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Research Article

Increased PD-1þ and TIM-3þ TILs duringCetuximab Therapy Inversely Correlate withResponse in Head and Neck Cancer PatientsHyun-Bae Jie1, Raghvendra M. Srivastava1, Athanassios Argiris2,Julie E. Bauman3, Lawrence P. Kane4, and Robert L. Ferris1,4,5

Abstract

Despite emerging appreciation for the important role ofimmune checkpoint receptors in regulating the effector func-tions of T cells, it is unknown whether their expression isinvolved in determining the clinical outcome in response tocetuximab therapy. We examined the expression patterns ofimmune checkpoint receptors (including PD-1, CTLA-4, andTIM-3) and cytolytic molecules (including granzyme B andperforin) of CD8þ tumor-infiltrating lymphocytes (TIL) andcompared them with those of peripheral blood T lymphocytes(PBL) in patients with head and neck cancer (HNSCC) duringcetuximab therapy. The frequency of PD-1 and TIM-3 expres-sion was significantly increased in CD8þ TILs compared withCD8þ PBLs (P ¼ 0.008 and P ¼ 0.02, respectively). This

increased CD8þ TIL population coexpressed granzyme B/per-forin and PD-1/TIM-3, which suggests a regulatory role forthese immune checkpoint receptors in cetuximab-promotingcytolytic activities of CD8þ TILs. Indeed, the increased fre-quency of PD-1þ and TIM-3þ CD8þ TILs was inversely corre-lated with clinical outcome of cetuximab therapy. These find-ings support the use of PD-1 and TIM-3 as biomarkers toreflect immune status of CD8þ T cells in the tumor microen-vironment during cetuximab therapy. Blockade of theseimmune checkpoint receptors might enhance cetuximab-based cancer immunotherapy to reverse CD8þ TIL dysfunc-tion, thus potentially improving clinical outcomes of HNSCCpatients. Cancer Immunol Res; 5(5); 1–9. �2017 AACR.

IntroductionImmune checkpoint receptors expressed by T cells can nega-

tively control their activation, expansion, and effector functionsthrough inhibitory signals generated by interacting with theircognate ligands. Particularly, many immune checkpoint receptorsare significantly upregulated by tumor-infiltrating lymphocytes(TIL), indicating that these inhibitory receptors are induced bycomplex components distinctively formed at the tumor micro-environment (TME; refs. 1–3). In the TME, T cells with low T-cellreceptor (TCR)binding affinities are often insufficiently primedorboosted by antigenic stimulation and the cytokine milieu, con-tributing to T-cell exhaustion, by upregulating immune check-point receptors (4). For example, upregulation of programmedcell death 1 (PD-1) correlates with suppressed effector functionsof T cells, such as cytolytic activity against tumors, cytokinerelease, and expansion in the TME, thus enabling tumors to evade

immunosurveillance (5). Upon interaction with its ligands PD-L1/-L2, expressed by tumor cells or inflammatory immune cells,PD-1 ligation inhibits TCR signaling by recruiting phosphatasesSHP-1 and SHP-2 (6). mAbs to PD-1 or PD-L1 can block thenegative signals and enhance T-cell functions, restoring antitumorimmunity, and have emerged as an effective immunotherapy forpatients with various cancers (7, 8). T-cell immunoglobulin andmucin domain 3 (TIM-3) is predominantly expressed by PD-1þ

CD8þ TILs in both tumor-bearing animals and cancer patients(1, 9). Three different ligands, including galectin-9, HMGB1, andCEACAM1, can interact with TIM-3 and suppress T-cell activities(10–12). In spite of this, TIM-3þ CD8þ TILs isolated from mel-anoma patients more efficiently lysed autologous tumor cell linesthan did their TIM-3�CD8þ TILs counterparts (1). Blockade ofTIM-3 can increase cytokine production, including IFNg andTNFa production, enhance expansion of tumor antigen NY-ESO-1–specific CD8þ T cells upon antigen stimulation, and actsynergistically when combined with PD-1 blockade (13). Thus,blockade of TIM-3 has therapeutic potential in treating cancerpatients by impairing the antitumor activities of tumor antigen–specific CD8þ TILs (14, 15).

