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Research Article

Nonclassical Antigen-Processing Pathways Are Required forMHC Class II–Restricted Direct Tumor Recognition byNY-ESO-1–Specific CD4þ T Cells

Junko Matsuzaki1,2, Takemasa Tsuji1,4, Immanuel Luescher5, Lloyd J. Old4, Protul Shrikant3,Sacha Gnjatic4, and Kunle Odunsi1,2,3

AbstractTumor antigen–specific CD4þ T cells that directly recognize cancer cells are important for orchestrating

antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II)presentation of intracellular tumor antigen by cancer cells are poorly understood.We found that two functionallydistinct subsets of CD4þ T cells were expanded after HLA-DPB1�04 (DP04)-binding NY-ESO-1157-170 peptidevaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 proteinpulsed on DP04þ target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. Thetumor-recognizing CD4þ T cells more efficiently recognized the short 8–9-mer peptides than the non–tumor-recognizing CD4þ T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-IIantigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomaldegradation and transporter-associated with antigen-processing–mediated peptide transport, were also involvedin the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly byprimaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologicinhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancercells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4þ T cells could be a promisingstrategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment. CancerImmunol Res; 2(4); 341–50. �2013 AACR.

IntroductionTumor antigen–specific CD4þ helper T cells play important

roles in the induction and maintenance of antitumor immuneresponses. The roles of antigen-specific CD4þ T cells includeproviding help to CD8þ T cells during the primary and sec-ondary immune responses, inducing the activation/matura-tion of antigen-presenting cells (APC), producing cytokinesthat are essential for differentiation or maintenance of long-lasting T-cell responses, and activating B cells to producetumor antigen–specific antibodies (1, 2). In addition,

immune-potentiating cytokines from CD4þ T cells may helpother immune cells to overcome the actions of immunosup-pressive factors (3, 4). In the classical view of antigen presen-tation, intracellular and extracellular proteins are presented toCD8þ and CD4þ T cells via the MHC class I (MHC-I) and MHCclass II (MHC-II) pathways, respectively (5, 6). Therefore,although tumor antigen–specific CD8þ T cells efficiently rec-ognize intracellular antigen-expressing cancer cells, the acti-vation of tumor antigen–specific CD4þ T cells generallyrequires professional APCs that take up and cross-presenttumor antigen proteins. Recently, accumulating evidence hasdemonstrated that APCs at the tumor microenvironment arefrequently immunosuppressive and lead to unresponsivenessof T cells (7, 8). The absence of functional APCs that cross-present tumor antigen protein to CD4þ T cells may limit CD4help at the local tumor sites and could partly explain the rapidexhaustion of tumor antigen–specific CD8þ T cells. An alter-native path by which tumor antigen–specific CD4þ T cellscould overcome the requirement for APCs within the tumormicroenvironment is direct recognition of tumors.

In contrast with murine cancer cells, many types of humancancers constitutively express MHC-II or are induced toexpress MHC-II in an IFN-g–dependent manner (9). Tumorantigen–specific CD4þ T cells that directly recognize cancercells have been described (10–13); however, the mechanisms

Authors' Affiliations: 1Center for Immunotherapy; 2Departments of Gyne-cologic Oncology and 3Immunology, Roswell Park Cancer Institute, Buf-falo; 4Ludwig Institute for Cancer Research, New York Branch at MemorialSloan-KetteringCancerCenter, NewYork,NewYork; and 5Ludwig InstituteforCancer Research, LausanneBranch,University of Lausanne, Epalinges,Switzerland

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

J. Matsuzaki and T. Tsuji contributed equally to this work.

Corresponding Author: Kunle Odunsi, Roswell Park Cancer Institute, Elmand Carlton Streets, Buffalo, NY 14263. Phone: 716-845-8376; Fax: 716-845-1595; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0138

�2013 American Association for Cancer Research.

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relating to antigen specificity and antigen processing that theCD4þ T cells use for direct tumor recognition are unknown.Although several distinct nonclassical antigen presentationpathways have been identified for the presentation of intra-cellular proteins on MHC-II of professional APCs (14–17), it isnot clear whether these pathways are functional in cancer cellsfor MHC-II presentation of intracellular tumor antigens.Recently, we have identified a novel nonclassical antigenpresentation pathway for the presentation of intracellularNY-ESO-1 to HLA-DRB1�01 (DR01)-restricted NY-ESO-1–spe-cific CD4þ T cells by cancer cells (18). Two DR01-restrictedCD4þ T-cell lines that recognize the NY-ESO-187-98 and NY-ESO-195-106 peptides were studied for the recognition of exog-enously pulsed and intracellularly expressed NY-ESO-1 pro-teins. Although both CD4þ T cell lines similarly recognizedrecombinant NY-ESO-1 protein pulsed on APCs, only the NY-ESO-195–106–specific CD4

