PD-L1MediatesDysfunctioninActivatedPD-1þ NK Cells in Head … · tumor-inﬁltrating PD-1þ NK...

Research Article

PD-L1MediatesDysfunction inActivatedPD-1þNKCells in Head and Neck Cancer PatientsFernando Concha-Benavente1,2, Benjamin Kansy3, Jessica Moskovitz1,Jennifer Moy1, Uma Chandran4, and Robert L. Ferris1,2,5

Abstract

Inhibitory immune-checkpoint receptors (ICRs), includingprogrammed death 1 (PD-1), have been characterized asexhaustion markers on T cells that infiltrate the tumor micro-environment (TME) ofmany cancer types, including head andneck cancer (HNC). However, expression and function ofICRs, including PD-1, on natural killer (NK) cells remains lessdefined. NK cells are innate immune effector cells that lyseepidermal growth factor receptor–overexpressing HNC cellsvia cetuximab-mediated antibody-dependent cytotoxicity.Cetuximab is clinically effective but only in 10% to 15%of patients. Therefore, it is necessary to investigate how immu-nomodulation with cetuximab or PD-1 blockade mightenhance NK cell responses in the TME and improve mono-clonal antibody therapeutic efficacy. We observed that expres-sion of PD-1 on NK cells marks an activated phenotype,

whichwas suppressed only after binding programmed deathligand-1 (PD-L1). HNC patients who exhibit higher circu-lating PD-1þ NK cells associate with better clinical outcome,and these cells are enriched in the TME. Cetuximab-mediated NK cell activation increased PD-1 expression onNK cells in vitro,which was confirmed in vivo in a prospectiveneoadjuvant cetuximab trial. In contrast, PD-L1 ligationof PD-1þ NK cells diminished their activation status, where-as PD-1 blockade increased cetuximab-mediated NK cellactivation and cytotoxicity, but only against HNC targetswith high PD-L1 expression. Therefore, blocking the PD-1–PD-L1 axis may be a useful strategy to reverse immuneevasion of HNC tumors with high PD-L1 expression duringcetuximab therapy by reversing NK cell dysfunction.Cancer Immunol Res; 6(12); 1548–60. �2018 AACR.

IntroductionInhibitory immune-checkpoint receptors (ICRs) such as PD-1,

T-cell immunoglobulin andmucin-domain containing-3 (TIM-3),and cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) havebecome important targets in cancer immunotherapy. PD-1has been studied in several immune cell subsets, includingCD8þ T cells, B cells, and dendritic cells (DCs), in the tumormicroenvironment (TME; refs. 1, 2). PD-1 expression on T cells iscoexpressed with activation markers such as Th1 transcriptionfactors STAT1 and T-bet, and cytokines IFNg and IL12 afterCD3/CD28 stimulation (3). However, binding of PD-1 with itscognate ligands, programmed death ligand 1 and 2 (PD-L1 andPD-L2), mediates T-cell exhaustion and immuno-escape (4–7).In the setting of head and neck cancer (HNC), we previouslydocumented that the majority of tumors express PD-L1 (8) and

harbor a high frequency of PD-1þ T cells (9–12). Blocking thePD-1–PD-L1 axis has shown encouraging results in the treatmentof several cancers, including melanoma, lung cancer, and HNC(13, 14), and PD-1 expression has been characterized in tumor-infiltrating T cells. However, less is known about PD-1 expressionand function on NK cells, despite their importance in bridginginnate and adaptive immunity and mediating monoclonal anti-body (mAb)–specific antitumor responses (15).

NK cells play a crucial role in tumor immunosurveillance,with a capacity of killing cancer cells without prior sensitiza-tion. NK cell dysfunction has been associated with increasedrisk of leukemia, gastric cancers, and HNC (16–19) and poorclinical prognosis (20–22). Therefore, reversing NK cell dys-function should improve cancer immunotherapy. NK cellsmediate cytotoxicity via CD16-mediated antibody-dependentcellular cytotoxicity (ADCC), particularly in the setting ofHNC, where the majority of tumors overexpress EGFR (15).In this setting, NK cells bind the Fc portion of cetuximab, anEGFR-specific IgG1 mAb, lyse tumor targets, and secreteTh1 cytokines. These effects activate DCs and promote cross-presentation of tumor antigen (TA)–specific cytotoxic Tlymphocytes (CTLs; refs. 15, 23). However, the benefit ofcetuximab-mediated immunotherapy is seen only in 10% to20% of patients (23–25). One explanation may be PD-L1–mediated suppression of tumor-infiltrating PD-1þ NK cells.

Circulating and tumor-infiltrating PD-1þ NK cells are foundin higher frequency in sarcoma, multiple myeloma, and ovar-ian cancer patients, and PD-1 blockade reversed their dysfunc-tional phenotype (26–28). However, whether PD-1 expressionon NK cells represents a dysfunctional subset in HNC patientsis still unclear. Therefore, we investigated circulating and

1Department of Otolaryngology, University of Pittsburgh, Pittsburgh,Pennsylvania. 2University of Pittsburgh Hillman Cancer Center, Pittsburgh,Pennsylvania. 3Department of Otorhinolaryngology, University HospitalEssen, Essen, Germany. 4Department of Biomedical informatics, Universityof Pittsburgh, Pennsylvania. 5Department of Immunology, University ofPittsburgh, Pittsburgh, Pennsylvania.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Robert L. Ferris, University of Pittsburgh CancerInstitute, 5117 Centre Avenue, Room 2.26b, Hillman Cancer Center ResearchPavilion, Pittsburgh, PA 15213. Phone: 412-623-3205; Fax: 412-623-4840; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-18-0062

�2018 American Association for Cancer Research.

