ex vivo pd-l1/pd-1 pathway blockade reverses dysfunction of...

Cancer Therapy: Preclinical

Ex Vivo PD-L1/PD-1 Pathway Blockade ReversesDysfunction of Circulating CEA-Specific T Cells inPancreatic Cancer PatientsYuan Chen1, Shao-An Xue1,2, Shahriar Behboudi3, Goran H. Mohammad4,5,Stephen P. Pereira6, and Emma C. Morris1

Abstract

Purpose:Carcinoembryonic antigen (CEA) is a candidate targetfor cellular immunotherapy of pancreatic cancer. In this study, wehave characterized the antigen-specific function of autologouscytotoxic T lymphocytes (CTL) specific for the HLA-A2–restrictedpeptide, pCEA691-699, isolated from the peripheral T-cell reper-toire of pancreatic cancer patients and sought to determine if exvivo PD-L1 and TIM-3 blockade could enhance CTL function.

Experimental Design: CD8þ T-cell lines were generated fromperipheral bloodmononuclear cells of 18 HLA-A2þ patients withpancreatic cancer and from 15 healthy controls. In vitro peptide-specific responses were evaluated by flow cytometry after stainingfor intracellular cytokine production and carboxy fluoresceinsuccinimydyl ester cytotoxicity assays using pancreatic cancer celllines as targets.

Results: Cytokine-secreting functional CEA691-specific CTLlines were successfully generated from 10 of 18 pancreatic cancerpatients, with two CTL lines able to recognize and kill bothCEA691 peptide–loaded T2 cells and CEAþ HLA-A2þ pancreaticcancer cell lines. In the presence of ex vivo PD-L1 blockade,functional CEA691-specific CD8þ T-cell responses, includingIFNg secretion and proliferation, were enhanced, and this effectwas more pronounced on Ag-specific T cells isolated from tumordraining lymph nodes.

Conclusions: These data demonstrate that CEA691-specificCTL can be readily expanded from the self-restricted T-cellrepertoire of pancreatic cancer patients and that their functioncan be enhanced by PD-L1 blockade. Clin Cancer Res; 1–12. �2017AACR.

IntroductionPancreatic cancer remains a highly aggressive and difficult-to-

treat malignancy. As such, it is one of the leading causes of cancerdeaths worldwide (1). At the time of diagnosis, approximately30% of patients have locally advanced disease and a further 50%already have evidence ofmetastatic disease. Aminority of patients(approximately 15%) are eligible for potentially curative surgery(such as pancreatico-duodenectomy, or the Whipple procedure),but the majority will die from recurrent disease (2, 3). Current 5-year overall survival (OS) rates are in the order of 5% with onlymarginal improvements in prognosis having been achieved in thelast few decades (4–6).

For patients with unresectable pancreatic cancer, gemcitabine-and folfirinox-based chemotherapeutic regimens are the treat-ment of choice; however, efficacy is limited, and drug resistanceand disease relapse are very common (7–9). There is therefore anurgent need to develop alternative therapeutic approaches for thetreatment of pancreatic cancer.

Targeted immunotherapeutic approaches aim to improve thetherapeutic index by maximizing tumor cell death while min-imizing side effects. Promising results have recently been dem-onstrated for various advanced cancers, including melanoma,metastatic non–small cell lung cancer, renal cell cancer, andovarian cancer (10) using a number of strategies includingmonoclonal antibodies, checkpoint inhibitors, vaccination,and genetically modified immune cells (11–13). Cytotoxic Tlymphocytes (CTL) play a pivotal role in cellular immunityand, by recognizing tumor-associated antigen (TAA)–derivedshort peptide epitopes presented by MHC-class I molecules,they can mediate target cell killing and induce protectiveantitumor immune responses (14).

Although a number of TAAs have been identified in the contextof pancreatic cancer, including mesothelin (15) and the carci-noembryonic antigen (CEA; ref. 16), both of which are widelyexpressed in pancreatic cancer cells, pancreatic tumors are gener-ally considered to be nonimmunogenic and resistant to immu-notherapies (10, 17, 18). In addition, pancreatic cancer cellsactively contribute to local immune suppression in the tumormicroenvironment through the production of anti-inflammatorycytokines such as TGFb, IL10, and IL6 and/or the expression ofnegative regulatorymolecules (18). Specifically, pancreatic cancercells express high levels of programmed death ligand-1 (PD-L1;ref. 19), an immunosuppressivemolecule that, upon engagement

1Institute of Immunity and Transplantation, University College London, London,United Kingdom. 2Genetic Engineering Laboratory, School of Biological andEnvironmental Engineering, Xi'An University, Xi'An, P. R. China. 3The PirbrightInstitute, Woking, Pirbright, United Kingdom. 4Department of Physiology,Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. 5Chem-istry Department, College of Science, University of Sulaimani, Sulaimanyah,Kurdistan Region, Iraq. 6Institute for Liver and Digestive Health, UniversityCollege London, London, United Kingdom.

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

S.P. Pereira and E.C. Morris are joint senior authors.

Corresponding Author: E.C. Morris, Institute of Immunity and Transplantation,University College London, Second Floor, Royal Free Hospital, Pond Street,London NW3 2QG, United Kingdom. Phone: 44(0)7753 745489; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-1185

�2017 American Association for Cancer Research.

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with its receptor programmed death-1 (PD-1) on the surface ofCTLs, delivers inhibitory signals impairing T-cell effector function(20). In murine models, it has been shown that anti–PD-L1 andanti–CTLA-4 blocking antibodies, commonly referred to as check-point inhibitors, improved T-cell–mediated antitumor immunityand prolonged survival (21–23). Although supporting evidencefrom human clinical trials is currently lacking (24), it is expectedthat T-cell–based immunotherapies in combination with check-point inhibitors may circumvent the immunosuppressive prop-erties of the pancreatic tumor microenvironment, and such com-binatorial approaches are likely to be required.