þ T cells directly recognized NY-ESO-1–expressing DR01þ melanoma cell lines in a HSP90-depen-dent manner. Such a tumor-recognizing CD4þ T-cell subset islikely selectively activated by the MHC-II–binding peptidesthat are naturally presented on cancer cells and could havesignificant potential as a strategy for cancer immunotherapy.In this regard, although DR01 is relatively frequent (8%–21% ofindividuals in population groups in the United States; ref. 19),additional MHC-II–binding epitopes for other HLAs will berequired for the development of immunotherapeutic strategiesusing tumor-recognizing CD4þ T cells. In addition, identifica-tion of the mechanisms of antigen processing for endogenousMHC-II presentation is critical for the understanding of directtumor recognition by CD4þ T cells.

In a previous clinical trial of NY-ESO-1 peptide vaccination(20), patients who were HLA-DPB1�04:01/�04:02 (DP04)þ andhad NY-ESO-1–expressing ovarian cancer were repeatedlyvaccinated with a DP04-binding peptide, NY-ESO-1157-170. Wefound a subset of vaccine-induced CD4þ T cells that secretedcytokines when they were cocultured with NY-ESO-1þDP04þ

cancer cells (20). In the present study, we identified the uniquepeptide specificity and antigen-processing pathways that allowdirect recognition of cytoplasmic protein presented byMHC-IIon cancer cells by the human CD4þ T-cell subset. Because ofthe frequent expression of DP04 (present in 43%–70% ofCaucasians; ref. 21), our observations will be useful for thedevelopment of novel immunotherapies that harness directtumor-recognizing ability of CD4þ T cells.

Materials and MethodsNY-ESO-1–specific T cells and cell lines

Peripheral blood mononuclear cells (PBMC) and tumortissues were obtained from patients with epithelial ovariancancer under an approved protocol from the institutionalreview board at Roswell Park Cancer Institute (Buffalo, NY).NY-ESO-1 expression in tumor tissues was determined byimmunohistochemistry and/or semiquantitative reverse-tran-scription PCR (RT-PCR), and anti-NY-ESO-1 antibody responsein serum was analyzed by ELISA, as described previously (22).NY-ESO-1–specific CD4þ and CD8þ T cells in PBMC wereamplified by in vitro presensitization from patients whoreceived NY-ESO-1 vaccination (20). NY-ESO-1157–170–specific

CD4þ T cells in tumor-infiltrating lymphocytes (TIL) from 4patients who were HLA-DP04þ and had spontaneous anti-NY-ESO-1 antibody response were also expanded by stimulationwith g-irradiated and peptide-pulsed CD4�CD8� cells derivedfrom autologous PBMCs. HLA-A�02:01 (A02)-restricted NY-ESO-1157–165–specific CD8

þ T cells were isolated using a FAC-SAria instrument (BD Biosciences) with HLA-A02/NY-ESO-1157-165 tetramer. DP04-restricted NY-ESO-1157–170–specificCD4þ T cells were isolated by a FACSAria instrument by gatingon IFN-gþ cells (Miltenyi Biotec) or CD40Lþ cells followingpeptide restimulation (23). For TILs, NY-ESO-1157–170–specificCD4þ T-cell lines were established from 3 patients. Amongthem, NY-ESO-1–specific CD4þ T-cell line from one patientcontained TR-CD4. CD4þ T cells derived from PBMCs werecloned by limiting dilution and periodic phytohemagglutinin(PHA, Remel) stimulations in the presence of feeder cells(irradiated allogeneic PBMCs) and interleukin (IL)-2 (RocheMolecular Biochemicals).

Melanoma cell lines and EBV-transformed B-cell lineswere from our cell bank. Establishment and characterizationof SK-MEL-37 clones expressing ICP47 have been described(18). Cells were cultured in RPMI-1640 medium supplemen-ted with 10% fetal calf serum (FCS), penicillin, streptomycin,and L-glutamine.

Generation of monocyte-derived dendritic cellsCD14þ monocytes were magnetically isolated from DP04þ

healthy donor PBMCs using anti-CD14 microbeads (MiltenyiBiotech). Monocytes were cultured for 6 days in RPMI-1640medium supplemented with 10% FCS, penicillin, streptomy-cin, and L-glutamine in the presence of 1,000 U/mL granu-locyte macrophage colony-stimulating factor (GM-CSF), and20 ng/mL IL-4 (CellGenix).