CancerImmunologyResearch

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tumor-infiltrating PD-1þ NK cells in HNC patients and alsodetermined the expression and correlation of the NK cellmarker NKp46 (NCR1), as well as PD-1, TIM-3, and CTLA-4,in tumors and paired control tissues from a large cohort ofHNC specimens in The Cancer Genome Atlas (TCGA). Wetested whether cetuximab-mediated NK cell activation wouldfurther increase PD-1 in vitro and in vivo, testing specimens froma neoadjuvant single-agent cetuximab clinical trial. We proposethat activated PD-1þNK cells might become dysfunctional onlyafter PD-L1 ligation. Taken together, our findings support theuse of combinational anti-EGFR and anti–PD-1 therapy in theclinic to enhance NK cell–mediated cytotoxicity.

Materials and MethodsPatients and specimens

All patients included in this report (n ¼ 74) gave writteninformed consent, as approved by the institutional review board(IRB #99-06). Peripheral blood samples were obtained fromnontrial HNC patients or stage III/IVA trial patients receivingneoadjuvant cetuximab (400 mg/m2/day on day 1, then 250mg/m2/day on days 8–15) on a prospective phase II clinicaltrial (UPCI 08-013, NCT 01218048) conducted in accordancewith the ethical standards of the Declaration of Helsinki.Tumors were biopsied before and after 4 weeks of cetuximabtherapy. Clinical response was analyzed by comparing pairedCT scans of tumors pre/post-cetuximab and quantifying tumormeasurement by a head and neck radiologist blinded to patientstatus. Anatomic tumor measurements were recorded in twodimensions, and the cohort segregated into clinical "responders,"who showed a reductionof 10% to30% in tumor volume, "partialresponders"whose tumors showeda reductionof less than10%ofvolume and "nonresponders," whose tumors grew during thistherapy. Tumor biopsies (pretreatment) or surgical tumor speci-mens (posttreatment) were preserved for amaximumof 12 hoursin complete media until tumor-infiltrating lymphocytes wereisolated and cryopreserved until analysis.

Tumor-infiltrating lymphocyte (TIL) isolationFresh tumors fromHNCpatientswereminced into small pieces

manually or using a gentleMACS dissociator using the preloadedprogram h_tumor_03.01 (Miltenyi Biotec) with no enzymaticsolution added, then transferred to 70-mm cell strainers (BD) andmechanically separated using the plunger of a 5-mL syringe. Thecells passing through the cell strainer were collected and washedtwice with RPMI media (Sigma-Aldrich) plus 10% fetal bovineserum (Corning). After centrifugation, mononuclear cells wererecovered and used immediately or cryopreserved in the case ofperipheral blood lymphocytes (PBLs) from active disease patientsor PBL and TIL samples from the UPCI clinical trial #08-013 andthawed immediately before phenotyping experiments.

Peripheral blood mononuclear cells (PBMC) and NK isolationfrom healthy donors and HNC patients

Blood from healthy donors (Central Blood Bank), patientswith active HNC, or from the UPCI clinical trial #08-013NCT#01218048 was withdrawn, and PBMCs were purified byFicoll-Paque PLUS centrifugation following a standard protocol(Amersham Biosciences). PBMCs from healthy donors used in allin vitro experiments in this report were used fresh after isolation,and NK cells were purified using a NK cell–negative selection

magnetic EasySep kit (catalog# 19051) following the manufac-turer's protocol (Stem Cell Technologies). Purity of the selectionwas >95% CD3�CD56þ CD16þ as assessed by flow cytometry.PBMCs from active disease HNC patients with at least 3-yearsurvival data available (n¼50)orUPCI clinical trial #08-013wereused from our cryopreserved inventory, thawed for 1 minute at37�C, washed twice with warm FBS-enriched RPMI medium, andused for flow cytometry staining.

Coculture of NK cells using hIgG1 or PD-L1–coupled beadsPD-L1–hIgG1 Fc fusion protein (R&D Systems) or control

human IgG1 (Southern Biotech) were covalently coupled toDynabeads M-450 according to the manufacturer's protocol (LifeTechnologies). We kept constant the total amount of protein at5 mg per 107 beads as previously described (29). Briefly, 107 beadswere coated with 50 mg/mL of either PD-L1–hIGg1 Fc fusionprotein or control human IgG1. NK cells were freshly isolatedfrom healthy donors' PBMCs and subjected to coculture experi-ments. NK cells were cultured with beads at a fixed cell:bead ratioof 1:20 for 24 hours and analyzed by flow cytometry.

Tumor cell linesJHU029 cells were a kind gift from Dr. James Rocco (Harvard

Medical School, Boston, MA) in January 2006. 93-VU-147 T(called 93VU in this report) was a kind gift from Dr. HenningBier (Technische Universitat Munchen, Munich, Germany) inOctober 2013. SCC90 cells were isolated from patients treatedat the University of Pittsburgh through the explant/culturemethod, authenticated, and validated using short-tandemrepeat profiling and HLA genotyping. All cell lines were routinelytested every 6months and found to be free ofMycoplasma infectionand were cultured in IMDM (Invitrogen) supplemented with 10%FBS (Mediatech), 2% L-glutamine, and 1% penicillin/streptomycin(Invitrogen Corp).