In this study, we examined the ex vivo functional and pheno-typic properties of CEA-specific T cells isolated from 18 consecu-tive HLA-A2þ pancreatic cancer patients.

Materials and MethodsPatients and samples

This studywas approved by theCentral LondonResearch EthicsCommittee (Study no. 06/Q0512/106) and conducted in accor-

dance with the Declaration of Helsinki. Written, informed con-sent was obtained from all patients.

Peripheral blood sampleswere collected frompancreatic cancerpatients at three central hospitals: University College LondonHospitals NHS Foundation Trust (UCLH); Royal Free LondonHospital NHS Foundation Trust (RFH); and Charing Cross Hos-pital - Imperial College Healthcare NHS Foundation Trust.Detailed patient demographics and tumor characteristics aresummarized in Table 1. In all cases, the diagnosis of pancreaticcarcinoma was confirmed by standard cytopathology or histopa-thology after biopsy, and the clinical stage was assigned usingstaging criteria described in the World Health Organizationhistologic classification of tumors of the exocrine pancreas(25). Anonymized peripheral blood mononuclear cells (PBMC)were obtained from the National Blood Service from healthycontrols.

PBMCs were isolated by density gradient centrifugation usingstandard methodology (Ficoll, Lymphoprep-Apogent Discover-ies). Where possible, lymph node (LN) samples were collectedfrom patients undergoing surgery and then mechanically dis-rupted to generate a single-cell suspension prior to PBMCisolation.

Patient HLA-A2 status was determined by flow cytometricanalysis after staining with a phycoerythrin (PE)-conjugated,anti–HLA-A2 antibody (clone BB7.2; BD BioSciences).

Peptides and tetramersThe HLA-A2–restricted peptides CEA691 (IMIGVLVGV), CMV

pp65 (NLVPMVATV), and Telomerase540 (ILAKFLHWL) weresynthesized by Mimotopes Pty Limited and dissolved in PBS at astock concentration of 2 mmol/L prior to use. APC-labeledpCEA691/HLA-A�0201 tetramers (TCMetrix) were used to detectCEA-specific T cells. Other HLA-A2–restricted peptides used in

Table 1. Patient demographic information (PBMC samples, N ¼ 18; LN samples, N ¼ 3)

ID of PC patients Age (year) Sex Histologic diagnosis Stage TreatmentOS sincerecruitment (months)

PBMC samplesCA01 51 M AC IV Untreated 14CA02 37 M PDAC IIB Untreateda 24CA03 86 F AC III Untreated 1CA04 56 F Poorly differentiated AC IV Resected þ FOLFOX 3CA05 67 M AC IV GemCap 8CA06 66 M Moderately differentiated PDAC IIB Resected, Pre-chem 15CA07 60 M Ductal AC IIA Resected, Pre-chem 23CA08 73 F Mucinous carcinoma IIA Resected, Gemcitabine 25CA09 45 M AC IIB FOLFIRINOX 18CA10 72 F AC IIB Resected þ Gemcitabine 14CA11 68 F Moderately differentiated AC IIB Resected, pre-chem 28CA12 69 F Adenocarcinoma IIB Resected þ GemCap 20CA13 60 M Adenocarcinoma IV Untreated 6CA14 46 M Adenocarcinoma III Untreated 21CA15 52 M AC IV Gemcitabine 1CA16 60 M AC IV Laparotomy, GemCap 14CA17 45 F Adenocarcinoma IIB FOLFIRINOXa 16CA18 55 M Moderately differentiated AC III Resected, pre-chem 31

ID of PC patients Age (year) Sex Histologic diagnosis Stage HLA-A2LN samplesCA13 60 M Adenocarcinoma IIB þCA19 52 M Moderately differentiated AC IIB �CA20 51 F Adenocarcinoma IIB �Abbreviations: AC, adenocarcinoma; chem, chemotherapy; FOLFIRINOX, FOLFOXþIrinotecan; FOLFOX, folinic acid, fluorouracil and oxaliplatin; GemCap,Gemcitabine and capecitabine; PDAC, pancreatic ductal adenocarcinoma.aStatus of patients when PBMCs were collected. Eight patients had already undergone surgical resection and one laparotomy alone, when the blood was taken.Patients CA02 and CA17 had PBMC collected prior to surgical resection of tumor.

Translational Relevance

This article describes the isolation of functional CEA691-specific CD8þ T cells from the peripheral blood and/or drain-ing lymph nodes of 18 consecutive patients with carcinoma ofthe pancreas. We demonstrate for the first time that theantigen-specific function of these self-restricted tumor-reactiveT cells can be enhanced by PD1/PDL1 pathway blockade.These findings support the clinical development of T-cell–mediated therapies in combination with checkpoint inhibi-tors for patientswith an extremely poor prognosismalignancy.

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this study are detailed in Supplementary Table S1 and were alsosynthesized by Mimotopes Pty Ltd.

Short-term T-cell expansion culturesShort-term primary T-cell lines were generated as previously

described (26). Briefly, isolated PBMCswere cultured at 1.5� 106

cells/mL in normal growth medium (NGM) consisting of RPMI1640 (Invitrogen) supplemented with 2 mmol/L glutamine, 1%penicillin/streptomycin (Sigma-Aldrich), and 10% heat-inacti-vated FCS (BioWest). Peptide stimulation was performed at afinal concentration of 2 mmol/L, in the presence of rhIL2 (20U/mL), and the cells were harvested after 9 to 10 days of culture.

