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Microenvironment and Immunology

PD-1–Expressing Tumor-Infiltrating T Cells Are a FavorablePrognostic Biomarker in HPV-Associated Head and NeckCancer

C�ecile Badoual1,2,3, St�ephane Hans4, Nathalie Merillon1,2, Cord�elia Van Ryswick1,2, Patrice Ravel11,Nadine Benhamouda1,2,5, Emeline Levionnois1,2,5, Mevyn Nizard1,2, Ali Si-Mohamed6, Nicolas Besnier1,2,Alain Gey1,2,5, Rinat Rotem-Yehudar12, H�el�ene Pere1,2,6, Thi Tran1,2, Coralie L. Guerin1,2, Anne Chauvat1,2,Estelle Dransart8, C�ecile Alanio5, Sebastien Albert13, Beatrix Barry13, Federico Sandoval1,2,Francoise Quintin-Colonna1,2,14, Patrick Bruneval1,2,3, Wolf H. Fridman5, Francois M. Lemoine9,10,Stephane Oudard1,2,7, Ludger Johannes8, Daniel Olive15,16, Daniel Brasnu4, and Eric Tartour1,2,5

AbstractHead and neck cancers positive for human papillomavirus (HPV) have a more favorable clinical outcome than

HPV-negative cancers, but it is unknown why this is the case. We hypothesized that prognosis was affected byintrinsic features ofHPV-infected tumor cells or differences in host immune response. In this study, we focused ona comparison of regulatory Foxp3þ T cells and programmed death-1 (PD-1)þ T cells in the microenvironmentof tumors thatwere positive or negative forHPV, in two groups thatwerematched for various clinical and biologicparameters. HPV-positive head and neck cancers were more heavily infiltrated by regulatory T cells and PD-1þ Tcells and the levels of PD-1þ cells were positively correlated with a favorable clinical outcome. In explaining thisparadoxical result, we showed that these PD-1þ T cells expressed activation markers and were functional afterblockade of the PD-1–PD-L1 axis in vitro. Approximately 50% of PD-1þ tumor-infiltrating T cells lacked Tim-3expression andmay indeed represent activated T cells. Inmice, administration of a cancer vaccine increased PD-1on T cells with concomitant tumor regression. In this setting, PD-1 blockade synergized with vaccine in elicitingantitumor efficacy. Our findings prompt a need to revisit the significance of PD-1–infiltrating T cells in cancer,where we suggest that PD-1 detection may reflect a previous immune response against tumors that might bereactivated by PD-1/PD-L1 blockade. Cancer Res; 73(1); 128–38. �2012 AACR.

IntroductionRecent studies have detected oncogenic HPV in 25.9% of

head and neck cancers (mostly HPV16) and this prevalenceincreases to more than 50% for cancers of the oropharynx(tonsil, base of tongue, etc.) with a growing worldwide inci-dence (1).

Although these tumors frequently have aggressive histo-pathological features (2), the presence of HPV DNA is afavorable prognostic factor with regard to recurrence andsurvival (3–5). Themechanisms underlying the more favorableoutcome of HPV-associated head and neck cancer have notbeen clearly elucidated.

These tumors are more responsive to chemotherapy andradiotherapy, but even patients with HPV-positive oropharyn-geal cancers treated by surgery alone have a better prognosisthan those with HPV-negative tumors after adjustment fortumor stage (6, 7).

HPV E6-E7 expressing tumors grew slower in immunocom-petent mice but not in nude mice, than HPV-negative tumorssuggesting a role of adaptive immunity and not simply anintrinsic biologic property of HPV-positive tumor cells toexplain this difference (8).

In human, T cells directed against HPV derived proteins (E2,E6, and E7) have been described in the blood and in the tumor

Authors' Affiliations: 1INSERM U970 PARCC; 2Universit�e Paris Des-cartes, Sorbonne Paris-Cit�e; 3Department of Pathology, Hopital Euro-p�een Georges Pompidou (HEGP), Assistance Publique Hopitaux deParis (AP-HP), 4Service d'oto-rhino-laryngologie et de chirurgie cer-vico-faciale, HEGP, 5Service d'immunologie biologique, HEGP, 6Servicede virologie, HEGP, and 7Service d'oncologie m�edicale, HEGP; 8Traffic,Signaling, and Delivery Laboratory, Institut Curie-Centre de Recherche,CNRS UMR 144; 9Department of Biotherapies and the Clinical Inves-tigation Center in Biotherapy AP-HP, Groupe Hospitalier Piti�e-Sal-petri�ere; 10Universit�e Pierre et Marie Curie Universit�e Paris 06, UMRCNRS 7211/INSERM 959, Paris; 11CNRS UMR5048 Centre de Biochi-mie structurale, INSERM U554, Universit�e de Montpellier 1 et 2, Mon-tpellier, France; 12CureTech Ltd., Yavne, Israel; 13Service de chirurgieORL et cervicofaciale, Hopital Bichat-Claude-Bernard, AP-HP; 14EcoleNationale V�et�erinaire d'Alfort, Maisons Alfort; 15Institut Paoli-Calmettes,Laboratoire d'Immunologie des Tumeur; and 16Centre de Recherche enCanc�erologie de Marseille, INSERM U 1068, Marseille, France

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

N. Merillon and C. Van Ryswick contributed equally to this work.

Corresponding Author: Eric Tartour, Hopital Europ�een GeorgesPompidou. Service d'Immunologie Biologique. 20 Rue Leblanc75015 Paris. Phone: 33-156-093-942; Fax: 33-156-092-080. E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-2606

�2012 American Association for Cancer Research.