Antibodies and treatmentsMouse anti-human CD3-PerCPCy5.5 or AF700 (clone

UCHT1), CD56-FITC (clone B159 or NCMA16.2), PD-1-APC(clone MIH4), PD-L1-PE (clone MIH1), CD16-PECy7 (clone3G8), CD107a-PE (clone H4A3), GranzymeB-PE-TxRed orCF594 (clone GB11), IFNg-APC-Cy7 (clone 4SB3), TIM-3-PE(clone F382E2), CTLA-4-PECy5 (clone BNI3), CD69-PerCpCy5.5, KIR-PE (clone DX27), CD69-PECy7 (clone FN50),CD96-APC (clone NK92.39), and NKp46-PE (clone 9E2) werepurchased from BD Pharmingen. Zombie aqua viability dyewas purchased from BioLegend. Human recombinant IL2 waspurchased from R&D Systems reconstituted according the man-ufacturer's instructions. For NK cell activation, rhIL2 was usedat 130 IU/mL for 24 hours following a previously validatedprotocol (28). NKp46-Fc chimera and IgG1-Fc control werepurchased from R&D Systems. Mouse anti-human anti-IFNgblocking antibody was purchased from R&D Systems and usedat 50 ng/mL. Cetuximab and nivolumab were kindly providedby Bristol-Meyers Squibb and used at 10 and 20 mg/mL,respectively. Panitumumab was kindly provided by Amgen andused at 10 mg/mL in all in vitro experiments.

Flow cytometryCells were harvested and resuspended in PBS containing a 1:50

dilution of viability dye Zombie Aqua (30) for 10 minutes atroom temperature and washed following the manufacturer's

PD-1/PD-L1 Axis Prevents NK Cell Cytotoxicity in HNC

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protocol (BioLegend). Cells were then resuspended in 50 mL offluorescence-activated cell sorting (FACS) buffer (BD Bioscience),and fluorophore-conjugated antibodies were added at 1:25dilution, incubated for 15 minutes at 4�C. Antibodies werewashed away twice by sequential centrifugation at 1400 RPMwith FACS buffer and acquired in a BD Fortessa flow cytometerusing FACSDiva software. Analysis of flow cytometry files wasperformed using FlowJo v 10.7 software.

ELISADetermination of IFNg in cell culture supernatants was per-

formed using the human IFNg ELISA kit (catalog# DIF50; R&DSystems) and used according to the manufacturer's protocol.

ADCC assayCytotoxicity was determined using a 51Cr release assay. Briefly,

target cells were incubated in 100 mL of media with 25 mCi ofNa2

51CrO4 (PerkinElmer) for 60 minutes at 37�C and resus-pended in RPMI 1640 medium supplemented with 25 mmol/LHEPES. Cells were thoroughly washed and plated at 20:1 effector-to-target ratio in 96-well plates, and then treatments (mAbs,10 mg/mL) and freshly purified NK cells were added. Plates wereincubated for 4 hours at 37�C in a 5% CO2 atmosphere. Controlsfor spontaneous (cells only) andmaximal lysis (cells treated with1% Triton-X) were included. Each reaction was done in triplicateand repeated three times. The supernatants were collected andanalyzed with a PerkinElmer 96-well plate gamma counter. Per-cent specific lysis ¼ (experimental lysis � spontaneous lysis)/(experimental lysis � maximal lysis) � 100.

TCGA data retrieval and analysisTCGA data for HNC gene expression by RNA-seq were down-

loaded from the c-Bio portal (http://www.cbioportal.org) or theUCSC cancer genomics browser (https://genome-cancer.ucsc.edu). TheHNC gene-expression profile wasmeasured experimen-tally using the Illumina HiSeq 2000 RNA-seq platform by theUniversity of North Carolina TCGA genome characterizationcenter. Data were downloaded from the TCGA data coordinationcenter and showed the gene transcription estimates, as in RSEMnormalized count, percentile ranked within each sample. Geneswere mapped onto the human genome coordinates using UCSCcgData HUGOprobeMap. The RSEMunits to quantitate RNA-seqexpression data were described and validated previously (31).Correlations and linear regression curve fits from TCGA data werecalculated using GraphPad PRISM software version 6 and valueswere plotted into graphs.

IHC protocolThe University of Pittsburgh IRB #99-069 approved the use of

clinical samples, and written informed consent was obtained.Slides were deparaffinized and rehydrated. Antigen retrieval wasperformed using Diva Retrieval (Biocare Medical) and a decloak-ing chamber at 124�C, 3 minutes, and cooled for 10 minutes.Slides were placed on an Autostainer Plus (Dako) using a TBSTrinse buffer (Dako) and stained using 3% H2O2 (Thermo FisherScientific) for 5 minutes, CAS Block (Invitrogen) for 10 minutes,and the primary antibody for PD-L1 (6.2 mg/mL final concentra-tion, clone 405.9A11, kindly provided byGordon J. Freeman)wasused as previously reported (32). The secondary consisted ofEnvision Dual Link þ (Dako) polymer for 30 minutes, rinsed,then a TBSTholding rinsewas applied for 5minutes. The substrate

used was 3,3,-diaminobenzidine þ (Dako) for 7 minutes andcounterstained with hematoxylin. PD-L1 staining was quantifiedby positive pixel count v9 algorithm (Aperio). A head and neckpathologist blinded to clinical patient data examined tumorsections. Scoring was determined by the percentage of tumorstained for PD-L1. Tumors with

Statistical analysisNonnormally distributed data were analyzed with nonpara-

metric tests. For two groups of independent variables, the Mann–Whitney test was used. For three or more groups of independentvariables, the Kruskal–Wallis test. For two groups of pairedsamples, the Wilcoxon test was used. For normally distributeddata, parametric tests were used: for two groups of independent orpaired variables, we used the Student t test. For three or moregroups of independent variables, we used ANOVA. Kaplan–Meiersurvival curves were plotted and analyzed using log-ranktest (Mantel–Cox). Correlations were analyzed using Personr coefficient test. For all statistical analyses, differences wereconsidered statistically significant when P < 0.05.