Pretreatment of target cellsSynthetic peptides were pulsed on target cells overnight at

10 mmol/L unless otherwise specified. Recombinant NY-ESO-1protein was expressed in Escherichia coli and purified by astandard method. NY-ESO-1 protein was pulsed overnight onSK-MEL-29 at a concentration of 10mg/mLor on dendritic cells(DC) at different concentrations. Peptide or recombinantprotein-pulsed and -unpulsed target cells were extensivelywashed before coculture with T cells. To determine HLArestriction of T-cell recognition, target cells were treated with10 mg/mL anti-HLA-ABC monoclonal antibody (W6/32;eBioscience) and/or 20 mL of anti-HLA-class II antibody super-natant for 1 hour before addition of T cells. Culture super-natants from anti-DP (B7/21), anti-DQ (SPV-L3), and anti-DR(L243) hybridomas were used as sources for anti-HLA-class IIantibodies. In some experiments, target cells were pretreatedwith 1,000 U/mL (50 ng/mL) IFN-g (Peprotech) for 2 days.Treatment of SK-MEL-37 with inhibitors for the antigen-pro-cessing pathway was performed as described (18). All inhibi-tors were water soluble except for lactacystin and epoxomicin,which were dissolved in dimethyl sulfoxide. After treatment,SK-MEL-37 was fixed with 1% paraformaldehyde, quenchedwith glycine, and extensively washed in PBS and culturemedium. For mRNA electroporation, EBV-transformed B-cell

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line (1 � 106) was mixed with 5 mg in vitro-transcribed mRNA(Ambion) in 50 mL X-Vivo15 (Lonza) andwas applied at a pulseof 1.25 kV/cm for 700 msec using the ECM 830 Electroporationsystem and cuvettes (Harvard apparatus-BTX). Cells wereincubated overnight in a culture medium until T-cell recog-nition assays. Electroporation of SK-MEL-37 with syntheticsiRNA (Integrated DNA Technologies or Origene) was per-formed as described (18).

Intracellular cytokine stainingBrefeldin A (BFA) and monensin were added 2 hours after

coculturing T cells and target cells. After 6 hours of coculture,cells were fixed with 2% paraformaldehyde and permeabilizedusing FIX&PERMreagents (Invitrogen-CALTAG) according tothe manufacturer's instructions (20). Cytokine production wasassessed by intracellular staining measured by flow cytometry.Antibodies were obtained from BD Biosciences.

ELISPOT assayThe number of IFN-g–secreting antigen-specific T cells was

assessed by ELISPOT assays as described (20). The dark-violetspots were counted by an automated ELISPOT reader (Zeiss orCTL).

Measurement of cytokinesCD4þ T-cells (5 � 104) were cultured with SK-MEL-37 (3 �

104), protein-pulsed monocyte-derived DCs (2.5 � 104) orsingle-cell suspensions of ovarian tumor tissues (2.5 � 104)in a 96-well culture plate. The culture supernatant was col-lected 20 to 24 hours after the coculture and stored at �20�Cuntil measurement of cytokines by ELISA according to themanufacturer's instructions. Unconjugated and biotin-labeledantibody pairs for human IFN-g and GM-CSF were obtainedfrom BD Biosciences and HRP-labeled avidin D and 3,30,5,50-tetramethylbenzidine substrate solution were obtained fromeBioscience.

Statistical analysesError bars in the graphical data representmeans� SD. All in

vitro experimentswere performed at least in duplicate. P valuesof less than 0.05 were considered statistically significant by theStudent t test. All statistical analyses were performed usingPrism 5 software (GraphPad Software).

ResultsCharacterization of tumor recognition by CD4þ T cellsWe established DP04-restricted NY-ESO-1157–170 peptide–

specific CD4þ T-cell clones from a patient with ovarian cancerwho was repeatedly vaccinated with DP04-binding NY-ESO-1157–170 peptide (20). For this patient, NY-ESO-1157–170peptide–specific CD4þ T cells were not detected in prevaccinePBMCs, but were significantly expanded by the vaccinations(data not shown). One of 4 established NY-ESO-1–specificCD4þ T-cell clones produced IFN-g against a DP04þNY-ESO-1þ melanoma cell line (20). The other 3 clones showedno direct reactivity against the melanoma cell line. To gain aninsight into the different ability in direct tumor recognition byCD4þ T cells, we characterized target recognition by represen-

