Preclinical models of HPV+ and HPV− HNSCC in mice: An immune clearance of HPV+ HNSCC

ORIGINAL ARTICLE

PRECLINICAL MODELS OF HPV1 AND HPV2 HNSCC INMICE: AN IMMUNE CLEARANCE OF HPV1 HNSCC

Robin Williams, MS,1 Dong Wook Lee, PhD,1 Bennett D. Elzey, PhD,2,3

Mary E. Anderson, BS,1 Bruce S. Hostager, PhD,4 John H. Lee, MD1

1Department of Otolaryngology, University of Iowa, Iowa City Veterans Administration Medical Center,Iowa City, Iowa. E-mail: [email protected] of Comparative Pathobiology, Purdue University, West Lafayette, Indiana3Department of Urology, Indiana University, Indianapolis, Indiana4Department of Pediatrics, University of Iowa, Iowa City, Iowa

Accepted 3 October 2008Published online 12 March 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/hed.21040

Abstract: Background. To investigate whether human pap-

illoma virus (HPV)-specific immune mechanisms can result in

tumor clearance, we have created HPVþ and HPV� tonsil cells

that form squamous cancers in immune-competent mice. Here,

we determine that an immune-specific response can clear

HPVþ tumor cells and the cellular requirements to mediate this

tumor clearance.

Methods. Through the benefit of this model, we use in vitro

and in vivo methods to better understand how HPVþ cells are

rejected.

Results. The data show that an in vivo antigen-specific

antitumor response is generated to HPVþ transformed cells

and that this response requires CD4þ and CD8þ cells to

mount this antitumor response.

Conclusion. The findings from this preclinical model will

have implications not only in understanding human disease but

also as a valuable model for testing immunotherapeutic strat-

egies for HPVþ head and neck cancer. VVC 2009 Wiley Peri-

odicals, Inc. Head Neck 31: 911–918, 2009

Keywords: HPV; squamous cell cancer; head; antitumor

immune response; mouse model

The incidence of head and neck squamous cellcarcinoma (HNSCC) has increased worldwide.1

Despite improved surgical and combined chemo/radiation therapies, the overall survival ratehas not increased during the last 30-year pe-riod.2 Human papillomavirus (HPV) 16 is thecause of carcinogenic progression in a substan-tial number of HNSCCs.3,4 Specifically, the oro-pharynx has a very high proportion (60%) ofcases that are HPVþ, and these present withmore metastatic and advanced disease thantheir HPV� counterparts.3,5 In particular, theincidence of HNSCC of the tonsillar region isincreasing.3,6,7 Therefore, understanding themost effective manner to treat this type ofHNSCC will be of great importance.

Currently, all treatment decisions of HNSCCare based on a histological diagnosis. When onecorrelates HPV status of HNSCC to treatmentefficacy, an intriguing result is observed: HPV-related HNSCC present at an advanced stagewith more lymph node metastasis, yet are 30%to 40% more curable than their corresponding

Correspondence to: J. H. Lee

VVC 2009 Wiley Periodicals, Inc.

In Vivo Immune Response of HPVþ Cancers HEAD & NECK—DOI 10.1002/hed July 2009 911

HPV� counterparts.5,8 To better understand thecellular mechanisms that lead to this change insurvival, we have transduced primary mouse ton-sil epithelial cells (MTECs) using the HPV16 E6/E7 viral oncogenes HPVþ or an shRNA strategyHPV� that mimics the actions of viral genes.9 Therole the immune system plays in the treatment ofHPVþ head and neck cancer has not beendelineated. The cell lines we developed have givenus the ability to better understand the response ofthe immune system to tonsil cells transformed byHPV oncogenes when compared with tonsil cellstransformed by similar molecular mechanismsutilized by HPV oncogenes, yet do not express vi-ral proteins.9 These HPVþ - and HPV� -trans-formed tonsil cells allow us to study growthdifference based on the presence or absence of vi-ral proteins in an immune-competent animalmodel and may give key insight in understandingthe curability of HPVþ tumors in humans.