CancerResearch

Cancer Res; 73(1) January 1, 2013128

of patients with head and neck cancers (9–12). However, thecapacity of tumor-infiltrating lymphocytes (TIL) to act aseffector cells may be affected by the tumor microenvironment.Immunosuppressive cells or molecules (13, 14) or chronicinflammation in head and neck cancer predispose to a tumorpromoting microenvironment (15, 16). Analysis of differencesin the type of immunity within the tumormicroenvironment ofHPV-associated head and neck cancer compared with HPV-negative tumors may, therefore, provide new clues to explainthe more favorable clinical behavior of this type of cancer. Inthe present study, we addressed the clinical significance ofregulatory Foxp3þT cells and programmed death (PD-1)-pos-itive T-cell infiltration in the tumor microenvironment ofHPV-positive and HPV-negative head and neck cancers. Theseimmunosuppressive cells were selected because regulatoryCD4þCD25þFoxp3þT cells have emerged as the dominantT-cell population governing peripheral tolerance by inhibitingeffector T cells. We and other authors have shown that thefrequency of intratumoral Treg is increased in head and neckcancer patients and in HPV-associated cervical carcinoma (17,18). PD-1, a member of the CD28 receptor family, is expressedby activated lymphocytes and inhibits their proliferation andeffector functions after binding to PD-1 ligands such as B7-H1(PD-L1; ref. 19). PD-1 expression is indicative of chronic anti-gen stimulation and contributes to T-cell dysfunction (exhaus-tion; ref. 19). Interferencewith PD-1–PD-L1 signaling, via eitherantibody blockade or PD-1 deficiency has been shown toimprove clinical outcome and restore functional T-cell respon-ses in chronic viral diseases and in several types of cancer (20).Preliminary results documented that more than 60% of freshlyisolated human head and neck tumors express PD-L1 (21) andin early stage tongue cancer, intraepithelial T cells frequentlyexpress PD-1 (22). All these data, therefore, support a com-prehensive analysis of PD-1–PD-L1 expression in head andneck cancers associated with chronic viral infection.As expected, HPV-associated head and neck cancers were

found to be associated with a good prognosis compared withHPV-negative tumors. HPV-positive tumors weremore heavilyinfiltrated by regulatory T cells and PD-1 expressing T cellsthan HPV-negative tumors. Surprisingly, levels of PD-1þ T-cellinfiltration positively correlated with a favorable clinical out-come in HPV-associated cancers and, in a preclinical HPVtumor model, predicted the clinical response to anti–PD-L1antibody.

Materials and MethodsPatientsSixty-four newly diagnosed untreated patients with primary

histologically proven head and neck squamous cell carcinoma(HNSCC) were included in this study. Patient characteristicsare presented in Supplementary Table S1. Each patient'sdisease was staged according to the 7th edition of the Inter-national Union Against Cancer/American Joint Committee onCancer system for head and neck cancer. Treatmentmodalitiesconsisted of chemoradiotherapy without surgery (organ pres-ervation) or surgery combined or not with radiotherapy andchemotherapy. This study was conducted in accordance with

French laws and after approval by the local ethics committee(ID RCB 2007-A01128-45).

Patients were divided in 2 groups depending on the presenceor absence of oncogenicHPV. These 2 groupswerematched forvarious parameters (gender, primary tumor site, tumor stag-ing, lymph node involvement, presence of metastasis, andtreatment modalities; Supplementary Table S1).

MiceSix- to eight-week-old female C57BL/6 (H-2b; B6) mice were

purchased fromCharlesRiver Laboratories (L'Arbresle, France).All mice were kept under specific pathogen-free conditions atthe INSERMU970 animal facility. Experiments were conductedaccording to institutional guidelines after acceptance by theVeterinary School of Maisons-Alfort ethics committee.

CellsTC-1 cells are transformed murine (H2b) epithelial cells

cotransfected with HPV-16 E6/E7 genes and the activatedhuman Ha-ras (G12V) oncogene DNA. They were obtainedfromDr. TCWu's laboratory (Department of Pathology, Schoolof Medicine, Johns Hopkins University, Baltimore, USA).Cells were grown in RPMI 1640 medium supplemented with10% fetal calf serum, 2 mmol/L L-Glutamine, antibiotics and0.4 mg/mL G418 at 37�C with 5%CO2.

