pd-1 status in cd8þ t cells associates with survival and ... · microenvironment and immunology...

Microenvironment and Immunology

PD-1 Status in CD8þ T Cells Associates withSurvival and Anti-PD-1 Therapeutic Outcomes inHead and Neck CancerBenjamin A. Kansy1,2, Fernando Concha-Benavente1, Raghvendra M. Srivastava1,Hyun-Bae Jie1, Gulidanna Shayan3, Yu Lei4, Jessica Moskovitz1, Jennifer Moy1, Jing Li3,Sven Brandau2, Stephan Lang2, Nicole C. Schmitt5, Gordon J. Freeman6,William E. Gooding7, David A. Clump8, and Robert L. Ferris1,9

Abstract

Improved understanding of expression of immune checkpointreceptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facil-itate more effective immunotherapy in head and neck cancer(HNC)patients.Ahigher frequencyofPD-1þ TILhasbeen reportedin human papillomavirus (HPV)þ HNC patients, despite the roleof PD-1 inT-cell exhaustion. This discordance led us tohypothesizethat the extent of PD-1 expression more accurately defines T-cellfunction and prognostic impact, because PD-1high T cells may bemore exhausted than PD-1low T cells and may influence clinicaloutcome and response to anti-PD-1 immunotherapy. In this study,PD-1 expression was indeed upregulated on HNC patient TIL, andthe frequency of these PD-1þ TIL was higher inHPVþ patients (P¼0.006), who nonetheless experienced significantly better clinical

outcome. However, PD-1high CD8þ TILs were more frequent inHPV� patients and represented a more dysfunctional subset withcompromised IFN-g secretion. Moreover, HNC patients withhigher frequencies of PD-1high CD8þ TIL showed significantlyworse disease-free survival and higher hazard ratio for recurrence(P < 0.001), while higher fractions of PD-1low T cells associatedwith HPV positivity and better outcome. In a murine HPVþ HNCmodel, anti-PD-1 mAb therapy differentially modulated PD-1high/low populations, and tumor rejection associated with loss ofdysfunctional PD-1high CD8þ T cells and a significant increase inPD-1low TIL. Thus, the extent of PD-1 expression on CD8þ TILprovides a potential biomarker for anti-PD-1–based immunother-apy. Cancer Res; 77(22); 6353–64. �2017 AACR.

IntroductionImmune checkpoint receptor (ICR) blockade has become a

major focus of investigation in the field of cancer immunother-apy. Importantly, ICRblockade has shownbeneficial results in theclinic for certain patient populations; however, biomarkers ofresponse have not been clearly identified. Programmed death-1(PD-1; ref. 1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) are

two of the main clinical targets (2). While these have beenincreasingly investigated, less is known about their expression intumor-infiltrating lymphocytes (TIL) during anti-PD-1 therapeu-tic interventions. Given the promising data that have beenreported in several malignancies (3–5), we investigated ICR levelson specific TIL subsets in patients with head and neck squamouscell carcinoma (HNC).

An increasingly important prognostic marker for HNCpatients is HPV status, because the percentage of HPVþ oro-pharyngeal squamous cell carcinoma in North America hasincreased from 30% in the 1980s to 80% at present (6).Although it is known that HPVþ HNC has a better prognosisthan HPV� HNC, and better response to anti-PD-1–basedimmunotherapy (7), the mechanism(s) underlying these clin-ical differences remain elusive, as do differences in immuneescape strategies (8). Because of these prognostic differencesbetween HPVþ and HPV� in HNC patients, the distinct immu-nologic features of the two groups should be compared in orderto advance knowledge regarding tumor immune evasion.Therefore, we investigated differences in ICR expression at themRNA and protein level and characterized immunologic prop-erties of tumor-associated T lymphocytes, including CD8þ Teffector cells, with respect to HPV status.

Promising results using PD-1 or PD-L1 blocking monoclonalantibodies (mAb) have emerged for advanced recurrent/metastatic HNC (7, 9), but still only a minority of patients(15%–20%) respond, despite elevated expression of PD-L1 in>50% of HNC patients (8). On the one hand, PD-1 ligation has

1Department of Otolaryngology, University of Pittsburgh, Pittsburgh, Pennsyl-vania. 2Department of Otorhinolaryngology, University Hospital Essen, Ger-many. 3School of Medicine, Tsinghua University, Beijing, China. 4Department ofPeriodontics and Oral Medicine, University of Michigan School of Dentistry,Graduate Program in Immunology, University of Michigan Medical School, AnnArbor, Michigan. 5Department of Otolaryngology, Johns Hopkins University,Baltimore, Maryland, and National Institute on Deafness and CommunicationDisorders, National Institutes of Health, Bethesda, Maryland. 6Department ofMedical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. 7Bio-statistics Facility, University of Pittsburgh Cancer Institute, Pittsburgh, Penn-sylvania. 8Department of Radiation Oncology, University of Pittsburgh, Pitts-burgh, Pennsylvania. 9Cancer Immunology Program, University of PittsburghCancer Institute, Pittsburgh, Pennsylvania.

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

CorrespondingAuthor:Robert L. Ferris, University of PittsburghHillman CancerCenter, 5117 Centre Avenue, Suite 2.26b, Pittsburgh, PA 15213. Phone: 412-623-0327; Fax: 412-623-4840; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-3167

�2017 American Association for Cancer Research.

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been demonstrated to have a negative impact on T cells, and theblockade of this ligation results in improvement of their function(10). On the other hand, PD-1 positivity has been shown torepresent antigen experienced, TA-specific T cells (11) and hasbeen correlated with better clinical outcome (12). Additionally,other checkpoint receptors such as T-cell immunoglobulin-3(Tim-3; refs. 13, 14), lymphocyte activation gene-3 (LAG-3) andB and T lymphocyte attenuator (BTLA) are under investigation.Tim-3 has been identified as a specific marker of fully differen-tiated IFN-g producing CD4þ and CD8þ T cells (15). Its expres-sion is regulated by the transcription factor T-bet (16) andnegatively regulates Th1 and CD8þ cytotoxic T-cell responses(17). LAG-3 is upregulated on activated CD4þ and CD8þ T cellsas well as in a subset of activated natural killer (NK) cells (18).Besides its capacity to bind to major histocompatibility com-plexes (MHC) class II and its role in T helper cell and regulatoryT-cell (Treg) signaling, direct inhibitory effects of LAG-3 on CD8þ

T effector cells have been demonstrated (19). BTLA negativelyregulates T-cell activation by inhibiting T-cell proliferation andcytokine production. In contrast to other checkpoint receptors,BTLA is expressedonna€�veT cells andonly transiently upregulatedupon TCR engagement (17). Thus, BTLA is downregulated onhighly activated T cells (20).