ResultsMore PD-1þNK cells in circulation and tumors ofHNCpatientsassociates with better outcome

Using PBLs isolated from new active disease HNC patients,we observed that circulating NK cells (live CD3–CD56þ) weresignificantly more frequent in early-stage HNC patients [T1(44%) N0 (77%; Table 1 and Fig. 1A, right, n ¼ 9, black solidcircles)] compared with patients with late-stage disease [T1(24%) N0 (24%; Table 1 and Fig. 1A, n ¼ 41, dark gray circles)]who had fewer circulating NK cells. Significantly higher fre-quencies of circulating PD-1þ (Fig. 1B) and TIM-3þ cells, butnot CTLA-4þ NK cells (Supplementary Fig. S1A), were observedin HNC patients. Patients with a frequency of PD-1þ NK cellsabove the mean value (>9%) showed significantly better sur-vival than those with PD-1þ NK cells below the mean (

Figure 1.

Frequency of total and PD-1þ NK cells in the circulation of HNC patients, association with clinical outcome, and enrichment of PD-1þ NK cells in HNC tumors. A,Frequency of total circulating NK cells (live CD3–CD56þ cells) in early-stage and late-stage HNC patients and healthy donors (HD). Representative gating strategydot plots shown (left) and summary of 15 HD and 50 HNC patients (right). Black circles, early-stage HNC patients; dark gray circles, late-stage HNC patients(ANOVA, �� , P < 0.001). B, Percentage of circulating PD-1þ NK cells in HNC patients and HD. Representative gating strategy dot plots shown (left) and summaryof 15 HD and 50 HNC patients (Student t test; �� , P < 0.01; mean: 9%). Black circles, values above the mean; dark gray circles, values below the mean.C, Kaplan–Meier survival curve of HNC patients shown in B segregated by higher/lower frequency of PD-1þ NK cells (above and below the mean, respectively).Mean ¼ 9% PD-1þ NK cells, log-rank test, P ¼ 0.03. D, Frequency of PD-1þ NK cells in TILs and paired PBLs from HNC patients (ANOVA; �� , P < 0.0001,ns P > 0.05; PD-1þ n ¼ 12, TIM-3/CTLA4þ n ¼ 4).

Concha-Benavente et al.

Cancer Immunol Res; 6(12) December 2018 Cancer Immunology Research1552




Figure 2.

Elevated expression of NCR1 in HNC specimens (TCGA) is associated with better survival and correlates with PDCD1 expression. A, NCR1mRNA expression (RSEMunits) in HNC tumor specimens (gray box) ranks third highest among all solid tumors (n ¼ 500, TCGA). B, Kaplan–Meier survival curve of HNC patientswith high versus low tumor NCR1 expression. NKp46 (NCR1)hi versus NKp46 (NCR1)low (n¼ 604, TCGA c-Bio portal, log-rank test, P¼ 0.016). C, NCR1, CD4, CD8A,CD8B, FOXP3, and CD14 mRNA expression in HNC tumor specimens and matched control mucosa (n ¼ 43, TCGA, Kruskal–Wallis test; �� , P < 0.001).D, PDCD1, HAVCR2, and CTLA4 expression (RSEM units) in tumor specimens and matched control mucosa (n ¼ 43, TCGA, Kruskal–Wallis test; �� , P < 0.001).E, Correlation ofNCR1 expression and PDCD1 (PD-1), CTLA4 (CTLA-4), or HAVCR2 (TIM-3) in HNC specimens (Pearson r test, graphs show linear regression curve fit.PD-1–positive correlation P < 0.001; CTLA-4, negative correlation P > 0.05; TIM-3, negative correlation P < 0.01). F, Correlation of NCR1 expression with thatof PDCD1, CTLA4, and HAVCR2 in head and neck control mucosa (Pearson r test, graphs show linear regression curve fit; n ¼ 43, TCGA).






before (8). In order to test this hypothesis, we investigatedwhether activated NK cells concomitantly upregulated PD-1expression (or TIM-3, CTLA-4) and activation markers CD69 andIFNg in vitro.We cocultured purifiedNK cellswith tumor cellswithand without cetuximab and determined PD-1, TIM-3, CTLA-4,and CD69 expression by flow cytometry and IFNg secretion byELISA. Cetuximab-activated NK cells significantly upregulatedPD-1 (Supplementary Fig. S2A) and CD69 but not TIM-3 orCTLA-4 expressionwhen comparedwith baseline or the untreatedconditions (Fig. 3A). PD-1þCD69þ NK cells were significantlyhigher than the PD-1þCD69– subset after cetuximab activation(Fig. 3B). Likewise, cetuximab-activated PD-1–upregulated NKcells showed high IFNg secretion (Supplementary Fig. S2B), andIL2-mediated NK cell activation upregulated expression of PD-1,CD107a, IFNg , granzyme B, and CD16 on NK cells (Fig. 3C).PD-1þ NK cells had significantly higher expression of IFNg ,granzyme B, and CD16 when compared with PD-1– NK cells(Fig. 3D). We did not find differences in granzyme B or CD107aexpression between the PD-1low and PD-1hi NK cells (Supple-mentary Fig. S2C). These findings provide experimental evidencethat both cetuximab and IL2-mediated activation induce PD-1upregulation on NK cells. To test whether PD-L1 ligation coulddiminish the activation status of PD-1 upregulated NK cells,aliquots of IL2-activated NK cells were cocultured with eitherIgG1 control mAb or PD-L1 Ig–conjugated beads. We found thatCD16, CD107a, IFNg , and granzyme B expression were down-regulated by PD-L1 ligation compared with control beads orbaseline activation (Fig. 3E). CD16 MFI expression also showedsignificant downregulation (Fig. 3F), suggesting that PD-L1 liga-tion of PD-1þ NK cells would also prevent further cetuximabactivation signals through downregulation of CD16 surfaceexpression.