Peptide, vaccine, and antibodiesPool of 15 mer peptides spanning the entire HPV16 E7

protein were designed with a 4 mer overlap between eachpeptide. They were obtained from PolyPeptide Laboratories(Strasbourg, France), reconstituted in PBS and stored at�20�C.

The STxB-E743–57 vaccine was produced by chemical cou-pling between the N-bromoacetylated E743–57 peptide and thesulfhydryl group of STxB-Cys recombinant protein, as previ-ously described (23). After purification, endotoxin concentra-tions determined by the Limulus assay test (Lonza) were lessthan 0.5 EU/mg.

The invariant natural killer T-cell ligand a-GalCer(KRN7000) was purchased from Funakoshi (Tokyo, Japan).

CT-011 is a humanized monoclonal anti-PD-1 antibodymanufactured by CureTech LTD. This antibody has alreadybeen validated in ex vivo functional assays in humans (24).

Anti-mouse PD-L1 and isotype control antibodies werepurchased from BioXcell. Mice received intraperitoneal injec-tions of 200 mg mAb/mouse as previously described (25).

For immunofluorescence analysis, an anti-PD-1 mAb gen-erated by D Olive's group was selected and used (see Supple-mentary Table S2) as previously described (26).

HPV detectionThe presence of HPV in head and neck cancer biopsies was

detected using the INNO-LIPA genotyping Extra assay(Innogenetics).

Immunofluorescence staining and flow cytometryanalysis

Immunofluorescence staining and flow cytometry analysiswere conducted as previously described (16) and detailed in

PD-1–Expressing T Cells in HPV-Associated Oral Cancers

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 129

Supplementary Data. The antibodies used for the variousimmunofluorescence stainings are described in Supplemen-tary Table S2.

Immunization of mice and in vivo tumor protectionC57BL/6 mice were immunized via the intraperitoneal

route at days 0 and 14 with the STxB-E7 vaccine (20 mg)combined with 1 mg of aGalCer during the first immuniza-tion.

In a therapeutic setting experiment, 105 TC-1 tumor cellswere injected subcutaneously (s.c.) in the rightflank of B6mice.They were then vaccinated or not at days 9 and 18 after tumorgraft in combination with anti-PD-L1 or isotype control anti-bodies. Mice were monitored every 48 to 72 hours for tumorgrowth.

Statistical analysisSurvival variables were estimated using the Kaplan–Meier

method and compared by the log-rank test for categoricalvariables and the Cox model and associated Wald �2 statisticfor quantitative variables.

Overall survival was defined as the time from initialdiagnosis until death or until last follow-up (right censoreddata).

Locoregional control was calculated from the end of treat-ment and defined as the absence or either persistent orrecurrent disease at the primary site or in the cervical lymphnodes. Patients with persistent disease at the end of treatmentwere considered to have experienced failure at time 0. Patientswith no signs of relapse were censored at the time of last-followup or death. The median follow-up for the overall populationwas 26 months.

The Mann–Whitney U test was used to assess whether 2samples of observations were derived from the same distri-bution. The c2 test with Yates correction was used to analyzethe relationship between tumor cell infiltration and the tumorHPV status.

ResultsHPV-associated head and neck cancer has a goodprognosis

In a retrospective study, we divided 64 head and neck cancerpatients into 2 groups differing according to presence orabsence of oncogenic HPV (30 HPV16, 1 HPV18 and 1 HPV33,and 32 HPV-negative) in their tumors. These 2 groups werebalanced for gender, primary tumor sites, tumor (T) staging,lymph node involvement, and treatment modalities (Supple-mentary Table S1). On the basis of Kaplan–Meier estimates, theoverall survival for patients with HPV-positive cancer wassignificantly better (median survival not reached) than forpatients with HPV-negative cancer (median survival: 16months; P ¼ 0.001; log rank test; Fig. 1). Patients with HPV-positive tumors also had a significantly better disease-freesurvival (P ¼ 0.003; log rank test) than patients with HPV-negative cancer (Fig. 1).

Intratumoral Foxp3þ T cells and PD-1þ T cells differbetween HPV-positive and HPV-negative head and neckcancers

HPV-positive head and neck cancers were more heavily in-filtrated by CD8þ cells (P ¼ 0.01), PD-1þCD4þ T cells (P ¼0.045), and the total number of PD-1þCD4þ and PD-1þCD8þ

T cells (P ¼ 0.045). A trend was also observed between highlevels of total PD-1þ cell (P ¼ 0.079), PD-1þCD8þ cell (P ¼0.078), and Foxp3þCD4þ T-cell (P¼ 0.059) infiltration and theHPV detection in the tumors (Fig. 2 and Table 1). PD-1expression was higher in CD4þ T cells (median of PD-1þCD4þ

cells¼ 5.5), than in CD8þ T cells (median of PD-1þCD8þ ¼ 1).As PD-1þCD4þ cells and PD-1þCD8þ cells could also re-

present macrophages and NK cells respectively, tumor dissoci-ation was conducted for 14 tumors and cytometry analysisconfirmed that more than 95% of PD-1þCD45þ cells wereCD3þ T cells (Supplementary Fig. S1).