tative tumor-recognizingCD4þT-cell clone (TR-CD4) and non–tumor-recognizing CD4þ T-cell clone (NTR-CD4). Whereas TR-CD4 specifically recognized NY-ESO-1–expressing DP04þ mel-anomacell lines, all DP04þmelanomacell lineswere recognizedby both TR-CD4 and NTR-CD4 after pulsing with the NY-ESO-1157–170 vaccine peptide (Fig. 1A and B). In subsequent experi-ments, we selected the NY-ESO-1–expressing melanoma cellline, SK-MEL-37 (SK37) because (i) DR01þDP04þ SK37 wasextensively characterized for the recognitionbyDR01-restrictedNY-ESO-1–specific CD4þ T cells in our previous study (18); and(ii) SK37 was efficiently recognized by A02-restricted NY-ESO-1–specific CD8þ T-cell clone (18). SK37 expressed both HLAclass I and class II molecules and CD40 but no costimulatorymolecules, CD80 and CD86 (Supplementary Fig. S1). Recogni-tion of SK37 by TR-CD4 was specifically inhibited by anti-HLA-DP blocking antibody, demonstrating DP04-restricted targetcell recognition (Fig. 1C). As expected, recognition of SK37 bycontrol NY-ESO-1–specific A02-restricted CD8þ T cells wasinhibited by anti-HLA class I blocking monoclonal antibody.Electroporation of NY-ESO-1–specific siRNA, which efficientlysilencedNY-ESO-1 expression in SK37, significantly reduced therecognition by TR-CD4 (Fig. 1D), supporting the NY-ESO-1specificity in the direct tumor recognition by TR-CD4.

We next asked whether the differential ability to recognizeNY-ESO-1–expressing cancer cells by TR-CD4 and NTR-CD4indicates differences in the recognition of intracellular NY-ESO-1 protein in these cells. The NY-ESO-1–non-expressing DP04þ

melanoma cell line, SK-MEL-29 (SK29), was either infectedwithadenovirus carrying the NY-ESO-1 gene for intracellular NY-ESO-1 protein expression or pulsed with recombinant NY-ESO-1 protein or synthetic NY-ESO-1157–170 peptide as a positivecontrol and the recognition by TR-CD4 and NTR-CD4 wascompared.Whereas exogenousNY-ESO-1 protein was efficient-ly recognized by both TR-CD4 and NTR-CD4, only TR-CD4recognized intracellular NY-ESO-1 ectopically expressed byadenovirus in SK29 (Fig. 1E). As expected from the classicalantigen-processing pathways, CD8þ T cells efficiently recog-nized intracellularly expressed NY-ESO-1 but not exogenousNY-ESO-1 protein. It is possible that the presentation of ade-novirally expressed intracellular NY-ESO-1may differ from thatof the physiologically expressed NY-ESO-1. As an alternativemethod to induce intracellular NY-ESO-1, EBV-transformedDP04þ B cells were electroporated with in vitro transcribedNY-ESO-1 mRNA. As shown in Fig. 1F, TR-CD4 efficientlyrecognized target cells electroporated with NY-ESO-1 mRNA,indicating that TR-CD4 can recognize exogenous and endog-enous intracellular NY-ESO-1 antigen presented on MHC-II.

Determination of minimal epitopesWe investigated the mechanism for the differential recogni-

tion of intracellular NY-ESO-1 by TR-CD4 and NTR-CD4 interms of peptide recognition by T-cell receptor (TCR). Thetitration curves for TR-CD4 and NTR-CD4 to recognize thevaccine peptide, NY-ESO-1157-170, were similar (Fig. 2A). Inaddition, two long 20-mer peptides, NY-ESO-1151-170 and NY-ESO-1161-180, were similarly recognized by TR-CD4 and NTR-CD4 (Fig. 2B and C). Recognition of naturally processed exog-enous NY-ESO-1 protein by TR-CD4 and NTR-CD4 was tested.

Mechanism of Tumor Recognition by CD4þ T Cells

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TR-CD4 more efficiently recognized NY-ESO-1 protein-pulsedmonocyte-derived DCs than NTR-CD4 (Fig. 2D), which poten-tially explains the more efficient tumor recognition by TR-CD4.To investigate the mechanism(s) by which TR-CD4 recognizescancer cells, short overlapping vaccine peptides were tested forrecognition by TR-CD4 andNTR-CD4 (Fig. 2E). In contrast withthe similar recognition of 10-mer peptide NY-ESO-1160-169 byTR-CD4 and NTR-CD4, recognition of 9-mer peptide NY-ESO-1161-169 by NTR-CD4 was significantly reduced, whereas it wasfully recognized by TR-CD4 (Fig. 2E). In addition, the recogni-tion of 8-mer peptide NY-ESO-1161-168 wasmore efficient by TR-CD4 than byNTR-CD4 (Fig. 2E). There is a significant differencein the titration curves for the recognition of NY-ESO-1161–169peptide by TR-CD4 and NTR-CD4 (Fig. 2F). The recognitionof NY-ESO-1161–169 by NTR-CD4 was barely detectable at 10nmol/L, whereas the recognition by TR-CD4was still detectableat 0.01 nmol/L concentration. Taken together, these resultsindicate that naturally processed intracellular and extracellularNY-ESO-1 proteins are loaded on DP04 in a manner that ispreferentially recognized by TR-CD4 rather than NTR-CD4.

