells, day 7; L, microglia cells, day 12; M, MHC class I,
day 3; N, MHC class II, day 1. Magnification ⫽ original magnification ⫻ 20 ⫻ objective.
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Fig. 4. Percentages of infiltrating immune cells. Positively
stained cells and tumor cells were counted, and the percentage of positively stained cells per total tumor cells is reported. Results are the average of counts from two slides
with two sections from three mice for each time point. A,
percentages of inflammatory cells after i.c. implantation of
S91 melanoma cells into DBA/2 mice. B, percentages of
inflammatory cells after implantation of S91 melanoma cells
followed 14 days later with 5 ⫻ 104 pfu HSV-1 1716.
Prior Immunity to HSV Increases Survival Time of Viraltreated Mice. Because the immune response to viral administration plays an important role in viral-mediated tumor cell destruction, we wanted to determine the effect of prior immunity to HSV
on tumor cell destruction. For immune mice, animals were infected
ocularly with HSV 17⫹ and allowed to become latent for 28 days
before tumor implantation. Control mice were mock-infected at the
same time. Tumors were implanted, and 14 days later, HSV-1 1716
was administered. Immune (latent) viral-treated tumor-bearing
mice had a significant increase in survival when compared with
naı̈ve viral-treated tumor-bearing mice (Fig. 7). Specifically, immune mice mean survival was 35.5 days, whereas naı̈ve mice mean
survival was 28.8 days (P ⬍ 0.0001). To insure that previous
immunity to HSV-1 had no effect on tumor growth, immune
tumor-bearing mice were mock-treated as described before. These
mice had a mean survival of 25 days (P ⫽ 0.0001 to naı̈ve and
immune-treated survival). Immunity had no effect on mortality in
nontreated groups. These results not only demonstrate that prior
immunity is not detrimental to viral therapy but also reiterate the
importance of the immune response in terms of cell destruction. If
virus administration accelerates the immune response to the tumor,
in the presence of prior immunity, the immune response will be
up-regulated and will aid in better tumoricidal activity.
DISCUSSION
Although the brain is historically considered an immune privileged
site, recent studies in cases of infection or damage have shed light on
the ability of the immune response to infiltrate into the CNS. The
concept of immune privilege of the CNS is based on the low levels of
resident lymphocytes, low levels of MHC expression on resident
microglia, and the presence of the BBB, which blocks immunoglobulin and complement access to the CNS (41– 43). Many recent studies
have shown that lymphocytes are able to migrate into the CNS and
that many cytokines are expressed within the CNS in response to
inflammatory or injury markers (41). Therefore, it is important to
determine the importance of the immune response to viral (HSV)
replication during tumor destruction and the contribution of the immune response to tumor cell destruction.
Neuro-attenuated HSV-1 strains are able to destroy human xenograft tumors in immunodeficient mice (27–29). However, it has been
shown that HSV-1 replicates with better efficiency in human tissue
than in rodent tissues (30). In the absence of an immune response to
eliminate replicating virus, it was shown that neuro-attenuated HSV-1
is able to spread through xenograft tumors and destroy them. Because
HSV-1 is currently being studied in clinical trials as a treatment for
human tumors in patients (who have probably been immunocompromised due to prior treatment) with this virus, it is important to
determine the ability of neuro-attenuated HSV-1 to destroy syngenic
murine tumors in immunodeficient mice.