Little is known about the role of checkpoint receptors in TILfrom HPVþ versus HPV� HNC. Interestingly, a prior reportsuggested that the presence of PD-1 T cells in HPVþ patients wasassociated with a beneficial effect on survival (12). Given thatHPVþ patients demonstrate a much better outcome in the clinic(12), we hypothesized that intratumoral ICR expression andfunction might provide immunologic insight into this differencein prognosis. Thus, as opposed to solely analyzing the frequencyof PD-1þ versus PD-1� TIL, we investigated whether the intensityof PD-1 expression on TIL influences clinical outcome, providinga potential prognostic biomarker and for monitoring response toanti-PD-1–based immunotherapy.

Materials and MethodsThe Cancer Genome Atlas data retrieval and analysis

RNAseq data from queried genes were downloaded from theUCSC cancer genomics browser (https://genome-cancer.ucsc.edu). TheHNC gene expression profile from 500HNC specimenswas measured experimentally (21). The RSEM units to quantitateRNAseq expression data were described previously (22). Correla-tions fromTheCancerGenomeAtlas (TCGA)datawere calculatedusing Pearson r test, and linear regression curve fits were graphedusing GraphPad PRISM software v6.

Patients and specimensPeripheral venous blood samples and tumors were obtained

from HNC patients seen in the Department of Otorhinolaryn-gology at the University of Pittsburgh Medical Center. No activepatient exclusion to clinical stage or HPV status was performedother than restriction to all consented, surgically treated patientswith primary tumors and curative treatment intention. Specimenacquisition was continuously performed by the same physicianduring 2011–2013, the patients' HPV status was defined fromsurgical specimen by IHC determination of the p16 status (pos-itive p16 expressionwas defined as strong and diffuse nuclear andcytoplasmic staining in at least 70% of the tumor cells). Allsubjects signed written informed consent approved by the Insti-tutional Review Board of the University of Pittsburgh, patient

studies were conducted in accordance with the Declaration ofHelsinki.

Collection of PBMC and TILBlood samples and tumor specimenwere collected at the day of

and prior to surgery and, therefore, at the beginning of thetreatment. Blood samples from cancer patients (30–40 mL) weredrawn into heparinized tubes and centrifuged on Ficoll–Hypaquegradients (GE Healthcare Bioscience). Peripheral blood mono-nuclear cells (PBMC) were recovered, washed in RPMI-1640(Invitrogen), and immediately used for experiments or stored at�80 �C until further analysis. For TIL isolation, freshly isolatedtumors from HNC patients were minced into small pieces inRPMI-1640 (Invitrogen), which then were transferred to a cellstrainer (70 mm Nylon) and mechanically separated using asyringe plunge. The cells passing through the cell strainer werecollected and subjected to Ficoll–Hypaque gradient centrifuga-tion. After centrifugation, mononuclear cells were recovered andstored at �80 �C until flow cytometry analysis.

Quantitative PCR of ICRQuantitative (q) PCR was performed to investigate tumor

specimen from20HPVþ and 20HPV�HNCpatients. Acquisitionof patient material was separately performed during 2007–2011,following the consistent standards as described in patients andspecimens. RNA was extracted using the RNeasy Kit (Qiagen)following the manufacturer's protocol. RNA concentration wasdetermined by spectrophotometer measurement (NanoVue Plus,GE Healthcare). Random hexamers and MultiScribe ReverseTranscriptase (Applied Biosystems) were used for first-strandcDNA synthesis according to the manufacturer's instructions. Thesamples were added to a 20-mL reaction using 2X Taqman MasterMix (Applied Biosystems) and analyzed on a StepOne Real-TimePCR System (Applied Biosystems) using the following PCRprobes: Hs00169472_m1 for PD-1, Hs00158563_m1 for LAG-3, Hs00262170_m1 for TIM-3, Hs03044418_m1 for CTLA-4,Hs99999905_m1 for GAPDH as a reference gene (all LifeTechnologies).

Flow cytometryFor cell surface staining, PBMCs and TILs were washed twice in

staining buffer (2% w/v fetal bovine serum) and stained for cellsurfacemarkers. Cells were incubated with relevant antibodies for30 minutes at 4�C in the dark, washed twice and resuspended instaining buffer. Flow cytometry was performed using a CyAn flowcytometer (Dako) and Fortessa cytometry (Becton Dickinson),anddatawere analyzed using FlowJo software (TreeStar, Inc.). Theacquisition and analysis gates were restricted to the lymphocytegate based on characteristic properties of the cells in the forwardand side scatter.

Antibodies and reagentsThe following anti-human mAbs were used for staining: PD-1-

APC (eBioscience), CD3-PerCP-Cy5.5, CTLA-4-PE, PD-1-PerCPCy5.5 (all Biolegend), CCR7-FITC (R&D Systems Inc.) CD3-AlexaFluor 405, CD4-PE-Texas Red, CD-8-PE-TR, CD45RA-PE-TR (allLife Technologies), CD3-APC-Cy7, CD8-APC, CD8-PE-Cy7 (allBD Biosciences), granzyme B FITC (clone GB11, Biolegend)including their respective isotypes, which served as negativecontrols for surface as well as intracellular staining. Antibodieswere pre-titrated using activated as well as nonactivated PBMC to

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determine optimal staining dilutions. Viability was assessed byZombie aqua (Biolegend).

Identification of PD-1 subsetsIsolated T cells of healthy donors were stimulated for 5 days

withCD3/CD28 beads. CD3þCD8þ T cells were analyzed byflowcytometry for expression of PD-1 as demonstrated in Supplemen-tary Fig. S1. Accordingly, TILs were analyzed by flow cytometryfor CD8þ PD-1þ cells in reference to isotype controls. Rangingfrom highest detected PD-1 expression to the lower limit ofPD-1þ, the gated PD-1þ cells were divided into cells with highPD-1 (PD-1high), intermediate (PD-1int) and low (PD-1low)expression levels.