Thirty-three (51.5%) of the 64 head and neck tumorsexpressed significant levels of PD-L1 (score þþ/þþþ) with

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Figure 1. HPV-associated headand neck cancers are associatedwith a good prognosis.Relationships between thepresence or absence of oncogenicHPV in head and neck tumors andoverall survival (A) or locoregionalcontrol (B).

Badoual et al.

Cancer Res; 73(1) January 1, 2013 Cancer Research130

homogeneous staining (Table 1 and Supplementary Fig. S2),but no correlation was observed between PD-L1 expressionand HPV status of these head and neck cancer patients(Table 1).

PD-1–positive infiltrating T cells are associated withbetter overall survival in HPV-positive head and neckcancer

Surprisingly, we showed that HPV-positive tumors, infiltrat-ed by large numbers of PD-1–positive cells or total number ofPD-1þCD4 and PD-1þCD8 T cells, were correlated with betteroverall survival of these patients. Indeed, patients with PD-1–positive tumor-infiltrating T cells above the median value(15 PD-1 cells/fields) had a 93.9% 60-month overall survival,whereas patients with low PD-1–positive cell infiltration had a63.6% 60-month overall survival [P ¼ 0.025; HR, 0.13; 95%confidence interval (CI), 0.02–0.067] (Fig. 3A). In line withthese results and with the previous demonstration that mostPD-1 cells are T cells (Supplementary Fig. S1), we showed thattumors infiltrated by high levels of PD-1þCD4þ and PD-1þCD8þ cells had a better survival than tumors withlow PD-1þ T-cell infiltration (P ¼ 0.045; HR 0.15; 95% CI,0.03–0.7). Patients with high infiltration (>median value) byPD-1þCD4þ T cells (P ¼ 0.046) but not by PD-1þCD8þ T cellsalso had a better overall survival.

A correlation was also shown between the total numberof PD-1þCD4þ and PD-1þCD8þ infiltrating T cells and 18-month overall survival in the whole population of headand neck patients whatever their HPV status (P ¼ 0.009;Fig. 3B).

In contrast, the levels of infiltration by CD8þ T cells or CD4þ

T cells or Foxp3 regulatory CD4þ T cells infiltration were notcorrelated with overall survival in HPV-positive head and necktumors (Fig. 3A). However, as previously reported by us and

Table 1. Correlation between HPV status andT-cell infiltration andPD-L1 expression by tumorcells

HPV-positivehead andneck cancer

HPV-negativehead andneck cancer

CD8 High 17 7Low 15 25 P ¼ 0.01

CD4 High 22 16Low 10 16 P ¼ 0.12

PD-1þCD4þ High 20 12Low 12 20 P ¼ 0.045

PD-1þCD8þ High 18 11Low 14 21 P ¼ 0.078

PD-1þCD4þ andPD-1þCD8þ

High 20 12

Low 12 20 P ¼ 0.045PD-1 High 19 12

Low 13 20 P ¼ 0.079Foxp3þ CD4þ High 20 12

Low 12 19 P ¼ 0.059PD-L1 High 20 13

Low 12 19 P ¼ 0.08

NOTE: For quantitative parameters, low and high levels ofeach cell type were defined by using the median level oftumor infiltration by these cells as cut-off. For semiquanti-tative parameters (CD4, CD8, and PD-L1), a score of 0 or þcorresponds to low levels, whereas a score of þþ/þþþcorresponds tohigh levels.All parametersweremeasuredon64 head and neck tumor biopsies (32 HPV-positive and 32HPV-negative) except for Foxp3þCD4þ cells, which werecounted on 63 biopsies.

Figure 2. PD-1þ and Foxp3þ T cellsinfiltrate HPV-associated head andneck cancer. Tissue derived frombiopsies of HPV-positive head andneck cancers were stained withantibodies to human CD4, CD8,Foxp3, and PD-1. Left and secondcolumn from the left, results ofsimple immunofluorescenceacquisitionwith each antibody. Thirdcolumn from the left, doubleimmunofluorescence staining.Isotype control antibodies were alsoincluded in each experiment (right).Arrows indicate colocalizationbetween the markers recognized bythe specific antibodies. (Originalmagnification, �400).

CD4 Foxp3 CD4-Foxp3 Isotype control CD4

CD8 PD-1 CD8–PD-1 Isotype control CD8

CD4 PD-1 CD4–PD-1 Isotype control Foxp3 and PD-1



other authors (17, 27), the number of Foxp3 regulatory T cellswas correlated with better 18-month overall survival in thewhole population of head and neck cancer patients (P¼ 0.0033;Supplementary Fig. S3).