Although the efficacy of cetuximab (an mAb to the EGF recep-tor) therapy is limited to 15% to 25% in treating patients withhead and neck squamous cell cancer (HNSCC), the immunologicmechanisms underlying this moderate response rate have notbeen well characterized. It has been proposed that immunetolerance in the TME is inversely associated with efficacy andcorrelated with resistance to anticancer drugs and cancer recur-rence (16). We have demonstrated that CTLA-4þ and Foxp3þ

regulatory T cells (Treg) are significantly increased in the

1Department of Otolaryngology, University of Pittsburgh, Pittsburgh, Pennsyl-vania. 2Division of Hematology/Oncology, Department of Medicine, UT HealthScience Center at San Antonio, Cancer Therapy & Research Center, San Antonio,Texas. 3Division of Hematology/Oncology, Department of Medicine, Universityof Pittsburgh, Pittsburgh, Pennsylvania. 4Department of Immunology, Universityof Pittsburgh, Pittsburgh, Pennsylvania. 5Cancer Immunology Program, Univer-sity of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.

Corresponding Author: Robert L. Ferris, Hillman Cancer Center ResearchPavilion, University of Pittsburgh, 5117 Centre Avenue, Room 2.26b, Pittsburgh,PA 15213. Phone: 141-2623-0327; Fax: 141-2623-4840; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-16-0333

�2017 American Association for Cancer Research.

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microenvironment of patients with HNSCC during cetuximabtherapy, suppress cetuximab-induced natural killer (NK) cellcytotoxicity, and correlate with poor prognosis (17). We alsoproposed that treatment with cetuximab can stimulate CD8þ Tcells through cross-presentation following tumor cell death,which is a critical process to induce expansion of tumor-derivedantigen, EGFR-specific, CD8þ T cells (18). Because TCR activationinduces immune checkpoint receptors, we hypothesized thatcross-presentationmediated by cetuximab could upregulate theseinhibitory receptors, thus contributing to T-cell dysfunction in theTME, subsequently affecting efficacy and resistance to cetuximabtherapy. In this report, we demonstrate that the frequency of PD-1þ and TIM-3þ cells was significantly increased on CD8þ TILs,which also expressed granzyme B and perforin. These resultssuggest that therapeutic mAb targeting PD-1 or TIM-3 moleculesshould be combined with cetuximab to improve clinical out-comes for HNSCC patients.

Materials and MethodsPatients and specimens

Patients were seen in the Outpatient Clinic of the DepartmentofOtolaryngology at theUniversity of Pittsburgh (Pittsburgh, PA)Medical Center, and all subjects signed an informed consentapproved by the Institutional Review Board of the University ofPittsburgh (IRB #99-06). Peripheral venous blood samples wereobtained from cetuximab-treated patients with previouslyuntreated stage III/IV HNSCC, receiving cetuximab monotherapyon a prospective phase II clinical trial (UPCI #08-013, NCT01218048; ref. 17). Tumor and blood were collected beforeand after a 4-week window of weekly cetuximab. Cetuximab250 mg/m2 was weekly injected and 400 mg/m2 after an initialdose. Two specimens were collected from larynx, 8 from oralcavity, and 8 from oropharynx specimens for this study. We alsosummarized the samples used in each figure in Table 2.

Collection and processing of PBMCs and TILsBlood samples from cancer patients (40 mL) were drawn into

heparinized tubes and centrifuged on Ficoll–Hypaque gradi-ents (GE Healthcare Bio-Sciences). PBLs were recovered,washed in RPMI1640 or AIM-V medium (Invitrogen), andstored at �80�C for FACS analysis. For TIL isolation, tumortissues obtained from HNSCC patients undergoing surgerywere collected, placed in sterile medium, and delivered to thelaboratory for processing. Tumor tissues were sliced into smallpieces, which then were transferred to a cell strainer (70-mmnylon) and mechanically minced using a syringe plunger. Noenzymes were used for TIL recovery. The cells that passedthrough the cell strainer were collected and subjected toFicoll–Hypaque gradient centrifugation. Mononuclear cellswere recovered and stored at �80oC for FACS analysis. Forpre- and post-cetuximab treatment, peripheral blood mono-nuclear lymphocytes (PBMC) and TILs were isolated fromHNSCC patients before and after therapy, according to theprotocol for the UPCI #08-013 trial.

mAbs and flow cytometryThe mAbs used to stain human immune cells included PD-1-

APC (clone: EH12.2H7, eBioscience), CTLA-4-PE (clone: L3D10),TIM-3-Brilliant violet 421 (clone: F38-2E2), granzyme B-FITC(clone:GB11), perforin-APC (clone: dG9, BioLegend),CD3-Alexa