Granzyme B expressionIsolated TIL (n ¼ 3 HNC patients) were stained for PD-1

expression of CD3þ CD8þ T cells and then compared for gran-zyme B positivity of PD-1negative, PD-1low, PD-1int, and PD-1high

subgroups.

Sorting of TIL subsets and IFN-g ELISPOTAfter TIL isolation from HNC tumors (n ¼ 3), T cells were

purified using EasySep Human T Cell Enrichment Kit (StemcellTechnologies). After staining, CD8þ T cells were sorted usingMoFlo Astrios (Beckman Coulter) in PD-1–negative, PD-1low–intermediate, and PD-1–high groups. Cells were rested over-night in human serum and compared for functional differences inan IFN-g ELISPOT assay. Therefore, MultiScreen-IP filter plates(Millipore) were coated overnight with anti-human IFN-g mAb1-D1K (Mabtech; 10 mg/mL in PBS) at 4�C. After washing withPBS, plates were blocked for 1 hour at 37�C with 10% humanserum in RPMI. Sorted cells were added to wells in duplicates(5 � 103) and stimulated with anti-CD3/CD28 beads (bead:cell ¼ 5:1). After incubation for 18 hours at 37�C, plates werewashed with PBS/0.5% Tween 20, and incubated with biotiny-lated anti-IFN-g mAb (Mabtech; 2 mg/mL in PBS/0.5% BSA) for 2hours at 37�C. Plates were washed again and incubated withstreptavidin–HRP (Mabtech, 1:500 in PBS/0.5% BSA) for 1 hourat 37�C. After washing, TMB substrate solution (Vector Labora-tories, Inc.) was added. Color development was stopped byextensive washing in tap water after 5 minutes. Plates were driedand spots were counted using CTL ImmunoSpot Analyzer (CTL)and evaluated by CTL Professional Double Color Software.

Murine HNC modelAll animal experiments were performed following institutional

guidelines for animal experimentation. The murine in vivo HNCmodel was established as previously described by Hoover andcolleagues (23). Briefly, C57BL/6 mice were obtained from TheJackson Laboratory (age 1–2 months). Immortalized, E6/E7 plusH-ras–transduced mouse tonsil epithelial cells (MTEC) wereobtained from Hoover (23). After subcutaneous injection of1 � 106 cells into the neck of the mice, 33 mice with tumorgrowth were randomized and assigned into different treatmentgroups. The purpose was to compare the radiation therapy (RT)þanti-PD-1mAb therapy group (n¼ 17) to the RTþ isotype control(clone MOPC-21, mouse IgG1k, BioXcell) group (n ¼ 16).Anti-PD-1 mAb (clone 4H2, IgG1 isotype) was obtained fromBristol-Myers Squibb (24). Anti-PD-1 therapy (3 mg/kg bodyweight) was administered on days 1, 4, 11, and 15. Radiotherapywas administered to induce PD-L1 expression (25) in the tumor

microenvironment in order to enlighten potential protectiveeffects of anti-PD-1mAb therapy. In addition, RT increases tumordeath, phagocytosis, and antigen presentation. Fractionatedradiotherapy was administered for 5 consecutive days at 2 Gy/dayduring week 1 (D4-D8) and week 2 (D11-D15). Treatmentresponse was analyzed by measurement of the tumor volume atintervals between 3 and 18 days. Additionally, mice spleenwere collected and processed as described for lymphocyte isola-tion. PD-1high and PD-1int/low fractions were compared fromCD3þ CD8þ PD-1þ cells and analyzed according to differenttreatment groups. Different clones for treatment (clone 4H2) andstaining (BV421 conjugated anti-PD-1 Ab, IgG2a isotype, clone29F.1A12) were utilized.

Statistical analysisDifferences in phenotypic counts byHPV statuswere calculated

by the Wilcoxon signed rank test for paired data or the Wilcoxontest for independent groups. PD-1þ TILs were divided by flowcytometry into subsets of the proportions of cells classified asPD-1high, PD-1int, and PD-1low. We investigated the impact of theproportions of PD-1high and PD-1low in a cohort of 56 HNCpatients having curative resection and followed up for diseaserecurrence. We used Cox proportional hazards models andcheckedmodels for linearity and for adequacy of the proportionalhazards assumptions. As a heuristic device Kaplan–Meier plots oftime to recurrence were prepared by dividing proportions ofPD-1high and PD-1low cells into tertiles. For the murine experi-ment, tumor volumes of day 18 were compared. By modeling thefixed and random coefficients of a polynomial regression model,we compared the slopes between the two treatment groups. Datarepresent observed mean with bootstrap 95% CI.

Study approvalApproval for the study of specimens from informed and con-

sented HNC patients was obtained by the institutional reviewboard of University of Pittsburgh (#99-069). Study approval foranimal experiments was obtained by the University of Pittsburgh(IACUC# IS00005860).

ResultsExpression of PD-1 and CD8A, CD8B is significantly higher inHPVþ than in HPV� tumor specimens

To investigate the quantitative expression of ICR in HNCpatients, a cohort of 20 HPVþ and 20 HPV� tumors was usedfor analysis by qRT-PCR. ICR expression was normalized to theaverage level of pharyngeal mucosa of healthy donors (n ¼4, Fig. 1A). ICRs show highly variable expression for HPVþ andHPV� tumors in qPCR in relation to reference tissue. In order toconfirm and further investigate these results in a larger cohort, ICRexpression data from TCGA was retrieved, and mRNA expressionof the ICR shown in Fig. 1B–D was analyzed. We found thatexpression of all ICRs and CD8A, CD8B, and CD4 was higher inHPVþ tumors than in HPV� ones. However, more importantlywas the finding that PD-1 was the only ICR that was significantlyhigher inHPVþ tumors alongwith CD8T-cellmarkers: CD8A andCD8B (Figs. 1E and F). In addition to comparing expression datafrom the two cohorts of patients, we correlated the expression ofPD-1, CTLA-4, TIM-3, and LAG-3 with that of CD8A, given thatthismarker of CD8T cells was highly upregulated inHNC tumors,especially inHPVþ samples.We found that all ICRsqueried highlycorrelated with CD8A expression regardless HPV status. However,

Prognostic and Predictive Impact of PD-1high T Cells

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Figure 1.

