[CANCER RESEARCH 60, 5714–5722, October 15, 2000]

Role of the Immune Response during Neuro-attenuated Herpes SimplexVirus-mediated Tumor Destruction in a Murine IntracranialMelanoma 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)g34.5 mutants canslow progression of preformed tumors and lead to complete regression ofsome tumors. However, the role of the immune response in this process ispoorly understood. Syngenic DBA/2 tumor-bearing mice treated withHSV-1 1716 fourteen days after tumor implantation had significant pro-longation in survival when compared with mice treated with viral growthsera (mock; 38.9 6 2.3 versus 24.9 6 0.6, respectively; P < 0.0001).Additionally, 60% of the animals treated on day 7 had complete regressionof the tumors. However, no difference was observed in the mean survivalrates of viral- or mock-treated tumor-bearing SCID mice (156 1.7versus14.86 2.2, respectively). When DBA/2 mice syngenic for the tumor weredepleted of leukocytes by cyclophosphamide administration (before andduring viral administration), there was again no significant differenceobserved in the survival times (19.06 1.9versus19.56 2.7, respectively).These data demonstrate that the immune response contributes to theviral-mediated tumor destruction and the increase in survival. Immunecell infiltration was up-regulated, specifically CD41 T cells and macro-phages (which are found early after viral administration). Prior immunityto HSV-1 increased survival times of treated mice over those of naive mice,an important consideration because 50–95% of the adult human popula-tion is sero-positive for HSV-1. Our results imply that the timing of viraladministration and the immune status of the animals will be an importantconsideration in determining the effectiveness of viral therapies.

INTRODUCTION

Previous reports have demonstrated the ability of a replicationcompetent neuro-attenuated HSV-13 to slow the progression of pre-formed tumors in experimental animals and, in some cases, to lead tocomplete regression of tumors (1–3). One concern regarding the useof neuro-attenuated HSV treatment for tumors is the effect of priorimmunity on viral-mediated tumor destruction because 50–90% of theadult population is sero-positive for HSV-1 (4). If the immune re-sponse contributes to tumor destruction, prior immunity to HSV maydecrease the effectiveness of the treatment by eliminating the virusbefore it is able to spread throughout the tumor and destroy it.

The immune response to HSV has been characterized in the contextof both the peripheral nervous system and the CNS (5–12). Both armsof the immune response, humoral and cellular, have been shown to beimportant in limiting the severity of acute HSV infections. The hu-moral response limits the manifestation of CNS disease, althoughantibody alone cannot protect mice from reactivation (13). CD41 Tcells and CD81T cells clear the virus and prevent it from spreading

to the CNS (11, 14, 15). T cells secrete antiviral cytokines in the CNSand thus limit HSV infection by primarily noncytolytic mechanismswithin the CNS (16). The primary cytokines secreted are IFN-g andtumor necrosis factora, with interleukin 6 also having a proposed role(17). Additionally, microglia cells are activated, and macrophages arerecruited during HSV infection of the nervous system (18).

The immune response to tumors has been studied in detail, andmuch is dependent on the model and therapeutic modality used in thestudy. It is generally accepted that many tumors are recognized by theimmune system but that tumor suppression factors, such as interleukin10, are released and down-regulate MHC class I expression throughvarious mechanisms, allowing the tumor to escape immune monitor-ing by CTLs (19–21). A useful property for cancer therapeutics is tonot only destroy local treated tumor masses but to also develop newtumor-specific immune responses to destroy distal tumor metastases.Neuro-attenuated HSV-1 may have the potential to do this. Whereasspecific lytic infection of tumor cells will destroy these cells at the siteof injection, this lysis may also expose new tumor cell antigens toimmune cells infiltrating the tumor mass due to viral infection, po-tentially including CD41and CD81T cells. However, the immuneresponse could also interfere with the viral therapy by preventing thespread of the virus throughout the tumor mass, thus reducing theability of the virus to destroy the tumor.

We have determined that the immune response can contribute totumor lysis and that the response is characterized by an early influx ofmainly CD41 T cells, NK cells, and macrophages (although mosttypes of immune cells are found in the tumor mass after viral therapy).Interestingly, prior immunity to HSV seems to aid in tumor lysis. Thisnovel result underscores the importance of patient selection in clinicaltrials for viral therapies of tumors because it predicts that an immune-compromised patient with a large tumor mass will have inferiorresults compared with a non-immune-compromised patient who has asmaller tumor mass.

