Intratumoral HPV16-Speciﬁc T Cells Constitute a Type I ......Biology of Human Tumors Intratumoral HPV16-Speciﬁc T Cells Constitute a Type I–Oriented Tumor Microenvironment to

Biology of Human Tumors

Intratumoral HPV16-Specific T Cells Constitutea Type I–Oriented Tumor Microenvironment toImprove Survival in HPV16-DrivenOropharyngeal CancerMarij J.P.Welters1,WenboMa1, Saskia J.A.M. Santegoets1, RenskeGoedemans1, IlinaEhsan1,Ekaterina S. Jordanova2, Vanessa J. van Ham1, Vincent van Unen3, Frits Koning3,Sylvia I. van Egmond4, Pornpimol Charoentong5, Zlatko Trajanoski5,Lilly-Ann van der Velden4, and Sjoerd H. van der Burg1

Abstract

Purpose: Human papillomavirus (HPV)–associated oropha-ryngeal squamous cell cancer (OPSCC) has a much betterprognosis than HPV-negative OPSCC, and this is linked todense tumor immune infiltration. As the viral antigens maytrigger potent immunity, we studied the relationship betweenthe presence of intratumoral HPV-specific T-cell responses,the immune contexture in the tumor microenvironment, andclinical outcome.

Experimental Design: To this purpose, an in-depth anal-ysis of tumor-infiltrating immune cells in a prospectivecohort of 97 patients with HPV16-positive and HPV16-negative OPSCC was performed using functional T-cellassays, mass cytometry (CyTOF), flow cytometry, and fluo-rescent immunostaining of tumor tissues. Key findings werevalidated in a cohort of 75 patients with HPV16-positive

OPSCC present in the publicly available The Cancer GenomeAtlas database.

Results: In 64% of the HPV16-positive tumors, type IHPV16-specific T cells were present. Their presence was not onlystrongly related to a better overall survival, a smaller tumor size,and less lymph node metastases but also to a type I–orientedtumor microenvironment, including high numbers of activatedCD161þ T cells, CD103þ tissue-resident T cells, dendritic cells(DC), and DC-like macrophages.

Conclusions: The viral antigens trigger a tumor-specific T-cellresponse that shapes a favorable immune contexture for theresponse to standard therapy. Hence, reinforcement of HPV16-specific T-cell reactivity is expected to boost this process.ClinCancerRes; 24(3); 634–47. �2017 AACR.

See related commentary by Laban and Hoffmann, p. 505

IntroductionThe incidence of oropharyngeal squamous cell cancer (OPSCC)

is rising, especially in younger adults (1). Classically, the devel-opment of OPSCC is related to p53 mutations, but currently,more than half of all OPSCCs are caused by a high-risk humanpapillomavirus (HPV), most often type 16 (HPV16; ref. 1).Although HPV-associated OPSCCs are more often diagnosedwith TNM stage III to IV, consisting of an earlier T stage andmore advanced N stage, than HPV-negative OPSCCs (2), theydisplay a much better prognosis than HPV-negative tumors after(chemo)radiotherapy. This is independent of many commonhistopathologic parameters (2, 3) but associated with the pres-ence of a strong adaptive immune response gene signature (4)and dense tumor infiltration by activated CD4þ and CD8þ T cells(3, 5, 6), suggesting a role for the adaptive immune systemin the response to therapy. Notably, HPV-associated OPSCCsexpress viral proteins, and we have shown that they may functionas tumor-specific antigens for OPSCC-infiltrating T cells (7).Clear evidence for a protective role of tumor-infiltrating, HPV-specific T-cells in OPSCC, however, is lacking. Hence, it is impor-tant to evaluate whether HPV-positive OPSCCs are commonlyinfiltrated by HPV-specific T cells and, specifically, how thispertains to the composition of the tumor microenvironment andsurvival. We focused purely on the analysis of HPV-specific T-cell

1Department of Medical Oncology, Leiden University Medical Center, Leiden, theNetherlands. 2Department of Pathology, Leiden University Medical Center,Leiden, the Netherlands. 3Department of Immunohematology and Blood Bank,Leiden University Medical Center, Leiden, the Netherlands. 4Department ofOtorhinolaryngology and Head and Neck Surgery, Leiden University MedicalCenter, Leiden, the Netherlands. 5Division for Bioinformatics, Innsbruck MedicalUniversity, Innsbruck, Austria.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address for R. Goedemans: Genmab, Utrecht, the Netherlands; currentaddress for E.S. Jordanova, Center for Gynaecologic Oncology, Free UniversityMedical Center, Amsterdam, the Netherlands; current address for L.-A. van derVelden, Department of Head and Neck Oncology and Surgery, NetherlandsCancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam, the Nether-lands; and current address for P. Charoentong,Department ofMedicalOncology,National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg,Germany.

Corresponding Author: Sjoerd H. van der Burg, Department of Medical Oncol-ogy, Leiden University Medical Center (LUMC), PO Box 9600, Building 1, Leiden2300 RC, the Netherlands. Phone: 31 71 526 1180; Fax: 31 71 526 6760; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-2140

�2017 American Association for Cancer Research.

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reactivity within the tumor-infiltrating lymphocyte (TIL) popu-lation, as detection of circulatingHPV-specific T cells might reflecta response to past infections (8), potentially even in other ana-tomic locations (8) and, thus, was less relevant to our study. Incase of such a relation, reinforcement of HPV-specific T-cellreactivity becomes highly attractive for treatment of OPSCC.

Materials and MethodsPatients

Patients with histology-confirmed OPSCC were included aftersigning informed consent. This study is part of a larger observa-tional study, P07-112 (7), approved by the local medical ethicalcommittee of the Leiden University Medical Center (LUMC; Lei-den, the Netherlands) and in agreement with Dutch law. Patientenrollment was from November 2007 to November 2015. Bloodand tumor tissue samples were taken prior to treatment andhandled as described previously (9) and in Supplementary Meth-ods. Peripheral blood mononuclear cells (PBMC) and TILs werestored until use. HPV typing and p16ink4a IHC staining wasperformed on formalin-fixed paraffin-embedded (FFPE) tumorsections at the LUMCDepartment of Pathology. Immunofluores-cent staining of FFPE tumor sections for CD8 and Tbet wasperformed as described previously (10) and in SupplementaryMethods. The patients received the standard-of-care treatment,which could consist of surgery, radiotherapy, chemotherapy,treatment with mAb, or combinations hereof. Staging of thetumor was done according to the National ComprehensiveCancer Network (https://www.nccn.org/professionals). Patientcharacteristics are provided in Supplementary Table S1.

Cancer cell linesThe OPSCC cell lines were obtained from the University of

Michigan (Ann Arbor,MI) and calledUM-SCC.We obtainedUM-SCC4 (passage 22), UMC-SCC6 (passage 33), UM-SCC19 (pas-sage 17; all three HPV negative), UM-SCC47 (passage 98), andUM-SCC104 (passage 15; bothHPV16positive) in 2012. The cellswere cultured in RPMI 1640 (Gibco/Thermo Fisher Scientific)

with 10% FCS (PAA Laboratories) and penicillin/streptomycin(Thermo Fisher Scientific). Tumor cell supernatant (TSN) wasprepared after 5 days of culture as described previously (11).Microsatellite analysiswas performed in July 2016byBaseClear toassure cell line authentication when the experiments were per-formed. Mycoplasma was tested on a monthly basis.