Fluor 405 conjugate (clone:UCHT1, Invitrogen), andCD4-AF700(clone RPA-T4, BD Biosciences) and their respective isotypecontrols. All mAb preparations were pretitrated using activatedas well as nonactivated PBMCs to determine the optimal stainingdilution for each. Intracellular staining for granzyme B andperforin was performed as follows: PBMCs or TILs were stainedwith mAb for surface markers and subsequently fixed and per-meabilized (eBioscience). After washing, cells were subjected tointracellular staining for Foxp3, granzyme B, and perforin. Flowcytometry was performed using an EPICS XL-MCL flow cytometerequipped with Expo32 software, a CyAn flow cytometer (Dako),or Fortessa (Becton Dickinson) machine; data were analyzedusing Summit V4.3 software or the FlowJo software (TreeStar,Inc.). The acquisition and analysis gates were restricted to thelymphocyte gate based on characteristic properties of the cells inthe forward and side scatter. At least 1� 105 events were acquiredfor analysis.

Statistical analysisAverages were calculated as means. For nonparametric dis-

tribution of samples, P values were calculated by Kruskal–Wallis and two-tailed exact Wilcoxon–Mann–Whitney tests ornonparametric paired test (Wilcoxon matched paired test).P values <0.05 were considered to be significant.

ResultsIncreased frequency of PD-1- and TIM-3–expressing TILsafter cetuximab monotherapy

When TILs isolated from HNSCC patients were analyzed forexpression of the immune checkpoint receptors CTLA-4, PD-1,and TIM-3, CD8þ TILs had an expression pattern distinct fromCD4þ TILs. PD-1 and TIM-3 were more predominantly express-ed by CD8þ TILs than CTLA-4 (P < 0.05 and P < 0.01, respec-tively), whereas CTLA-4 was more frequently expressed by CD4þ

TILs, than were PD-1 and TIM-3 (P < 0.01 for both; Fig. 1A).Analysis of coexpression pattern of these immune checkpointreceptors showed that PD-1 and TIM-3 were highly coexpressedby CD8þ TILs, whereas CTLA-4 was rarely coexpressed with PD-1or TIM-3 on these two TIL subsets (Fig. 1B; Table 1). This tightcoexpression pattern between PD-1 and TIM-3 was more tightlycorrelated on CD8þ TILs compared with CD4þ TILs (R2 ¼0.7576 vs. R2 ¼ 0.501, Table 1). Previously, we demonstratedthat the majority of CTLA-4–expressing CD4þ TILs were Foxp-3þ

Tregs in the TME (17).Because cetuximab induces clinical activity with a response rate

of about 15% to 20% for patients with HNSCC, we postulatedthat these immune checkpoint receptors are involved in limitingthe efficacy of cetuximab therapy by inhibiting T-cell effectorfunctions in the TME. On the basis of the observation that PD-1and TIM-3 are predominantly expressed by CD8þ TILs that areeffectors to eliminate tumors, we examined the frequency ofPD-1- and TIM-3–expressing CD8þ TILs isolated from a cohortof patients (n¼ 18)who received only single-agent cetuximab in aprospective phase II clinical trial (UPCI #08-013). As expected,CTLA-4þ, PD-1þ, and TIM-3þCD8þ T cells are significantly andconsistently enriched in TILs compared with PBLs (Fig. 2A).Treatment with single-agent cetuximab for 4 weeks significantlyincreased the frequency of CTLA-4þ, PD-1þ, and TIM-3þCD8þ

TILs in the patients with HNSCC, as compared with baseline(pre-cetuximab; P < 0.05, P¼ 0.008, and P¼ 0.021, for CTLA-4þ,

Jie et al.

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PD-1þ, and TIM-3þCD8þ TILs, respectively; Fig. 2B), EachCTLA-4þ, PD-1þ, and TIM-3þCD8þ TIL subset increased up to14.3%, 35.7%, and 29.8%, respectively, during cetuximabmono-therapy, indicating that PD-1 and TIM-3molecules were predom-

inantly expressed by CD8þ TILs. In addition, the average frequen-cyof PD-1�TIM-3� (double negative)CD8þTIL subsets decreasedfrom 67.0% to 56.9%, suggesting that PD-1 and TIM-3 single-positive and double-positive cells comprise a significant propor-tion of CD8þ TILs during cetuximab monotherapy. However,these CD8þ T-cell subsets were not increased in PBLs during thetherapy. This result indicates that cetuximab therapy increasedCTLA-4þ, PD-1þ, and TIM-3þCD8þ T cells locally in the TME,which potentially could impair effector functions such as cytokinerelease and cytolytic activities of CTL, contributing to suppressionof cetuximab-induced antitumor immunity.