Expression of PD-1 and CD8A and CD8B is significantly higher in HPVþ than in HPV� tumor specimens. A, qRT-PCR from clinical HNC tumor samples(n ¼ 40) was performed for several ICRs: PD-1, CTLA-4, LAG-3, TIM-3, BTLA, and the Treg transcription factor FOXP3. As a reference, the fold change ofaverage expression of each ICR from healthy donor pharynx mucosa tissues (n ¼ 4) was utilized. ICRs show highly variable expression for HPVþ and HPV�

tumors in qPCR in relation to reference tissue. B–D, Additionally, ICR mRNA expression data retrieved from TCGA were analyzed and compared withhealthy donor mucosa. Expression of all ICRs and CD8A, CD8B, and CD4 was higher in tumors as compared with normal TCGA reference mucosa. E and F,The analysis of the TCGA database revealed that PD-1 was the only ICR that was significantly higher in HPVþ tumors along with CD8 T cells markers CD8Aand CD8B. All ICRs highly correlated with CD8A expression regardless of HPV status. However, HPVþ tumors showed a higher correlation coefficient thanHPV� ones for all ICRs (G). PD-1 expression highly correlated with that of CD8A in both HPVþ and HPV� tumors; CTLA-4 being the other ICR with a slightlyhigher correlation coefficient in HPVþ tumors (G and H).

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HPVþ tumors showed a higher correlation coefficient than HPV�

samples for all ICRs (Fig. 1G). Interestingly, PD-1 expression washighly correlated with that of CD8A in both HPVþ and HPV�

tumors, with CTLA-4 being the other ICR have a similar correla-tion coefficient in HPVþ tumors (Figs. 1G andH). BecausemRNAexpression of ICRs was significantly higher in tumors versuscontrol mucosa, and because PD-1 was a highly expressed genecommon to both HPVþ and HPV� tumors and highly correlatedwith CD8A expression, we decided to further investigate proteinexpression of these ICRs by flow cytometry in different subsetsof T cells in PBL and TIL from HNC patients.

PD-1 is predominantly expressed by CD8þ TEM and tumor-associated antigen-specific CD8þ T cells in the circulation ofHPVþ and HPV� head and neck cancer patients

In order to consider interindividual ICR expression of HPVþ

andHPV�HNC patients' cellular subsets, we analyzed CD8þ PBLfor expression of PD-1 and CTLA-4, the best characterized ICR, inrespect to traditional phenotypic markers (26). We investigatedthe proportions of native and memory T cells of HNC patientsbased on CCR7 (lymphoid tissue homing receptor) and CD45RA(a transmembrane tyrosine phosphatase) expression (Fig. 2A). Asshown in Fig. 2B, the frequency of effector memory (TEM:CCR7�CD45RA�) was significantly higher than na€�ve(CCR7þCD45RAþ), central memory (TCM: CCR7þCD45RA�),and terminal effectors (TEMRA: CCR7

�CD45RAþ) in both HPVþ

and HPV� patients (P < 0.001). The frequency of TEM wascomparable between HPVþ and HPV� patients. Approximately20% of TEM expressed PD-1, which was significantly higher thanthat expressed by na€�ve T cells in both HPVþ and HPV� patients(Fig. 2C, P < 0.05 and P < 0.01 respectively). In HPV� patients,PD-1þ cells in TCM were significantly higher than in na€�ve T cells(P < 0.05). PD-1 single-positive (PD-1þ) T cells from both HPVþ

andHPV� patients were significantly more frequent than double-positive (PD-1þCTLA-4þ) T cells or CTLA-4 single-positive(CTLA-4þ) T cells, indicating that PD-1 was more frequentlyexpressed by T cells in peripheral blood than CTLA-4(P < 0.001, Fig. 2D). However, in peripheral blood, the frequen-cies of PD-1þ T cells between HPVþ and HPV� patients weresimilar. Taken together, these results indicate that PD-1 ratherthan CTLA-4 is frequently expressed by CD8þ effector T cells inboth HPVþ and HPV� patients. These results led us to investigateTILs by flow cytometry from these patient subgroups.

Significantly higher frequencies of PD-1þ CD8þ TIL in HPVþ

HNC patients' tumorsTo evaluate the relevance of ICR in effector cells infiltrating

HPVþ and HPV� HNC, we compared CTLA-4 and PD-1 expres-sion on CD8þ TIL isolated from both HPVþ and HPV� patienttumors. As shown in Fig. 3A, the frequencies of CTLA-4þ andPD-1þ cells were notably higher in CD8þ TIL for HPVþ andHPV�

patients compared with those on CD8þ PBL. Most notably, thefrequency of PD-1þ cells of CD8þ TIL was significantly higherin HPVþ patients than that in HPV� patients (P < 0.006, Fig. 3B).PD-1 positivity was defined by isotype controls through flowcytometry as shown in Fig. 4A.

HPV� tumors contain significantly more PD-1high CD8þ TIL,which are functionally impaired

Others have suggested that higher numbers of intratumoralPD-1þ T cells may correlate with better prognosis in HPVþ HNC

(12), and the role of HPV in HNC patients' response to anti-PD-1therapy is controversial. Furthermore, Wherry has shown that thehighest level of PD-1 expression reflects T-cell exhaustion status(27). Thus, based on positive PD-1 expression, CD8þ TIL weredivided into three subgroups, from highest PD-1 expressing cells(PD-1high), intermediate (PD-1int) to lowest PD-1 positive cells(PD-1low; Fig. 4A). Definition of subgroups was confirmed withPD-1þ subpopulations of CD3/CD28 bead-stimulated healthydonor CD8þ T cells (Supplementary Fig. S1). We previouslyshowed that PD-1 expression level correlates with higher SHP-2 phosphatase, more potent dysfunction, and impaired Th-1signaling (28). Thus, we investigated the activation status andfunctional differences in subsets of PD-1-negative, PD-1low,PD-1int, and PD-1high T cells, taken directly from freshly excisedHNC tumors. As a marker of activation, granzyme B expressionin each of the subsets was analyzed, indicating the highestactivation for PD-1high subsets (Fig. 4B), enriched in HNCpatient TIL (29). To analyze functional capabilities of thesegroups, sorted T-cell populations of negative, moderate(PD-1lowþPD-1int), or high (PD-1high) PD-1 expression werecompared for their IFN-g secretion capacity, after TCR stimu-lation using CD3/CD28 beads. ELISPOT analysis revealedsignificantly compromised IFN-g secretion only in PD-1high

CD8þ T cells, as compared with robust IFN-g secretion byPD-1–negative and moderate PD-1(low/int) T cells (Fig. 4C).HPVþ and HPV� patients were compared based on the propor-tions of PD-1high and PD-1low TIL subsets. The fraction ofPD-1high T-cells was greater in HPV� patients (7.3% vs. 2.1%of PD-1high cells, P ¼ 0.0005 Fig. 4D). In contrast, the propor-tion of PD-1low expressing T cells was higher in HPVþ patientsthan in HPV� patients (65.7% vs. 53.0% P ¼ 0.047, Fig. 4E).