Establishment of primary T-cell linesPBMCs were expanded over four rounds of peptide-specific

stimulation, with cells being analyzed by flow cytometry at theend of rounds one and four. Briefly, 3� 106 PBMCs were initiallyresuspended in NGM at 1.5 � 106 cells/mL in a 24-well plate, inthe presence of rhIL2 (20 U/mL; Roche), rhIL7 (2 ng/mL; R&DSystems), rhIL15 (5 ng/mL; R&D Systems), and rmIL21 (0.5ng/mL; R&D Systems). Peptide-specific stimulation was per-formed by adding pCEA691 or CMV pp65 directly into specificwells, at a final concentration of 10 mmol/L. After a first round of7 to 9 days of duration, cells were moved to restimulation. Threerounds of restimulation were performed as described below,each of which was 7- to 9-day duration.

Restimulation was conducted by resuspending 5� 105 cells in2 mL of cytokine-supplemented NGM in a new 24-well plate,and stimulating them with irradiated (70 Gy) T2 cells (2 � 105)pulsed with the appropriate peptide, at a final concentration of10 mmol/L. Irradiated (35Gy) autologous PBMCs or PBMCs fromhealthy HLA-A2þ donors obtained from the National BloodService were used as feeder cells (2 � 106).

Cell linesSix pancreatic cancer cell lines (MiaPaca-2, PK-45, Panc-1, KLM-

1, Bx-Pc-3, and PK-1) were obtained from PIKEN BioResource-centre (PIKEN BRC). FITC-CEA antibody (clone B1.1; BDSciences) was used to detect CEA expression on the surface ofthese cell lines, which was analyzed by flow cytometry. Amongthem, Panc-1 (HLA-A2þ, CEAþ), MiaPaca2 (HLA-A2�, CEA�),PK-1 (HLA-A2þ, CEAþþþ), andBx-Pc-3 (HLA-A2þ, CEAþþþ)wereused as target cells in cytotoxicity assays. Apart fromMiaPaca2, allcell lines were maintained in NGM containing RPMI 1640 (Invi-trogen) supplemented with 2 mmol/L glutamine, 1% penicillinplus streptomycin (Sigma-Aldrich), and 10% heat-inactivatedFCS (BioWest). MiaPaca2 cells were cultured in DMEM (Invitro-gen) with 1% penicillin plus streptomycin and 10% heat-inacti-vated FCS. The HLA-A2–positive T2 cell line was loaded withspecific peptides and used as target cells were indicated. TAP-deficient T2 cells have impaired presentation of HLA moleculeswith endogenous peptide, but can be efficiently loaded withexogenously peptides (27). The T2 cell line was maintained inNGM.

Cytotoxicity assaysA carboxy fluorescein succinimydyl ester (CFSE) cytotoxicity

assay was used to determine the antigen-specific cytotoxicity ofexpanded T-cell lines. pCEA691 peptide–loaded T2 cells or HLA-A2–positive pancreatic cancer cell lines (known to express CEA,CEAþ) were used as target cells. T2 cells pulsed with irrelevant

peptides and HLA-A2–negative pancreatic cancer cell lines wereused as control target cells. Target cells (1� 106) were suspendedin PBS/1%FCS at a concentration of 106/mL and then stainedwith CFSE. For sensitive targets, 0.5 mL of CFSE stock solution(5 mol/L) was added to 1 mL of cell suspension, whereas forcontrol targets, 0.5 mL of diluted CFSE at 500 mmol/L was used.After 4 minutes' incubation at room temperature, 9 mL ofPBS/1%FCS was added to stop the reaction. The cells werewashed with PBS, and then resuspended at 5 � 104 cells/mLprior to setting up cocultures with the effector cells. T cells(effectors) were added to round-bottomed 96-well plates toobtain a total volume of 200 mL/well (28). Various effector:target(E:T) ratios were tested, including 100:1, 50:1, 20:1, 10:1, 5:1,2.5:1, 1.25:1, and 0.625:1, respectively. Assay plates were incu-bated for 4 hours at 37�C, 5% CO2. Cells were washed in PBSprior to FACS analysis (FACSCalibur). For peptide titrationassays, CFSE-stained T2 cells were loaded with variable con-centrations of peptides, at 10�5, 10�6, 10�7, 10�8, 10-9, 10�10,10�11, and 10�12 mol/L, respectively (29).

In vitro PD-L1 and TIM-3 blockadePBMC from 11 pancreatic cancer patients (where sufficient cells

were available) and LN-derived lymphocytes from 1 pancreaticcancer patient were cultured in the presence of CEA691 peptidesand rhIL2 as described above, at a concentration of 2� 106/mL in200 mL of NGM. On day one, anti–PD-L1 and anti–TIM-3 anti-bodies (Mouse IgG, eBioscience) were added to the wells, eitherseparately, or in combination, at afinal concentration of 10mg/mL.After 7 days of incubation at 37�C, the cells were harvested forfunctional analysis using intracellular cytokine staining.

Flow cytometryThe following antibody–fluorochrome combinations were

used: CD3-PE-Cy7, CD8-Horizon v450, CD4-Horizon v500,IFNg–FITC, PD-1–PE,CD45RO-BV650 (all fromBDBiosciences);CD62L-APC-Cy7 (eBioscience); LAG-3–FITC (R&DSystems); andTIM-3-AF700 (eBioscience). Ex vivo surface staining was per-formed on 1 � 106 freshly isolated PBMC. Briefly, one microliterof a 1:50 dilution of each antibody was added to the cells andincubated for 30 minutes, at 4�C, in the dark. Cells were washedtwice with PBS/1% FCS and then resuspended in 200 mL PBS/1%FCS for data acquisition. Flow cytometry data acquisition wasperformed using a FACSCalibur. Propidium iodide (10 mg/mL)was added immediately prior to acquisition to discriminate deadcells from viable cells. Data analysis was performed using FlowJo(Treestar Inc.; version v10).