TIL PD-1þ NK cells display an exhausted phenotype whenPD-L1 is highly expressed in the tumor

On the basis of our in vitro findings, we hypothesized that TILPD-1þ NK cells would interact with PD-L1 expressed in thetumor, which may provide a suppressive signal. Therefore, TILPD-1þ NK cells would have lower expression of activation/effector molecules than PD-1– NK cells. We sorted TIL PD-1þ

and PD-1– NK cells from a PD-L1-high–expressing tumor (85%PD-L1þ as determined by IHC) and analyzed their transcrip-tome profile (Supplementary Fig. S3A and S3B). We found atotal of 3,391 differentially expressed genes, from which 1,745were downregulated in TIL PD-1þ NK cells. Among the differ-entially downregulated transcripts (>2-fold) in PD-1þ versusPD-1– TIL NK cells were Th1 transcription factors, EOMES,BLIMP-1, and TBET. Likewise, NK cell activation markers suchas CD69, CD38, 2B4, NKG2D, NKG2C, CD56, CD94, andKLRG1; upstream activation molecules ZAP70, CD247; cytolyt-ic molecules such as GZMA, GZMK, PRF1, GNLY, and NKG7;RAS/MAPK, PI3K, and JAK–STAT signaling pathway moleculeswere also significantly downregulated. NK cell activating cyto-kine receptors such as IL2RB, IL12RB1, IL18RAP, and IL9R werealso found to be downregulated in this subset (SupplementaryTable S1). We did not find changes in expression of theinhibitory receptor KIR in TIL PD-1þ versus PD-1� NK cells(by transcriptome sequencing) or PBLs (by flow cytometry;Supplementary Fig. S4A). Other inhibitory receptors such asTIGIT, CD96, and HAVCR2 were found downregulated inPD-1þ NK cells.

Cetuximab increased circulating and tumor-infiltrating PD-1þ

NK cells in PD-L1low/– tumorsIn order to extend our in vitro findings, we used a cohort of

advanced (stage III/IV) HNC patients who were treated withcetuximab in a prospective phase II neoadjuvant trial (clinicalfeatures shown in Table 2). Prior to and after 4weeks of cetuximabtherapy, PBLs and TILs were collected, andNK cells were analyzedby flow cytometry. We found a significant increase of PD-1þ NKcells in PBLs in cetuximab responders when compared with theirrespective matched pretreatment samples (Fig. 4A). ActivatedPD-1þIFNgþ and PD-1þCD69þNK cells were significantly higherin the circulation of responders post-cetuximab treatment (Fig.4B; �, P > 0.05; ��, P > 0.01). Similar to PBLs, we found that TILPD-1þ NK cells were upregulated in post-cetuximab responders(Fig. 4C; left, representative gating strategy; right, summary of 5patients with matched pre/post TILs). We characterized CD69expression on PD-1þNK cells pre/post-cetuximab treatment fromthree trial patients who also had tumor PD-L1 expression andresponse status available. We found that TIL PD-1þCD69þ NKcells were higher post-cetuximab treatment in a responder patientwhose tumor was PD-L1–negative. In contrast, when tumorPD-L1 expression post-cetuximab was upregulated, we saw lessenrichment of CD69þPD-1þ NK cells, a response that was asso-ciated with poor clinical outcome. Lower expression of tumorPD-L1 and CD69þPD-1þ NK cells post-cetuximab were seen in apartial responder (Fig. 4D and E). Because PD-L1 ligation of PD-1upregulated NK cells reduced their activation status in vitro and areduced activation status was associated with poor response tocetuximab therapy in vivo, we hypothesized that PD-1–PD-L1 axisblockademight reverseNK cell dysfunction. PD-1 blockademightenhance cetuximab-mediated tumor lysis, particularly becausePD-L1 is expressed in the majority of HNC tumors (8).

Cetuximab-activated NK cells increase IFNg-mediated PD-L1upregulation on tumor cells

We previously reported that IFNg induces PD-L1 upregulationin HNC tumor cells (8). Because cetuximab-activated, PD-1upregulated, NK cells increased IFNg secretion (SupplementaryFig. S2B), we hypothesized that NK cell-derived IFNg couldupregulate PD-L1 expression on HNC cells in the coculture. Wefound that cetuximab-activated NK cells significantly upregulatedPD-L1 expression on HNC cells in an IFNg-dependent fashionbecause PD-L1 upregulation was completely abolished by anIFNg-blocking mAb (Fig. 5A). To test whether nivolumab, aPD-1–blocking mAb, could further increase PD-L1 expression byenhancing cetuximab-dependent NK cell activation and IFNgsecretion, we cocultured purified NK cells and HNC cells withcetuximab, nivolumab, or the combination. An even higherPD-L1 upregulation on tumor cells was seen when NK cells weretreated concurrently with cetuximab plus nivolumab (Fig. 5B).

PD-1 blockade enhanced cetuximab-mediated NK cellcytotoxicity against PD-L1high tumor cells

Next, we determined whether PD-1–PD-L1 axis blockade withnivolumab could enhance cetuximab-mediated NK cell cytotox-icity of tumor targets with either negative, low, or high PD-L1expression. We hypothesized that the extent of tumor PD-L1expression would be the limiting factor for the efficacy of PD-1blockademediating enhancement ofNK cell activation, especiallybecause higher PD-1 expressionwasnot seenonNKcells activatedwith cetuximab plus nivolumab compared with cetuximab alone






Figure 3.