PD-1high cells are associated with worse disease-free survivalBased on the higher frequency of PD-1high T cells in HPV�

patients, we investigated the impact of the extent of PD-1 expres-sion by CD8þ T cells on time to disease recurrence. A cohort ofHNC patients (n ¼ 56, 36 HPV� and 20 HPVþ) all of whom hadcurative therapy for locally advanced disease, were followed fordisease recurrence. With a median follow-up 19 months, 40patients remained free of disease and 16 recurrences wereobserved, yielding a 2-year probability of recurrence-free survivalof 70%, which is typical for our institution (30). A proportionalhazards regression analysis revealed that the proportion ofPD-1high CD8þ T cells potently increased the risk of diseaserecurrence in this cohort of HNC patients (hazard ratio ¼ 2.15,95% CI ¼ 1.46–3.15, P < 0.0001). To validate this finding, weexamined the effect of the proportion of PD-1low cells uponrecurrence and found the opposite impact. Indeed, greater frac-tions of PD-1low CD8þ T cells were protective of recurrence(hazard ratio ¼ 0.19, 95% CI ¼ 0.07–0.49, P ¼ .0006). Figure5A–D illustrates these findings showing Kaplan–Meier curvesarbitrarily divided into tertiles of the proportions of PD-1high

(Fig. 5A) and predicted log hazard ratio (Fig. 5B) and Kaplan–Meier curves arbitrarily divided into tertiles of the proportions ofPD-1low (Fig. 5C) and predicted log hazard ratio (Fig. 5D).

PD-1high CD8þ T cells are dramatically reduced in the setting ofbest treatment response to anti-PD-1 therapy

To investigate if PD-1 targeted Ab therapy influences PD-1T-cell fractions and how therapeutic modalities influence PD-1






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(CCR7- CD45RA-)

TCM

(CCR7+CD45RA-)

Naïve

Naïv

e

Naïv

e

Naïv

e

Naïv

e

(CCR7+CD45RA+)

HPV+ HPV-

Pro

po

rtio

n o

f e

ach

su

bse

t (%

) P < 0.001

P < 0.001

P < 0.001

P < 0.001

HPV+ HPV-

PD

-1+

Ce

lls

(%)

P < 0.05

P < 0.05

P < 0.01

HPV+ HPV-

Pro

po

rtio

n o

f e

ach

su

bse

t (%

) P < 0.001

P < 0.001

P < 0.001

P < 0.001

A

B C

D50

40

30

20

10

0

100

80

60

40

20

0

60

40

20

0

Figure 2.

PD-1 is predominantly expressed on antigen-experienced peripheral TEM cells in both HPVþ and HPV� HNC patients. A, PBL from HNC patients wereanalyzed for coexpression of PD-1 and CTLA-4 and phenotypic markers: effector memory (TEM: CCR7

�CD45RA�) na€�ve (CCR7þCD45RAþ), central memory(TCM: CCR7

þCD45RA�), and terminal effectors (TEMRA: CCR7�CD45RAþ). B, Comparison of phenotypic proportions between HPVþ and HPV� T cells

(one-way ANOVA, Bonferroni's multiple comparison test, HPVþn ¼ 11, HPV�n ¼ 15). C, Percentage of PD-1þ T cells was measured by flow cytometrydepending on phenotype and HPV status (one-way ANOVA, Bonferroni's multiple comparison test, HPVþn ¼ 11, HPV�n ¼ 15). D, Comparison of single-positive (PD-1 or CTLA-4) and double-positive T cells in PBL (one-way ANOVA, Bonferroni's multiple comparison test, HPVþn ¼ 20, HPV�n ¼ 22).

Kansy et al.





expression levels, we used a murine HNC model investigatingtreatment response to anti-PD-1 mAb or its isotype in combina-tion with radiation therapy (RT). Figure 6A demonstrates thedevelopment of tumor volume for the different therapeuticgroups. As expected, better treatment response (i.e., lowest tumorvolumes at day 30)was observed in the combination therapywithanti-PD-1 mAb. Moreover, anti-PD-1 mAb therapy plus RT sig-nificantly reduced the fraction of PD-1high CD8þ T cells andenhanced the frequency of PD-1low cells (Figs. 6B and C), inassociationwith the best treatment response. This beneficial effectreversed the enrichment of PD-1high fractions thatwas observed inthe RT þ isotype control mAb group. This result demonstrates avaluable effect of anti-PD-1 mAb therapy, by dramatically reduc-ing the poor prognostic subpopulation of PD-1high TIL cells, andpermitting potent antitumor activity to develop via PD-1low/int,IFN-g–positive T cells.

DiscussionIn peripheral blood, we observed that PD-1 ismainly expressed

on effector T cells rather than by na€�ve T-cell populations. Despitean overall higher PD-1þCD8þ T-cell frequency in the TIL ofHPVþ

HNC patients, HPV� patients had larger fractions of PD-1high

CD8þ T cells. We demonstrate that these PD-1high expressing CTLwere highly activated (expressing the most granzyme B), butshowed a severely dysfunctional phenotype, with compromisedIFN-g secretion capacity and negative prognostic impact in theclinic. In a murine PD-1 targeting immunotherapy model, wewere able to observe that anti-PD-1 therapy overcomes theincrease of PD-1high expressing cells that could be observed inmice treated with RT alone, which drives PD-L1 levels within24 hours of treatment (25). Furthermore, in contrast to PD-1high

CD8þ T cells, frequencies of PD-1low/int T cells were associatedwith a better disease-free survival (DFS) in our cohort of HNCpatients. These beneficial, activated T cells were significantlyincreased upon anti-PD-1 mAb therapy in the murine HNCmodel, correlating with clinical efficacy.