MATERIALS AND METHODS

Animals. Female DBA/2 mice (4–6 weeks old; body weight, approxi-mately 20 grams) were obtained from Taconic (Germantown, NY). Immuno-deficient female SCID (CB-17 scid/scid) mice, originally obtained from M.Bosma (Fox Chase Cancer Center, Philadelphia, PA), were bred and main-tained 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 themice were 6–7 weeks old, and only non-IgM-producing mice were used in ourstudies. All animal work was approved by the University of Pennsylvania’sand the Wistar Institute’s Institutional Animal Care and Use Committee(IACUC).

Tumor Cells. S91 Cloudman M3 melanoma cells (22) were obtained fromthe American Type Culture Collection (Manassas, VA). Cells were grownusing DMEM containing penicillin, streptomycin, and 15% fetal bovine serum.When originally obtained, cells were first implanted s.c. in the flanks ofsyngenic DBA/2 mice. Tumors that arose in these mice were removed asep-tically, grown, and then frozen in 95% culture media/5% DMSO so that allexperiments could be initiated with cells of a similar passage number. On theday of i.c. injection, cells in subconfluent monolayer culture were passagedwith 0.25% trypsin solution in EDTA, washed once in cell culture medium,

Received 2/4/00; accepted 8/17/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Funded by NIH Grant NS39546. C. G. M. was supported by NIH post-doctoraltraining grant CA77903.

2 To whom requests for reprints should be addressed, at Department of Microbiology,University of Pennsylvania School of Medicine, 319 Johnson Pavilion, 3610 HamiltonWalk, Philadelphia, PA 19104-6076. Phone: (215) 898-3847; Fax: (215) 898-3849;E-mail: nfraser@mail.med.upenn.edu.

3 The abbreviations used are: HSV-1, herpes simplex virus-1; CNS, central nervoussystem; NK, natural killer; pfu, plaque-forming unit; BBB, blood-brain barrier; i.c.,intracranial.

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counted using trypan blue, resuspended at the appropriate concentration inmedium 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 smallanimal stereotactic apparatus (Kopf Instruments, Tujunga, CA). Injectionswere done with a Hamilton syringe through a 30-gauge needle. The needle waspositioned 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 of2 mm from the skull surface and then extracted 0.5 mm to create a potentialspace.

Cells (53 104, DBA/2; 5 3 102, SCID) in a total volume of 10ml wereinjected over 2 min. After the injection, the needle was left in place for 2 minand then slowly withdrawn. The skin was sutured closed.

Virus. To produce virus stocks, subconfluent monolayers of baby hamsterkidney 21 clone 13 cells were infected with HSV-1 strain 1716 (stock titer,1 3 108 pfu/ml). The creation of HSV-1 strain 1716 has been describedpreviously (23). Briefly, HSV-1 1716 has a 759-bp deletion that deletes part ofthe genes encoding ICP34.5, mLAT, and orfP (23). Virus was concentratedfrom the culture and titrated by plaque assay as described previously (24). Allviral stocks were stored frozen in viral culture medium (serum-free DMEMcontaining penicillin and streptomycin) at270°C and thawed rapidly justbefore use. Serum-free medium was used for control (mock) inoculationstudies 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 aHamilton syringe with a 30-gauge needle, the appropriate amount of virus wasinjected in a total volume of 10ml through a midline incision at the samestereotactic coordinates used for tumor cell injection. The needle was passedthrough the hole punctured previously for tumor implantation. The injectionwas performed over a 2-min period, and after the injection, the needle was leftin place for 2 min and then slowly withdrawn. The amount of HSV-1 1716used in all experiments was 53 104 pfu/mouse, the amount determined to givethe best survival at the lowest dosage.4

Interocular Viral Inoculation. For latent mice, DBA/2 mice were anes-thetized i.p. with ketamine/xylazine as described above. Eyes were scarified ina cross pattern 10 times, and HSV 171(5 3 103 pfu) was pipetted onto theeyes in 10ml of viral culture media. Mice were monitored daily for signs ofinflammation, and proparacaine hydrochloride was administered ocularly asneeded. Virus infection was allowed to become latent for 28 days before tumorinjections were done as described above.