PD-L1 downregulates the activation status of PD-1þ–activated NK cells in vitro. A, Healthy donor-purified NK cells were stained for PD-1, CTLA-4, TIM-3, and CD69expression at baseline and after coculture with tumor cells (JHU029 and 93VU, NK:tumor ratio 1:1, 24 hours) in the absence of mAb or with cetuximab (10 mg/mL).n ¼ 6, Kruskal–Wallis test, �� , P < 0.001; �� , P < 0.01. B, Frequency of PD-1þCD69þ and PD-1þCD69– NK cells after cetuximab activation. NK cells fromexperiment shown in A were analyzed for CD69 and PD-1 coexpression by flow cytometry (n ¼ 6, Kruskal–Wallis test, � , P < 0.05). C, IL2-activated NK cellsconcomitantly upregulate PD-1, CD107a, IFNg , granzymeB, andCD16. PurifiedNK cellswere analyzed at baseline (grayboxes) or after rhIL2 treatment (130 IU/mL, 24hours, dark gray boxes). n ¼ 6, Kruskal–Wallis test; �� , P < 0.001; �� , P < 0.01. D, Expression of IFNg , GrzB, and CD16 in PD-1þ versus PD-1� NK cells. Healthy donorpurified NK cells were treated with rhIL2 (130 IU/mL/24 hours) and markers were determined by flow cytometry (n ¼ 6; Mann–Whitney test, �� , P < 0.001;� , P < 0.05). E, PD-1þ NK cells downregulate expression of activation markers after PD-L1 ligation. NK cells were activated as in D (light gray boxes), then incubatedwith either IgG1 control (dark gray boxes) or PD-L1–conjugated beads (darkest gray boxes) for additional 24 hours; cells were harvested, and CD16, CD107a,IFNg , and granzyme B expression were determined by flow cytometry (n¼ 6, Kruskal–Wallis test, �� , P < 0.01; �, P < 0.05). F, CD16 expression on activated NK cellsafter PD-L1 ligation. IL2-activated NK cells were cultured under the same conditions as in E and analyzed for CD16 expression (MFI) by flow cytometry (Student t test,n ¼ 6; �� , P < 0.001).






(Supplementary Fig. S5A). In order to test our hypothesis, wecocultured PD-1 upregulated NK cells (IL2 pretreated) with eitherPD-L1 negative (SCC90), PD-L1low (JHU029), or PD-L1high

(93VU) tumor target cells (Supplementary Fig. S5B). Under theseconditions, we found that nivolumab enhanced cetuximab-mediated NK cell ADCC only when tumor targets expressedhigher PD-L1, given that cetuximab cytotoxicity was only signi-ficantly increased by nivolumab in 93VU but not SCC90 orJHU029 cells (Fig. 5C). Therefore, NK cells upregulate PD-1 uponactivation and concomitantly induce PD-L1 on tumor cells viaIFNg secretion, upregulating the suppressive PD-1–PD-L1 axisleading to NK cell dysfunction. PD-1 blockade appeared toenhance cetuximab-mediatedNK cell cytotoxicity primarily whenPD-L1 was highly expressed on tumor targets (Fig. 5D).

DiscussionAlthough the suppressive PD-1–PD-L1 axis is well documen-

ted in T-cell tumor immune evasion (35, 36), informationregarding ICR on NK cells is still sparse. Here, we report thatearly-stage HNC patients (T1, 44%; N0, 77%) have highercirculating NK cells compared with late-stage HNC patients(T1, 24%; N0, 24%), findings that agree with previous reportsfrom breast cancer patients (37, 38). Lang and colleagues alsofound a high frequency of NK cells in HNC patients (39). Wereport that circulating PD-1þ NK cells are significantly higher inHNC patients than in healthy controls, and higher frequency ofcirculating PD-1þ NK cells (mean value >9%) was associatedwith better overall survival. PD-1þ NK cells were significantlyenriched in tumors when compared with paired PBLs, suggest-ing that PD-1þ NK cells traffic to the TME or are activated there.As part of our characterization of ICR expression of NK cells, wealso analyzed TIM-3 and CTLA-4 expression. We found higherfrequencies of TIM-3þ NK cells in PBLs of HNC patients.Previous reports showed that PBL TIM-3þ NK cells appearedto be dysfunctional because they had lower expression of T-bet,

EOMES, IFNg , CD107a, and impaired cytotoxicity could bereversed by TIM-3 blockade (40–42). However, we did not findenrichment of TIM-3þ NK cells in HNC tumors nor a positivecorrelation between HAVCR2 (TIM-3) and NCR1 (NKp46)expression in TCGA HNC specimens. We also found thatCTLA-4 expression on NK cells was low or undetectable inPBLs and TILs, as well as in healthy donors, as previouslyreported (39, 43).

Because NCR1 (NKp46) is a specific marker of NK cells (44),we analyzed NCR1 mRNA expression as a surrogate indicativeof NK cell infiltration in HNC specimens. The high expressionof NCR1 in HNC compared with all other solid malignanciessuggests an antitumor role of NK cells, especially because wefound higher tumor NCR1 expression (>median: 1.03 RSEM)was associated with better patient survival (n ¼ 604). However,NCR1 expression was lower than that of CD4, CD8, FOXP3,and CD14, which may suggest a lesser extent of NK cellinfiltration in HNC tumors than adaptive lymphocytic ormyeloid immune cell subsets. Likewise, a higher expressionof HAVCR2 and CTLA4 mRNAs was noted in HNC tumorsthan in paired control mucosa. However, such high expressioncorrelated negatively with NCR1 expression, whereas PDCD1expression showed a positive correlation specific to tumorspecimens and not healthy tissue, indicating that the higherHAVCR2 or CTLA4 expression detected in our TCGA analysismay correspond to other immune subsets that infiltrate thetumor such as CD8þ T cells or regulatory T cells as we reportedpreviously (45–47).