Clinical trials targeting ICR have shown substantial results formultiple cancer subsets including melanoma, renal cell carcino-ma, non–small cell lung cancer (31, 32) and recently HNC (7).Nevertheless, the knowledge about ICR inHNC tumors and TIL islimited, but is of interest due to emerging immunotherapeutictargeting of these molecules in several cancer types (33). Whilecancer immunotherapy targeting ICR has emerged as a majortherapeutic advance (34), biomarkers of response are inadequateand have so far focused on ligand expression (i.e., PD-L1) by

CD3+CD8+

CTLA-4

PD

-1

0.1 0.2

0.7

4.0 0.2

0.6

7.2 13.4

14.3

0.2 0.2

0.4

8.5 0

2.3

12.4 20.0

17.1

HPV+

HPV-

TILPBLControl

PD

-1+

Ce

lls

(%)

CT

LA

-4+

Ce

lls

(%)

P = 0.006 P = 0.0004

ns

P = 0.002 P < 0.0001

P = 0.006

TILPBL

HPV+

TILPBL

HPV-

TILPBL

HPV+

TILPBL

HPV-

A

B60

40

20

0

80

60

40

20

0

Figure 3.

HPVþ HNSCC patient TIL demonstratehigher overall expression of PD-1. Thefrequency of CTLA-4þand PD-1þ cellswas compared by flow cytometry inCD8þ TIL and PBL from HPVþ and HPV�

patients. A, Representative gating andstratification and sorting of TIL by extentof the PD-1 level of expression. B,Comparison of CTLA-4þ and PD-1þ inPBMC and TIL of HPVþ and HPV�

(matched-pair Wilcoxon test forcomparison of TIL vs. PBL, Mann–Whitney test for comparison HPVþ vs.HPV� TIL, HPVþn ¼ 10, HPV�n ¼ 16).






tumor or immune cells. Here, we propose that response toanti-PD-1 mAb therapy may be influenced by levels of PD-1on effector T cells in the circulation or the microenvironment.The effect we observe here may reflect salvage and protection

of activated functional T cells during tumor response to anti-PD-1 therapy.

Upon ligation, PD-1 directly inhibits TCR-mediated effectorfunctions and thereby causes T-cell dysfunction (35). Despite the

CD3+CD8+ TIL

CD8

PD

-1

Control PD-1+ cells Level of PD-1 expression

PD-1high

PD-1low

PD-1int

3.6%

39.3%

58.4%

HPV- HPV+ HPV- HPV+

PD

-1h

igh

(%)

PD

-1lo

w(%

)

P = 0.047P = 0.005

20

10

30

40

50

0

20

60

80

40

100

A

0

100

200

300

400

500

IFN

g Sp

ots

/10

0,0

00

ce

lls

CD8+ TIL ELISPOTCB

Gra

nzy

me

B (

%)

CD8+ TIL

ED

60

40

20

0

Figure 4.

HPV� tumors contain significantly more PD-1high CD8þ TILs that manifest an exhaustion phenotype and produce little IFN-g . A, The range from highestdetected PD-1 to the negative control (PD-1negative) was divided into the three subgroups with low (PD-1low), intermediate (PD-1int), and high (PD-1high)PD-1-expressing cells. B, Flow cytometric staining for CD3þ, CD8þ, and granzyme B on representative TIL (n ¼ 3, two HPVþ and one HPV�), gated on PD-1negative,PD-1low, PD-1int, and PD-1high (n ¼ 3, one-way ANOVA, P ¼ 0.03). C, ELISPOT analysis for IFN-g secretion per 100,000 cells of CD8þ TIL (n ¼ 3, two HPVþ

and one HPV�, distinct tumor samples from B), sorted into PD-1negative-, PD-1lowþint-, and PD-1high-expressing subsets (n ¼ 3, one-way ANOVA P ¼ 0.01). D and E,Box and whiskers plots for PD-1high (D) and PD-1low (E) fractions of HPVþ and HPV� patients (Mann–Whitney comparison, HPVþn ¼ 20, HPV�n ¼ 36).

Kansy et al.





immune-inhibitory pathways that are activated upon PD-1engagement, reports of PD-1 positivity in TIL and its prognosticimpact have been controversial to date. PD-1 expression reflectsT-cell activation but means only that a T cell is receptive to animmunoinhibitory signal, not that the T cell is necessarilyexhausted and dysfunctional. Our results demonstrate that partof this controversy might be attributed to the limitations ofgrouping PD-1 positive cells despite heterogeneous functionalcapacity. Indeed, we show that PD-1high versus PD-1low T cellspossess distinct and important functional differences with signif-icant prognostic importance.

In a recent meta-analysis of 29 studies investigating PD-1expression and overall survival (OS) in patients with epithelialmalignancies, PD-1 expression by TIL was associated with ashorter OS (36). In contrast, Badoual and colleagues reportedPD-1-expressing tumor-infiltrating T cells as a favorable prognostic

marker in HPVþ HNC (12). This conundrum intrigued us due tothe potential for the level of PD-1 expression to more accuratelymark different TIL subsets with unique behavior. Also, distinctetiologies of HNC such as HPVþ/� disease, with such dramaticallydifferentprognoses (37), providedanunparalleledwindowon thispotential biomarker.HPVþHNCrepresents a unique cancer subset(38), given its location in lymphoepithelial tissue with specificimmunologicalproperties (39), virally associated immuneevasionproperties (40) and yet better clinical prognosis than non–HPV-associated HNC (37). Therefore, we investigated immunologicalproperties like ICR expression of both patient subgroups in anti-gen-specific cells from peripheral blood and tumor tissue. Ourfindings in CD8þ TIL confirm an overall increased number ofPD-1þ T cells for HPVþ patients. Most importantly, when inves-tigating the extent of PD-1 expression in PD-1þ cells, we observedsignificant differences between HPVþ and HPV� patients. HPVþ

Disease-free survival

by % PD-1low tertile

Pro

po

rtio

n w

ith

ou

t re

curr

en

ce

Lower PD-1low tertile

Middle PD-1low tertile

Upper PD-1low tertile

Months

Pro

po

rtio

n w

ith

ou

t r

ecu

rre

nce

Disease-free survival

by % PD-1high tertile

Months

Lower PD-1high tertile

Middle PD-1high tertile

Upper PD-1high tertile

PD-1high (%)

3020100

0Re

lati

ve

ha

zard

(lo

g)

–0.5

Relative risk of recurrence in

dependence of PD-1high

2.0

1.5

1.0

0.5

PD-1low (%)

Re

lati

ve

ha

zard

(lo

g)

80604020

Relative risk of recurrence in

dependence of PD-1low

–1

–2

0

1

2

0 6 12 18 24 30 36

363024181260

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

A B

C D

Figure 5.