In previous studies from our lab that focusedon viral oncogene mechanisms, we noted a dis-crepancy in growth between HPVþ and HPV�cells after implantation.9 A fraction of the miceimplanted with HPVþ cells cleared their tumorcells and survived, while all the mice implantedwith HPV� cells developed tumors and died. Inthe following studies, we utilize immune deple-tion strategies, knockout mice, and in vitroimmune assays to explore whether HPV-specificimmune responses could explain the survivaladvantage in the mice receiving HPVþ cells.The implications from this preclinical modelmay help us better understand the increasedsurvival of humans with HPVþ HNSCCC.

PATIENTS AND METHODS

Mice and Animal Care. Male C57BL/6, both wild-type and B6.129S7-rag1tm1Mom/J (rag1), micewere purchased at 6 to 8 weeks from JacksonLaboratories (Bar Harbor, Maine). On arrival,the animals were acclimated for 1 week prior totumor challenge or immune transfer therapy.All animals were maintained in a pathogen-freefacility and cared for and used in accordancewith protocols approved by the Animal Care andUse Committee of the University of Iowa (IowaCity, IA), which follows the U.S. Public HealthService’s guide for the care and use of animals.

Cell Lines. The HPVþ and HPV� cell lineswere established in previous work by our labusing primary MTECs from C57BL/6 mice to

ensure histocompatibility among all the miceused in our study.9,10 Both cell lines were grownin E media containing Dulbecco’s modifiedEagle’s medium (DMEM), Ham’s F12, 10% FBS,1% pen/strep, EGF (10 lg/mL), insulin (10 mg/mL), transferrin (25 lg/mL), chorera toxin (0.25lg/mL), hydrocortisone (25 lg/mL), and tri-iodo-thyronine (0.2 lg/mL). To aid in visualizingclearance of tumor cells, we also expressed lucif-erase in our HPVþ cell line. To create thesecells, E6/E7/H-ras MTECs were transduced witha retroviral vector that expressed luciferase andhygromycin resistance genes. A pool populationof cells expressing luciferase was obtained byselecting the cells with hygromycin, but individ-ual clones were not developed. Growth of E6/E7/H-ras/Luc MTECs and E6/E7/H-ras MTECs areidentical in mice (data not shown).

Splenocyte Preparation. Immediately followingeuthanization, spleens were removed andground into a single cell suspension using thefrosted ends of the 2 glass slides. After centrifu-gation, the red blood cells were removed usingammonium chloride lysis buffer. The remainingsplenocytes were centrifuged and resuspendedin RPMI-1640 medium containing 10% fetal calfserum and 1% pen/strep.

Antibodies. All monoclonal antibodies includingCD4, CD8, MHCI, and interferon (IFN)-c werepurchased from either Pharmingen (San Diego,CA) or eBioscience (San Diego, CA).

Intracellular Staining and Flow Cytometry

Analysis. Fresh or cultured cells were labeledwith fluorochrome-conjugated mouse antibodies(mAbs) specific for different surface markers for20minutes at 4�C. After binding, cells were washedand then fixed with formalin in phosphate-buf-fered saline (PBS) for flow cytometry analysis. Forintracellular staining, cells were stained for sur-face markers as indicated earlier, fixed with eBio-science Fixation buffer (Cat. No. 00-8222) for20 minutes, and then permeabilized with eBio-science permeabilization buffer (Cat. No. 00-8333)for 5 minutes. Both steps were performed at roomtemperature and in the dark. Cells were thenstained with fluorochrome-conjugated monoclonalantibodies (mAbs) for an intracellular IFN-c for20 minutes at room temperature in the dark. Af-ter washing away the excess antibodies with per-meabilization buffer, cells were resuspended inflow cytometry staining buffer for analysis. Data

912 In Vivo Immune Response of HPVþ Cancers HEAD & NECK—DOI 10.1002/hed July 2009

were analyzed using CellQuest Pro software (BDBiosciences, San Jose, CA). Quadrant markerswere set according to isotype control antibodies.

In Vivo Bioluminescent Imaging. Mice were anes-thetized with a mixture of isoflurane and O2, andthe hair from the tumor site was removed usingcommercially available Nair. The mice thenreceived a 150 mg/kg intraperitoneal injection ofD-luciferin (Xenogen Corporation, Alameda, CA).Ten minutes after substrate injection, the ani-mals were imaged for 5 minutes.

Bioluminescence images were obtained usingan IVIS Imaging System 100 Series (Xenogen)consisting of a 1-in. charge-coupled device cam-era cooled to �105�C. All specimens wereimaged at a range of 25 cm. Acquired imageswere analyzed using Living Image Software ver-sion 2.50 (Xenogen). Bioluminescence measure-ments were recorded as photons per second.