When we analyzed the differential transcriptome expressionof paired TIL PD-1þ versus PD-1– NK cells from a specimen thatexpressed high PD-L1, we noted >2-fold downregulation of NKcell activation/effector molecules such as EOMES, BLIMP-1,TBET, CD69, 2B4, NKG2D, NKG2C, and NKG2E in the PD-1þ

subset. KLRG1, which constitutes a marker of responsiveness ininnate lymphoid cells that is downregulated by PD-1 (48), wasfound to be downregulated in our study, providing furtherevidence of a less responsive phenotype of TIL PD-1þ NK cells.Likewise, PI3K and MAPK pathway components that are crucialfor NK cell activation downstream NKG2D, 2B4, and IFNGproduction were downregulated (49–54). IL2RB1, IL12RB,IL18RAP, and IL9R, which are essential for NK cell activation,proliferation, and survival (55–57), were also found to bedownregulated in TIL PD-1þ NK cells. The inhibitory receptorNKG2A was found to be neither downregulated nor upregu-lated in our study, an observation concordant with that of Pesceand colleagues, where PD-1þ NK cells were NKG2A– (27).mRNA expression of other ICR such as TIGIT, HAVCR2, andCD96, which inhibit NK cell responses in mice (58), was foundto be downregulated in TIL PD-1þ NK cells when comparedwith the PD-1– subset, suggesting that compensatory ICRupregulation may not happen on human TIL PD-1þ NK cells.

In the light of these findings, we hypothesized that PD-1þ NKcells are activated and would become dysfunctional whenexposed to PD-L1 ligation in the tumor.We tested this hypothesisin vitro, where CD16- or IL2-activated NK cells upregulated PD-1along with CD69, CD107a, IFNg , and granzyme B, which weresubsequently downregulated after ligation by PD-L1. ActivatedPD-1þ NK cells had significantly higher coexpression of IFNg ,granzyme B, and CD16 than PD-1– NK cells. PD-L1 ligation ofPD-1–upregulated NK cells induced a significant downregulationofCD16 surfacedensity in vitro (MFI), suggesting that PD-1–PD-L1

Table 2. Clinical features of HNC patients from the clinical trial UPCI #08-013

Patients, n (%) 6 (100)Gender, n (%)Male 4 (66.6)Female 2 (33.3)

Age, years; median (range) 57.5 (47–74)T stage, n (%)T1 1 (16.6)T2 1 (16.6)T3 1 (16.6)T4 3 (50)

N stage, n (%)N0 2 (33.3)N1 2 (33.3)N2 2 (33.3)

Tumor site, n (%)Oral cavity 2 (33.3)Tonsil 2 (33.3)Larynx 2 (33.3)

HPV status, n (%)Positive 2 (33.5)Not evaluated 4 (66.5)

Response status, n (%)Responder 3 (50)Partial responder 1 (16.6)Nonresponder 1 (16.6)Not available 1 (16.6)






Figure 4.

Invivo cetuximabrespondershavehigherPD-1þNKcells inPBLsand inTILsonlywhen tumorcells expressed little tonoPD-L1.A,FrequencyofPBLPD-1þNKcells invivo.PBLs were isolated from stage III/IVA HNC tumor specimens pre- and post-cetuximab single-agent treatment (clinical trial UPCI #08-013) and PD-1 expression onNK cells was determined by flow cytometry and matched with response status (n ¼ 4 responders, n ¼ 4 nonresponders. Wilcoxon test; ns, P > 0.05; � , P < 0.05;�� , P < 0.001). B, PD-1þ IFNgþ and PD-1þCD69þ NK cells were determined in the same cohort of patients (Wilcoxon test; ns, P > 0.05; �, P < 0.05; �� , P < 0.001). C, TILswere isolated from tumor specimens pre- and post-cetuximab single-agent treatment and PD-1 expression on NK cells was determined by flow cytometry. Left,representative gating strategy; right, summary of 6 paired pre–post specimens (R, responder; PR, partial responder; NR, nonresponder; Wilcoxon test; � , P < 0.01).D, Frequency of PD-1þCD69þ NK cells in TIL post-cetuximab treatment and correlation with clinical response. Fold change of % PD-1þCD69þ NK cells POST/PREcetuximab was calculated and matched with response status. R, responder; PR, partial responder; NR, nonresponder. E, PD-L1 expression (IHC) in tumor specimensin the same cohort shown in D. Fold change of PD-L1þ tumor cells POST/PRE cetuximab was determined and correlated with response status. R, responder;PR, partial responder; NR, nonresponder.






interaction could prevent cetuximab cytotoxicity not only byblocking CD16 intracellular activation cascade via PD-1/SHP2,but also by diminishing the number of CD16 receptors capable ofbinding to cetuximab. Our observation that PD-1þ NK cellsdisplay an activated status is concordant with those found ingastric cancerwhere PBLPD-1þNKcells expressedhigherCD107a

and NKG2D than the PD-1– subset and that PD-1 blockadeincreased IFNg and CD107a expression (59). Similarly, Pesceand colleagues demonstrated higher expression of perforin, gran-zyme B, and CD16 in PD-1þ than in PD-1–NK cells from PBLs inovarian cancer patients (27). Previous studies showed that TILPD-1þ T cells coexpress Th1 transcription factors T-bet and STAT1

Figure 5.