Although many reports of the tumoricidal activity of neuro-attenuated HSV have been published (1, 2, 3, 29), few studies have
investigated the effect of the immune response on replication-competent viral treatment of tumors (44). M3 Cloudman S91 melanoma cell
tumor-bearing mice treated with HSV-1 1716 virus have a significant
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IMMUNE RESPONSE TO HSV-1 1716-MEDIATED TUMOR DESTRUCTION
Fig. 5. A–H, detection of positive staining for HSV-1 antigen in the tumor mass of DBA/2 mice after i.c. implantation of S91 melanoma cells followed 14 days later by 5 ⫻ 104
pfu HSV-1 1716. A, day 0; B, day 1, C, day 3; D, day 5; E, day 7; F, day 12; G, day 15; notice the lack of staining in mock-treated tumor (H, day 3). Magnification ⫽ original
magnification ⫻ 4 ⫻ objective. No staining was seen in any areas of the surrounding normal brain tissue. I–N, detection of positively stained apoptotic tumor cells within tumor mass
after viral administration of DBA/2 tumor-bearing mice. Mice were injected with 5 ⫻ 104 S91 melanoma cells followed 14 days later with 5 ⫻ 104 pfu HSV-1 1716. I, day 0; J, day
3; K, day 7; magnification ⫽ original magnification ⫻ 4 ⫻ objective. Higher magnification of the above-mentioned pictures detailing apoptotic cells. L, day 0; M, day 3; N, day 7;
magnification ⫽ original magnification ⫻ 20 ⫻ objective.
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IMMUNE RESPONSE TO HSV-1 1716-MEDIATED TUMOR DESTRUCTION
Fig. 6. Percentages of apoptotic cells. Positively stained cells and tumor cells were
counted, and the percentage of positively stained cells per total tumor mass cells is
reported. Results are the average of counts from two slides with two sections from three
mice for each time point. u, mock-treated mice; 䡺, HSV-1 1716-treated mice.
prolongation in mean survival over mock-treated mice (Fig. 1A).
Examination of the contribution of the immune response and the cells
that mediate this response indicates that it is important for tumor
destruction. The depletion of leukocytes by cyclophosphamide administration demonstrated that a cellular immune response played a role.
Furthermore, the immune cell infiltration, mainly CD4⫹ T cells and
macrophages, seen early after viral administration demonstrated that
immune cells are present within the tumor. Finally, previous immunity to HSV-1 was found to prolong the survival of viral-treated
tumor-bearing DBA/2 mice (Fig. 7).
Both humoral and cellular immune responses have been demonstrated to be important in HSV clearance and prevention of viral
spread to the CNS. The humoral response is important in protecting
the CNS from disease (13). CD4⫹ T cells reduce primary replication
and protect against latent infection (45), whereas CD8⫹ T cells
prevent the immunopathological activity of CD4⫹ T cells (31). Interestingly, our results closely parallel those seen previously using
nonreplicating HSV-1 as a gene therapy vector (43). In these studies,
MHC class I and II expression was up-regulated along with the
infiltration of macrophages, T cells, and NK cells soon after viral
administration (43). However, our response is accelerated and is seen
before that of other models (43, 46, 47). During gene therapy, the goal
is generally long-term gene expression from a viral vector; therefore,
an immune response to the virus or the recombinantly expressed
protein is undesirable. However, during cancer therapy, the goal is
tumor cell destruction; therefore, an immune response to the virus,
which is expressing its genome only in tumor cells, may help tumor
destruction by leading to a tumor-specific immune response that may
aid in destruction of distal metastases. Additionally, nonreplicating
viruses used for gene therapy carry a foreign gene of therapeutic
interest, which may alter the immune response to the virus. In the
present study, the virus is restricted to infection in dividing tumor cells
by its ICP34.5 deletion. The accelerated increase in infiltration of
immune cells seen in viral-treated tumors is due in part to an immune
response to viral proteins expressed on infected tumor cells but may
also be due to the prior immune response elicited by the tumor itself,
suggesting that virus injection either up-regulates the expression of
tumor antigens or that lytic infection increases tumor antigen expression. Additionally, viral injection may up-regulate the expression of
new tumor antigens, helping in the treatment of highly metastatic
tumors. Unfortunately, the predominant cells that are seen in our
model after HSV-1 1716 therapy are CD4⫹ T cells, NK cells, and
macrophages, of which only CD4⫹ T cells will exert antitumor
effects at metastatic sites.