In Vivo Tumor Growth Studies. Six- to 8-week-oldwild-type C57BL/6 or rag1 mice (Jackson Labo-ratories) were anesthetized using a 75% CO2/25% O2 gas mixture and received 1 � 106 HPVþor HPV� cells subcutaneously on the rightflank. Tumor growth was followed until themass was cleared or until it reached 20 mm in 1direction. The 2 longest surface dimensions ofthe tumor were measured with calipers. Thelongest was squared and multiplied to the sec-ond longest and that was multiplied to 0.52 toyield a tumor volume in mm3. Mice that clearedtheir initial tumor challenge were rechallengedwith 2 � 106 cells in the left flank.

To test long-lasting immunity, tumor nodulesfrom animals that did not clear their tumor bur-den were extracted from the right flank andprocessed to produce a single cell suspension.These cells were then reinjected into the leftflank of cleared mice.

In Vivo Adoptive Transfer. A single cell suspen-sion of splenocytes was produced as describedearlier using spleen from naı̈ve mice, tumor-bear-ing mice, and mice that cleared their tumor chal-lenge. Cells were resuspended in PBS at aconcentration of 2.5 � 105 cells/lL. Six- to 8-week-old rag1 mice were anesthetized with keta-mine/xylazine, and 200 lL of cells (5 � 107 totalcells) or 200 lL of PBS was injected through theretroorbital venous plexus. One week afterimmune transfer, the animals underwent thesame tumor challenge described earlier.

In Vitro IFN-c Stimulation and Production. Spleno-cytes from naı̈ve, cleared, and tumor-bearingmice were cocultured with either the HPVþ orHPV� cells at an effecter to target ratio of 50:1.After 36 hours, supernatants from the cocul-tures were isolated for analysis using an R&DSystems ELISA kit (Minneapolis, MN).

Depletion Protocol. CD4 antibody (GK1.5) andCD8 antibody (2.43) were grown and purified byISU Hybridoma Facility (Ames, IA). T-cell deple-tion was started by intraperitoneal injectionswith 500 to 600 lg of purified antibody in100 lL total volume. Injections were given on 3consecutive days and twice a week thereafterfor approximately 40 days throughout thecourse of tumor growth. Effectiveness of deple-tion was confirmed by flow cytometry on periph-eral blood leukocytes after 3 days of depletion.

Statistical Methods. The statistical analysesfocused on the effects of different treatments oncancer progression. The primary outcomes of in-terest are time to death and tumor growth overtime. The log-rank test was used to compare sur-vival times between treatment groups. Kaplan–Meier survival plots were constructed to esti-mate the survival functions. Tumor sizes (mm3)were measured throughout the experiments,resulting in repeated measurements across timefor each mouse. Linear mixed effects regressionmodels were used to estimate and compare thegroup-specific tumor growth curve. All testswere 2-sided and carried out at the 5% level ofsignificance. Analyses were performed with theSAS and R statistical software packages.

RESULTS

In Vivo Tumor Growth and Survival in Immune-

Competent and Immune-Incompetent C57BL/6

Mice. Prior to testing this system in vivo, theHPVþ and HPV� cell lines were characterizedextensively in vitro. These cells were derivedfrom the same primary mouse tonsil keratino-cytes and have similar mechanisms that allowinvasive growth.9 In preliminary studies, wenoted that the HPVþ cells were at times clearedby mice. To examine this observation moreclosely, we grew parallel cultures of cells andinjected 1 � 106 of either the HPVþ or theHPV� cells into wild-type C57BL/6 mice. Weobserved a difference in growth, both within the


HPVþ group and between the HPVþ and HPV�groups.