PD-1 blockade onNK cells enhances cetuximab-mediated ADCCof PD-L1high tumor cells.A,Cetuximab-activatedNK cells upregulate PD-L1 expression on tumor cellsin an IFNg-dependent fashion. Healthy donor NK cells were coculturedwith JHU029, JHU022, or SCC90 tumor targets for 24 hours in the absence ofmAb, cetuximab(10 mg/mL), panitumumab (10 mg/mL), and cetuximab plus IFNg-blocking antibody (anti-IFNg , 50 ng/mL), harvested and PD-L1 expression on tumor cellswas determined by flow cytometry (n¼ 3, ANOVA, �� , P < 0.01; �� , P < 0.001). B, Expression of PD-L1 on tumor cells after cetuximab activation and PD-1 blockadeon NK cells. NK cells were cocultured with tumor cells (JHU029 or 93VU, 1 to 1 ratio, 24 hours) in the absence of mAb or with IgG1 control (10 mg/mL), cetuximab(10 mg/mL), nivolumab (20 mg/mL), or cetuximab plus nivolumab, tumor cells were harvested and PD-L1 expression was determined by flow cytometry (n ¼ 6,ANOVA, �� , P < 0.001; � , P < 0.05). C, Cetuximab-mediated ADCC of target cells with negative, low, or high PD-L1 expression. IL2-pretreated NK cells (130 IU/mL,24 hours) were cocultured with PD-L1–negative (SCC90), PD-L1–low (JHU029), or PD-L1–high (93VU) tumor targets (51Cr labeled, 20:1 ratio) with no mAb,IgG1 isotype control (10 mg/mL), cetuximab (10 mg/mL), nivolumab (20 mg/mL), or cetuximabþ nivolumab for 4 hours. 51Cr release was determined and % specificlysis was calculated (n¼ 3, ANOVA; ns, P > 0.05; �� , P < 0.001). D, PD-1–PD-L1–mediated tumor immune evasion of NK cell cytotoxicity. (1) HNC cells express basalPD-L1, cetuximab induces CD16-mediated NK cell activation and basal lysis. (2) Cetuximab activated NK cells increase PD-1 expression and secrete IFNg that in turninduces PD-L1 upregulation on tumor cells. (3) Tumor PD-L1 binds upregulated PD-1 on NK cells, providing an inhibitory signal for NK cell–CD16 mediatedactivation. (4) Nivolumab-mediated PD-1 blockade restores NK cell activation and enhances cetuximab-mediated ADCC.






and correlate with better clinical outcome (3, 9).We also reportedthat TIL CD8þ T cells with PD-1hi expression hadhigher granzymeB than the PD-1low subset (9, 45). In contrast, we did not finddifferences in granzyme B or CD107a expression between thePD-1low and PD-1hi NK cells.

Because cetuximab treatment induced upregulation of PD-1þ

NK cells in vitro and in vivo, the otherwise beneficial effect ofcetuximabmay constitute amechanism of tumor immune escapedominated by PD-1–PD-L1 axis interaction in the TME. Nivolu-mab significantly increased cetuximab-mediated killing onlywhen PD-L1 had high expression in tumor targets. Therefore,tumor PD-L1 expressionwas the singlemost important factor thatprovided suppressive signals to NK cells since PD-1 expressionremained constant on NK cells in both conditions. These findingsagree with reports that demonstrate a better clinical outcome ofanti–PD-1 therapy when tumors had robust PD-L1 expression,presenting PD-L1 expression as a predictor of response to anti–PD-1 therapy (13, 60). Further highlighting the role of NK cells inanti–PD-1 immunotherapy, a study reported significant associa-tion of an NK cell gene signature with better clinical outcome ofnon–small cell lung cancer, melanoma, andHNCpatients treatedwith pembrolizumab and nivolumab (61). Overall, our resultsshowed a better prognosis for HNC patients who have higherfrequency of circulating PD-1þ NK cells and that PD-1þ NK cellsdisplay an activated status and become dysfunctional only whenPD-1 is bound by its cognate ligand PD-L1 in the tumor. PD-1blockade restored NK cytotoxicity against PD-L1high expressingtumor targets and enhanced cetuximab-mediated ADCC (Fig.5D). Our findings support the combination of PD-1 and EGFRblockade in HNC patients with high PD-L1 tumor expression inorder to improve their clinical response.

Disclosure of Potential Conflicts of InterestR.L. Ferris has received compensation for consulting/advisory boards from

Astra-Zeneca/Medimmune, Bristol-Myers Squibb, Merck, Pfizer, and Tesaro.Through contracts with the UPMC Hillman Cancer Center, his laboratory hasalso been funded to perform sponsored research by Astra-Zeneca/Medimmune,Bristol-Myers Squibb, and Tesaro. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: F. Concha-Benavente, R.L. FerrisDevelopment of methodology: F. Concha-Benavente, R.L. FerrisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Concha-Benavente, J. Moskovitz, J. Moy, R.L. FerrisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Concha-Benavente, B. Kansy, J. Moskovitz, J. Moy,U. ChandranWriting, review, and/or revision of the manuscript: F. Concha-Benavente,B. Kansy, J. Moy, R.L. FerrisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Concha-Benavente, B. Kansy, R.L. FerrisStudy supervision: R.L. Ferris

AcknowledgmentsThis work was supported by NIH grants R01 DE19727, P50 CA097190,

and CA110249 and University of Pittsburgh Cancer Center Support GrantP30CA047904.

We thank Dr. Angen Liu for his kind help with scoring IHC images from thePD-L1–stained tumor specimens included in this report.

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 February 6, 2018; revised July 25, 2018; accepted September 27,2018; published first October 3, 2018.

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2018;6:1548-1560. Published OnlineFirst October 3, 2018.Cancer Immunol Res Fernando Concha-Benavente, Benjamin Kansy, Jessica Moskovitz, et al. and Neck Cancer Patients

NK Cells in Head+PD-L1 Mediates Dysfunction in Activated PD-1

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http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-18-0062http://cancerimmunolres.aacrjournals.org/content/suppl/2018/10/03/2326-6066.CIR-18-0062.DC1http://cancerimmunolres.aacrjournals.org/content/6/12/1548.full#ref-list-1http://cancerimmunolres.aacrjournals.org/content/6/12/1548.full#related-urlshttp://cancerimmunolres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerimmunolres.aacrjournals.org/content/6/12/1548http://cancerimmunolres.aacrjournals.org/

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PD-L1MediatesDysfunctioninActivatedPD-1þ NK Cells in Head … · tumor-inﬁltrating PD-1þ NK...

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