PD-1high TILs are associated with worse DFS, whereas high levels of PD-1low TILs portend better clinical outcome. A, Kaplan–Meier curves portray DFS for HNCpatients (HPVþn ¼ 20, HPV�n ¼ 36) in relation to PD-1high vs. PD-1low fractions of CD8þ TIL. Tertiles for PD-1high fractions were distinguished and survivalplotted as shown (ranges of lower tertile, 0–1.75; middle tertile, 1.75–8.7; upper tertile, 8.7–51). Cox proportional hazardsmodels were used to investigate the relativerisk and models were checked for linearity and adequacy of the proportional hazards assumptions. B, Hazard ratio ¼ 2.25 (95% CI, 1.46–3.15),P < 0.0001. C, Tertiles for PD-1low fractions were distinguished and survival plotted as shown (ranges of lower tertile, 9.3–49.5; middle tertile, 49.5–78.5;upper tertile, 78.5–99.4). D, Hazard ratio ¼ 0.19 (95% CI, 0.07–0.49, P < 0.0006).






patients had a significant lower fraction of the PD-1high-expressingcells in comparison with the HPV� patients. In contrast, thePD-1low fraction was significantly elevated in HPVþ patients incomparison with HPV� patients. In combination with the shownfunctional differences of PD-1low and PD-1high CD8þ TIL, theseresults might contribute to a better understanding of prognosticdifferences between HPVþ and HPV� patients, and additionally,account for correlations of PD-1þ cells and favorable prognostics.Therefore, our analysis ofDFS focused on the impact of fractions ofPD-1high and PD-1low CD8þ TIL.

Here, we show that the patients with higher PD-1high fractionshad a significantly higher risk of recurrence (2.25-fold increasedrisk as this fraction of cells increased). On the contrary, highPD-1low fractions were associated with improved DFS. Thesedata support an important role for T-cell exhaustion in progres-sion of disease, with the possibility that rescue of the PD-1low/int

cells represents the mechanism of clinical benefit as well as amore accurate biomarker of clinical response to anti-PD-1/PD-L1targeting immunotherapy. In the setting of chronic viral infection,Blackburn and colleagues were able to identify a subset ofexhausted T cells, which can be rescued through anti-PD-1 target-ing therapy (10). The data from ourmurine HPVþmodel supportthese observations in the case of cancer-associated T-cell exhaus-tion as demonstrated in treatment groups with anti-PD-1 treat-ment, which showed an increase of PD-1low-expressing T-cellfractions. Moreover, radiotherapy treatment response wassignificantly increased through the addition of PD-1 targetingimmunotherapy.

Our data show that the effector memory cell subset of HNCpatients more highly expresses PD-1. As PD-1 has been shown tonegatively regulate T-cell phenotype, proliferation, and insteadinduces apoptosis (13, 14, 28), the combination of our observa-tions linking PD-1 and peripheral effector memory subsets in vivosupports the association of PD-1 with clonally expanded, tumor-reactive populations (11). In this situation, ligation of PD-1 by itsligands is likely, and even more so for the PD-1high-expressingpopulations. This may explain the vulnerability and functionalinsufficiency of antigen-specific T-cell expansion in the tumormicroenvironment. Indeed, Fourcade and colleagues reportinverse correlations of expansion and PD-1 levels in a melanomavaccine study (41). In our study, we observed a protective effectfrom these negative regulatory pathways in case of anti-PD-1treatment, as demonstrated by the significant increase of thePD-1low/int fractions in the murine anti-PD1 therapy model. Theantitumor activity of the PD-1low/int populations is also supportedby the improved treatment response in this group. Additionally,analyzed CD8þ T cells were obtained from splenocytes, whichrepresent circulating T cells. Therefore, in our point of view, theincrease in PD-1low fractions represents more likely a clonalexpansion of PD-1þ antitumor effective T-cell populations, per-haps detectable in the circulation of anti-PD-1–treated patients.

By separating different extent of PD-1 expression levels inCD8þ

TIL, we observed that cells of high PD-1expression levels possess aseverely impaired IFN-g secretion capacity, in contrast to PD-1low

and PD-1int T cell fractions. This is supported by the fact that infreshly isolated TIL, the PD-1neg groups secrete lower IFN-g after

250

500

750

1816141141Days

Tu

mo

r v

olu

me

(m

m3)

Anti-PD-1 Ab (n = 17)

Isotype Ab (n = 16)

P = 0.003

P = 0.002P = 0.002

% C

D3

+P

D-1

hig

h T

Ce

lls

0

10

20

30

% C

D3

+P

D-1

low

/in

tT

Ce

lls

RT + Anti-PD-1Ab

RT + Isotype100

90

80

70

RT + Anti-PD-1Ab

RT + Isotype

A

B C

Figure 6.

Dysfunctional PD-1high CD8þ T cells aredramatically reduced in the treatment responsegroup to anti-PD-1 plus RT targeted therapy.A, 33 mice with positive tumor growth wererandomized into two treatment groups (n¼ 16 forisotype þ RT; n ¼ 17 for anti-PD-1 Ab þ RT).Treatment response was analyzed bymeasurement of the tumor volume on differentdays. A comparison of radiotherapy alone(isotype) and radiotherapy in combination withanti-PD-1 Ab treatment was performed. Bymodeling the fixed and random coefficients of apolynomial regression model, we conclude thatthe slopes between two treatment groups differand that RT þ anti-PD-1 Ab-treated tumors grewmore slowly than RT þ isotype–treated tumors.Data represent observed mean with bootstrap95% CI (P ¼ 0.0039). Anti-PD-1 Ab or isotypecontrol (3 mg/kg body weight) was administeredat days 1, 4, 11, and 15; radiotherapy wasfractionated in 2 Gy�10 days (on days 4, 5, 6, 7, 8,11, 12, 13, 14, and 15). B and C, PD-1 expression ofisolated splenic CD3þ PD-1þ lymphocytes,PD-1high (B), and PD-1int/low (C) fractions of RT þisotype vs. RT þ anti-PD-1 Ab was comparedwith nonparametric Mann–Whitney test, data arerepresentative of � SEM.