Within the HPVþ group, 2 growth pheno-types were noted among the initial 40 micetested: (1) growth (n ¼ 27), and (2) clearance (n

¼ 13) (Figure 1A), suggesting that while theanimals are syngeneic, they differ in how theyhandle this particular tumor burden. Addition-ally, as noted earlier, the HPVþ and HPV�tumors grew differently in comparison to each

FIGURE 1. Expression of HPV-associated antigens on tumor improves survival and decreases tumor growth in immune-competent

mice. Age-metched, male C57BL/6 mice were implanted with 108 HPV antigen positive (HPVþ) or negative (HPV�) tumor cells subcu-

taneously in the flank. (A) Bioluminescent imaging of HPVþ luciferase producing tumor cells on the days indicated following implanta-

tion. (B) Average tumor growth (tumor growth was measured by calipers and expressed as an elliptical volume) from the mice that

developed tumors. Error bars represent SE of the mean for each group. HPV� tumors had a statistically significantly more rapid rate

of growth in vivo (p < .0001). (C) Mouse survival following implantation of HPVþ and HPV� tumor cells shown an increased survival

for HPVþ tumors. (p < .0001) Mice living last 80 days did not develop tumors.

FIGURE 2. HPV(þ) and HPV(�) tumor cells have similar growth and pathogenicity in immune-incompetent mice. HPV(þ) and HPV(�)

tumor cells (108) were implanted in C57BL/6 mice. Growth and survival were calculated as indicated in Figure 1. Results from experi-

ments with wild-type mice are presented for comparison. (A) The difference in tumor growth rate between HPV(þ) cells in C57BL/6

mice compared with Rag mice was found to be statistically significant (p < .0002), while the difference between Rag mice with

HPV(þ) cells compared to Rag mice with HPV(�) cells and HPV(�) cells in C57BL/6 mice compared to Rag mice with HPV(�) cells

were not. (B) The difference in survival rate between HPV(þ) cells in C57BL/6 mice compared to Rag mice injected with these cells

was found to be significant (p < .0001), while the difference between Rag mice with HPV(þ) cells and those with HPV(�) cells and

HPV(�) cells in C57BL/6 mice compared to Rag mice with these same cells again was not.


other. While the HPVþ tumors varied in growth,the HPV� cells universally and uniformly formedtumors (Figure 1B). Despite the fact thatapproximately 25% to 30% of HPVþ tumor-bearing mice cleared their tumor burden, not asingle mouse bearing the HPV� cells did.

These 2 groups differed with respect to sur-vival as well (Figure 1C). As indicated earlier,approximately 25% to 30% of mice injected withHPVþ cells cleared their tumor burden and sub-sequently remained disease free. The other 70%that maintained a tumor burden survived any-where from 50 to 75 days. Alternatively, all ofthe HPV� mice had succumbed to their illnesswithin 45 days, with the first animals dying at37 days.

To follow-up on the variation of growth phe-notype reported earlier, we next asked whetherthe presence of functional B and T lymphocyteswere required for the noted tumor clearance ofHPVþ cells. C57BL/6 rag1 mice were injectedwith the same 2 cell lines described previously.Rag1 mice are B- and T-cell deficient owing to anabsence of functional recombinases, leading todefects in initial phases of V(D)J recombina-tion.11 Other immune cells such as macrophages,NK cells, and complement activity are unaf-fected11 Unlike wild-type C57BL/6 mice, HPVþcells in all rag1 mice grew at the same rate anddid not have variations in growth rate. None ofthe rag1 mice were able to clear their tumor bur-den, and all of these animals died as a result oftheir disease. Additionally, in rag1 mice therewas no growth rate difference between theHPVþ and HPV� groups (Figure 2A) and no dif-ference in survival (Figure 2B). This evidencesuggests that the difference in growth of HPVþcells compared to HPV� cells in wild-type miceis immune mediated and not simply an intrinsicresult of the HPVþ cells themselves.

In Vivo Clearance of HPV1 Cells Provides Long

Lasting Tumor Immunity. As a means of investi-gating whether the clearance of HPVþ cells isimmune mediated or alternatively caused bysome change in the tumor cells themselves (ie,tumors which escape clearance are variants thathave lost antigen expression), we implanted tu-mor cells isolated from mice unable to rejecttheir tumor burden into mice that had previ-ously cleared their HPVþ tumor burden (seeFigure 3). Specifically, 2 � 106 reconstituted tu-mor cells were injected into the contralateralflank of each cleared mouse. Even up to 6

months after the initial clearance, all animalsrejected such a rechallenge. These data suggestthat tumor cells remain antigenic, and the vari-ation in response is a result of a variableimmune response to the cells in the mice. Figure3A is the average clearance in these mice follow-ing the initial exposure and the subsequentrechallenge. During the initial exposure toHPVþ cells, it took 14 to 21 days for clearanceto occur; clearance after rechallenge took onlyhalf that amount of time, roughly 5 to 7 days(Figure 3B). The more rapid clearance of the tu-mor rechallenge as compared to the primaryinoculation likely represents an immune-medi-ated ‘‘recall’’ response.