PD-L1 expression was not correlated with overall survival ordisease free survival in either the whole head and neck cancercohort or in HPV-positive patients (Supplementary Fig. S2). Inbivariate analysis including PD-L1 and various subpopulationof PD-1 cells each analyzed separately, we showed that onlyPD-1þCD4þ T cells, the total number of PD-1þCD4þ andPD-1þCD8þ T cells and the total number of PD-1 cellsremained significant (data not shown).

Phenotypic and functional characterization of PD-1þ

T cells infiltrating head and neck cancerPD-1þ T cells infiltrated both the stroma and the nest of

tumor cells. As shown in Fig. 4A, close contact was shownbetween PD-L1 tumor cells and PD-1þ cells strongly suggestingthat a negative signal was delivered to these PD-1þ tumor–infiltrating T cells. Interestingly, in the whole population, acorrelation was observed between PD-L1 expression and the

number of tumor infiltrating CD4þPD-1þ T cells (P¼ 0.029). Atrend was also observed between PD-L1 expression andthe number of PD-1þ CD4þ T cells and PD1þCD8þ T cells(P ¼ 0.07) and also the total number of PD-1þ T cells (P ¼0.089; Supplementary Fig. S4).

PD-1 is a hallmark of both activated and exhausted T cells(28). We found that PD-1 positive T cells, either CD4 or CD8þ

T cells, expressed higher levels of HLA-DR and CD38 thanPD-1 negative T cells (Fig. 4B and C). No difference in thisactivation state appeared to occur between HPV-positive orHPV-negative tumors (data not shown). Tim-3 expressionwas also assessed, as it has been associated with an exhaust-ed phenotype of PD-1þ T cells (29, 30). One-half of PD-1þ

T cells in head and neck cancer were found to express Tim-3(Fig. 4D and Supplementary Fig. S5A). The same number ofPD-1þ T cells in HPV-positive and HPV-negative tumors pre-sented hallmarks of exhausted T cells (Fig. 4D).

We also showed that PD-1–positive cells did not expressthe Foxp3 marker of regulatory T cells, despite the fact that,in some cases, PD-1–positive cells and Foxp3 regulatory T cellswere in close contact (Supplementary Fig. S5B).

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HR = 0.15(95% CI, 0.03–0.77)P = 0.045

HR = 0.13(95% CI, 0.02–0.67)P = 0.025

P = 0.079 Wald chi-square test P = 0.009 Wald chi-square test

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Figure 3. PD-1–positive infiltratingT cells positively correlate withsurvival in both HPV-positive headand neck cancer and in the overallhead and neck cancer population.A, tissues derived from biopsies ofHPV-positive head and neckcancers were stained withantibodies to human CD8,CD4, PD-1 by simpleimmunofluorescence analysis.Double immunofluorescencestainings for CD4 and PD-1, CD8and PD-1, CD4 and Foxp3 werealso conducted. For overallsurvival analysis, high and lowlevels of these various populationswere defined using the median oftumor infiltration for these cells ascut-off values. B, PD-1þ cells andtotal number of PD-1þCD4þ andPD-1þCD8þ cells were measuredin biopsies derived from all headand neck cancer patients,regardless of their HPV status. Therelationship between the totalnumber of these cells selected as aquantitative variable and the 18-month survival is shown. The blueline corresponds to thisrelationship, whereas the red linerepresents theupper or lower limitsof the 95% CI.

Badoual et al.


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Figure4. Phenotypic analysis ofPD-1–positive Tcells in headandneckcancer patients. A, headandneckcancer biopsieswere stainedwith anti–PD-1 (secondimage from the right) andanti–PD-L1antibodies (second image from the left). Double immunofluorescencestaining forPD-1 andPD-L1are shownon the right.Isotype control antibodies for anti–PD-L1 (left) and PD-1 (shown in Fig. 2) were included in each experiment. B, head and neck cancer biopsies weredissociatedbyDNAse andcollagenase andcellswere stainedwithCD45, Dye780, andCD3. Live T cellswere then stainedwithCD4,CD8,PD-1,HLA-DR, andCD38. After gating on PD-1þCD4þ or PD-1�CD4þ or PD-1þCD8þ or CD8þPD-1� cells, expression of the activation markers (HLA-DR and CD38) werecomparedon the variousPD-1–positive andPD-1–negative T-cell populations. A representative experiment is shown inB.Mean (�SD) expression ofHLA-DRand CD38 on CD3þ T cells, CD4þ T cells, and CD8þ T cells from 14 patients is shown in C. D, twenty PD-1þ T cells were selected at random from 16 HPV-positive (black square) or HPV-negative (white square) head and neck cancers. The number of cells costainedwith Tim-3 is shownwith themean indicated forthe PD-1þTim-3� and PD-1þTim-3þ groups.