0CTLA-4

CTLA-4+PD-1–

CTLA-4–PD-1+

CTLA-4+PD-1+

CTLA-4–PD-1–

CTLA-4+TIM-3–

CTLA-4–TIM-3+

CTLA-4+TIM-3+

CTLA-4–TIM-3–

PD-1+TIM-3–

PD-1–TIM-3+

PD-1+TIM-3+

PD-1–TIM-3–

CTLA-4+PD-1–

CTLA-4–PD-1+

CTLA-4+PD-1+

CTLA-4–PD-1–

CTLA-4+TIM-3–

CTLA-4–TIM-3+

CTLA-4+TIM-3+

CTLA-4–TIM-3–

PD-1+TIM-3–

PD-1–TIM-3+

PD-1+TIM-3+

PD-1–TIM-3–

5.4±1.0

2.3±1.0

5.5±1.2

18.1±3.8

63.7±4.0

2.7±0.9

71.6±4.3

57.7±4.2 17.5±3.3

8.2±1.216.6±

1.6

22.2±3.7

3.4±1.3

6.3±1.4

12.4±1.6

7.7±1.36.6±1.417.0±1.9

63.8±2.7

68.7±3.0

74.6±3.1

7.9±1

.0

11.3

±1.1

18.5±1.9

CTLA-4PD-1 PD-1TIM-3 TIM-3

CD4+ TIL cells

Gated on CD4+ TIL cells Gated on CD8+ TIL cells

CD8+ TIL cells

20

40

60

80

% P

ositi

ve c

ells

100A

B

P < 0.01

P < 0.001

P < 0.001

P < 0.05

Figure 1.

PD-1/TIM-3 are coexpressed by CD8þ TILs, whereas CTLA-4 are predominantly expressed by CD4þ TILs. CD4þ and CD8þ TILs isolated from HNSCC patients(n ¼ 23) were analyzed for CTLA-4, PD-1, and TIM-3 expression by flow cytometry. A, Comparison analysis of CTLA-4, PD-1, and TIM-3 expression on CD4þ

and CD8þ TILs subsets. B, Coexpression pattern of CTLA-4, PD-1, and TIM-3 expression on CD4þ and CD8þ TIL subsets. Proportion of each subset coexpressingCTLA-4, PD-1, and/or TIM-3 is indicated in the pie diagram.

Table 1. Correlation among CTLA-4, PD-1, and TIM-3 expressed on TILs

CD4þ T cells CD8þ T cells

CTLA-4 and PD-1 R2 ¼ 0.0095 R2 ¼ 0.002CTLA-4 and TIM-3 R2 ¼ 0.0375 R2 ¼ 0.0093PD-1 and TIM-3 R2 ¼ 0.501 (P ¼ 0.0002) R2 ¼ 0.7576 (P < 0.0001)

TIM-3 and PD-1 on CD8þ TIL Induced by Cetuximab

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Gated on CD8+ PBL

Pre

A

B

0.0

3.2 0.2 5.3 0.1

2.91.6

0.1

1.4 2.6

0.1 0.17.6

8.5 16.0

20.5

6.2 35.3

30.3

4.5 32.2

33.4

20.4

15.9

PD

-1

TIM-3

TIM-3-Violet 1 log comp

P = 0.008

P = 0.01P < 0.005

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n.s.

n.s.

n.s.n.s.

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PBL TIL

Post Pre PostPre

PBL TIL

Post Pre PostPre

PBL TIL

Post Pre Post

Pre

PBL TIL

Post Pre Post Pre

PBL TIL

Post Pre Post

10

20

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40

50P = 0.02

P < 0.05

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Figure 2.

Cetuximab monotherapy significantly increases frequency of PD-1þ and TIM-3þCD8þ TILs isolated from 18 HNSCC patients. PBLs and TILs were isolatedfrom blood and tumors, respectively, of HNSCC patients at pre- and post single-agent cetuximab therapy. A, Representative flow cytometry analysisof CTLA-4, PD-1, and TIM-3 expression on CD8þ PBLs and TILs isolated from an HNSCC patient (pre- and post-cetuximab treatment). The numbersrepresent the percentages of CTLA-4, PD-1, TIM-3 single-, and double-positive cells in CD8þ T cells. B, Percentages of CTLA-4þ, PD-1þ, TIM-3þ,PD-1þTIM-3þ, and PD1�TIM-3� cells in CD8þ T cells were compared in HNSCC patients at pre- and post single-agent cetuximab therapy for 18HNSCC patients. n.s., not significant.