In Vivo Adoptive Transfer. To further assesswhether this clearance represented an immunereaction to the tumor cells, we tested whetherimmune clearance capacity could be transferredfrom a cleared mouse into a mouse unable toclear tumor. In this test, splenocytes from wild-

FIGURE 3. Mice that have previously cleared tumor rapidly clear

a second tumor challenge. C57BL/6 mice that cleared their first

tumor challenge were rechallenged with 106 HPV(þ) tumor cells

in the opposite flank 3 months later. (A) The clearance of HPVþtumor in a representative mouse using IVIS imaging. (B) Tumor

size following primary and secondary implantation of tumor. Sym-

bols are the mean (�SEM) tumor volume in mice at the indi-

cated day after tumor implantation. Second exposure had a

more rapid rate of clearance (p < .0028).


type ‘‘naı̈ve’’ animals and wild-type ‘‘cleared’’animals were injected retroorbitally into rag1mice that were not expected to clear thesetumors based on Figure 2. Seven days postsple-nocyte adoptive transfer, the mice were chal-lenged with 1 � 106 HPVþ tumor cellssubcutaneously in the right flank. All of themice initially developed tumor (Figure 4A).Within 14 to 21 days, all of the mice thatreceived ‘‘cleared’’ splenocytes and 30% of themice that received ‘‘naı̈ve’’ splenocytes hadcleared their tumor burden (Figure 4B). Theremaining animals, which received ‘‘naı̈ve’’ sple-nocytes, survived approximately 54 days (Figure4C). The rag1 mice that received PBS only (noimmune transfer) developed tumors that grewprogressively and resulted in death within 40days (Figures 4B and 4C). These data demon-strate that lymphocytes from mice that rejectedtumor can confer protective tumor immunity toimmune-incompetent animals. This holds truefor the ‘‘naı̈ve’’ spleens as well; however, theseanimals had to mount a primary immuneresponse to the tumor challenge, whereas theanimals receiving the ‘‘cleared’’ splenocytesmounted a recall response as indicated by thedifference in clearance kinetics.

In Vitro Stimulation of Splenocyte IFN-c Production

in Response to HPV1 Cells. To determinewhether we could detect an immune-specific

reaction to E6/E7 tumor cells, we tested whetherlymphocytes from mice that cleared their tumorswould produce an IFN-c response in vitro. Previ-ous reports have shown that IFN-c production invitro correlates with tumor clearance in vivo12

The results shown in Figure 5 indicate that theantitumor response from the mice that cleartheir tumor is directed toward the viral antigenssince the HPV� cells do not produce a response.Interestingly, even mice that do not clear theirtumor produce a response, albeit less than themice that cleared their tumor. This result com-bined with the growth rate difference of HPVþcells in rag1 mice versus wild-type mice suggeststhat HPVþ antitumor responses do occur even inmice that are unable to clear their tumors. SinceIFN-c response is not produced exclusively dur-ing an immune-specific CTL response, furthertesting for immunophenotypes involved in thiscellular response will be required in the future.

Immune Clearance Requires Both CD41 and CD81

Lymphocytes. To better understand what cellsare required for the immune-mediated clear-ance, CD4þ and CD8þ cells were depleted invivo via injection with CD4- and CD8-specificmonoclonal antibodies prior to tumor implanta-tion. Groups of 3 mice were given either 100 lgof anti-CD4 (clone GK1.5), anti-CD8 (clone2.43), an isotype IgG, or PBS via IP injection.After 24 hours, depletion was verified by flow

FIGURE 4. Adoptive transfer of splenocytes from tumor-free mice protects Rag mice from tumor. Rag mice were injected with PBS or

106 splenocytes from naive or tumor clearing mice. Seven days after transfer, mice were implanted with HPV(þ) tumor cells, and tu-

mor growth was determined by IVIS imaging (A) or calipers (B). Survival is indicated in (C). Survival and growth rate was statistically

different between all groups tested (p < .0001).


cytometry, yielding better than 99% specificdepletion of the targeted cells (CD4 and CD8)compared to the controls that received nonspe-cific IgG or PBS (data not shown). After deple-tion was verified, all mice again received 1 �106 E6/E7/Ras/Luc cells. Growth rate increasedwhen either CD4þ or CD8þ cells were removed(see Figure 6). In addition, none of the mice inthe depleted groups were able to clear the tumorand survive (see Figure 6). These data indicatethat CD4þ and CD8þ lymphocytes play a rolein inhibiting the growth of HPVþ cells.