To assess whether the functionality of these T cells could beenhanced, fresh TIL were cocultured with tumor cells in thepresence or absence of anti-PD-1 mAb and a pool of E7peptides. In 3 (2HPVnegative and 1HPVpositive) of 4 patients,a significant increase of IFNg positive T cells was shown byElispot in the presence of CT-011, an anti–PD-1 mAb (Fig. 5).An isotype control antibody had no effect on enhanced IFNgproduction (Fig. 5).

In 1 HPV-positive patient, E7 peptides slightly increased thefrequency of CT-011 induced IFNg secretion (Fig. 5).

Vaccination increases PD-1 and PD-1 blockadesynergizes with the vaccine antitumor effect

To explain this paradoxical good prognostic value ofPD-1þ T-cell infiltration in HPV-associated head and neckcancer, we modelized this phenomenon in a preclinicalmodel.

When mice were grafted with an epithelial tumor cell line(TC-1) expressing the E7 protein derived from HPV, tumorswere poorly infiltrated by CD8þ T cells that expressed lowlevels of PD-1 (6%; Fig. 6A).

When mice were vaccinated with a nonreplicative deliveryvector targeting dendritic cells, the B subunit of Shiga toxin,coupled to E7, 44% of CD8þ T cells expressed PD-1 (Fig. 6A andB). In addition, 89% of specific anti-E749–57 CD8þ T cells,induced by the vaccine, also expressed PD-1 (Fig. 6A and B).The vaccine therefore appeared to elicit PD-1 expression inboth total and E749–57 specific CD8

þ T cells. The vaccine alonehad a partial inhibitory effect on the growth of establishedTC-1tumors (Fig. 6D). This effect was significantly enhanced by theaddition of anti-PD-L1 (Fig. 6D). In contrast, the anti-PD-L1antibody alone had no effect on tumor growth (Fig. 6D), whichmay be explained by the low (6%) PD-1 expression in infiltrat-ing T cells in the absence of vaccination (Fig. 6A). Similarresults were obtained, when overall survival or the percentageof tumor-freemice were selected as endpoints (SupplementaryFig. S6).

DiscussionThis study shows that HPV-positive head and neck tumors

were heavily infiltrated by PD-1þ cells, as the number of PD-1þCD4þ infiltrating T cells and the total number of PD-1þCD4þ and PD-1þCD8þ T cells were significantly higher inHPV-positive than in HPV-negative head and neck cancers.The relevance of these results is strengthened by the matchingbetween HPV-positive and HPV-negative tumors for variousconfounding parameters (tumor–node–metastasis, treatmentmodalities, etc).

Surprisingly, we found that high levels of PD-1þ cells and thetotal number of PD-1þCD4þ andPD-1þCD8þT cells correlatedwith better survival compared with low levels of infiltration bythese cells in primary HPV-positive head and neck cancers.

These results were unexpected because of the known inhib-itory function of PD-1 on T cells and immune cells (20, 31). Inaddition, in renal cell carcinoma, nasopharyngeal cancer andHodgkin lymphoma, PD-1 expression on immune cells wascorrelated with poor prognosis and was associated withshorter survival (32–34). However, Hsu and colleagues showedthat the prognostic value of PD-1 differed between CD4þ andCD8þ T cells (33). The other studies did not conduct doubleimmunofluorescence staining or cytometry analysis to moreprecisely characterize PD-1 positive cells that can be expressedby many types of infiltrating cells (NK, B cells, macrophages,etc.). In the present study, we showed that most (>95%) PD-1positive cells corresponded to T cells (Supplementary Fig. S1)and that the prognostic value of PD-1 was associated with thetotal number of CD4þ andCD8þT cells expressing thismarker.It should also be mentioned that in contrast to many tumors,CD8þ T cells are associated with bad prognosis in renal cellcarcinoma and hodgkin lymphoma supporting the fact that theclinical significance ofmolecules expressed by these cells couldalso vary depending on the tumor histology (35, 36).

Interestingly, recent studies have reported that follicularlymphomas infiltrated by PD-1þ T cells were associated with alower risk of transformation, a higher progression-free survival

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Figure 5. Functional analysis ofintratumoral PD-1þ T cells afterblockade of the PD-1/PD-L1pathway. Head and neck cancerbiopsies from 4 head and neckcancer patients (3 HPV-negativeand 1 HPV-positive) weredissociated by DNAse andcollagenase, resulting in cellsuspensions containing bothtumor cells and stromal cellsincluding T cells. These cells(2 � 105) were transferred to IFNgElispot plates incubated withmedium, anti-PD-1, or isotypecontrol antibodies in the presenceor absence of a pool of E7 peptidesfor 48 hours. All tests wereconducted in triplicate. Thenumber of spots (mean� standarddeviation) is shown. �, P < 0.05.