We searched for the presence of TR-CD4 in DP04þ ovariancancer patientswhodid not receive peptide vaccinationbut hadspontaneous NY-ESO-1–specific serum antibody. As we foundpreviously that ovarian tumors are highly enriched with NY-ESO-1–specific CD8þ T cells (24), ovarian TILs were assessed toincrease the possibility of detecting NY-ESO-1–specific T cells.We tested TILs from 4 patients and found NY-ESO-1–specificCD4þ T cells that recognize both NY-ESO-1161–169 and SK37 inone TIL-derived NY-ESO-1–specific CD4þ T-cell line (Fig. 3A),indicating that TR-CD4 is not induced only by synthetic peptidevaccinationsbut is also induced spontaneously by theNY-ESO-1protein expressed in cancer cells and infiltrates in the tumorsites. Next, we tested whether freshly isolated ovarian cancercells from patients can stimulate TR-CD4 ex vivo when theyexpress NY-ESO-1 and DP04. Single-cell suspensions of 5 NY-ESO-1–expressing and 5 NY-ESO-1–negative tumor specimensfromDP04þpatientswere tested for recognitionbyTR-CD4andNTR-CD4. Notably, TR-CD4 but not NTR-CD4 specifically pro-duced high amounts of IFN-g when cocultured with 2 of 5 NY-ESO-1þ ex vivo ovarian cancer cells (Fig. 3B). Together, the

Figure 1. Characterization of NY-ESO-1–specific tumor-recognizing (TR-CD4) and non–tumor-recognizing (NTR-CD4) CD4þ T-cell clones. A, recognition ofDP04þNY-ESO-1þ/� melanoma lines was tested by ELISPOT assays. B, DP04þ melanoma cells were pulsed overnight with NY-ESO-1157–170 peptideand recognition by T cells was evaluated by intracellular IFN-g staining. C, HLA restriction of SK37 recognition was determined using blockingantibodies by intracellular IFN-g staining. A02-restricted NY-ESO-1157–165–specific CD8þ T-cell clones (ESO-CD8) were used as control tumor-recognizingT cells. D, antigen specificity of tumor recognition. NY-ESO-1 (ESO), pan-MAGE (MAGE), or GFP-specific siRNA was electroporated into SK37.Recognition was evaluated by IFN-g-ELISPOT assays. E, recognition of NY-ESO-1157–170 (peptide), NY-ESO-1 protein (protein), and adenovirally inducedNY-ESO-1 (Adeno) in SK29 was tested by IFN-g ELISPOT assays. F, recognition of mRNA-induced intracellular NY-ESO-1. HLA-DP04þ EBV-transformedB cells were electroporated with ESO or GFP mRNA. Reactivity of TR-CD4 was measured by IFN-g-ELISPOT assays. Statistical significance wascalculated by the Student t test and is shown as �, P � 0.05; ��, P � 0.01; and ��, P � 0.001.

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infiltration of TR-CD4 at theovarian tumor site and the antigen-specific direct stimulation of TR-CD4 by ex vivo ovarian cancercells indicate thatTR-CD4plays a role in antitumor immunity inthe tumor microenvironment.

Antigen-processing pathway of endogenous MHC-IIpresentationTo elucidate the antigen-processing pathway for the NY-

ESO-1/DP04 epitope in SK37, we treated SK37 with inhibitorsof antigen processing at doses that did not inhibit MHC-IIexogenous peptide presentation by SK37 (18). As shown inFig. 4A, treatment of SK37 by a proteasome inhibitor, epox-omicin, substantially inhibited the recognition by TR-CD4. Theinhibitory effect by another proteasome inhibitor, lactacystin,was negligible, indicating that generation of the peptide rec-ognized by TR-CD4 is dependent on the chymotrypsin-likeactivity of the proteasome (25). Treatment of melanoma celllines with IFN-g did not inhibit recognition by TR-CD4 (Sup-

plementary Fig. S2), indicating that both standard- andimmuno-proteasomes similarly processed the epitope. In addi-tion, two endosomal/lysosomal protease inhibitors, chloro-quine and leupeptin, significantly inhibited the presentation,suggesting the role of the proteases in antigen processingand/or loading on MHC-II in endosomes/lysosomes (Fig.4B). As the generation ofMHC-II–binding peptide is dependenton proteasome, which is generally involved in the generation ofMHC-I–binding peptides, we investigated the involvement ofother enzymes that were reported to be involved in thegeneration of MHC-I–binding peptides. As shown in Fig. 5A,AAF-CMK that inhibits tripeptidyl peptidase II (TPPII; refs. 26,27) partially inhibited the presentation at the high dose,whereas aminopeptidase inhibitor (bestatin; refs. 28–30) andmetalloprotease inhibitor (1,10-phenanthroline; ref. 31) had noeffect. TPPII is a cytosolic peptidase that trims peptidesgenerated by the proteasome. The involvement of TPPII inaddition to the cytosolic proteasome in the endogenous MHC-