Jie et al.





Increased frequency of granzyme B- and perforin-expressingCD8þ TILs after cetuximab

To characterize cytolytic phenotypes of CD8þ T cells induc-ed during cetuximab therapy, we first analyzed baseline ofgranzyme B and perforin expressed by CD8þ PBLs and TIL. Atbaseline, perforin expression was lower on CD8þ TILs com-pared with PBLs (P ¼ 0.002), whereas granzyme B expressionwas comparable with TILs and PBLs. To investigate the effect ofcetuximab monotherapy on granzyme B and perforin expressedby CD8þ T cells, paired PBLs and TILs were isolated prospec-tively from HNSCC patients (n ¼ 12) treated before and after4 weeks of single-agent cetuximab therapy (Fig. 3C–E). Cetux-imab monotherapy significantly and consistently increasedthe frequency of granzyme Bþ and perforinþ cells in CD8þ

TILs (P ¼ 0.001). This result suggests that cetuximab therapy

can significantly enhance cytolytic capacity of CD8þ T cells inthe TME by increasing granzyme B and perforin expression.

Cetuximab increased granzyme B and perforin expression onCD8þ TILs positive for PD-1/TIM-3

Tumor antigen–specific T cells can be generated through cetux-imab-mediated cross-presentation and subsequent TCR trigger-ing, which induces granzyme B/perforin as well as PD-1/TIM-3molecules in T cells (19, 20). Indeed, we observed that cetux-imab therapy concomitantly increased the frequency of granzymeBþ/perforinþ and PD-1þ/TIM-3þ cells only on CD8þ TILs (Figs. 2and 3). Next, we investigated whether the cetuximab-increasedgranzyme B/perforin–expressing CD8þ TILs also coexpressedPD-1/TIM-3 molecules on their cell surface. Indeed, most gran-zyme B–expressing CD8þ TILs coexpressed PD-1 and/or TIM-3

Per

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Pre Post Pre Post Pre Post Pre Post

Pre Post

1.10.80.51.0 7.0

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13.4

8.8

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39.929.8

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Perforin–17.2

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80100n.s. P = 0.001

P = 0.03

P = 0.001

TILs

Figure 3.

Cetuximab monotherapy significantly increases the frequency of granzyme Bþ and perforinþCD8þ TILs isolated from HNSCC patients. A–C, Representativeflow cytometry analysis of granzyme B and perforin on CD8þ PBLs and TILs isolated from an HNSCC patient (A), granzyme B and perforin expression comparison ofpaired CD8þ PBLs and TILs isolated from 10 HNSCC patients (B), and representative flow cytometry analysis of granzyme B and perforin expression onCD8þ PBLs and TILs isolated from an HNSCC patient at pre- and post-cetuximab treatment (C). The numbers represent the percentages of granzyme Bþperforin�,granzyme B�perforinþ, and granzyme Bþperforinþ cells in CD8þ T cells. D and E, Percentages of granzyme Bþ and perforinþ cells in CD8þ T cells werecompared in a cohort of HNSCC patients (n ¼ 12) pre- and post cetuximab neoadjuvant therapy. n.s., not significant.






on their surface, which is more evident than perforinþCD8þ

TILs (Fig. 4A and B). Because of the observation that this coexpres-sion pattern was not detected in CD8þ PBLs, we further analyzedonly CD8þ TILs to investigate the correlation between PD-1/TIM-3and granzyme B/perforin pre- and post-cetuximab monotherapy.For TIM-3þCD8þ TILs, the frequency of both granzyme Bþ andperforinþ cells was increased during cetuximab monotherapy (Fig.4C andD, P < 0.01). Regarding TIM-3�CD8þ TILs, the frequency ofperforinþ cells but not granzyme Bþ cells was also significantlyincreased during cetuximab monotherapy. Compared with TIM-3�CD8þ TILs, TIM-3þCD8þ TILswere significantly and consistentlyhigh in granzyme B and perforin expression before and aftercetuximab monotherapy (P ¼ 0.001). Cetuximab monotherapyalso significantly increased the frequency of granzyme Bþ cells andperforinþ cells in both PD-1þCD8þ (P < 0.05) and PD-1�CD8þ

TILs (P < 0.05 and P < 0.005; Fig. 4E and F). Compared with PD-1�CD8þ TILs, PD-1þCD8þ TILswere consistently high in granzymeB but not perforin expression before cetuximabmonotherapy (P¼0.005). Taken together, granzyme Bþ/perforinþ CD8þ TILsincreased during cetuximab monotherapy coexpressed TIM-3 andPD-1, indicating potential roles of PD-1 and TIM-3 molecules incontrolling cytolytic activities of these cetuximab-expandedCD8þ Tcells and inducing immune tolerance in the TME.