Immunohistochemistry. Mice were sacrificed by cervical dislocation ondays 0, 1, 3, 5, 7, 10, 14, 15, 17, 19, and 21 after tumor implantation, and thebrains were dissected for histological and immunohistochemical analysis.These time points were chosen to provide information involving the immediateimmune response after tumor cell and viral injection and to provide informa-tion on the specific immune response seen later after tumor cell and viralinjection. The methods for tissue processing and light microscopic immuno-histochemical 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 monoclonalantirat TRIBu (class II polymorphic) and mouse H-21-A (Biosource Interna-tional, Camarillo, CA; 1:5 dilution); (c) MHC I, biotin-conjugated mouseantimouse H-2Kb/H-2Db monoclonal antibody (PharMingen, San Diego, CA;1:1000 dilution); (d) secondary antibody, biotin-conjugated goat antirat immu-noglobulin (Biosource International; 1:1000 dilution); (e) CD8a, purified ratantimouse 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 (poly-morphic; Biosource International; 1:1000 dilution); (h) macrophage, rat mono-clonal antimouse pan macrophage marker (Biosource International; 1:25 dilu-tion); (i) B cells, purified rat antimouse CD19 monoclonal antibody(PharMingen; 1:1000 dilution); (j) NK cells, purified rat antimouse pan-NKcell monoclonal antibody (PharMingen; 1:1000 dilution); (k) IgG2a k isotype,

purified mouse IgG2a monoclonal immunoglobulin isotype standard(PharMingen; 1:1,000 dilution); (l) IgG2b k isotype, purified mouse IgG2bmonoclonal immunoglobulin isotype standard (PharMingen; 1:1000 dilution);and (m) IgG2c k isotype, purified mouse IgG2c monoclonal immunoglobulinisotype standard (PharMingen; 1:1000 dilution). Cells were detected by anindirect avidin-biotin immunoperoxidase method (Vectastain ABC kit; VectorLaboratories, Burlingame, CA) as specified by the manufacturer, with a slightmodification. 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 primaryantibody, used at concentrations stated above. Next, the tissues were incubatedat room temperature with biotinylated goat antirabbit IgG or goat antirat IgG,the avidin-biotin horseradish peroxidase complex, and 3,39-diaminobenzidineas the chromagen. Sections were counterstained with hematoxylin and exam-ined under the light microscope. Sections were washed twice with 0.01M

Tris-HCl (pH 7.9) and then washed twice with 0.01M Tris-HCl containing 5%goat serum between every step except addition of chromagen, in whichsections were washed twice with 0.01M Tris-HCl only. As an additionalcontrol for the specificity of immunostaining, tissues were processed as de-scribed above, except that 5% nonimmune goat serum alone was substitutedfor the primary antibody for the overnight incubation and was then followedwith secondary antibody. Quantification of positively stained cells was doneusing the Phase 3 Imaging program (Phase 3 Imaging, Glen Mills, PA).Briefly, the positively stained cells and tumor cells were counted, and thepercentage of positively stained cells per number of total tumor cells wasdetermined. Two fields per tumor were counted for each antibody. Eachtreatment includes three mice, and two slides were counted for each mouse, fora total of 12 fields per antibody.

Apoptosis Detection.We used the Dead End Colorimetric Apoptosis De-tection Assay from Promega (Madison, WI) per the manufacturer’s directions.Briefly, slides were deparaffinized and rehydrated with sequential ethanolwashes. Slides were then incubated in 0.85% NaCl and washed with PBS, andtissue was fixed with 4% paraformaldehyde. The slides were washed again andthen incubated with proteinase K. After an additional wash, slides werecovered with equilibration buffer, and the biotinylated nucleotide mixture wasmade. Terminal deoxynucleotidyltransferase reaction mixture was added toslides, and slides were incubated overnight at 4°C. The reaction was stoppedby incubating slides in SSC, and the slides were washed and colorized using3,39-diaminobenzidine as described above. Negative controls were prepared bytreating a sample with DNase I, and these slides were treated as describedabove but in separate jars to prevent contamination of sample slides withDNase I. Positive cells were counted as described above.

Cyclophosphamide Administration. Mice for cyclophosphamide experi-ments were implanted with tumors as described above. On days 6 and 8 aftertumor implantation, 75 mg/kg cyclophosphamide (Sigma Chemical Co.) wasinjected i.p., followed every 2 days by 50 mg/kg cyclophosphamide. Micewere bled to insure leukocyte depletion, which was determined to be.97% insampled mice throughout the study period. Viral or mock therapy was admin-istered as described above on day 10, and mice were followed for signs ofmorbidity or neurological symptoms, at which point they were sacrificed.