DISCUSSION

In this study, we evaluated the in vivo growthphenotypes of MTECs with and without HPV16E6/E7 oncogenes. We examined both immune-competent and immune-incompetent animals.We observed a difference in growth and survivalrate within the HPVþ tumor-burdened group, aswell as between the HPVþ and HPV� cell lineswhen they were given to the wild-type animals,but found no such differences when they wereinjected into mice lacking B and T cell immunity.Specifically, CD4þ and CD8þ T cells were foundto be required to mount this immune response.A subset of immune-competent animals that

received HPVþ tumor cells cleared their initialtumor burden and was found to be immune tofuture HPVþ tumor cell challenge. In an adop-tive transfer experiment, the whole splenocytesfrom this same group of mice transferred toimmune-incompetent animals provided HPVþantitumor immunity to 100% of recipients. Thisdata presents a testable model for comparingHPVþ and HPV� and may prove to be a valua-ble source to better understand the role theimmune response plays in HPVþ HNSCC.

A major question regarding HPV-relatedHNSCC in humans is why HPVþ cancers havegreater cure rates. At least 2 different hypothe-ses could potentially explain why HPV-relatedcancers are more curable. First, it is possiblethat the mechanism of HPV oncogenesis makesthe epithelial cells themselves more sensitive toeither radiation or chemotherapy that is used for

FIGURE 5. Tumor-cleared mice have higher anti-HPV IFN-y

responses. Mice were implanted with 106 HPV(þ) MTEC cells

or left untreated (naive), and 40 days later, spleens were har-

vested. Total splenocytes (1 � 106 per well) from naive, tumor-

bearing, and tumor-cleared mice were cocultured with HPV(þ)

MTECs (2 � 104 per well) for 36 hours. Culture supernatants

were analyzed for IFN-y production by ELISA. The black bar is

IFN-y production from cocultures of splenocytes from tumor-

cleared mice and HPV� MTECs. Bars represent the mean �SE from duplicate experiments. Differences in IFN-y production

was statistically significant for all conditions (p < .01).

FIGURE 6. Tumor growth in mice after CD4 and CD8 depletion.

After CD4 and CD8 cell populations were depleted as described

in the Methods section. 1 � 106 tumor cells were implanted

subcutaneously in the right flank. Growth after depletion was

compared with growth in wild-type and Rag 1 mice. CD4-, and

CD8-depleted animals had statistically faster growth and worse

survival when compared with wild-type mice (p < .05). Tumor

growth was followed using calipers and expressed as mm3. (A)

Average tumor growth � SE and (B) survival.


treatment. However, preliminary data from ourlab has shown that HPVþ cell lines are moreradioresistant than the HPV� cell lines, sug-gesting that the increased response for HPVþtumors is not due to intrinsic sensitivity to radi-ation and cisplatinum (unpublished). A secondhypothesis is that, since HPV tumors expressforeign antigens, an immune-mediated mecha-nism may be activated during therapy toimprove tumor clearance. This hypothesis is sup-ported by the observation that immune-sup-pressed individuals have a propensity to developHPVþ HNSCC,13,14 and these patients fre-quently have a worse response to therapy.Besides determining mechanisms required forimmune-related clearance of HPVþ HNSCC,this model may also serve as a valuable preclini-cal tool testing how therapies alter the immuneresponse during therapy.

Previous studies have produced similarHPVþ tumor cells through the transformationof primary cells with E6, E7, and H-Ras.15 Ourmodel is unique, however, in the cell type usedto produce the HPVþ cell line and in the devel-opment of a comparative HPV� tumor cell line.Prior studies used primary lung cells, while wechose to use primary tonsil epithelia, a cell typevery commonly associated HPVþ HNSCC. Addi-tionally, we constructed an HPV� cell linewhich utilizes similar mechanisms of transfor-mation, yet lacks viral oncogene expression.