Kansy et al.





stimulation in the ELISPOT assays, with increasing differences ofIFN between PD-1low/int and PD-1high after 18 hours of stimula-tion during ELISPOT. These results are in congruence with reportsof Flecken and colleagues, who showed reduced IFNg secretionafter expansion of tumor-associated antigen-specific CD8þ T-cells(42). This impairment did not resolve after Treg depletion andsupports direct involvement of PD-1 ligation and thereby down-regulation of functional cytokine production. A study of Zhangand colleagues identified alteration of IFNg levels depending onPD-1 polymorphisms, guiding to a direct involvement of PD-1receptor pathways on IFNg production (43). Additionally, PD-1blockade has been associated with an increased IFNg productionand elevation of IFNg–dependent associated chemokines (44).These relationships may contribute to difficulties in separatingreasons for—and effects of—both PD-1 and PD-L1 expression.PD-1–positive cells have been associated with high TA specificity.The release of cytokines like IFNg of these TA-specific cells in thetumor microenvironment has been shown to induce PD-L1 withnegative impact on function and cytokine production of PD-1þ

cells. Therefore, a more sophisticated view of different PD-1expression levels is needed.

In their analysis of tissue-specific differences in PD-1 expressionof T cells, Blackburn and colleagues describe that their ability todegranulate is dependent on the PD-1 expression levels, inde-pendent of anatomical location and thereby PD-L1 expression(45). This is supported by our findings of higher granzyme Bpositivity for PD-1high-expressing TIL. Therefore, the consider-ation of different PD-1 expression levels exemplifies the twodisparate interpretations of PD-1 expression as a marker ofactivated, competent tumor reactive T cells, on the one hand,and PD-1high expression as a marker of exhausted, dysfunctionalcells with a negative influence in the tumormicroenvironment onthe other hand.

Our results are supported by the fact that anti-PD-1 therapy hasbeen reported to be effective in HPV� than HPVþ patients(46, 47). Thus, it is possible that higher frequencies of PD-1high

T cells fromHPV� patients may contribute to worse prognosis, aswell as to a good response to anti-PD-1 immunotherapy. Inconsequence, we demonstrate that PD-1 expression in CD8þ TILof HNC represents (over-) activation and—at highest expressionlevels—exhaustion of effector CD8þ T cells that is associated withbetter clinical outcome. Thereby, we conclude two essential con-sequences for immunotherapy: first, low/intermediate but posi-tive PD-1 expression levels are favorable in termsof tumor-specificfunctionality and prognosis, and, second, these data support thatPD-1 blockade may allow the expansion of TA-specific T cells,keeping their PD-1 expression at lower levels with enhanced

antitumor response and reduced susceptibility to PD-L1 ligation,indicating a possible predictive role of the level of PD-1 expressionon TIL. Whether this effect is due to active restoration of prolif-eration of PD-1high cells or enhanced proliferation and responsesof PD-1low cells has to be investigated. Further studies mustvalidate ourfinding using anti-PD-1mAb–treated cancer patients,includingHNC. Baseline PD-1high versus PD-1low levels should bemeasured and correlated with responder status.

Disclosure of Potential Conflicts of InterestG.J. Freeman has ownership interest (including patents) in Roche, Merck,

Bristol Myers Squibb, Novartis, EMD-Serono, AstraZeneca, Boehringer-Ingel-heim, Roche, and Novartis and is a consultant/advisory board member forRoche, Novartis, Eli Lilly, Bristol-Myers Squibb, Seattle Genetics, Bethyl Lab-oratories, Surface Oncology, and Novartis. R.L. Ferris is a consultant/advisoryboard member for Astra-Zeneca/MedImmune, Bristol-Myers Squibb, Lilly,Merck, and Pfizer. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: B.A. Kansy, H.-B. Jie, Y. Lei, R.L. FerrisDevelopment of methodology: B.A. Kansy, R.M. Srivastava, H.-B. Jie, D.A.Clump, R.L. FerrisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.A. Kansy, R.M. Srivastava, H.-B. Jie, G. Shayan, Y.Lei, J. Moy, J. Li, N.C. Schmitt, D.A. Clump, R.L. FerrisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):B.A. Kansy, F. Concha-Benavente, R.M. Srivastava,H.-B. Jie, G. Shayan, J. Moy, J. Li S. Lang, G.J. Freeman, W.E. Gooding, R.L. FerrisWriting, review, and/or revision of the manuscript: B.A. Kansy, F. Concha-Benavente, H.-B. Jie, J. Moy, S. Brandau, S. Lang, N.C. Schmitt, G.J. Freeman, W.E. Gooding, D.A. Clump, R.L. FerrisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Moy, J. Li, R.L. FerrisStudy supervision: R.L. Ferris

AcknowledgmentsWe would like to thank Bratislav Janjic and Michael Meyers from the Flow

Cytometry Facility for excellent technical assistance.

Grant SupportThis work was supported by National Institute of Health grants R01

CA206517, DE019727, P50 CA097190, T32 CA060397 (R. L. Ferris), theUniversity of Pittsburgh Cancer Institute award P30 CA047904 (R. L. Ferris),P50CA101942 (G. J. Freeman), DE024173 (Y. Lei), and the IFORES program ofUniversity of Duisburg-Essen (B. A. Kansy).

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 November 26, 2016; revised May 10, 2017; accepted August 24,2017; published OnlineFirst September 13, 2017.

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Kansy et al.




2017;77:6353-6364. Published OnlineFirst September 13, 2017.Cancer Res Benjamin A. Kansy, Fernando Concha-Benavente, Raghvendra M. Srivastava, et al. Therapeutic Outcomes in Head and Neck Cancer

T Cells Associates with Survival and Anti-PD-1+PD-1 Status in CD8

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