Intracellular cytokine staining assayIntracellular cytokine staining was performed on cultured cells,

either after short-term stimulation or after four rounds of antigen-specific stimulation during the primary T-cell line establishmentprotocol (described above). Upon harvest, cultured cells wererestimulated with 10 mmol/L of relevant peptide for a further 5hours in the presence of 10 mg/mL Brefeldin A. Cells stimulatedwith an irrelevant peptidewere used as negative controls, and cellsstimulatedwith PMA(50ng/mL)þ Ionomycin (500ng/mL)wereused as positive controls. Cells were surface stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, as described above,then permeabilized and fixed using FACS fix/perm solution(Invitrogen) prior to staining for intracellular cytokines withFITC-conjugated anti-IFNg , for 20 minutes, at 4�C in the dark.

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Cells werewashedwith PBS/1%FCS and then resuspended in 200mL PBS/1% FCS.

An immunological "response/responder" was defined as a 2-fold increase in the frequency of cytokine-producing cells inrelation to that observed with the irrelevant peptide (Telomerase540). For example, if the frequency of IFNg-producing CD8þ Tcells induced by CEA691 doubled that stimulated by controlpeptide at the end of 4 rounds of pCEA691-specific stimulation,the response was defined as positive (i.e., a "responder").

Statistical analysisStatistical analysis was conducted using the SPSS software

(SPSS for windows, version 21). Data sets were first tested forparametric distribution using the Skewness–Kurtosis and thehomogeneity of variance tests. For parametric data, the T test wasused to determine statistical significance; for nonparametric datadistributions, the Mann–Whitney U test was applied. Whencomparing data sets between more than two groups, the one-wayANOVA test (for parametric data) or Kruskal–Wallis test (fornonparametric data) was used. Whenever an overall P value wasstatistically significant, post hoc pairwise comparisons were per-formed with the Tukey Honest Significant Difference method. Pvalues < 0.05 were considered statistically significant. For survivalanalyses, the Mann–Whitney U test was applied.

ResultsCEA691-specific CTL responses can be generated from PBMCsof pancreatic cancer patients

We previously investigated antigen-specific CTL responses gen-erated by short-term culture of PBMCs isolated from13pancreaticcancer patients (Supplementary Table S1) and 10 healthy con-trols. These were stimulated with 3 CEA HLA-A2–restricted pep-tides (CEA605-613 YLSGANLNL, CEA691-699 IMIGVLVGV, andCEA694-702 GVLVGVALI) and one irrelevant peptide Telome-rase540-548 ILAKFLHWL over 9 to 10 days. Antigen-specific IFNgproduction was most frequently observed in CEA691-responsiveCD8þ T cells, with a higher percentage of both patients andhealthy controls generating functional responses than to othertested epitopes (Supplementary Fig. S1A). In responders, thefrequency of IFNg-producing cells within the CD3þCD8þ T-cellsubset was not significantly higher in pancreatic cancer patientsthan in healthy controls (Supplementary Fig. S1B and S1C).Thesedata were in agreement with previous findings indicating thatimmune tolerance to particular self-peptides may be incomplete(30).

Here, we demonstrate in Fig. 1A the gating strategy for analysisof PBMCs from a single pancreatic cancer patient (CA11) afterfour rounds of expansion in vitro. An increase in the percentage ofantigen-specific IFNg-producing CD8þ T cells was observed. Afterthe 4th round of peptide stimulation, IFNg-producing CD8þ Tcells were detectable in 5 of 15 healthy controls and 10 of 18pancreatic cancer patients, with 4 examples of each shown in Fig.1B (healthy controls H01,H04,H07,H13) andC (cancer patientsCA07, CA11, CA12, CA18), respectively.

Pancreatic cancer disease progression is associated withimpaired CEA691-specific CTL responses

Six patients recruited for this studywere classifiedashaving stageIV pancreatic cancer (metastatic disease at presentation), whereasthe rest were classified stages II (N¼ 10) and III (N¼ 2) pancreaticcancer (Fig. 2A). CEA691-specific CTL responses were detected in 9

of 12 patients with stage II–III disease, whereas only 1 of 6 patientswith stage IVdiseasehaddemonstrable functionalCEA691-specificT-cell responses as determined by IFNg secretion upon four-roundantigen-specific stimulation (Fig. 2B). Relative frequencies ofCEA691-specific CD8þ T cells were also significantly lower thanthat in patients with earlier stage disease II–III (Fig. 2B). Pancreaticcancer patients whohad been treatedwith chemotherapy (Fig. 2C)and patients with inoperable tumors (Fig. 2D) also had lowerfrequencies of CEA691-specific CTLs compared with the non-chemo and surgical patient groups, respectively.

No statistically significant difference inmedianOS fromdate ofrecruitment to the study was observed between patients whogenerated functional CD8þ T-cell responses to CEA-691-699and those with no responses. Median OS was 20.5 months(range, 1–31 months) for responders versus 14 months (range,1–24months) for nonresponders, P¼ 0.079 (Table 1). However,the median OS of the 3 patients with more than 20% of func-tional CD8þ T cells at the end of stimulation (CA07, CA11, andCA18) was significantly longer than the other patients (28 vs.14 months, P ¼ 0.004). When only patients treated with surgi-cal resection were analyzed (n ¼ 10), those with functionalCEA-specific CTL had a significantly longer median OS of38.5 months (23–59 months, n ¼ 6) compared with 22 months(6–31 months, n ¼ 4), P ¼ 0.039.