Figure 2. Determinationofminimumepitopes for TR-CD4 and NTR-CD4. A–C, dose dependence ofNY-ESO-1 peptide recognition byTR-CD4 and NTR-CD4. Indicatedpeptides were pulsed overnight onSK29 at indicated concentrations.Recognition by TR-CD4 and NTR-CD4 was evaluated by IFN-gELISPOT assays. D, dosedependence of NY-ESO-1 proteinrecognition. NY-ESO-1 proteinwas pulsed on DP04þ immaturemonocyte-derived DCs overnightat indicated concentrations.TR-CD4 and NTR-CD4 werestimulated with these DCs for24 hours. IFN-g production inthe supernatant was measuredby ELISA. E, recognition ofoverlapping NY-ESO-1 peptide-pulsed SK29 was evaluated byintracellular IFN-g staining. F, dosedependence of NY-ESO-1161-169recognition. NY-ESO-1161-169 waspulsed on SK29 at indicatedconcentrations. Recognition wasevaluated by intracellular IFN-gstaining. All experiments wererepeated at least twice withconsistent results. Error barsindicate SD from duplicated wells.






II presentation supports the cytosolic degradation of intracel-lularNY-ESO-1. In addition, SK37 clones expressing viral ICP47,which inhibits transporter-associated with antigen-processing(TAP)-mediated peptide transport, showed reduced stimula-tory activity compared with parental SK37, indicating a role forTAP-mediated peptide transport into the endoplasmic retic-ulum during the presentation (Fig. 5B).

Next, we investigated the involvement of nonclassical anti-gen-presentation pathways for the presentation of intracellularprotein to CD4þ T cells by professional APCs and B cells. Wefound that macroautophagy and chaperone-mediated autop-hagy were not involved in this presentation by treatment withmacroautophagy inhibitor, 3-methyladenine, and by siRNA-mediated silencing of LAMP-2, respectively (Fig. 6A and B;refs. 14, 16). Intracellular viral protein has been presented onrecycled MHC-II, which is efficiently inhibited by primaquine(15, 32). As shown in Fig. 6C, treatment with primaquinesignificantly inhibited the presentation to TR-CD4, indicatingthe involvement of endosomal recycling, presumably theMHC-II molecules. No significant inhibition by cycloheximide andbrefeldin A, which inhibit new protein synthesis and vesiculartrafficking, respectively, supported the presentation by recy-cled but not newly synthesized MHC-II (Fig. 6D). Significantenhancement of presentation by treatment with cyclohexi-mide suggests that inhibiting MHC-II synthesis enhancedcell surface expression of recycled MHC-II loaded with pep-tides from intracellular NY-ESO-1. Treatment with selectiveinhibitors (17-DMAG and radicicol) and siRNA-mediatedsilencing indicated that the presentation did not requirechaperoning by HSP90 (Supplementary Fig. S3A and S3B),which played a critical role in the presentation of NY-ESO-195–106 to HLA-DR01–restricted TR-CD4 by SK37 (18, 33, 34). Inaddition, treatment with PFT-m and quercetin, which inhibitHSP70 chaperoning and stress-induced HSP expression (35,36), respectively, enhanced the presentation, suggesting aninhibitory role for HSP70 in this presentation (SupplementaryFig. S3A).

DiscussionIn classical antigen-processing pathways, intracellular pro-

teins are not efficiently loaded ontoMHC-II for presentation toCD4þ T cells unless the protein is localized in or targeted toendosomal or lysosomal compartments (37–40). Becausemostimmunogenic tumor antigens, including NY-ESO-1, are intra-cellularly expressed, tumor antigen–specific CD4þ T cells

Figure 3. Detection of TR-CD4 at the local tumor site. A, tumor-infiltratinglymphocytes from a patient with ovarian cancer were stimulatedwith NY-ESO-1157–170. After 20 days, NY-ESO-1157–170–reactive CD4þ T cellswere isolated and expanded. Recognition of SK37, NY-ESO-1157–170,and NY-ESO-1161–169 was tested by intracellular staining. Values inquadrants indicate percentages of cells. B, TR-CD4 or NTR-CD4 cellswere cocultured with NY-ESO-1þ/� tumor single-cell suspensions (TSC)obtained fromDP04þ patients for 24 hours. IFN-g level in the supernatantwas measured by ELISA.