Statistics. Data analysis, including calculations of means, SDs, repeatedmeasures ANOVA, Fisher’s PLSD for survival, and unpairedt test, wasperformed 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 Immuno-competent Mice. We have previously shown that neuro-attenuatedHSV-1 1716 is able to significantly prolong survival of i.c. Harding-Passey tumor-bearing C57/Bl6 mice when the virus is administered atthe midpoint of survival (1). However, the Harding-Passey tumor lineis not a syngenic tumor line because it is derived from an outbredmouse strain. Because we are interested in the role of the immuneresponse in viral-mediated tumor destruction, we have used the M3Cloudman S91 melanoma (S91) model in syngenic DBA/2 mice. Herewe demonstrate that HSV-1 1716 is also able to significantly prolongsurvival of i.c. S91 tumor-bearing immunocompetent DBA/2 mice.4 B. Randazzo, personal communication.

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Specifically, 6-week-old DBA/2 mice were injected i.c. with S91cells as described in “Materials and Methods.” Fourteen days after thisimplantation, the animals were split into two groups, with half of theanimals receiving HSV-1 1716 treatment, and the other half of theanimals receiving mock treatment. Animals were followed for signs ofsevere illness and sacrificed according to IACUC guidelines. A sec-ond group of mice was treated similarly to the above-mentionedgroup, but virus or mock treatment was administered 7 days aftertumor cell implantation.

Tumor-bearing mice treated with HSV-1 1716 fourteen days aftertumor implantation had a mean survival of 38.96 2.3 days, whereastumor-bearing mice mock-treated with viral growth media had a meansurvival of 24.9 6 0.6 days (P, 0.0001; Fig. 1A). To furthercharacterize this model, we determined whether the previously seenincrease in survival with earlier (day 7) administration of virus (1) wasalso seen. When HSV-1 1716 or mock treatment was administered onday 7 after tumor implantation, 60% of the HSV-1 1716-treated miceshowed complete regression of tumors. There was also a significantprolongation of mean survival in the remaining mice (546 9.1 days),whereas mock-treated mice had a mean survival of 27.86 1.1 days(P , 0.0003; Fig. 1B). Although a more significant prolongation in

survival of mice after viral treatment than mock treatment is seenwhen virus is administered on day 7 after tumor implantation, the sizeof the tumor was small at this time, and it was difficult to insure thatvirus was administered within the tumor mass. Therefore, the resultswere not as consistent as those of mice with treatment administered atday 14, and we have used this model for further studies. These datademonstrate the feasibility of using this model to study the role of theimmune response in neuro-attenuated HSV-1 therapy of murine i.c.melanoma.

HSV-1 1716 Does Not Prolong Survival of i.c. Tumor-bearingImmunodeficient Mice. We have previously shown that HSV-11716 is able to decrease human tumor cell growth in SCID and nudemice (27–29). The ability of HSV-1 1716 to destroy tumors inimmunodeficient mice implies that the immune system does notcontribute substantially to viral-mediated tumor destruction. How-ever, nonspecific immune responses to the xenographic tumor cellsmay be triggered by viral administration, and these responses, alongwith unchecked viral replication throughout the tumor mass, may besufficient to mediate tumor destruction. Additionally, it has beenshown that HSV-1 replicates more efficiently in human tissue than inrodent tissue (30). This fact may make the viral-mediated destructionof tumors more effective in the human tumor/mouse host models thanin mouse tumor/mouse host models.

We have evaluated whether a specific immune response contributesto viral-mediated tumor cell destruction using an allogenic immuno-deficient model. Six-week-old SCID mice were implanted with S91cells as described in “Materials and Methods.” Due to the acceleratedgrowth of S91 melanoma cells in SCID mice as compared withsyngenic DBA/2 mice, we injected 2-fold less tumor cells and admin-istered the virus on day 7. Fig. 2A shows the mean survival for HSV-11716-treated and mock-treated S91 i.c. tumor-bearing SCID micetreated on day 7. No significant difference in survival was seenbetween mock- and HSV-1 1716-treated mice (14.86 2.2 and15.0 6 1.7 days, respectively;P 5 0.95). This may be due to theaccelerated growth kinetics of the S91 melanoma cells in SCID micebecause the tumor may have grown too large for the virus to kill asignificant portion of the tumor and thus have an effect on survival.We have shown previously that administration of the virus at a laterperiod during tumor growth decreases its effectiveness in tumordestruction and decreases survival times (30).