PD-1 and TIM-3 are associated with the clinical outcome ofcetuximab monotherapy

On the basis of the observation that PD-1 and TIM-3 arecoexpressedwith granzyme B andperforin onCD8þ T cells, whichis further evident during cetuximab monotherapy (Fig. 4), andpotentially negatively regulate antitumor immunity of CD8þ Tcells (21), we hypothesized that PD-1 and TIM-3 could correlatewith clinical outcome inHNSCC patients treated with cetuximab.To assess this, we compared the frequency of TIM-3þCD8þ TILsand PD-1þCD8þ TILs in objective clinical responders, based ontumor size shrinkage by comparing pre- and posttreatment CTscan, with clinical nonresponders during cetuximab monother-apy (Fig. 5A). In clinical nonresponders, the frequency of TIM-3þCD8þ TILs and PD-1þCD8þ TILs was significantly increasedduring cetuximab neoadjuvant therapy (P¼ 0.016 and P¼ 0.009,respectively; Fig. 5B andC). However, the frequency of these sameCD8þ TILs subsets in clinical responders was not significantlychanged during cetuximab monotherapy. Taken together, theseresults suggest that frequency of TIM-3þCD8þ TILs and PD-1þCD8þ TILs negatively impacts on the efficacy of cetuximabmonotherapy and provides a rationale to combine TIM-3 or PD-1blockade with cetuximab therapy to improve clinical outcome forHNSCC patients.

Per

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Gated on CD8+ T cells

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Per

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cells

Gated on CD8+ TIL

PBLA C D

E F

B

Pre

9.8

7.3

9.0

6.4 14.0 0.8 6.0 11.2

22.4

14.4 14.7

20.91.9

0.5

1.5

21.6 0.3 21.9 38.7 30.8

4.70

0 0

20

40

60

80

50

100

20

40

60

0

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40

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25.5

8.00.2

0.7

1.3

0.2 13.3 0.2 2.9 4.6 20.7

35.3

12.4

21.50.60.9

0.2 22.0 0.2 15.8 29.7 15.9 51.3

4.84.20.60.9

Post Pre Post

Pre Post Pre PostPre

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PD-1– PD-1+

Post Pre Post

Pre Post Pre Post Pre Post Pre Post

P = 0.005

P = 0.001

P < 0.01P = 0.01

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P < 0.05

P < 0.005

n.s.

n.s.

P < 0.05 P < 0.05P < 0.05

TIL

TIM-3

TIM-3

PD-1

PD-1

Figure 4.

Granzyme Bþ and perforinþCD8þ TILs coexpress with PD-1/TIM-3 and further increased during cetuximab monotherapy. Representative flow cytometryanalysis of coexpression of granzyme B/perforin and TIM-3 (A) or PD-1 (B) on CD8þ PBLs and TILs isolated from an HNSCC patient. C–F, Percentagesof granzyme Bþ (C and E) and perforinþ (D and F) in TIM-3� and TIM-3þ (C and D) or PD-1� and PD-1þ (E and F) CD8þ T-cell subsets werecompared in 11 HNSCC patients at pre- and post single-agent cetuximab therapy. n.s., not significant.

Jie et al.





DiscussionIn this report, we demonstrate that the frequency of PD-

1þCD8þ T and TIM-3þCD8þ T cells in the TME of the HNSCCpatients increased during cetuximab monotherapy. We alsoobserved that the frequency of granzyme Bþ and perforinþ cellswas also concomitantly increased in these CD8þ TILs, which also

coexpressed PD-1 and TIM-3. These results indicate that activatedCD8þ TILs are increased at the tumor sites during cetuximabtherapy and may be controlled or suppressed of their cytolyticactivity by PD-1 and/or TIM-3 in the TME. More importantly, PD-1þCD8þ and TIM-3þCD8þ TILs increased after cetuximab treat-ment were strongly associated with clinical outcome in these

Table 2. Summary for patients and figures using their specimens (UPCI #08-013)