Figure 4. Effect of inhibitors for antigen degradation on the recognition of SK37 by TR-CD4. A and B, SK37 was cultured for 16 to 20 hours with or withoutindicated proteasome inhibitors (A) or protease inhibitors (B) followed by fixing with paraformaldehyde and extensive washes. SK37 was coculturedfor 20 hourswith TR-CD4. IFN-g level in the supernatantwasmeasuredbyELISA.Results are shown aspercentage of inhibition of IFN-g production comparedwith untreated target cells. Statistical significance was calculated by the Student t test and is shown as �, P � 0.05 and ��, P � 0.01.

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generally are not considered to recognize MHC-II-expressingcancer cells efficiently. Nevertheless, many tumor antigen–specific CD4þ T cells have been reported to directly recognizecancer cells (10–13). However, in contrast to the detailedstudies of endogenous MHC-II presentation pathways inexperimental systems using model antigens such as viralantigens and ectopically expressed self-antigens and profes-sional APCs (14–17), mechanisms of endogenous MHC-IIpresentation by cancer cells are poorly understood. It isimportant to characterize antigen specificity and antigen-processing pathways that are responsible for the direct rec-ognition of cancer cells by CD4þ T cells.In the present study, we provide several lines of evidence

that indicate the requirements for antigen (NY-ESO-1) spec-ificity and MHC-II (DP04) restriction for direct tumor recog-nition by TR-CD4. Although both TR-CD4 and NTR-CD4similarly recognized the 14-mer NY-ESO-1157–170 vaccine pep-tide, TR-CD4 efficiently (103–104 fold) recognized 9-mer NY-ESO-1161–169 peptide. The differences in the minimal epitopefor TR-CD4 and NTR-CD4 suggest that the unique peptide–TCR interaction is responsible for the tumor-recognizingability by TR-CD4. Indeed, the sequences of the genes encodingthe TCRa and b chains for TR-CD4 and NTR-CD4 are different(ref. 20 and data not shown). A future approach to test whetherthe TCR is solely responsible for the ability of TR-CD4 torecognize tumor cells will be to retrovirally transduce TCRgenes from TR-CD4 and NTR-CD4 into polyclonal expanded Tcells and test for the recognition of intracellular NY-ESO-1.We have characterized the antigen-processing mechanisms

for the endogenous MHC-II presentation of intracellular NY-ESO-1 to DR01-restricted NY-ESO-195–106–specific CD4þ Tcells by SK37 (18). The use of the same cancer cell line (SK37)as well as pharmacologic inhibitors for antigen processingenabled us to directly compare pathways for DR01- and DP04-restricted endogenous MHC-II presentation of intracellularNY-ESO-1. Both presentations were efficiently inhibited byepoxomicin, an inhibitor of proteasome. Interestingly, another

proteasome inhibitor, lactacystin, only inhibited DR01-restricted presentation, potentially indicating that there aredifferences in the enzymatic activities required for the gener-ation of the DR01- and DP04-binding epitopes (25). Thecytosolic proteasome is generally involved in the generationof MHC-I–binding short peptides, which supports our findingthat TR-CD4 cells efficiently recognize short 8-9-mer peptides.Both presentations were efficiently decreased by inhibitors forendosomal/lysosomal proteases, indicating that the peptidesare loaded on MHC-II in the endosomal or lysosomalcompartments.

Although there are multiple shared processing pathwaysbetween DR01- and DP04-restricted endogenous MHC-II pre-sentations of intracellular NY-ESO-1, there are also distinctdifferences. DP04-restricted presentation required TAP-medi-ated peptide transport into endoplasmic reticulum and endo-somal recycling, whereas DR01-restricted presentationrequired vesicular trafficking through the trans-golgi networkand chaperoning by HSP90. As HSP90 is known to facilitate thetranslocation from endosomal/cytosomal compartments tocytosol (41), it is likely that HSP90 plays a role in the reversetranslocation of proteasome-dependent cytosolic peptides toendosomal/lysosomal compartments for the loading onMHC-II as seen for constitutive HSP70 (HSC70)-dependent chaper-one-mediated autophagy (16). However, in the case of DP04-restricted presentation, the route for the cytosolic peptides toendosomal/lysosomal compartments is yet to be determined.In the classical MHC-I antigen-presentation pathway, peptidesgenerated by the proteasome are translocated into endoplas-mic reticulum through TAP and loaded onto MHC-I. Becauseof the TAP dependency of the DP04-restricted endogenousMHC-II presentation (Fig. 5B), the DP04-binding peptide isconsidered to enter the endoplasmic reticulum. We proposethat there are two potential mechanisms for the peptide in theendoplasmic reticulum to be loaded onto MHC-II in theendosomal/lysosomal compartments: (i) fusion between endo-some/lysosome and endoplasmic reticulum (42); and (ii)