To readdress whether a specific immune response is important toviral-mediated tumor cell destruction, we used a second murine tumormodel using i.p. cyclophosphamide injections to deplete leukocytes insyngenic DBA/2 mice (31–34). After implantation of 53 104 S91melanoma cells, cyclophosphamide administration was initiated onday 6 and continued until the mice were moribund, at which point theywere sacrificed. On day 10 after tumor implantation, viral therapy wasadministered. After HSV-1 1716 or mock treatment, leukocyte-de-pleted mice showed no significant difference in mean survival(19.06 1.9 and 19.56 2.7 days, respectively;P 5 0.6702; Fig. 2B).Mice were bled before viral treatment and throughout the posttreat-ment monitoring period to determine the degree of leukocyte deple-tion (.97%; data not shown). Because there was some variance inaverage mean survival times between experiments and to assess theeffect of cyclophosphamide treatment on tumor growth, a third groupof mice was implanted with tumor cells concurrently with the othergroups but was not treated with cyclophosphamide or given the virus.These mice had a mean survival of 256 0.8 days. Therefore, theleukocyte depletion due to the cyclophosphamide therapy may con-tribute to a slightly accelerated tumor growth rate. Furthermore, wesaw no difference in the growth rates of s.c. S91 flank tumors betweencyclophosphamide-treated, HSV-1-treated, and mock-treated mice(data not shown). Taken together, these data suggest that an immune

Fig. 1. Survival of DBA/2 mice after i.c. injection of 53 104 S91 melanoma cellsfollowed by treatment injection.A, viral treatment on day 14. There is a significantincrease in survival of mice receiving 53 104 pfu HSV-1 1716 (E) compared with thosereceiving mock treatment (M).n 5 10 for HSV-1 1716-treated mice;n 5 10 formock-treated mice.P , 0.0001.B, viral treatment on day 7. There is a significant increasein survival of mice receiving 53 104 pfu HSV-1 1716 (E) compared with those receivingmock treatment (M).n 5 10 for HSV-1 1716-treated mice;n 5 10 for mock-treated mice.P , 0.0003.

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response develops after viral administration that aids in tumor celldestruction.

Immune Cells Infiltrate the Tumor but not the SurroundingBrain Mass. The S91 melanoma cell line used in these studies isimmunogenic (35). Thus we investigated the immune response to thei.c. implantation of tumor cells in DBA/2 mice to better understandthe response to the tumor and virus administration. The major class ofcells infiltrating into tumors after implantation was CD41 T lympho-cytes (10.6%; Fig. 4A). Significant numbers of CD81 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 edgeregions of the tumor mass, although positively stained cells werefound throughout the tumor masses. This response could be due to thebreach of the BBB during tumor implantation, although the recruit-ment of leukocytes into the CNS is now considered common inresponse to virus, bacteria, and so forth. However, no significantimmune cell infiltration was seen when mock-implanted mice wereexamined (Fig. 3E). Therefore, breaching the BBB is not enough tocause recruitment of immune cells into the CNS; stimulation isrequired. The infiltration into tumor-bearing mouse tissue disappearedafter several days; however, on day 12, a slight increase in infiltrating

cells was again seen for a short period of time. Specifically, CD41andCD81 T cells (4.1% and 3.7%, respectively), NK cells (3.5%), and Bcells (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 tumorafter viral administration. The main infiltrating cells early after viraltreatment were CD41T cells (11.7%) and macrophages (8.2%), butPMN (7%), CD81T cells (2.1%), B cells (3.2%), NK cells (4.4%),and microglia cells (3.6%) were also present (Fig. 4B). This immunecell infiltration was sustained until day 21, when tumor size in mostsampled mice was negligible. These mice would have survived untilthe maximum time (i.e., day 50 after tumor implantation) because asmall number of tumor cells escaped destruction and would haveresumed tumor growth. No infiltration or signs of inflammation oc-curred in the surrounding normal brain tissue on any day (data notshown). Infiltration into the tumor proceeded from small, localizedsites on days 1 and 3 to more widespread sites in the tumors on days7 and 12 (Fig. 3,F–N). Significant NK (16.3%) and polymorphonu-clear leukocytes (16.5%) infiltration was seen on day 7, with signif-icant CD41T cells (14.5%) again present on day 12. Staining forHSV antigen showed a growth curve throughout sampling, with thehighest staining on day 7 (15.3%; Fig. 4). HSV-1 staining was foundthroughout the tumor mass (Fig. 5,A–H). No staining was seen inmock-treated tumor-bearing mice (Fig. 5H). MHC class I expressionwas down-regulated 3 days after viral therapy in treated mice whencompared with mock-treated mice. This is in accordance with reportson the ability of HSV-1 to down-regulate MHC class I expressionthrough ICP47 (36–38). The down-regulation of MHC class I expres-sion also corresponds with the concurrent shift from CD41 andCD81 T cells to NK cell and PMN infiltration. This correlates withthe proposed escape from CTL recognition of tumors and the impor-tance of NK cells in tumor clearance (39). This corresponds thefindings of Lewandowskiet al. (40), who state that HSV-1 KOS isable 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 and171are both more pathogenic than strain F, it is interesting to suggestthat more pathogenic strains are able to down-regulate MHC class IIexpression through some as yet unknown mechanism.