Figures using specimens

Responder: before & afterHN12-6675 & HN12-6741 Fig. 1 Fig. 2 Fig. 3 Fig. 5HN12-6864 & HN12-6938 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-7031 & HN12-7089 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-7081 & HN12-7148 Fig. 1 Fig. 2 Fig. 5HN12-7131 & HN12-7197 Fig. 1 Fig. 2 Fig. 3 Fig. 5HN12-7252 & HN12-7308 Fig. 1 Fig. 2 Fig. 3 Fig. 5

Nonresponder: before and afterHN12-6453 & HN12-6487 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-6479 & HN12-6528 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-6631 & HN12-6687 Fig. 1 Fig. 2 Fig. 5HN12-6658 & HN12-6721 Fig. 1 Fig. 2 Fig. 5HN12-6465 & HN12-6517 Fig. 1 Fig. 2 Fig. 5HN12-6768 & HN12-6829 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-6839 & HN12-6901 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-6837 & HN12-6884 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-6933 & HN12-6983 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-6908 & HN12-6980 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-7036 & HN12-7091 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5HN12-7006 & HN12-7097 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

0Pre

Responders Nonresponders

Post Pre Post Pre

Responders Nonresponders

Baseline 2 Weeks 4 Weeks Baseline 2 Weeks 4 Weeks

A

B C

NonrespondersResponders

Post Pre Post

20

40

60

80 n.s.

n.s.

P = 0.016P = 0.009

Gated on CD8+ T cells

% P

D-1

+ Cel

ls

0

20

40

60

80

% T

IM-3

+ Cel

ls

Figure 5.

Increased frequency of PD-1þ and TIM-3þCD8þ TILs is inversely correlated with clinical outcome in cetuximab monotherapy. A, Representative PET/CTscanning results of a responder and a nonresponder during cetuximab monotherapy clinical trial (UPCI-#08-013). The frequency of PD-1þ (B) or TIM-3þ (C) cells inCD8þ TILs was measured using flow cytometry and compared in pre- and post single-agent cetuximab monotherapy for 5 responders and 13 nonresponders,respectively. n.s., not significant.






patients. Taken together, these results suggest that combinationtherapy using blockade of PD-1 or TIM-3 in the cetuximab therapymight improve clinical outcome of cetuximab-treated HNSCCpatients by breaking immune tolerance induced by these cetux-imab-induced immune checkpoint receptors on CD8þ TILs in theTME.

We speculate that T-cell activation takes places through TCRtriggering in the TME, which is presumably enhanced by cetux-imab-mediated cross-presentation (22), contributing toincrease of PD-1þ and TIM-3þCD8þ TIL subsets. Previously,we reported that cetuximab therapy significantly increases thefrequency of tumor antigen–specific CD8þ T cells in HNSCCpatients (20). In this report, we also demonstrated that cetux-imab-activated NK cells subsequently induced dendritic cell(DC) maturation by producing IFNg . On the basis of theseobservations, we expect that cross-presentation function of DCmight be enhanced by cetuximab treatment, thus contributingto activation and expansion of tumor antigen–specific T cells inthe TME of head and neck cancer patients. Indeed, we also haveobserved that the frequency of CD137þ and CD107aþ intra-tumoral NK cells was increased during cetuximab monother-apy, confirming that NK cells are activated in the TME of theHNSCC patients treated with cetuximab (17). Given that gran-zyme B/perforin as well as PD-1/TIM-3 are induced upon T-cellactivation, cetuximab therapy activates CD8þ T cells in theTME. Taken together, these results support the idea that CD8þ

T-cell activation actively takes place in the TME presumablythrough cetuximab-promoting cross-presentation.

It has been proposed that chronic antigen stimulation, such asin chronic virus infection or tumors, leading to upregulation ofimmune checkpoint receptors associatedwith T-cell exhaustion, ahyporesponsive status of T cells (23).However, it is challenging todefine themost important functions of T cells expressing immunecheckpoint receptors in artificial tumor models. Several lines ofresearch showed that tumor antigen–specific CD8þ TILs expres-sing high levels of PD-1 are defined as functionally impaired Tcells in the TME (24–26). However, it was reported that thefrequency of PD-1þ TILs positively correlatedwith a better clinicaloutcome inHPV-associatedheadandneck cancer patients (27). Inthis article, treatment with cancer vaccine increases frequency ofPD-1þ TILs and combination therapy using PD-1 blockadeenhances antitumor activity, suggesting that PD-1þ TILs couldbe reactivated by PD-1 blockade (27). Regarding the roles of TIM-3 on TIL functions, the majority of TIM-3þ TILs were found tocoexpress PD-1 in both mice bearing tumors (9) and melanomapatients (1). In the mouse tumor model, TIM-3þPD-1þ TILs weredefined as severely exhausted cells based on their impairedcapability to proliferate and produce cytokines, including IL2,TNFa, and IFNg . However, PD-1þCD8þ and TIM-3þCD8þ TILs inmelanoma patients, but not PD-1�CD8þ and TIM-3�CD8þ TIL,are capable to recognize and lyse autologous tumor and react totumor-derived mutated antigen. Importantly, blockade of TIM-3led to rejection of carcinogen-induced sarcomas by producingIFNg in a preclinical model (28), and in response to antigenstimulation, it enhanced cytokine productions and expansion oftumor antigen–specific CD8þ T cells isolated from melanomapatients (13). Although TIM-3þCD8þ T cells express high level ofperforin aswell as T-bet inHIV infection, they are impaired in theirdegranulation capacity (29). In this study, blockade of TIM-3increased the cytotoxicity of TIM-3þCD8þ T cells to lyse CD4 T