The decrease in MHC class I expression over time is interesting
because it appears that the tumor cells express MHC class I on
implantation but are down-regulating it as viral infection proceeds.
MHC class I peaks 3 days after viral administration and decreases
thereafter. Because HSV encodes a protein, ICP47, that interferes with
transporters associated with antigen processing (TAP) and thus downregulates the antigen presentation through MHC class I (37, 38), this
may also be due to ICP47 action. Although ICP47 has been shown to
not interact with murine TAP in vitro, a recent report by Goldsmith et
al. (48) demonstrates that it is able to interfere with antigen processing
in vivo. MHC class II expression is also down-regulated after viral
infection. It has been shown previously that after infection with
HSV-1 strain KOS, MHC class II cell surface expression is blocked
(40). This is not seen with HSV-1 strain F (40), a less pathogenic
strain. Because HSV-1 strains KOS and 17⫹ are both highly pathogenic, there may be a similar mechanism between the viruses, and this
may be occurring within the CNS in our model.
Up to 90% of the adult human population is sero-positive for HSV
antibodies. Our result that prior immunity aids in viral-mediated
tumor cell destruction suggests that HSV-1 therapies will have better
effectiveness in humans. The increased response to HSV may either
lead to an increased infiltration of immune cells into the tumor mass
or suggest a switch for the dominant immune cell type infiltrating into
the tumor mass. In contrast to these results, Herrlinger et al. (47)
observed no increase in survival in immune animals in their studies
and saw a decrease in gene transfer in immune mice, suggesting that
the response may be tumor or model specific. Their model uses rat
D74 gliomas in which the mutant HSV-1 is unable to replicate
sufficiently due to resistance of the rat cell line to HSV infection.
These authors did report more inflammatory infiltrates in the tumors
of immune mice at early time points compared with those of naı̈ve
mice. Therefore, prior immunity may aid in tumor destruction only in
the presence of a clearly replicating viral infection of the tumor cells.
In conclusion, the role of the immune response in viral-mediated
tumor cell destruction is important when the virus is administered late
in tumor progression. This is important when timing virus administration in clinical trials, considering that many current clinical trials
are being performed in which patients have their immune response
depressed (by chemotherapy and radiation treatment). Earlier administration of virus or administration of virus in the absence of immune
compromise may facilitate the usefulness of neuro-attenuated HSV-1
for i.c. tumors.
Fig. 7. Survival of immune and naı̈ve DBA/2 mice after i.c. injection of 5 ⫻ 104 S91
melanoma cells followed 14 days later by treatment injection. Note the significant increase
in survival of immune HSV-1 1716-treated mice (E) over naı̈ve HSV-1 1716-treated mice
(‚). No difference in survival of nontreated mice was seen (䡺, immune tumor control; 〫,
naı̈ve tumor control). n ⫽ 10 for all groups. P ⬍ 0.0001 for immune HSV-1 1716-treated
mice versus all other groups. P ⫽ 0.0122 for immune control versus naı̈ve treated mice.
P ⫽ 0.0001 for naı̈ve tumor treated mice versus naı̈ve tumor control. There is no
significant difference between the naı̈ve and tumor control groups (P ⫽ 0.0937). All
statistics were performed using the Fisher’s PLSD for survival.
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IMMUNE RESPONSE TO HSV-1 1716-MEDIATED TUMOR DESTRUCTION
ACKNOWLEDGMENTS
We thank Moira Brown for the HSV-1 1716 virus and for her help. We also
thank Priscilla Shaffer and Bill Halford for helpful discussion on immunosuppression, Tara Friebel and Vikram Suri for technical assistance in the laboratory, Kathy Molnar-Kimber for editorial review, and Jennifer Driscoll for help
with the manuscript.
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Role of the Immune Response during Neuro-attenuated Herpes
Simplex Virus-mediated Tumor Destruction in a Murine
Intracranial Melanoma Model
Cathie G. Miller and Nigel W. Fraser
Cancer Res 2000;60:5714-5722.
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