Apoptosis Increases within the Tumor Mass after Viral Admin-istration. An important factor in understanding the mechanism oftumor destruction is whether tumor cells are being destroyed vianecrosis and/or apoptosis. Necrosis leads to potentially undesirableinflammatory responses within the CNS. However, necrosis alsoincreases the potential development of a tumor-specific immune re-sponse because infiltrating immune responses clean up necrotic celldebris and present tumor cell and viral antigens to lymphocytes. Deaththrough apoptosis, on the other hand, would limit the inflammation atthe site of tumor destruction but may block the development of atumor-specific immune response that would be useful in potentiallyeliminating distant metastases. To determine whether tumor destruc-tion occurs by apoptosis, we used the Dead-End Colorimetric Apo-ptosis Detection System (Promega) to localize apoptotic cells withinthe tumor mass of viral-treated and mock-treated mice. Detection ofapoptotic cells increased throughout the monitoring period in viral-treated mice, beginning at 5% on day 0 and increasing to.30% byday 15 (Fig. 6). The apoptotic cells were found throughout theviral-treated tumor mass (Fig. 5,I–N). In mock-treated mice, baselineapoptotic cell detection was seen early in monitoring period (days1–6) but was not detected after day 7. These results suggest thatalthough apoptotic cell death is induced after viral administration,necrotic cell death may also be occurring because some cells inviral-treated tumor masses appear necrotic and do not stain for apo-ptosis (data not shown).

Fig. 2. Survival of immunodeficient mice after i.c. injection of tumor cells.A, survivalof CB17 (scid/scid) mice after i.c. injection of 53 102 S91 melanoma cells followed 7days later by treatment injection. There was no significant difference in survival of micereceiving mock treatment (viral culture media;M) compared with those receiving 53 104

pfu HSV-1 1716 (E).n 5 11 for HSV-1 1716-treated mice;n 5 10 for mock-treated mice.P 5 0.9484.B, survival of cyclophosphamide-treated DBA/2 mice after i.c. injection of5 3 104 S91 melanoma cells followed 14 days later by treatment injection. There was nodifference in survival of mice receiving mock treatment (viral culture media;M) com-pared to those receiving 53 104 pfu HSV-1 1716 (E).n 5 8 for HSV-1 1716-treatedmice; n 5 8 for mock-treated mice.P 5 0.6702.

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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, CD41T cells; B, CD81T 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 53 104 pfu HSV-11716.F, CD41 T cells, day 1;G, macrophages, day 1;H, PMN, day 7;I, CD81 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. Magnification5 original magnification3 20 3 objective.

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Prior Immunity to HSV Increases Survival Time of Viral-treated Mice. Because the immune response to viral administra-tion plays an important role in viral-mediated tumor cell destruc-tion, we wanted to determine the effect of prior immunity to HSVon tumor cell destruction. For immune mice, animals were infectedocularly with HSV 171and allowed to become latent for 28 daysbefore tumor implantation. Control mice were mock-infected at thesame time. Tumors were implanted, and 14 days later, HSV-1 1716was administered. Immune (latent) viral-treated tumor-bearingmice had a significant increase in survival when compared withnaı̈ve viral-treated tumor-bearing mice (Fig. 7). Specifically, im-mune mice mean survival was 35.5 days, whereas naı̈ve mice meansurvival was 28.8 days (P , 0.0001). To insure that previousimmunity to HSV-1 had no effect on tumor growth, immunetumor-bearing mice were mock-treated as described before. Thesemice had a mean survival of 25 days (P 5 0.0001 to naı̈ve andimmune-treated survival). Immunity had no effect on mortality innontreated groups. These results not only demonstrate that priorimmunity is not detrimental to viral therapy but also reiterate theimportance of the immune response in terms of cell destruction. Ifvirus administration accelerates the immune response to the tumor,in the presence of prior immunity, the immune response will beup-regulated and will aid in better tumoricidal activity.