cells expressing HIV antigens by enhancing degranulation(release) of granzyme B and perforin. Thus, TIM-3may negativelycontrol cytotoxicity ofCD8þT cells by inhibiting degranulation ofgranzyme B and perforin. These two studies imply that TIM-3þCD8þ T cells are activated cells but are suppressed in theircytolytic activity by TIM-3 molecules. Therefore, they might berecovered from the suppressed status and reactivated upon anti-genic stimulation by using blockade of TIM-3.

In our study, granzyme Bwas coexpressed with 60% to 70% ofTIM-3þCD8þ and PD-1þCD8þ TILs, whereas this cytolytic mol-ecule was not expressed by 20% to 30%of TIM-3þCD8þ and PD-1þCD8þ TILs. Given the amount of granzyme B indicates cytolyticability of activated CD8þ T cells, TIM-3þCD8þ and PD-1þCD8þ

TILs coexpressing granzymeB couldbe defined as activated cells inthe TMEandhave the potential to be reactivated using blockade ofPD-1 or TIM-3. In addition, PD-1þCD8þ and TIM-3þCD8þ TILs,compared with their negative counterparts, contain significantlymore granzyme Bþ and perforinþ cells both before and aftercetuximab monotherapy. Our results also show that these acti-vated CD8þ T cells increased and coexpressed PD-1 and TIM-3 inthe TME during cetuximab monotherapy, suggesting that theircytolytic activities are being dampened. Hence, these resultsprovide us with an insight into understanding dysfunction ofCD8þ T cells induced by PD-1 and TIM-3 in the TME duringcetuximab therapy as well as provide a rationale to combinecetuximab therapy with blockade of PD-1 and/or TIM-3 toimprove clinical outcome for HNSCC patients.

Disclosure of Potential Conflicts of InterestA. Argiris is a consultant/advisory board member for BMS. R.L. Ferris is a

consultant/advisory board member for AstraZeneca – MedImmune, Bristol-Myers Squibb, Lilly, and Pfizer. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: H.-B. Jie, J.E. Bauman, L.P. Kane, R.L. FerrisDevelopment of methodology: H.-B. Jie, R.M. Srivastava, R.L. FerrisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.-B. Jie, A. Argiris, J.E. Bauman, R.L. FerrisAnalysis and interpretation of data (e.g., statistical analysis, biostati-stics, computational analysis):H.-B. Jie, R.M. Srivastava, A. Argiris, L.P. Kane,R.L. FerrisWriting, review, and/or revision of the manuscript: H.-B. Jie, A. Argiris,J.E. Bauman, L.P. Kane, R.L. FerrisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H.-B. Jie, R.L. FerrisStudy supervision: R.L. Ferris

AcknowledgmentsWe acknowledge excellent technical assistance from Michael Meyer and

Bratislav Janjic, University of Pittsburgh Cancer Institute. We thank Ferrislaboratory members for helpful suggestions.

Grant SupportThis work was supported by NIH grants R01 DE019727, P50 CA097190,

CA206517, and University of Pittsburgh Cancer Institute grant P30CA047904.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 17, 2016; revised January 17, 2017; accepted April 7,2017; published OnlineFirst April 13, 2017.

Jie et al.





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Published OnlineFirst April 13, 2017.Cancer Immunol Res Hyun-Bae Jie, Raghvendra M. Srivastava, Athanassios Argiris, et al. Patients

CancerInversely Correlate with Response in Head and Neck TILs during Cetuximab Therapy+ and TIM-3+Increased PD-1

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