Badoual et al.


and a better overall survival than tumors not infiltrated by PD-1þ cells (37). PD-1 was preferentially expressed by infiltratingCD4þ T cells in these follicular lymphomas, as also observed inour study. In line with our results, PD-1 mRNA expressiondetermined by reverse transcriptase-PCR (RT-PCR) in colo-rectal carcinoma was also associated with a good prognosis(38). It is therefore noteworthy that the 3 tumors (lymphoma,colorectal cancer, and head and neck cancer), in which PD-1expression was significantly associated with a favorable out-come (37, 38), were also those with an unexpected clinical

benefit of Treg infiltration (17, 39, 40). The present study,conducted in an independent series of patients, confirms ourprevious findings that high levels of Treg in head and neckcancer patients were also positively correlated with a betterprognosis (17). These results concerning the good prognosticvalue of intratumor regulatory T cells in head and neck cancerhave subsequently been reproduced by various groups (27, 41).In addition, the frequency of regulatory Foxp3þCD4þ T cellshas been shown to be higher in patients with HNSCC with noevidence of disease after oncologic therapy, than in patients

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Figure 6. PD-1 induced onCD8þ T cells after vaccination is required for the efficacy of PD-1–PD-L1 blockade. A,micewere s.c. graftedwith the E7-expressingTC-1 epithelial cell line. At day 9, mice were vaccinated twice at a 9-day interval with a vaccine composed of a nonreplicative delivery vector, the B subunit ofShiga toxin, coupled to an E7 polypeptide (STxB-E7) used at 20 mg. aGalCer (1 mg) was mixed with the vaccine during the prime. PD-1 expression ontotal CD8 or E749–57 specific CD8

þ T cells was determined on dissociated cells by cytometry analysis from biopsies of mice tumors (previously vaccinated ornot) at a time corresponding to 5 to 6 days after the second vaccination. B, PD-1 expression on total CD8þ T cells and E749–57–specific CD8þ T cells in micevaccinated or not with STxB-E7. PD-1 expression on E749–57–specific CD8

þ T cells from the nonvaccinated group is not shown, as we could not detect thesecells in this group. This experiment is representative of 3 experimentswith 4mice per group. C, the epithelia tumor cell line, TC-1, was stained in vitrowith anti–PD-L1 or isotype control antibodies. D, mice were s.c. grafted with the E7-expressing TC-1 epithelial cell line. Mice were then either vaccinated or notvaccinated at day 9 and day 18 with STxB-E7mixed during the prime withaGalCer. The vaccine was combined with anti–PD-L1 or isotype control antibodies(200 mg/injection) that were administered 1, 4, and 9 days following the vaccine injection. A group of mice receiving the anti–PD-L1 mAb without thevaccine was also included. Tumor growth was monitored every 3 days. Results shown are representative of 3 experiments with 7 to 10 mice per group. Forstatistical analysis, each group was compared with the control group. �, P < 0.05; ��, P < 0.01; ��, P < 0.005.



with active disease (42). The plasticity of regulatory T cells,transient expression of Foxp3 by activated T cells and apossible role of regulatory T cells in the inhibition of delete-rious cancer inflammation could explain the paradoxicallygood clinical prognosis associated with regulatory T cells insome circumstances (43, 44).

Several hypotheses can also be proposed to explain thisparadoxical favorable outcome associated with PD-1 expres-sion on T cells. PD-1 is upregulated on lymphocytes followingT-cell receptor activation and remains elevated in the contextof persistent antigen-specific immune stimulation (45). Theabsence of co-expression of Tim-3 and PD-1 in one-half ofPD-1–positive cells strongly suggests that some PD-1þ T cellsin the tumor microenvironment of HPV-positive head andneck cancers are not exhausted T cells (Fig. 4). We did notassess the expression of other inhibitory receptors (Lag-3,CTLA-4. . .) on PD-1þ T cells, but Tim-3 is the most frequentcoinhibitory receptors associated with PD-1þ T cells in tumorand an hallmark of exhausted T cells (29, 46). It has beenreported that high PD-1 levels on T cells more closely reflecttheir "exhausted status" than intermediate PD-1 levels. How-ever, in our study, PD-1 expression was homogeneous and itwas, therefore, difficult to divide T cells into 2 groups. Some ofour PD-1 infiltrating T cells remained responsive to anti-PD-1in vitro (Fig. 5). From these results, PD-1 expression alsoindicates, that T-cell activation has occurred during thedevelopment of HPV-associated head and neck cancers andPD-1 could, therefore, also be viewed as a witness of theantitumor immune response.