Th clonesClonal dilution was performed using the TILs from patient

H68 as described previously (7). Their HPV specificity andcytokine production were determined. This resulted in multipleCD4þ Th cell clones of which Th1 (clones 78 and 97), Th2(clone 133), and Th17 (clones 12 and 103) were selected forthe experiments. T-cell supernatant was obtained after stimu-lation with cognate HPV peptide loaded on with Epstein–Barrvirus (EBV)–immortalized autologous B cells for 3 days.

TILs and tumor cell analysesThe phenotype and composition of dispersed tumors (and

expanded TILs) were analyzed by flow (9, 12–15) and time-of-flight mass cytometry (CyTOF; ref. 16; Supplementary Methods).Supplementary Table S2 shows the 36 markers used for CyTOFanalysis. The reactivity of TILs was determined in a 5-day prolif-eration assay (9) and by intracellular cytokine staining (15).Supernatant from the proliferation test was subjected to cytokineanalysis (15). The effect of TSN on dendritic cell (DC) differen-tiation was determined phenotypically and functionally(cytokine/chemokine production) upon LPS or agonistic CD40antibody stimulation in the presence or absence of INFg asdescribed previously (11, 13) and in Supplementary Methods.

Treatment of tumor cells.Tumor cells were seeded (15,000–27,500cells/well) in a flat-bottom, 96-well plate (Costar/Thermo FisherScientific) and allowed to adhere overnight at 37�C. The next day,the cells were incubatedwith the indicated concentrations of IFNgand/or TNFa for 48 hours at 37�C, followed by the MTT assay(Trevigen) according to the manufacturer's protocol to determinethe percentage of proliferating cells compared with the untreatedcells (set at 100%; ref. 13). Tumor cells (70,000–100,000) wereadhered in a 24-well plate overnight as described above, followedby treatment for 24hourswith afixeddoseof cisplatin (15mg/mL)in the presence or absence of indicated concentrations TNFa (0–30ng/mL). The cellswere harvested and analyzed for apoptosis byflow cytometry. In another experiment, tumor cells prepared in24-well plates were treated for 24 and 48 hours with IFNg (250IU/mL; ImmunoTools) and TNFa (30 ng/mL) or 20% of super-natant obtained from Th1 (H68 clone 97), Th2 (H68 clone 133),or Th17 (H68 clone 103) cells with or without the addition ofapoptosis inducer and cIAP1/2-interacting compound BV6(5 mmol/L smac mimetic; ApexBio) and pan-caspase inhibitorzVADfmk (20 mmol/L FMK001; R&D Systems), together knownto induce necroptosis (17–19). Necrostatin (Nec)-1s (2263-1;BioVision) was added to the conditions used for UM-SCC19 toinhibit necroptosis via inhibition of RIP1K (14). The treatedtumor cells were harvested and subjected to SYTOX green stainingto establish the percentage of dead cells and, in parallel, stainedfor flow-based apoptosis analysis using Annexin V (early apopto-sis) and 7-AAD (late apoptosis). As indicated, tumor cells werealso analyzed for RNA expression (quantitative PCR; ref. 14)and protein content (Western blot analysis; ref. 14; see alsoSupplementary Methods).

Translational Relevance

Anumber of studies have reported that T cells responding tothe two oncoproteins E6 and E7 of high-risk human papillo-mavirus (HPV) can infiltrate the tumor microenvironment ofpatients with HPV-driven tumors and speculated that theseT cells might be important for tumor control. This is the firststudy that really addresses this question by measuringthe T-cell response in the tumor, analyzing the influenceof these HPV16-specific T cells on the microenvironmentwithin the tumor, and then waiting for many years to definetheir impact on patient survival. Here, we show how thepresence of these HPV-specific T cells is associated with acompletely differentmicroenvironment and that intratumoralHPV-specific type I–polarized T cells provide patients withHPV16-positive oropharyngeal cancer with a 37-fold higherchance to respond excellently to standard therapy across allTNM stages. The results will fuel the discussion on deintensi-ficationof the standard therapy andpotential applicable formsof immunotherapy.

HPV-Specific T Cells Improve Oropharyngeal Cancer Survival

www.aacrjournals.org Clin Cancer Res; 24(3) February 1, 2018 635



https://www.nccn.org/professionals


A B

HPV– (n = 40)

HPV+ (n = 57)

HR = 5.5 95% CI (2.5–12.2)

0 20 40 60 80 1000

20

40

60

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Disease-specific survival (months)

% o

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Patient ID

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40

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Ex vivo (direct)

Expanded (cultured)

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HPV positive, IR+HPV positive, IR+HPV negative, IR–

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1-92

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20,000

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40

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Welters et al.

Clin Cancer Res; 24(3) February 1, 2018 Clinical Cancer Research636




Statistical analysisUnpaired parametric t test was used to determine the difference

between various treatments of the cells from the UM-SCC tumorcell lines. Dates of two groups of patients were analyzed using theunpaired nonparametric analysis (Mann–Whitney). Fisher exacttest was used to analyze categorical data in a contingency table.Data of the three groups of patients (p16�IR�, p16þIR�, andp16þIRþ) were analyzed using the unpaired nonparametric one-wayANOVA (Kruskal–Wallis).HRwith a 95%confidence interval(CI) was calculated to determine the difference in survival curves.The nonparametric log-rank test (Mantel–Cox test) was done tocompare the survival distribution of the two groups of patients. Inall cases, a P value of 0.05 and below was considered statisticallysignificant (�), with P < 0.01 (��) and P < 0.001 (��) as highlysignificant.

ResultsThe majority of HPV16-positive OPSCCs contain HPV16-specific Th1/Th17 cytokine–producing T cells

To interrogate the role of HPV-specific T cells in OPSCC, weprospectively assembled a cohort of 97 patients with OPSCC, 57of whom were HPV16 positive. Analysis of the patient character-istics showed the expected percentage of HPV-positive patients(2, 3) and the differences in smoking, N stage, and disease-specificsurvival when compared with HPV-negative OPSCC (Fig. 1A;Supplementary Table S1), indicating that our patient cohort doesnot differ from those reported in literature.

From each patient, freshly obtained and FFPE tumor materialwas stored (Supplementary Fig. S1). The presence, prolifera-tion, and cytokine production of HPV16-specific and otherOPSCC-infiltrating T cells in the dissociated OPSCC wereanalyzed either directly or following a 2- to 4-week expansionperiod (Supplementary Fig. S1). Reactivity to the HPV16 E6and/or E7 oncoproteins was detected directly ex vivo in 6 of 24samples, and in 29 of 45 of the expanded TIL HPV16-positivecases. All directly ex vivo detectable responses were confirmed inthe expanded TILs. None of the 23 tested TIL cultures obtainedfrom HPV-negative tumors displayed HPV-specific reactivity(Fig. 1B and C), showing the specificity of these types of TILanalyses (7) and demonstrating that HPV-specific T cells infil-trate only HPV-positive OPSCC.