CEA691-specific CTL from pancreatic cancer patients canrecognize and kill pancreatic cancer cell lines in vitro

T-cell lines were generated by stimulating the PBMCs of pan-creatic cancer patients using pCEA691-loaded T2 cells for fourrounds. As described above, over 30% of CD8þ T cells from 3patients (CA07,CA11, andCA18)produced cytokines in responseto pCEA691 restimulation after four rounds of expansion (Fig.1C). To examine their ability to recognize and induce tumor celldeath, we utilized CFSE killing assays from T-cell lines generatedfrom CA07, CA 11, and CA18.

T2 cells loaded with 1mmol/L (10�6 mol/L) pCEA691 peptide(CFSEhi) or irrelevant peptide-loaded T2 cells (CFSElo) werecocultured with CTL at different E:T ratios ranging from 100:1to 0.6:1. Unstimulated HLA-A2þ PBMCs were used as controleffector cells (Supplementary Fig. S2A). The results demonstratethat CEA691-specific CTLs generated from all these three patients(CA07, 11, and 18) were able to recognize and kill CEA691-loaded T2 cells (Supplementary Fig. S2B). Moreover, T cells fromCA11were able to kill T2 cells loadedwith 10�9mol/L CEA691 (1nmol/L), at an E:T ratio¼ 20:1, in peptide titration assay, inwhichT cell lines were stimulated with T2 cells loaded with 10�5 to10�12 mol/L (10 mmol/L to 1 pmol/L) CEA691 peptide for 4hours (E:T ¼ 20:1; Supplementary Fig. S2C and S2D). The T-cellline generated from CA11 was shown to have moderate-to-highavidity with recognition of nanomolar (10�9 mol/L) concentra-tions ofCEA,which are comparablewith the concentration of TAAexpression on the surface of tumor cells (31).

Subsequently, we investigated the cytotoxic effector function ofT-cell lines following stimulation with pancreatic cancer cell linesin vitro. The expression of CEA andHLA-A2 in six pancreatic cancercell lines was determined after staining with specific monoclonalantibodies and analyzed by flow cytometry. MiaPaca-2 was neg-ative for both CEA and HLA-A2; the Panc-1 cell line was HLA-A2positive, but expressed low levels of CEA; Bx-Pc-3 and PK-1 wereHLA-A2–positive cell lines with high expression of CEA (Fig. 3A).Therefore, MiaPaca-2 was used as the negative control for both

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HLA-A2 and CEA, and PK-1, Bx-Bc-3 (HLA-A2þ and CEAþ),together with Panc-1 (HLA-A2þ and CEA low) were used asspecific targets. Figure 3B shows the relative reduction in thefrequencies of PK-1 after coculture with the CTL line (frompatientCA11) and MiaPaca-2 cells, at different E:T ratios. T-cell linesgenerated from CA07 and CA11 patients lysed PK-1 and Bx-Pc-3cancer cell lines, but not Panc-1, demonstrating their CEA691-specific cytotoxicity (Fig. 3C). No cytotoxicity against pancreaticcancer cell lines was observed using unstimulated PBMCs from

the same donors (data not shown). These data suggest thatfunctional CEA-specific CTLs can be generated in pancreaticcancer patients and that these cells may have the potential tocontrol tumor growth.

Inhibitory pathways play a role in the modulation of antigen-specific CTL responses in pancreatic cancer patients

Possible explanations for the failure of antitumor T-cellresponses in patients include inadequate T-cell priming and

Figure 1.

CEA691-specific T cells isolated frompancreatic cancer patients andhealthycontrols produce type 1 cytokines.A,Atypical example of T cells expandedduring stimulation with pCEA691-loaded T2 cells. Between the second(2R) and fourth rounds (4R) ofstimulation, the percentage ofINFg�producing CD8þ T cellsgradually increased. By the end of the4th round stimulation, 77.9% of CD8þ

T cells were INFg�secreting,compared with 2% after 2 rounds. Dotblots representing IFNg andTNFa�producing CD8 T cells culturedwith CEA691-pulsed T2 cells andcontrol peptide–loaded T2 cells after 4rounds of stimulation (Gated on CD8 Tcells) in 4 of 15 healthy controls (B) and4 of 18 cancer patients (C) are shown.The percentages of IFNgþTNFaþ CD8T cells are shown in the upper-rightquadrants.






insufficient duration of the effector phrase, both of which can beregulated by the coinhibitory receptors, including CTLA-4, PD-1,TIM-3, and LAG-3 (32). For example, PD-1/PD-L1 signalinginhibits T-cell function via suppressing TCR-dependent activationof both CD4þ and CD8þ T cells, particularly, through the inhi-bition of their proliferation and cytokine production, includingIFNg , TNFa, and IL2 (33). PD-L1 expression by tumor cells hasbeen associated with a poorer prognosis or advanced disease (20,34–36). About 80% of pancreatic cancer cases express PD-L1, ofwhich 20% have upregulated expression of PD-L1 (comparedwith normal pancreatic tissue) and tend to be highly invasive andrecurrent (37, 38).

In order to investigate the relationship between CEA691-spe-cific CTL responses and the expression of negative regulatorymolecules on T cells, ex vivo phenotypic examination of pancreaticcancer patients' peripheral T cells was performed prior to stimu-lationwithCEA691-peptide. As shown in Fig. 4A, the frequency ofPD-1, TIM-3, and LAG-3 expressing CD8þ T cells was significantlyhigher in the nonresponder group of pancreatic cancer patientsthan in the healthy controls.

It is known that T-cell activation and differentiation statusinfluences the expression of some cell surface markers, such asPD-1. In order to examine the T-cell phenotype inmore detail, weidentified na€�ve, central memory (TCM), effector memory (TEM),and end-stage/effector phenotypes based on CD62L andCD45ROexpression (i.e., TCMwas defined asCD62LþCD45ROþ

T cells, TEM as CD62L�CD45ROþT cells, na€�ve T cells asCD62LþCD45RO�, and end-stage/effector T cells asCD62L�CD45ROþ). As shown inFig. 4B, therewas no statisticallysignificant difference in the percentage of gated CD4þ or CD8þ T

cells at various differentiation stages between pancreatic cancerresponders and nonresponders. The percentage of na€�ve T cells,however, in responders was observed to be higher than in non-responders, but this difference did not achieve statistically differ-ence (P ¼ 0.051).