Indeed, we showed that PD-1–positive T cells expressedhigher levels of theHLA-DR andCD38 activationmarkers, thanPD-1–negative T cells (Fig. 4). Although this response was noteffective to clear the tumor, it may have slowed tumor growthcompared with a clinical setting associated with absence ofhost response to the tumor. Although the tumor specificity ofPD-1 expressing T cells could not be shown because of thesmall number of cells present in tumor biopsies, it has beenpreviously shown that tumor-reactive CD8þ T cells generatedafter in vitro culture of dissociated biopsies of melanoma weremore frequently derived from PD-1 expressing T cells presentat the beginning of culture (47). In preclinical tumor models,the CD137þCD8þ T cells were enriched in antitumor effectorT cells (48).

Our preclinicalmodel showed that grafting of the E7-expres-sing TC-1 epithelial cell line, which constitutively expressesPD-L1 led to tumor growth without therapy. Analysis of itstumor microenvironment showed the absence of PD-1 expres-sion by CD8þT cells. In contrast, activation of an anti-HPVresponse by an anti-E7 vaccine, elicited PD-1 expression byCD8þTcells andE7-specificT cells, whichwere associatedwithpartial regression of the tumor. In humans, it has been shownthat TIL derived from spontaneously regressing melanomasalso express substantial levels of PD-1 (49).

Another important result of this study is that the efficacy ofanti-PD-L1 mAb depended on the presence of PD-1 on specificT cells induced after vaccination. In preclinical models, immu-notherapy approaches that should lead to upregulation ofPD-1 following activation of the immune system, such as

recombinant cytokines or stimulatory antibodies or transferof activated adoptive T cells, synergized with blockade of thePD-1–PD-L1 pathway (21, 25, 50).

Consistent with these results, a recent report showed thatexpression of PD-L1, the ligand of PD-1, which was consideredto be an immunosuppressive molecule, positively correlatedwith better survival in melanoma (51). In melanoma, PD-L1 ismainly induced by IFNg produced by infiltrating T cells and thedistribution of T cells within the tumor was correlated withthat of PD-L1 (51). In line with these results, in patients treatedwith anti-PD-1 mAb, tumor cell surface PD-L1 expressionappeared to be correlated with the likelihood of response totreatment, possibly because of a reprogramming of infiltratingantitumor PD-1þT cells (52). PD-1 and PD-L1 expression may,therefore, represent surrogate markers of endogenous antitu-mor immune response, explaining their unexpected associa-tion with good prognosis in some tumors in which PD-L1expression is not oncogene driven. Indeed, in glioma, loss oftumor suppressor PTEN function increased PD-L1 (53). In ourstudy, PD-L1 was not associated with a good prognosis in headand neck cancer patients, regardless of HPV status, but lowPTEN expression was detected in the majority of these tumors(54).

This study, therefore, revisits the significance of PD-1–positive tumor-infiltrating T cells in cancer. The inhibitoryfunction linked to PD-1 should not mask the fact that itsdetection could also reflect a past antitumor immune responseready to be reprogrammed by PD-1–PD-L1 blockade.

Disclosure of Potential Conflicts of InterestW.H. Fridman is a consultant/advisory board member of CureTech LTD.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: C. Badoual, S. Hans, E. TartourDevelopment of methodology: C. Badoual, N. Merillon, C. Van Ryswick, N.Benhamouda, E. Levionnois, A. Si-Mohamed, N. Besnier, A. Gey, H. Pere, C.L.Guerin, A. Chauvat, C. Alanio, D. Olive, D. Brasnu, E. TartourAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Badoual, S. Hans, N. Merillon, N. Benhamouda,E. Levionnois, M. Nizard, A. Si-Mohamed, N. Besnier, A. Gey, H. Pere, A. Chauvat,E. Dransart, C. Alanio, S. Albert, B. Barry, F. Sandoval, F. Quintin-Colonna,P. Bruneval, W.H. Fridman, F.M. Lemoine, S. Oudard, L. Johannes, D. Olive,D. Brasnu, E. TartourAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Badoual, S. Hans, N. Merillon, C. Van Ryswick, P.Ravel, N. Benhamouda, E. Levionnois, A. Si-Mohamed, A. Gey, R. Rotem-Yehudar,H. Pere, C.L. Guerin, A. Chauvat, C. Alanio, F. Sandoval, F. Quintin-Colonna,P. Bruneval, W.H. Fridman, F.M. Lemoine, S. Oudard, L. Johannes, E. TartourWriting, review, and/or revision of the manuscript: C. Badoual, S. Hans,R. Rotem-Yehudar, F. Sandoval, D. Olive, D. Brasnu, E. TartourAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C. Badoual, S. Hans, E. TartourStudy supervision: C. Badoual, E. Tartour

Grant SupportThis work was supported by grants from the Ligue contre le Cancer, the

Agence Nationale de la Recherche (ANR), the Association pour la Recherchecontre le Cancer (ARC), Labex, Immuno-Oncology, Canceropole Region Ile deFrance, Fonds d'amorçage AP-HP, and Institut National du Cancer (INCA).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 9, 2012; revised September 11, 2012; accepted October 7, 2012;published OnlineFirst November 7, 2012.

Badoual et al.


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