Subsequently, supernatants taken from the HPV-reactive cul-tures were assessed for the presence of Th1 (IFNg , TNFa, and IL2),Th2 (IL4, IL5, and IL10), and Th17 (IFNg and IL17) cytokines,revealing a Th1/Th17-like profile (Fig. 1D). Flow cytometry anal-ysis demonstrated that the population of activated and/or cyto-

kine-producing, HPV-specific T cells frequently comprised bothCD4þ and CD8þHPV-specific T cells (Fig. 1E; Supplementary Fig.S2), which targeted multiple epitopes simultaneously (Fig. 1F),albeit the percentage ofHPV-specific, cytokine-producing CD4þ Tcells oftenwas higher than that ofCD8þT cells (Fig. 1G). Thus, themajority of HPV16-positive OPSCC tumors are infiltrated byHPV16-specific CD4þ and CD8þ T cells with a Th1/Th17 profile.

The mechanisms underlying the failure to detect an intratu-moral HPV16-specific response can be manifold, but the firstrequirement is the presence of sufficient quantities of antigen tostimulate T cells. The expression of p16INK4a is a surrogate markerfor overexpressed, functionally active E7 oncoprotein (20). Fortyof the TIL-tested HPV16-positive OPSCC tumors could be ana-lyzed for p16INK4a overexpression, and, in contrast to immuneresponders (IRþ), 7 of the 15 immune nonresponders (IR�) failedto show a positive staining (Supplementary Fig. S3A). Further-more, tobacco smoking and, in particular, nicotine are known toimpair the responsiveness of T cells to antigenic stimulation (21).Althoughmany patients hadmore than 10 pack years of smoking(Supplementary Fig. S3B; ref. 2), this was not discriminative forthe detection of HPV16-specific immunity (SupplementaryFig. S3C). Hence, the failure to produce a T-cell reaction to HPVin HPV16-positive OPSCC most likely is due to the limitedquantities of viral proteins available to the immune system.

Tumor infiltration by HPV-specific T cells correlates withhigh numbers of type I–oriented T cells and professionalantigen-presenting cells in the tumor

On the basis of the observation that the major componentof OPSCC-infiltrating, HPV-specific T cells consists of CD4þ Tcells, and the known activity of tumor-specific CD4þ T cells torecruit, activate, and sustain other immune cells (22, 23), weperformed an in-depth analysis of the tumor microenviron-ment in the context of HPV-specific T-cell reactivity. As theabsence of overexpressed p16INK4a in HPV16-positive OPSCCmay indicate that their development was not driven by theHPV oncoproteins (24), we separated the HPV16-positivepatients into three groups: p16INK4a-negative, IR-negative(p16�IR�); p16INK4a-positive, IR-negative (p16þIR�); andp16INK4a-positive, IR-positive (p16þIRþ) patients.

An understanding of the general cytokine polarization in thetumors was obtained through analysis of cytokine productionfollowing the direct ex vivo activation of all tumor-infiltratingT cells using the mitogen phytohemagglutinin. Interestingly, theIFNg/IL17 cytokine polarization of HPV-specific T cells was mir-rored in the remainder of tumor-infiltrating cells (SupplementaryFig. S4). The production of IFNg and IL17 was lower in the

Figure 1.HPV-driven oropharyngeal cancer induces HPV-specific T cells and responds better to therapy. A, The Kaplan–Meier plot shows the survival of a cohort of97 treated patients with OPSCC divided by HPV status. B, Three representative examples of freshly dispersed OPSCCs as well as expanded (cultured) TILs for thesame patient subjected to a proliferation assay (in triplicate wells) to determine the specificity of the TILs [shown as counts per minute (CPM) with SEM]. Cells inmediumonly or stimulatedwith PHA served as a negative and positive control, respectively.C, In total, 23 patientswith anHPV-negativeOPSCC and 45 patientswithan HPV-positive OPSCC were tested in the proliferation assay as described in B. The percentage and number of patients showing an immune response (IRþ) or not(IR�) is depicted. D, Cytokine production was determined in supernatants of HPV-reactive cultures in the proliferation assay. The average production of 21 culturedTILs is shownwith SEM.E,The cultured TILswere stimulatedwith peptidepools or single peptides of theHPV16 E6orE7oncoprotein and analyzedbymultiparametricflowcytometry to determine the specific upregulation of activationmarkers (CD154 andCD137) and production of IFNg , TNFa, and IL2 by CD4þ andCD8þT cells. Thepercentage and number of patients demonstrating an HPV-specific T-cell response are given. F, Heatmap of the analysis as in E showing the specificity of HPV-specific responses (gray) to single peptides, pooled peptides, and proteins of HPV16 E6 and E7 for each individual patient. The percentage of total CD4þ and CD8þ Tcells among TILs is indicated at the top of the heatmap.G, The total frequency of HPV16-specific CD4þ and CD8þ T cells in cultured TILs, indicated by the cumulativepercentage of HPV-specific, cytokine-producing T cells to each single peptide or pool, is shown for individual patients. Box and whiskers are shown including theminimal and maximal value. CI, confidence interval; n.d., not detectable.






A

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1008060402000

20

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Log-rank P = **


% o

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100Tbet+CD8+ hi (n = 19)

Log-rank P = *

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Tbet+CD4 hi (n = 19)

Tbet+CD4 lo (n = 19)

HR = 7.1 95% CI (1.4–35.4)

HR = 8.8 95% CI (1.7–44.6)

Welters et al.





p16þIR� and the p16�IR� groups. Moreover, the production ofIL5 was increased in the latter two groups, suggesting a shifttoward a more type II cytokine profile.

In addition, we quantified the number of type I polarizedimmune cells in the HPV16-positive tumors using IHC for CD8and then with IFNg production–associated T-box transcriptionfactor TBX21 (Tbet). The numbers of tumor-infiltratingTbetþCD8þ T cells and TbetþCD8� T cells, based on our flowcytometry data, most likely CD4þ T cells, correlated with animproved survival (Fig. 2A) and were particularly high when theOPSCC contained HPV-specific T cells (Fig. 2B).