CD8þ T-cell priming and activation take place in draining LNswhere, upon interaction with antigen-presenting cells, na€�ve Tcells are primed and differentiate into fully functional antigen-specific CTLs. We were able to analyze the expression of PD-1,TIM-3, and LAG-3 inmatched peripheral- and LN-derivedCD8þ Tcells isolated from3of the pancreatic cancer patients (see Table 1).The proportion of LN-derived CD8þ T cells expressing PD-1 andTIM-3was higher than that observed in peripheral T cells, whereasmore LAG-3–positive cellswere identifiedwithin the peripheral T-cell repertoire than in the draining LNs (Fig. 4C, left). In addition,similar differences were observed when analyzing the expressionlevels of PD-1, TIM-3, and LAG-3, as determined by mean fluo-rescence intensity, MFI (Fig. 4C, right).

Blockade of the PD-1/PD-L1 pathway improves tumor antigen-specific CD8þ T-cell responses

To assess whether blockade of the PD-1/PD-L1–negative reg-ulatory pathway enhanced the function of patient-derived, self-restricted CEA691-specific CTL, PBMCs from patients with pan-creatic cancer (and LN cells from one patient) were cultured for 7days in the presence of pCEA691 or the control peptide, rIL2, withorwithout anti–PD-L1 and/or anti–TIM-3 antibodies. In total, thefrequency of peptide-specific cytokine-producing CD8þ T cellswas evaluated in 11 patients with pancreatic cancer (see Table 2for patient details). In addition, the frequency of CEA691

Figure 2.

Higher frequencies of CEA691-specificCD8þ T-cell responses are detected inearlier stages of pancreatic cancer.A,Piechart illustrates the frequency ofpancreatic cancer patients at differentdisease stages (II–IV). Frequencies ofIFNgþ cells within the CD8þ T-cellpopulation are shown for patientsstratified according to (B) disease stage,(C) prior administration ofchemotherapy, and (D) submission tosurgery. Each symbol represents oneindividual, and horizontal bars representmedian. P values < 0.5 were consideredstatistically significant and are shown inthe graphs.

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tetramer–positive CD8þ T cells was determined in 8 of the 11patients (where sufficient samples were available for analysis).

As shown in Table 1, a significant increase in the frequency ofCEA691 tetramer–binding CD8þ T cells was observed in cellscultured in the presence of anti–PD-L1 antibody (P ¼ 0.030, n ¼8) and the combination of anti–PD-L1þanti–TIM-3 antibodies(P ¼ 0.045, n ¼ 8), compared with nontreated cells. These find-ings suggest that PD-L1 and TIM-3 blockade can enhance theproliferation of pancreatic cancer patient–derived CEA691-spe-cific CTLs in vitro. Similarly, the frequency of IFNg-producing

CD8þ T cells was significantly higher in the cells treated withanti–PD-L1 or anti–TIM-3, alone or in combination (Fig. 5A–C).Even though both PD-1/PD-L1 and TIM-3–regulatory pathwayswere observed to be involved in the modulation of CTL function,our data did not reveal a significant synergistic effect when PD-1/PD-L1 and TIM-3 pathways were blocked at the same time.

To determine the relative function of negative regulatory path-ways in the inhibition of antigen-specificCTL responses in the LNsof patients with pancreatic cancer, MNCs isolated from LNs werecultured with the pCEA691 peptide in the presence or absence of

Figure 3.

Cytotoxic activity of CTL lines againstpancreatic cancer cell lines. A, CEAand HLA-A2 expression of sixpancreatic cancer cell lines. The resultswere analyzed by FACS and presentedin histogram (top). Isotype antibodieswere used to determine thebackground. Percentage and MFI ofHLA-A2/CEA expressions by differentpancreatic cancer cell lines was alsoshown (bottom). N.B. The unstainedcells were gated out. B, FACS analysisof CA11 T-cell killing activity inresponse to recognize and kill PK-1 cellline (HLA-A2þ, CEAþ, labeled withhigh dose CFSE), and MiaPaca-2 cellline (HLA-A2�, CEA�, labeled withlow-dose CFSE), at different E:T ratios.C, The percentage of relative killing ofPK-1, Panc-1, and Bx-Pc-3 by CTLsfrom CA07 or CA11, compared withMiaPaca-2, at different E:T ratios. Allthe experiments were repeated twice,and themean of the results was shownin the figure.






anti–PD-L1 and/or anti–TIM-3 blocking antibodies. Blockade ofthe PD-1/PD-L1 pathway, but not of the TIM-3 pathway, led to anincrease in the frequency of IFNg-producing cells in both circu-lating and LN-derived CTLs, suggesting a role for the PD-1/PD-L1axis in the modulation of IFNg responses in patients with pan-creatic cancer (Fig. 5C and D). We observed that the expression ofPD-1 and TIM-3 was higher in LN-derived CD8þ T cells than thatin peripheral T cells, and blockade of these inhibitory moleculesled to a more significant recovery of peptide-specific IFNg pro-duction by CD8þ T cells.

Our findings suggest that the expression level of negativeregulatory molecules, in circulating and LN-derived CTLs, mayinfluence themagnitude of the CTL-mediated antitumor responsein different tissues.