In this study, we utilized a number of differ-ent experimental strategies to address this hy-pothesis. We found evidence that clearance ofHPVþ tumor cells requires a full and intactimmune system. One of the interesting observa-tions is the dichotomy of tumor responses ingenetically inbred mouse strains. In these ex-periments, the exact same cells were implantedinto matched individuals, and yet some micedeveloped tumors and some mice clearedtumors. Our studies have shown some of theimmune requirements in the mice that clearedtumors but it would be equally as important tounderstand how the mice that allow tumorgrowth become tolerant to the antigenic cells.What are the required components of the sup-pressive immune response that allow growth?Such questions are currently being addressed inpresent studies. We hope that a greater under-standing of the requirement for clearance, aswell as an understanding of mechanisms of tol-

erance in mice, will be applicable for humans aswell, thus providing rationale for the develop-ment of therapeutic intervention by manipulat-ing a particular arm of the immune system andaugmenting survival.

REFERENCES

1. Johnson N. Tobacco use and oral cancer: a global per-spective. J Dent Educ 2001;65:328–339.

2. Buckley JG, Ferlito A, Shaha AR, Rinaldo A. The treat-ment of distant metastases in head and neck cancer—present and future. ORL J Otorhinolaryngol Relat Spec2001;63:259–264.

3. Smith EM, Ritchie JM, Summersgill KF, et al. Age, sex-ual behavior and human papillomavirus infection in oralcavity and oropharyngeal cancers. Int J Cancer 2004;108:766–772.

4. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Humanpapillomavirus types in head and neck squamous cellcarcinomas worldwide: a systematic review. Cancer Epi-demiol Biomarkers Prev 2005;14:467–475.

5. Ritchie JM, Smith EM, Summersgill KF, et al. Humanpapillomavirus infection as a prognostic factor in carci-nomas of the oral cavity and oropharynx. Int J Cancer2003;104:336–344.

6. Ioka A, Tsukuma H, Ajiki W, Oshima A. Trends in headand neck cancer incidence in Japan during 1965–1999.Jpn J Clin Oncol 2005;35:45–47.

7. Chaturvedi AK, Engels EA, Anderson WF, Gillison ML.Incidence trends for human papillomavirus-relatedand -unrelated oral squamous cell carcinomas in theUnited States. J Clin Oncol 2008;26:612–619.

8. Fakhry C, Westra WH, Li S, et al. Improved survival ofpatients with human papillomavirus-positive head andneck squamous cell carcinoma in a prospective clinicaltrial. J Natl Cancer Inst 2008;100:261–269.

9. Spanos WC, Hoover A, Harris GF, et al. The PDZ bind-ing motif of human papillomavirus type 16 E6 inducesPTPN13 loss, which allows anchorage-independent growthand synergizes with ras for invasive growth. J Virol2008;82:2493–2500.

10. Hoover AC, Spanos WC, Harris GF, et al. The role ofhuman papillomavirus 16 E6 in anchorage-independentand invasive growth of mouse tonsil epithelium. ArchOtolaryngol Head Neck Surg 2007;133:495–502.

11. Mombaerts P, Iacomini J, Johnson RS, Herrup K, Tone-gawa S, Papaioannou VE. RAG-1-deficient mice have nomature B and T lymphocytes. Cell 1992;68:869–877.

12. Welters M. Frequent display of human papillomavirustype 16 E6-specific memory t-Helper cells in the healthypopulation as witness of previous viral encounter.Cancer Res 2003;63:636–641.

13. Frisch M, Biggar RJ, Goedert JJ. Human papillomavi-rus-associated cancers in patients with human immuno-deficiency virus infection and acquired immunodeficiencysyndrome. J Natl Cancer Inst 2000;92:1500–1510.

14. Cobo F, Garcia C, Talavera P, et al. Human papillomavi-rus associated with papillary squamous cell carcinoma ofthe oropharynx in a renal transplant recipient. Infection2006;34:176–180.

15. Lin KY, Guarnieri FG, Staveley-O’Carroll KF, et al.Treatment of established tumors with a novel vaccinethat enhances major histocompatibility class II presenta-tion of tumor antigen. Cancer Res 1996;56:21–26.


Preclinical models of HPV+ and HPV− HNSCC in mice: An immune clearance of HPV+ HNSCC

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