To comprehensively analyze the composition and phenotypeof intratumoral immune cells directly ex vivo, a validated panel of36 antibodies adapted from a previous study (SupplementaryTable S2; ref. 16) was used in combination with mass cytometry(CyTOF) in 13 freshly dissociated OPSCCs. This showed thatthe HPV16-positive OPSCCs from HPV16 immune responderpatients were strongly infiltrated with CD4þ and CD8þ T cells(Fig. 2C and D) carrying an effector memory phenotype (Fig. 2E),whereas theHPV16-positiveOPSCCs inwhich noHPV16-specificT-cell reactivity was detected displayed a strong influx with B cells(Fig. 2D). Natural killer (NK) cells, which may also infiltratetumors and express Tbet, were virtually absent (Fig. 2D). Toautomatically discover stratifying biological signatures, we usedthe CITRUS algorithm with an FDR of 1%, resulting in fivedistinctive (groups of) populations of immune cells (Fig. 2F). Itconfirmed the differences in the percentages of tumor-infiltratingB cells and T cells (Fig. 2G) but also revealed the presence of threesubsets of T cells that were present at significantly higher levels inHPV16 immune responders (Fig. 2H). Inspection of these subsetsrevealed two subsets of activated CD4þ T cells and a subset oftissue-resident effector memory CD8þ T cells expressing CD103(Supplementary Fig. S5A and S5B). The two subsets of activatedCD4þ T cells expressed CD38, HLA-DR, and PD-1 but wereseparated on the basis of CD161 expression (Supplementary Fig.S5A and S5B). The CD161� subset of activated CD4þ T cells had ahigh expression of CD25 but also expressed CD127, whereas theCD161þ subset displayed an intermediate expression of CD25,making it unlikely that these two populations reflected regulatoryT cells. Comparison of the tSNE plots of each patient clearlyshowed the almost exclusive presence of CD103þCD8þ T cellsin the IRþ patient group (Supplementary Fig. S5C and S5D).Interestingly, part of the CD103þCD8þ T cells also expressedCD161. There was no difference between the different patientgroups with respect to the percentage of central memory

CD161þCD4þ T cells, but in each of the patients with an IRþ

HPV16-positive OPSCC, a clearly visible effector memoryCD161þCD4þ T-cell population was present (SupplementaryFig. S5C and S5D).

In parallel, we analyzed the tumor microenvironment in acohort of 75 patients with HPV16-positive OPSCC present in thepublicly available The Cancer Genome Atlas (TCGA) database(25) using our previously published analytic strategy to estimatesubpopulations of tumor-infiltrating immune cells (26). AsCD4þ

T cells formed the major component in IRþ patients, a gene setenrichment analysis of the TCGA RNA-sequencing data wasperformed to determine which immune cells were relativelyenrichedordepleted inHPV16-positiveOPSCCwith ahigh versuslow CD4 gene expression (Fig. 3A). The results confirmed theenrichment of activated and effector memory T cells but alsopointed at a potential enrichment in NK cells and activated DCand B cells as well as a decreased presence of MDSCs in tumorswith a high CD4 expression. Notably, an increased percentageof DCs/DC-like macrophages was observed among the HPVresponders when the dissociated HPV16-positive OPSCCs ofour cohort were analyzed by flow cytometry (n ¼ 18) or CyTOF(n ¼ 13; Fig. 3B and C). In vitro experiments suggest that theincreased percentages of these antigen-presenting cells (APCs) arecaused by the presence of the intratumoral, IFNg-producing HPV-specific T cells. Analysis of the impact of two different HPV16-positive head and neck squamous cell carcinoma (HNSCC) celllines (27, 28) on GM-CSF þ IL4-driven differentiation of mono-cytes to IL12p70-producing DCs showed that tumor-secretedcompounds skewed the monocytes toward type II–like macro-phages instead (Fig. 3D), which had a low capacity to produceIL12p70 after CD40 ligation unless IFNg was present (Fig. 3E).The resulting APCs also produced the T-cell–attracting chemo-kines CXCL9 and CXCL10 (Fig. 3F). Replacing IFNg by thesupernatant of genuine activated HPV-specific Th1 or Th17 T-cellclones (Supplementary Fig. S6A) also neutralized the M2-likemacrophage skewing effect of the tumor cells (SupplementaryFig. S6B). A similar effect of HPV-specific Th1 and Th17 cytokineswas observed on the direct M2-macrophage skewing effect oftumor cells (Supplementary Fig. S6C). In addition, the costimu-latory molecules were upregulated.

Thus, the infiltration of OPSCC by HPV16-specific Th1/Th17cells is associated with the presence of a highly active tumormicroenvironment consisting of a dense type I–oriented immunecell infiltrate, known to favor immune-mediated control of cancercells (29).

Figure 2.HPV16-positive OPSCCs harboring HPV16-specific T cells display a stronger and more activated immune infiltrate. A, The number of CD8þ and CD8� (CD4þ)T cells positive for Tbet/mm2 tumor as determined in OPSCC sections (5 high-power fields per patient were counted). The 38 HPV16-positive OPSCCpatients were grouped according to the number of Tbet-positive cells above (hi) or below (lo) the median and plotted in a Kaplan–Meier analysis for survival.B, The patients were grouped on the basis of the p16INK4a expression of the tumor and the detection of an HPV-specific immune response (IR). The number of Tbet-positive T cells, with each dot representing an individual patient sample and themedian plus interquartile range, is shown. Data of all three groups were analyzed byKruskal–Wallis test. Data of twogroupswere analyzed by unpaired nonparametric analysis (Mann–WhitneyU test).C, TheViSNEplots visualize the high-dimensionalCyTOF data in two dimensions. The different cell subsets are indicated. The frequency of CD4þ and CD8þ T cells in the freshly dispersed OPSCC samples asdetermined by CyTOF is shown in the graph. Data are expressed as average frequencies (�SEM). The three groups differed significantly in their CD8þ T-cellfrequency.D, Pie charts showing the composition of the immune cells and their relative contribution to the tumor microenvironment. E, The subdivision of the CD4þ

and CD8þ frequencies (�SEM) into na€�ve, central memory, and effector memory T cells. Significant differences in the three groups for effector memory CD4þ andCD8þ T cells and central memory CD8þ T cells were found. F, CITRUS analysis visualized four main populations. The CD4þ T-cell population included twosubpopulations (indicated by the numbers 1 and 2), and the parental T-cell node is indicated as total T cells. G, The differences in frequency of T and B cells aredepicted as box andwhiskers (plusmin–max) between the groups of patients.H, The frequency of the two subsets of CD4þ T cells and the CD8þ T cells (subset 3) asdetermined in F and similar to G. NK, natural killer; NS, not significant; � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.