DiscussionThe prognosis for patients with pancreatic cancer remains

extremely poor, and novel treatments such as immunotherapyare appealing alternatives to improve survival. However, there iscurrently little evidence from large clinical trials to demonstratethat pancreatic cancer is sensitive to checkpoint inhibition, andvaccine-based immunotherapies have largely failed to generatesignificant clinical results (10, 17, 18, 24). Meanwhile, someanimal experiments have suggested that the combination ofcheckpoint inhibitors with other immunotherapies, such as ther-

apeutic vaccines, may improve outcomes in pancreatic cancer(24). Our study has focused on exploring potential epitopes fortargeted immunotherapy in pancreatic cancer patients and hasevaluated the influence of blockade of PD-1/PD-L1 pathway orTIM-3 pathway in antitumor T-cell responses.

CEA is an 180 kDa glycosylated membrane protein that isoverexpressed in more than 90% of pancreatic cancer cases(16). As a self-antigen, CEA-specific T cells are expected to besubject to immunological tolerance. Although previous studiesdemonstrated that tolerance to CEA691 is incomplete (30), littleis known about the function of CEA691-specific CTL isolatedfrompatients with pancreatic cancer. Here, we identified themostimmunogenic CEA-derived epitopes using short-term in vitroPBMC stimulation followed by prolonged antigen stimulationto expandprimaryCEA691-specific T-cell lines. After one roundofin vitro peptide stimulation, we observed that the CEA691 pep-tide–specific IFNg-producing T-cell responses were more readilydetectable in pancreatic cancer patients compared with healthycontrols and that responding patients had higher percentages ofIFNg-producing T cells than those stimulated by other peptideepitopes. After a total of four rounds of in vitro stimulation, theCEA691-specific CTLs from pancreatic cancer patients were alsoable to produce TNFa and kill relevant target cells.

Despite the small sample size, wewere able to demonstrate thatT cells isolated from patients presenting with more advanceddisease were less likely to generate effective antigen-specific

Figure 4.

Expression of negative regulatory molecules in pancreatic cancer. PBMCswere isolated from pancreatic cancer patients and healthy controls, and surface stained toassess (A) the expression of PD1, TIM-3, and LAG-3 molecules; as well as (B) the relative proportions of na€�ve/memory subsets (based on the expression ofCD45RO and CD62L), within the CD8þ T-cell population. Pancreatic cancer patients were stratified according to their ability to mount CEA691-specificCD8þ T-cell responses (responders vs. nonresponders). Each symbol represents one individual, and horizontal bars represent mean. P values < 0.5 were consideredstatistically significant and are shown in the graphs. C, T cells were obtained from matching samples of peripheral blood (closed circles) and LNs (open circles)from 3pancreatic cancer patients. Graphs illustrate the levels of negative regulatorymolecule expressionwithin the CD8þ population, both in terms of frequency andnumber of molecules per cell, expressed as MFI. Each symbol represents one individual, and horizontal bars represent mean. P values < 0.5 were consideredstatistically significant and are shown in the graph.

Chen et al.





cytokine-producing responses. It is possible that prolonged TAAexposure in the tumormicroenvironmentmay induce exhaustionof antigen-specific T-cell responses. Unfortunately, we wereunable to test PBMCs at different time points over the course ofpancreatic cancer progression for individual patients. Thus, theevolution of phenotypic and functional changes in the anti-CEA691 T-cell responses in our patients may be related to mul-tiple factors including prior treatment, lymphopenia in advancedpatients, comorbidities including age, and/or the use of immu-nosuppressive drugs. Similarly, pancreatic cancer patientswithoutdemonstrable CEA691-specific T-cell responses may have pre-sented with more aggressive and/or quickly progressing cancers,hence having more advanced disease at diagnosis. To addressthese issues, a prospective longitudinal study with a larger samplesize is needed.

Interestingly, our preliminary findings suggest that surgicaltreatment, which had occurred prior to patients being evaluatedin the study (and therefore prior to T-cell isolation), may alsomodulate CD8þ T-cell responses, suggesting different modes oftreatment received may also affect TAA-specific immune

responses in pancreatic cancer. The interpretation of these datashould take into account that this study was not designed to lookat differences in clinical outcome and that the treatment modal-ities usedwere heterogeneous. Inour previous study, the influenceof medical interventions on T-cell response was observed dem-onstrating that embolization improved AFP-specific CD4þ T-cellresponses in patients with hepatocellular carcinoma (39). Again,to confirm these findings, a much larger study would be requiredto control for all the relevant variables.

Activation of the inhibitory PD-1/PD-L1 pathway has beenlinked to the poor prognosis of pancreatic cancer (40). Our datahave also shown that patients not responding to pCEA691 hadlarger numbers of PD-1–expressing T cells in their peripheral T-cell compartment, compared with the T cells isolated fromresponding patients or healthy controls. Also, T cells isolatedfrom the tumor draining LNswere enriched for PD-1– andTIM-3–expressing cells comparedwith T cells isolated from theperipheralblood. Not surprisingly, these findings support the existence of animmunosuppressive tumor microenvironment in pancreatic can-cer patients. It is therefore likely that combination therapies with

Figure 5.

PD-1/PD-L1 and TIM-3 blockade inpancreatic cancer. A, The percentageof CEA tetramer binding CD8þ T cellsafter 7 days of culture with CEA691 inthe presence or absence of anti–PD-L1and/or anti–TIM-3 blocking antibodies.B, CEA691 CD8þ T cells from 11pancreatic cancer patients wereexpanded for 7 days with or withoutanti–PD-L1 and/or anti–TIM-3 blockingantibodies, and intracellular stained toassess the levels of IFNg production.The graph shows frequencies of IFNgþ

cells within the CD8þ T-cell population.Each symbol represents one individual,and horizontal bars represent mean.P values < 0.5 were consideredstatistically significant and are shown inthe graph. PBMCs and MNCs isolatedfrom LNs of (CA13) were alsostimulated for 7 days by pCEA691with or without anti–PD-L1 and/oranti–TIM-3 blocking antibodies, andassessed for levels of IFNg production.A representative example of IFNgproduction is shown in the dot plots(C). D, The graph shows the frequencyof IFNgþ cells within the CD8þ

T-cell population. Experiments areduplicated, and symbols indicate meanand SD.






checkpoint inhibitors and antigen-specific T cells may be requiredto optimize immunotherapeutic approaches to pancreatic cancer.