Figure 5. Effect of inhibitors for MHC-I antigen-processing pathways on the recognition of SK37 by TR-CD4. A, SK37 was treated by the indicated inhibitorsfor 16 to 20 hours followed by fixing with paraformaldehyde and extensive washes. SK37 was cocultured for 20 hours with TR-CD4. IFN-g level in thesupernatant was measured by ELISA. B, role of TAP in the presentation. Untreated parental SK37 and SK37 clones stably expressing ICP47 gene wereusedas stimulator cells. GM-CSF level in the supernatantwasmeasuredbyELISA. Statistical significancewas calculatedby theStudent t test and is shownas�, P � 0.05; ��, P � 0.01; and ��, P � 0.001.






exchange of MHC-I- and MHC-II–loaded peptides duringendosomal recycling (43). Confirmation of these mechanismswill require the development of sensitive imaging experimentsusing epitope-specific antibodies to determine the route.

In summary, we have shown that human cancer cells canpresent intracellular tumor antigens on MHC-II by multiplenonclassical antigen-processing pathways, which results indirect tumor recognition by tumor antigen–specific CD4þ Tcells. It is likely that the use of multiple nonclassical proces-sing pathways increases the repertoire of intracellular tumorantigen–derived MHC-II–binding epitopes presented oncancer cells. Although we found only a minor subset ofNY-ESO-1–specific CD4þ T cells has direct tumor-recogniz-ing ability and the majority of these CD4þ T cells onlyrecognized exogenous NY-ESO-1 protein pulsed on APCs,the implications are significant. For example, direct tumorrecognition by CD4þ T cells may provide "CD4 help" in thetumor microenvironment, in which professional APCs arefrequently dysfunctional or immunosuppressive. Moreover,strategies for expansion and recruitment of tumor-recog-nizing CD4þ T cells at the local tumor sites, such as

vaccination and adoptive T-cell therapy, may enhance thetherapeutic effect of cancer immunotherapy. Therefore,identification of more MHC-II–binding tumor antigen pep-tides for other tumor antigens and HLA types is warrantedfor the development of more effective immunotherapy thatwill harness tumor-recognizing CD4þ T cells.

Disclosure of Potential Conflicts of InterestS. Gnjatic has ownership interest (including patents) in NY-ESO-1. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Matsuzaki, T. Tsuji, L. Old, S. Gnjatic, K. OdunsiDevelopment of methodology: J. Matsuzaki, T. Tsuji, I.F. Luescher, K. OdunsiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Matsuzaki, T. Tsuji, K. OdunsiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Matsuzaki, T. Tsuji, K. OdunsiWriting, review, and/or revision of themanuscript: J. Matsuzaki, T. Tsuji, S.Gnjatic, K. OdunsiStudy supervision: P. Shrikant, K. Odunsi

AcknowledgmentsThe authors thank A. Beck and A. Miliotto for assistance in experiments.

Figure 6. Effect of inhibitors forpreviously characterizedendogenous MHC-II presentationpathways on the recognition ofSK37 by TR-CD4. SK37 wastreated by the indicated inhibitorsfor 40 to 44 hours (A and C) or 16 to20 hours (D) followed by fixing withparaformaldehyde and extensivewashes. SK37 was cocultured for20 hours with TR-CD4. GM-CSFlevel in the supernatant wasmeasured by ELISA. A, effect of aninhibitor for macroautophagy. B,effect of siRNA-mediated silencingof LAMP2. SK37 waselectroporated with indicatedsiRNA and cultured for 3 days. C,effect of an endosomal/lysosomalrecycling inhibitor. D, effect ofvesicular transport and proteinsynthesis inhibitors. Allexperiments were repeated at leastthree times with consistent results.Error bars indicate SD fromduplicated wells. Statisticalsignificance was calculated by theStudent t test and is shown as�, P � 0.05 and ��, P � 0.01.

Matsuzaki et al.





Grant SupportThis work was supported by a Cancer Vaccine Collaborative grant (Cancer

Research Institute/Ludwig Cancer Research), Cancer Research Institute Anna-Maria Kellen Clinical Investigator Award, Ovarian Cancer Research Fund,Roswell Park Alliance Foundation, Ovarian Cancer Research Fund, NIH1R01CA158318-01A1, NIH 2P30 CA016056-36, and RPCI-UPCI Ovarian CancerSPORE NIH P50CA159981-01A1.

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 indicate thisfact.

Received August 30, 2013; revised November 16, 2013; accepted December 5,2013; published OnlineFirst December 17, 2013.

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2014;2:341-350. Published OnlineFirst December 17, 2013.Cancer Immunol Res Junko Matsuzaki, Takemasa Tsuji, Immanuel Luescher, et al.

T Cells+Specific CD4−Restricted Direct Tumor Recognition by NY-ESO-1−Class II

Nonclassical Antigen-Processing Pathways Are Required for MHC

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