CBA

% o

f CD

45+

cells

DC-like macrophage (CD14+CD11b+CD11c+)DC (CD14-CD11c+CD11b+/-)Langerhans-like DC (CD1a+HLA-DR+)

DC-like macrophage (CD14+CD11b+CD11c+)DC (CD14-CD11bdimCD11c+)

Act CD4

NKTMac

Tcm CD4Tgd

Tem CD4

Th2

CD56dim

Act CD8

Th1Th17

CD56bright

Treg Tcm CD8

Tem CD8

Imm DC

pDC Neu

Imm B

NK

Act B

Mem B

TfhAct DC

MDSC

MastMon

Eos

5.25.2−Immune cell type enrichment (NES)

0

1

2

3

4

−Lo

g 10(

q−va

lue)

CD4 high vs. low (n = 75)

moD

C

D

0

20

40

60

80

100

% C

D14

+ C

D20

6+

% C

D14

+ C

D16

3+

0

20

40

60

80

100

0

20

40

60

80

100

% C

D14

+ C

D1a

-

% C

D14

- CD

1a+

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

% C

D14

– H

LA-D

R−

% C

D14

+ H

LA-D

R+

0

20

40

60

80

100

% C

D14

- HLA

-DR

+

% C

D14

- CD

16+

SC

C47

SC

C10

4

moD

C

moD

C

moD

C

LPS

0

250

500

750

1,000

1,250

1,500Anti-CD40

0

1,000

2,000

3,000

4,000

5,000

6,000Anti-CD40 + IFNγ

0

5,000

10,000

15,000

20,000

IL12

p70

moD

C

moD

C

moD

C

E

p16+ IR+p16+ IR-p16- IR-0.0

0.5

1.0135

p16+ IR+p16+ IR-p16- IR-0.0

0.5

1.0

1.51030

% o

f CD

45+

cells

F

0

2,000

4,00010,00020,00030,00040,000

moDC SCC47 SCC104

0

1,000

2,000

3,000

4,000

5,000

Imm

atur

e

LPS

Ant

i-CD

40

Ant

i-CD

40 +

IFN

γ

Imm

atur

e

LPS

Ant

i-CD

40

Ant

i-CD

40 +

IFN

γ

Imm

atur

e

LPS

Ant

i-CD

40

Ant

i-CD

40 +

IFN

γ

CX

CL9

CX

CL1

0

SC

C47

SC

C10

4

SC

C47

SC

C10

4

SC

C47

SC

C10

4

SC

C47

SC

C10

4

SC

C47

SC

C10

4

SC

C47

SC

C10

4

0

2,000

4,00010,00020,00030,00040,000

0

1,000

2,000

3,000

4,000

5,000

0

2,000

4,00010,00020,00030,00040,000

0

1,000

2,000

3,000

4,000

5,000

* *

0.0

Welters et al.





Type I cytokines influence tumor cell proliferation andsynergize with cisplatin-induced cell death

The OPSCC-infiltrating, HPV-specific CD4þ T cells producedIFNg and TNFa, known to drive tumor cell senescence (30) and tosynergize with platinum-based therapy to kill tumor cells (31).We, therefore, studied whether similar mechanisms could playa role in controlling oropharyngeal cancer cell growth byHPV-specific CD4 T cells in vitro. We used our collection of threeHPV-negative and two HPV16-positive HNSCC cell lines toanalyze the expression of proteins involved in proliferation,apoptosis, and necroptosis following stimulation with IFNgand TNFa. All cell lines expressed IFNGR and TNFR1 (and wereresponsive to IFNg , evidenced by the phosphorylation of STAT1,and to TNFa, evidenced by RelA phosphorylation; Supplemen-tary Fig. S7A–S7C). Furthermore, they expressed the proteinsrequired for apoptosis and necroptosis, although the HPV16-positive tumor cells lacked expression of the necroptosis-essentialprotein RIPK3 (Fig. 4A). Stimulation of the tumor cells with IFNgand/or TNFa or culture supernatant from antigen-stimulatedHPV-specific Th1 or Th17 cells revealed a reduction in theirproliferation (Fig. 4B and C) and an increase in the expressionof the IFNg-responsive genes IFITM1 and RARRES. Both genesare known to stop the proliferative process in cells (refs. 32, 33;Fig. 4D and E), albeit these effects differed per cell line tested.Expression analysis of the relation between IFNg , IFITM1, andRARRES in the TCGA cohort of HPV16-positive patients showedthat especially IFNg and IFITM1were coexpressed (r¼ 0.475; P¼0.00060), suggesting that IFNg-induced arrest in proliferationoccurs in vivo. In line with the RIPK3 expression, only the HPV-negative cell lines were sensitive to necroptosis (Fig. 4F). Ascisplatin is the chemotherapeutic compound of choice for thetreatment of OPSCC, the induction of cell death by increasingdoses of TNFa in the presence of cisplatin was tested. Thecombination of TNFa and cisplatin resulted in an increasedpercentage of apoptotic tumor cells at 24 hours, specifically inthe HPV-positive cell lines, as no synergistic effect was observedin the HPV-negative cell lines (Fig. 4G), and resulted in a highpercentage of dead tumor cells at 48 hours (SupplementaryFig. S7D). These effects did not depend on necroptosis, as inhi-bition with necrostatin-1s did not prevent cell death (Supple-mentary Fig. S7D). Thus, apart from their role in changing themicroenvironment, IFNg and TNFa may also synergize withstandard therapy in controlling tumor cell growth and form oneof the underlying mechanisms explaining the good response rateof HPV-responding patients to (chemo)radiotherapy (2, 3).

Intratumoral activated effector memory CD161þCD4þ Th1/Th17 cells have a potential role in disease control

CD161þCD4þ T cells are the dominant subtype of T cellspresent in inflammatory diseases where CD4þ T cells have animportant role todrive acute inflammatory processes (34).Hence,a similar rolemay be expected in the rejection of cancer cells. First,CD161 expression among freshly and in vitro expanded TILs wasanalyzed. A large proportion of our fresh and in vitro expandedTILs expressed CD161. Importantly, in vitro expansion did notinduceCD161 expression (Supplementary Fig. S8A). Subsequent-ly, a flow cytometric analysis of eight in vitro expanded TILs wasperformed to assess the HPV-specific component among thesecells.On average, the percentage ofCD161þCD4þT cellswas 29%(Fig. 5A). The number of HPV-specific T cells producing TNFa(Fig. 5B) was a bit higher than those producing IFNg (Supple-mentary Fig. S8B), and, on average, 31% of the HPV-specificCD4þ T cells expressed CD161 (Fig. 5B). This indicates that therewas a sizeable CD161þ T-cell fraction among HPV-specific CD4þ

T cells in most of the patients and also that the distribution ofCD161þ cells among theseHPV-specific T cells is similar to that ofthe total population.

Subsequently, we analyzed the survival of the 75 patients withHPV16-positive OPSCC in the publicly available TCGA databasefocusing on the expression of CD4, CD8, CD103, and CD161. Ahigh expression of CD4, CD8, or CD161 was associated withbetter overall survival, but this was not the case for CD103expression (Fig. 5C–F), albeit the combined high expressionof CD103with CD8 resulted in a better segregation of the survivalcurves (Fig. 5G). This fits with the observation that the expressionof CD8 and CD103 was not strongly correlated (r ¼ 0.2559; P ¼0.0267) within this cohort. A high expression of CD161 witheither high CD4 or CD8 expression was also associated withbetter survival (Fig. 5H and I). Notably, the populations ofpatients within the group seem to overlap completely. Indeed,these markers were highly coexpressed (CD4 and CD161: r ¼0.8351, P ¼ 0.00E00; and CD8 and CD161: r ¼ 0.8363, P ¼0.00E00), suggesting that they predominantly single out thesame patients. As the HPV-specific T cells predominantly pro-duced IFNg , TNFa, and IL17 (Fig. 1D), we also analyzed thecontribution of the respective gene expression levels to survival.Specifically, a high expression of IFNg was associated withbetter survival, whereas a similar trend was visible for IL17(Fig. 5J and K). Combinations of two to three cytokines did notresult in better separation of the survival curves (Supplemen-tary Fig. S8C–S8G).