Recently, the efficacy of anti–CTLA-4 and anti–PD-1/PD-L1antibodies in treating several types of cancers has been demon-strated in early-phase clinical trials (41). However, these check-point inhibitors are considered most effective in treating cancerswith high mutational loads, e.g., melanoma and lung cancer,because such cancers typically generate more neoantigens andcheckpoint inhibitors reverse the inhibition of tumor-infiltratingneoantigen-specific T cells (42). On average, the neoantigenrepertoire of pancreatic cancer is 1mutation per megabase, whichis much less than that observed in melanoma and lung cancer(with more than 10 mutations per megabase on average; ref. 43).In theory, therefore, pancreatic cancer should not be very sensitiveto checkpoint blockade. To date, there have been a number ofclinical trials assessing the safety and efficacy of checkpointinhibitors in pancreatic cancer patients (44), but no positiveresults have yet been published.

However, our in vitro results suggest that the PD-1/PD-L1pathway may be an important factor in the inhibition of antitu-mor functions of CD8þ T cells from pancreatic cancer patients.Following the addition of PD-L1 blocking antibodies, antigen-specific T-cell proliferation and cytokine production (IFNg andTNFa) were improved, with a greater effect observed on T cellsisolated from the tumor draining LNs than from the peripheralblood. In addition, the percentage of T cells expressing PD-1 inLNs was higher than that observed in the peripheral blood. Thus,the limited impact to date of checkpoint inhibition in pancreaticcancer may be due to the relatively small neoantigen repertoire,and/or that TAA-specific T cells less readily enter the tumormicroenvironment due to associated desmoplasia. Finally, wedid not observe a synergistic effect when combining PD-L1blockade with TIM-3 blockade. This may have been becauseTIM-3 levels were generally low in freshly isolated T cells. Littleis known about the expression and role of TIM-3 (45, 46) andLAG-3 (47) in T cells frompancreatic cancer patients. In our study,the percentage of TIM-3– and LAG-3–positive CD4þ and CD8þ Tcells was higher in pancreatic cancer patients, compared withhealthy donors. Although the ex vivo culture of patient-derivedeffector T cells with PD-1 and Tim-3 blocking antibodies hasexperimental limitations, which cannot reflect the complexity ofin vivo environment,we believe it provides useful preliminary dataon which to base further studies.

In conclusion, here we describe the in vitro stimulation andexpansion of self-restricted, autologous, functional CEA691-spe-cific T cells, isolated from the peripheral blood and draining LNsof patients with pancreatic adenocarcinoma. This raises the pos-sibility that expansion and infusion of autologous CEA691-spe-cific T cells may be an effective immunotherapeutic approach topancreatic cancer. Although alternativeCEA-based immunothera-

pies are still being designed and optimized for pancreatic cancer(12, 48, 49), includingCEA-specific engineered T cells,whichhavebeen shown to eradicate pancreatic cancer tumors without induc-ing toxicity in mice (50, 51), in another study, the administrationof autologous T lymphocytes genetically engineered to express amurine TCR specific for CEA691 to colorectal cancer patients wasshown to induce severe transient colitis (52) resulting in suspen-sion of the clinical trial, raising questions about the suitability ofthis particular epitope for the design of novel TCR-based thera-peutic approaches. Compared with the CEA691-specific high-affinity TCR generated by others (52–54), our CEA691-specificT cell lines were isolated frompatients with pancreatic cancer, andit is possible that their antitumor efficacy can be improved by thein vivo reversal of dysfunctionmediated by increased expression ofPD-1 and TIM-3.

Disclosure of Potential Conflicts of InterestE.C. Morris reports receiving commercial research grants from Cell Medica,

and is a consultant/advisory board member for GE, GlaxoSmithKline, and UCLTechnology Fund. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: Y. Chen, S. Behboudi, E.C. MorrisDevelopment of methodology: Y. Chen, S.-A. Xue, S.P. PereiraAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Chen, G.H. Mohammad, S.P. PereiraAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Chen, E.C. MorrisWriting, review, and/or revision of the manuscript: Y. Chen, S.-A. Xue,S. Behboudi, S.P. Pereira, E.C. MorrisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Chen, G.H. MohammadStudy supervision: S.P. Pereira, E.C. Morris

AcknowledgmentsWe thank Mr. Kito Fusai for performing LN resections and providing clini-

cal care for surgically treated patients.

Grant SupportE.C. Morris was supported by the UCLH NIHR Biomedical Research Centre,

the CRUK UCL Experimental Cancer Medicine Centre, Bloodwise and theMedical Research Council. S.P. Pereira was supported by NIH (Grant: PO1CA084203) andby theNational Institute forHealthResearchUniversityCollegeLondon Hospitals Biomedical Research Centre. S.-A. Xue was supported by theLLRprogramgrant. S. Behboudiwas supported byBBSRCgrant (BB/N002598/1and BBS/E/I/00001825).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 25, 2017; revised June 20, 2017; accepted July 10, 2017;published OnlineFirst July 14, 2017.

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Published OnlineFirst July 14, 2017.Clin Cancer Res Yuan Chen, Shao-An Xue, Shahriar Behboudi, et al. Pancreatic Cancer PatientsDysfunction of Circulating CEA-Specific T Cells in

PD-L1/PD-1 Pathway Blockade ReversesEx Vivo

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