DISCUSSION

Although the brain is historically considered an immune privilegedsite, recent studies in cases of infection or damage have shed light on

the ability of the immune response to infiltrate into the CNS. Theconcept of immune privilege of the CNS is based on the low levels ofresident lymphocytes, low levels of MHC expression on residentmicroglia, and the presence of the BBB, which blocks immunoglob-ulin and complement access to the CNS (41–43). Many recent studieshave shown that lymphocytes are able to migrate into the CNS andthat many cytokines are expressed within the CNS in response toinflammatory or injury markers (41). Therefore, it is important todetermine the importance of the immune response to viral (HSV)replication during tumor destruction and the contribution of the im-mune response to tumor cell destruction.

Neuro-attenuated HSV-1 strains are able to destroy human xeno-graft tumors in immunodeficient mice (27–29). However, it has beenshown that HSV-1 replicates with better efficiency in human tissuethan in rodent tissues (30). In the absence of an immune response toeliminate replicating virus, it was shown that neuro-attenuated HSV-1is able to spread through xenograft tumors and destroy them. BecauseHSV-1 is currently being studied in clinical trials as a treatment forhuman tumors in patients (who have probably been immunocompro-mised due to prior treatment) with this virus, it is important todetermine the ability of neuro-attenuated HSV-1 to destroy syngenicmurine tumors in immunodeficient mice.

Although many reports of the tumoricidal activity of neuro-atten-uated HSV have been published (1, 2, 3, 29), few studies haveinvestigated the effect of the immune response on replication-compe-tent viral treatment of tumors (44). M3 Cloudman S91 melanoma celltumor-bearing mice treated with HSV-1 1716 virus have a significant

Fig. 4. Percentages of infiltrating immune cells. Positivelystained cells and tumor cells were counted, and the percent-age of positively stained cells per total tumor cells is re-ported. Results are the average of counts from two slideswith two sections from three mice for each time point.A,percentages of inflammatory cells after i.c. implantation ofS91 melanoma cells into DBA/2 mice.B, percentages ofinflammatory cells after implantation of S91 melanoma cellsfollowed 14 days later with 53 104 pfu HSV-1 1716.

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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 3104

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). Magnification5 originalmagnification3 4 3 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 massafter viral administration of DBA/2 tumor-bearing mice. Mice were injected with 53 104 S91 melanoma cells followed 14 days later with 53 104 pfu HSV-1 1716.I, day 0;J, day3; K, day 7; magnification5 original magnification3 4 3 objective. Higher magnification of the above-mentioned pictures detailing apoptotic cells.L, day 0;M, day 3;N, day 7;magnification5 original magnification3 20 3 objective.

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prolongation in mean survival over mock-treated mice (Fig. 1A).Examination of the contribution of the immune response and the cellsthat mediate this response indicates that it is important for tumordestruction. The depletion of leukocytes by cyclophosphamide admin-istration demonstrated that a cellular immune response played a role.Furthermore, the immune cell infiltration, mainly CD41 T cells andmacrophages, seen early after viral administration demonstrated thatimmune cells are present within the tumor. Finally, previous immu-nity to HSV-1 was found to prolong the survival of viral-treatedtumor-bearing DBA/2 mice (Fig. 7).

Both humoral and cellular immune responses have been demon-strated to be important in HSV clearance and prevention of viralspread to the CNS. The humoral response is important in protectingthe CNS from disease (13). CD41 T cells reduce primary replicationand protect against latent infection (45), whereas CD81T cellsprevent the immunopathological activity of CD41 T cells (31). In-terestingly, our results closely parallel those seen previously usingnonreplicating HSV-1 as a gene therapy vector (43). In these studies,MHC class I and II expression was up-regulated along with theinfiltration of macrophages, T cells, and NK cells soon after viraladministration (43). However, our response is accelerated and is seenbefore that of other models (43, 46, 47). During gene therapy, the goalis generally long-term gene expression from a viral vector; therefore,an immune response to the virus or the recombinantly expressedprotein is undesirable. However, during cancer therapy, the goal istumor cell destruction; therefore, an immune response to the virus,which is expressing its genome only in tumor cells, may help tumordestruction by leading to a tumor-specific immune response that mayaid in destruction of distal metastases. Additionally, nonreplicatingviruses used for gene therapy carry a foreign gene of therapeuticinterest, which may alter the immune response to the virus. In thepresent study, the virus is restricted to infection in dividing tumor cellsby its ICP34.5 deletion. The accelerated increase in infiltration ofimmune cells seen in viral-treated tumors is due in part to an immuneresponse to viral proteins expressed on infected tumor cells but mayalso be due to the prior immune response elicited by the tumor itself,suggesting that virus injection either up-regulates the expression oftumor antigens or that lytic infection increases tumor antigen expres-sion. Additionally, viral injection may up-regulate the expression ofnew tumor antigens, helping in the treatment of highly metastatictumors. Unfortunately, the predominant cells that are seen in ourmodel after HSV-1 1716 therapy are CD41 T cells, NK cells, andmacrophages, of which only CD41T cells will exert antitumoreffects at metastatic sites.