Figure 3.HPV16-specific T-cell–produced cytokines stimulate myeloid cells toward a type I phenotype. A, To identify immune cell types that are overrepresented inHPV16-positive OPSCC with CD4þ T-cell infiltrate, a gene set enrichment analysis was performed on a cohort of 75 patients with HPV16-positive OPSCC presentin the publicly available TCGA database. The expression level of each gene was z-score normalized across all patients. For each patient (or group of patients),genes were then ranked in descending order according to their z-scores (mean of z-scores). The association was represented by a normalized enrichmentscore (NES). An immune cell type was considered enriched in a patient or group of patients when the FDR (q-value) was �10%. The Volcano plot for theenrichment (dark gray) and depletion (white) of immune cell types in CD4þ-high versus CD4þ-low HPV16-positive OPSCC is shown. B, The DCs, Langerhans-likeDCs, andDC-likemacrophages in freshly dispersedOPSCCof 7 p16�IR�, 3 p16þIR�, and8p16þIRþpatientsweredeterminedbyflowcytometry (percentageofCD45þ

cells � SEM). C, As in B but analyzed by mass cytometry (CyTOF; p16�IR�, n ¼ 4; p16þIR�, n ¼ 4; p16þIRþ, n ¼ 5). Both in B and C, significant differences intotal DC population were observed between IR� and IRþ within the p16þ OPSCC patient group. D, Purified CD14þ cells from 5 healthy donors were culturedwith IL4 and GM-CSF for 6 days to differentiate them into monocytic DCs (moDC) in the presence/absence of 20% TSN obtained from UM-SCC47 or UM-SCC104(both HPV16-positive OPSCCs), stained and analyzed by flow cytometry. The percentages (�SEM) of cells stained for the different marker combinations areshown. E, As in D for the 5 healthy donors after the cells have been stimulated for an additional 2 days with LPS, agonistic anti-CD40 antibody, or thecombination of this antibody with IFNg . The IL12p70 production (in pg/mL; mean � SEM) is depicted. Nonstimulated cells (moDC) were taken along asnegative control. F, As in E for the 5 healthy donors showing the production of CXCL9 and CXCL10 (in pg/mL; mean � SEM) by these myeloid cells. NS,not significant; �, P < 0.05; �� , P < 0.01; and �� , P < 0.001.






IFNγ + TNFα IFNγ + TNFα

IFNγ + TNFα

IFNγ + TNFα

IFNγ + TNFα +

IFNγ + TNFα IFNγ + TNFα IFNγ + TNFαControl

0

1

2

3

Exp

ress

ion

of IF

ITM

0

5

10

15

01234

Exp

ress

ion

of R

AR

RE

S1

012345

0.00.51.01.52.0

0.00.51.01.52.0

0

2

4

6

01234

01020304050

012345

D

E

RIPK3

MLKL

cIAP2

Caspase-8

β-actin

IFNγ + TNFα----- +++++

SCC104SCC47SCC19SCC4SCC6

FADD

RIPK1

CYLD

TRAF2

cIAP1

FLIP

A

Control Control Control Control

B

C

SCC4

40

60

80

100

% P

rolif

erat

ing

cells

SCC6

40

60

80

1120SCC19

40

60

80

100SCC47

40

60

80

100

SCC104

00.

5 1 2 5 10 20 40

40

60

80

100

Control (no TNFα)TNFα 5 ng/mLTNFα 10 ng/mLTNFα 20 ng/mL

40

60

80

100

Th17 clone 103Th1 clone 97

40

60

80

1100

40

60

80

100

40

60

80

Con

trol

1%

40

60

80

100

00.

5 1 2 5 10 20 4000.

5 1 2 5 10 20 4000.

5 1 2 5 10 20 40

IFNγ (ng/mL) IFNγ (ng/mL) IFNγ (ng/mL) IFNγ (ng/mL) IFNγ (ng/mL)

00.

5 1 2 5 10 20 40

100

2% 5% 10%

20%

Con

trol

1% 2% 5% 10%

20%

Con

trol

1% 2% 5% 10%

20%

Con

trol

1% 2% 5% 10%

20%

Con

trol

1% 2% 5% 10%

20%

% P

rolif

erat

ing

cells

1100

Control

12.6

0 5,0M 10M 15M 20M

100

101

102

103

104

105

106

107 11.2

0 5,0M 10M 15M 20M

100

101

102

103

104

105

106

107

BV6 + zVADfmk BV6 + zVADfmk

50.0

0 5,0M 10M 15M 20M

100

101

102

103

104

105

106

107 76.2

0

100

101

102

103

104

105

106

107

5,0M 10M 15M 20M

0

20

40

60

80

100

++ --BV6 + zVADfmk++--

++ --

++--

++ --

++--

++ --

++--

++ --

++--

% S

YTO

X g

reen

pos

itive

cel

ls

SCC104SCC47SCC19SCC6SCC4

NS

NS

NS

******

FSC-A

SY

TOX

gre

en

cells46,5

0 5,0M 10M 15M 20M

0

5,0M

10M

15M

FSC-A

FSC

-H

FSC-A

SY

TOX

gre

en

GSCC4

0

10

20

30

40

% A

popt

otic

cel

ls

SCC6

0

10

20

30

40

0

10

20

30

40

Apoptosis

TNFαCisplatin

LateEarly Total

--- + + + +

+ + +--

-+ + + ++ + +

--- + + + +

+ + +

LateEarly Total

--- + + + +

+ + +--

-+ + + ++ + +

---+ + + +

+ + +

LateEarly Total

---+ + + +

+ + +--

- + + + ++ + +

--- + + + +

+ + +

SCC19

***

***

**

****

***

***

*** SCC47

0

10

20

30

40SCC104

0

10

20

30

40

LateEarly Total

--- + + + +

+ + +--

- + + + ++ + +

--- + + + +

+ + +

LateEarly Total

--- + + + +

+ + +--

-+ + + ++ + +

---+ + + +

+ + +

*

***

******

***

**

F


Welters et al.




In combination with the above, these data suggest that a denseinfiltration of HPV16-positive OPSSC with IFNg/IL17-orientedCD4þ and/or CD8þCD161þ T cells, including theHPV16-specificT cells, is important for superior disease control in HPV-drivenOPSCC. Therefore, we analyzed the disease-specific survival ofHPV-specific T-cell responders within the group of patients withHPV16-positive OPSCC. Patients with HPV-positive OPSCC dis-playing an HPV-specific T-cell reaction had a 37.8-fold (95% CI,7.1–199.9) higher chance to respond to therapy when comparedwith patients with HPV16-positive OPSCC lacking such a T-cellreaction (Fig. 6A). Especially in stage III to IV HPV16-positiveOPSCC, the local presence of an HPV16-specific T-cell responsewas a better prognostic parameter for long survival after therapythan staging (Fig. 6B). The differences in survival between thesetwo groups could not be attributed to a different cancer treatment(Supplementary Table S3). Intriguingly, also the T and N stagewere, on average, lower in the immune responders (Fig. 6C andD), suggesting that HPV16-specific T cells were especially presentin patients with a better control of tumor growth.

DiscussionThe improved clinical response of patients with OPSCC to

(chemo)radiotherapy has been associated with HPV and with adense, activated T-cell infiltrate, but the role of the immuneresponse against HPV in this was still not completely understood.Our findings demonstrate that the virally derived E6 and E7antigens make HPV-associated OPSCC highly visible to theimmune system and unleash an intratumoral, HPV-specific T-cellresponse. These cells are polyfunctional, detected among TILs inmany of the patients, and have the CD161þ phenotype oftenfound in acute rejection processes. They may locally facilitatethe development of a clinically favorable tumor microenviron-ment because their presence is associated with a stronger influx oftype I–oriented CD4þ and CD8þ T cells, as well as DCs and DC-like macrophages. Moreover, they produce cytokines that syner-gize with the platinum-based chemotherapy used to treat thesepatients, and their detection is highly predictive for the responseof patients to (chemo)radiotherapy.

HPV-specific CD4þ and CD8þ T cells were detected in 64%of the TILs derived fromHPV16-positive OPSCC, with a predom-inance ofHPV-specific CD4þ T cells—a result that closelymatchesan earlier study (35). We show that these HPV-specific tumor-infiltrating T cells as well as the other TILs predominantly pro-duced IFNg and IL17, suggesting the presence of Th1 andTh17 cells. In view of the accepted roles of Th1/Th17 CD4þ and

CD8þ T cells in tumor control (36, 37), the detection of these cellsin HPV16-positive OPSCC is likely to favor tumor control.Indeed, a high expression of IFNg and, to a lesser extent, IL17in HPV16-positive OPSCC was associated with superior survival.Furthermore, the detection of HPV-specific T cells singled outimmunologically "hot" tumors, with higher numbers of CD4þ

and CD8þ T cells expressing Tbet, effector memory T cells, DCs,and DC-like macrophages when compared with HPV16-positiveOPSCCwithoutHPV-specific T cells. A dense tumor infiltration byT cells (38) and DCs (39) and a predominant adaptive immunegene signature (4) have been associated with better survival inhead and neck cancer, indicating that HPV-specific T-cell–infil-trated tumors possess the right type of inflammation. Last but notleast, a dense infiltrate with T cells is foundmore often in patientswith superior local disease control (40), fitting with our obser-vation that the group of patients with a tumor-specific immuneresponse presented with lower T and N stages.

Concomitant with the detection of HPV16-specific TILs, wefound increased frequencies of CD161þ effector memory CD4þ

and CD8þ TILs as well as CD8þCD103þ TILs. The intratumoralpresence of CD8þCD103þ T cells is a beneficial prognosticfactor in a number of cancer types (41), and this would fit withthe fact that we detected a high frequency of these cells spe-cifically in T-cell inflamed tumors as well as with our analysis ofthe TCGA database, showing a survival advantage for patientswith HPV16-positive OPSCC with a strong expression of bothCD8 and CD103. Earlier reports showed that CD161þ ispredominantly detected on effector and central memory T cellsthat produce IFNg and/or TNFa (42), Th17 cells (43), andregulatory T cells (44). CD4þCD161þ T cells can drive acuteinflammatory processes (34), suggesting an important andsimilar role for them in cancer. Indeed, CD161 was amongthe top 10 of tumor leukocyte-associated genes associated withpositive prognosis for many human tumors (45). In our study,CD161 was expressed by tumor-specific IFNg- and/or TNFa-producing CD4þ T cells; higher frequencies of CD161 expres-sing CD4þ T cells were detected in T-cell inflamed tumors; and,finally, in the TCGA database, the expression between CD161and CD4 or CD8 was highly correlated and a high expression ofthese three genes was associated with a survival advantage forpatients with HPV16-positive OPSCC. Interestingly, mass cyto-metry showed that part of the CD8þCD103þ T cells alsoexpressed CD161.

In a large meta-analysis in head and neck cancer (MACH-NC)patients treated with radiotherapy alone had an overall 5-yearsurvival of 27.2%, whereas in patients receiving concomitant

Figure 4.Sensitivity and resistance of OPSCC cell lines to the antiproliferative and cytotoxic effects of proinflammatory cytokines and/or chemotherapy. A, Proteinexpression of the indicated proteins involved in the cell death pathway. B, Proliferation of tumor cells (from five different UM-SCC cell lines) treated with theindicated different concentrations of IFNg and TNFa as determined by MTT assay with untreated cells was set at 100%. C, As in B, but tumor cells werestimulated with different concentrations of culture supernatant from HPV-specific stimulated Th1 or Th17 clones. Tumor cells were left untreated (control) ortreated with 50 IU/mL IFNg and 30 ng/mL TNFa for 24 hours, and the expression of IFITM (D) and RARRES1 (E) was determined by qRT-PCR andnormalized to the GAPDH mRNA. The expression is given as mean (�SEM) for three independent experiments. F, The five different UM-SCC tumor cell lineswere treated (in triplicate wells) for 48 hours with 250 IU/mL IFNg and 30 ng/mL TNFa in the absence or presence of the necroptosis/apoptosis inducersBV6 (5 mmol/L) and zVADfmk (20 mmol/L). Untreated tumor cells were taken along as negative controls. Dead cells were stained positive using SYTOX green,and the mean percentage (�SEM) is depicted. Unpaired t test analysis was performed between IFNg þ TNFa treatment with or without BV6 þ zVADfmk.G, The tumor cells were left untreated, treated for 24 hours with 30 ng/mL TNFa, or treated with a fixed concentration of cisplatin (15 mg/mL) plus increasingconcentrations of TNFa (7.5, 15, or 30 ng/mL), as indicated by the triangle. The cells were stained for early apoptosis by Annexin V and late apoptosis by 7-AAD,and analyzed by flow cytometry. The mean percentage (�SEM) of the apoptotic cells in triplicate wells is shown. Total indicates the sum of percentage ofboth the early and late apoptotic cells. NS, not significant; �, P < 0.05; �� , P < 0.01; and ��, P < 0.001.