The decrease in MHC class I expression over time is interestingbecause it appears that the tumor cells express MHC class I onimplantation 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 withtransporters associated with antigen processing (TAP) and thus down-regulates the antigen presentation through MHC class I (37, 38), thismay also be due to ICP47 action. Although ICP47 has been shown tonot interact with murine TAPin vitro, a recent report by Goldsmithetal. (48) demonstrates that it is able to interfere with antigen processingin vivo. MHC class II expression is also down-regulated after viralinfection. It has been shown previously that after infection withHSV-1 strain KOS, MHC class II cell surface expression is blocked(40). This is not seen with HSV-1 strain F (40), a less pathogenicstrain. Because HSV-1 strains KOS and 171 are both highly patho-genic, there may be a similar mechanism between the viruses, and thismay be occurring within the CNS in our model.

Up to 90% of the adult human population is sero-positive for HSVantibodies. Our result that prior immunity aids in viral-mediatedtumor cell destruction suggests that HSV-1 therapies will have bettereffectiveness in humans. The increased response to HSV may eitherlead to an increased infiltration of immune cells into the tumor massor suggest a switch for the dominant immune cell type infiltrating intothe tumor mass. In contrast to these results, Herrlingeret al. (47)observed no increase in survival in immune animals in their studiesand saw a decrease in gene transfer in immune mice, suggesting thatthe response may be tumor or model specific. Their model uses ratD74 gliomas in which the mutant HSV-1 is unable to replicatesufficiently due to resistance of the rat cell line to HSV infection.These authors did report more inflammatory infiltrates in the tumorsof immune mice at early time points compared with those of naı¨vemice. Therefore, prior immunity may aid in tumor destruction only inthe presence of a clearly replicating viral infection of the tumor cells.

In conclusion, the role of the immune response in viral-mediatedtumor cell destruction is important when the virus is administered latein tumor progression. This is important when timing virus adminis-tration in clinical trials, considering that many current clinical trialsare being performed in which patients have their immune responsedepressed (by chemotherapy and radiation treatment). Earlier admin-istration of virus or administration of virus in the absence of immunecompromise may facilitate the usefulness of neuro-attenuated HSV-1for i.c. tumors.

Fig. 7. Survival of immune and naı̈ve DBA/2 mice after i.c. injection of 53 104 S91melanoma cells followed 14 days later by treatment injection. Note the significant increasein 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 (M, immune tumor control;L,naı̈ve tumor control).n 5 10 for all groups.P , 0.0001 for immune HSV-1 1716-treatedmice versusall other groups.P 5 0.0122 for immune controlversusnaı̈ve treated mice.P 5 0.0001 for naı̈ve tumor treated miceversusnaı̈ve tumor control. There is nosignificant difference between the naı̈ve and tumor control groups (P5 0.0937). Allstatistics were performed using the Fisher’s PLSD for survival.

Fig. 6. Percentages of apoptotic cells. Positively stained cells and tumor cells werecounted, and the percentage of positively stained cells per total tumor mass cells isreported. Results are the average of counts from two slides with two sections from threemice for each time point.u, mock-treated mice;M, HSV-1 1716-treated mice.

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ACKNOWLEDGMENTS

We thank Moira Brown for the HSV-1 1716 virus and for her help. We alsothank Priscilla Shaffer and Bill Halford for helpful discussion on immunosup-pression, Tara Friebel and Vikram Suri for technical assistance in the labora-tory, Kathy Molnar-Kimber for editorial review, and Jennifer Driscoll for helpwith the manuscript.

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2000;60:5714-5722. Cancer Res   Cathie G. Miller and Nigel W. Fraser  Intracranial Melanoma ModelSimplex Virus-mediated Tumor Destruction in a Murine Role of the Immune Response during Neuro-attenuated Herpes

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