H68

% o

f CD

3+ T

cel

ls

CD161–CD4+

CD161+CD4+

H10

3

H13

3

H13

6

H13

8

H14

8

H10

4

H18

0

% o

f CD

4+ T c

ells

% o

f TN

Fα+ C

D4+ T

cel

ls

% o

f CD

4+ T c

ells

CD161−TNFα+

CD161+TNFα+

A

B

CD161−

CD161+

CD161−

CD161+

EDC

HGF

I

Survival time (months)

Ove

rall

surv

ival

HR = 0.23 95% CI (0.10–0.64)Log-rank P = *

CD161+CD8 hi (n = 33)

other (n = 42)

20 40 60 80 100 1200

0 20

HR = 0.24 95% CI (0.10–0.62)Log-rank P = **

CD4 hi (n = 38)

CD4 lo (n = 37)Ove

rall

surv

ival

40 60 80 100

HR = 0.19 95% CI (0.10–0.51)Log-rank P = **

CD8 hi (n = 38)

CD8 lo (n = 37)

HR = 0.19 95% CI (0.08–0.51)Log-rank P = ***

CD161 hi (n = 38)

CD161 lo (n = 37)

HR = 0.72 95% CI (0.29-1.76)Log-rank P = NS

CD103 hi (n = 38)

CD103 lo (n = 37)

Ove

rall

surv

ival

HR = 0.29 95% CI (0.15–0.93)Log-rank P = *

CD103+CD8 hi (n = 25)

Other (n = 50)

HR = 0.24 95% CI (0.11–0.68)Log-rank P = **

CD161+CD4 hi (n = 32)

Other (n = 43)

0 20 40 60 80 100

0 20 40 60 80 100 0 20 40 60 80 100

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Figure 5.

Superior disease control correlates with the presence of intratumoral CD161þ T cells. A, The proportion of CD161þCD4 T cells among TILs (left) and theproportion of CD161þ cells among CD4þ T cells (right) where the frequencies of total CD4þ T cells is set to 100%.B,As forA but now for the HPV-specific CD4þ T cellsproducing TNFa upon stimulation with HPV16 E6 and/or E7 overlapping peptides. Kaplan–Meier survival plots of the 75 patients with HPV16-positive OPSCC inthe TCGA database grouped according to high and low gene expression using themedian value of CD4 (C), CD8 (D), CD161 (E; KLRB1), and CD103 (F). As in C–F, butnow, patients are grouped according to a high expression of two indicated genes versus all others based on the median expression levels of CD103 and CD8(G), CD161 and CD4 (H), CD161 and CD8 (I). J and K, As in C–F for the expression of IFNg (J) and IL17A (K). All graphs, the HR with the 95% CI as well as thelog-rank test P value are given. NS, not significant; � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.


Welters et al.




cisplatin chemotherapy and radiotherapy, an improvement inoverall survival of 6.5% was achieved (46). Potentially, this isexplained by studies showing that platinum-based chemotherapysynergizes with immune cell–produced IFNg and TNFa in killingtumor cells (31), including OPSCC cells (this study). Becauseof the described cisplatin toxic side effects, dose reductions incisplatin of 30% to 69% are often required for sustained concur-rent (chemo)radiotherapy treatment (47, 48), and deintensifica-tion protocols for these patients are being discussed. This shouldnot pose a major problem, as lower doses of cisplatin stillsynergize with T-cell responses in animal tumor models (31).

Finally, thequestion surfaceswhether reinforcement ofHPV16-specific T-cell reactivity in patients withHPV16-positiveOPSCC iswarranted, not only to convert nonresponders to HPV respondersbut also to boost existing responses. Clearly, the HPV16-positiveOPSCCs infiltrated by HPV16-specific T cells meet the criteria ofthe cancer immunogram for immunotherapy (49). The percen-tages of HPV-specific T cells among TILs are respectable, however,not enough to mediate full tumor regression. In parallel tomelanoma, where treatment with increased numbers of tumor-

specific T cells can mediate clinical responses, therapeutic vacci-nation is expected to increase the number of HPV16-specific Tcells andmay result in clinical benefit for patients withOPSCC. Inview of the expression of PD-1 by the effector memory CD4þ andCD8þ T cells (this study and ref. 6) and of PD-L1 (50) in tumortissue, a combination of therapeutic vaccination and PD-1/PD-L1blocking is expected to have the best outcome.

Disclosure of Potential Conflicts of InterestS.H. van der Burg reports receiving other commercial research support from

ISA Pharmaceuticals and Kite Pharma EU, is listed as a co-inventor on aprovisional patent application on the use of synthetic long peptide vaccinesfor therapeutic vaccination against chronic infections and cancer includingHPVthat is owned by the Leiden University Medical Center and licensed to ISAPharmacueticals, Leiden, the Netherlands, and is a consultant/advisory boardmember for ISA Pharmaceuticals. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design:M.J.P. Welters, L.-A. van der Velden, S.H. van der BurgDevelopment of methodology: M.J.P. Welters, W. Ma, E.S. Jordanova

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Figure 6.

HPV16-specific T cells control tumor growth in HPV16-positive OPSCC. A, Kaplan–Meier survival curves showing the outcome of the 45 tested HPV16-positiveOPSCC patients who harbored HPV16-specific T cells in their tumors (immune response positive, IRþ) versus those who did not display an immune response (IR�).The HR with 95% CI and log-rank P value are given. B, Kaplan–Meier survival curves when the 45 patients with HPV16-positive OPSCC were plottedaccording to the stage of the disease. Staging was done according to the National Comprehensive Cancer Network. C, The group of 45 patients withHPV16-positive OPSCC was split on the basis of p16INK4a expression in the tumor and having an immune response directed against HPV16 or not. Thetumor size (T stage) is depicted for each individual patient in the three groups of patients. D, As in C but now for the involvement of lymph nodes (N stage). NS,not significant; �, P < 0.05; �� , P < 0.01; and �� , P < 0.001.






Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.J.P. Welters, W. Ma, S.J.A.M. Santegoets,R. Goedemans, I. Ehsan, E.S. Jordanova, V.J. van Ham, F. Koning,S.I. van Egmond, L.-A. van der Velden, S.H. van der BurgAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.J.P. Welters, W. Ma, S.J.A.M. Santegoets,E.S. Jordanova, V. van Unen, F. Koning, P. Charoentong, Z. Trajanoski,S.H. van der BurgWriting, review, and/or revision of the manuscript: M.J.P. Welters, W. Ma,E.S. Jordanova, S.I. van Egmond, Z. Trajanoski, L.-A. van der Velden,S.H. van der BurgAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.J.P. Welters, L.-A. van der VeldenStudy supervision: M.J.P. Welters, S.H. van der BurgOther (mass cytometry analysis): F. Koning

AcknowledgmentsThe authors thank the patients for participating in this study. The authors

also acknowledge C.E. van der Minne for her technical assistance.This study was financially supported by a grant from the Dutch Cancer

Society 2014-6696 (to S.H. van der Burg, L.-A. van der Velden, and M.J.P.Welters).

The costs of publication of this articlewere defrayed inpart 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 24, 2017; revised September 15, 2017; accepted October 4,2017; published OnlineFirst October 10, 2017.

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2018;24:634-647. Published OnlineFirst October 10, 2017.Clin Cancer Res Marij J.P. Welters, Wenbo Ma, Saskia J.A.M. Santegoets, et al. Oropharyngeal CancerTumor Microenvironment to Improve Survival in HPV16-Driven

Oriented−Intratumoral HPV16-Specific T Cells Constitute a Type I

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