Self/Tumor Antigen Speci c CD4þ T Cells in Phase I ... · 10/9/2013 · Research Article Effect of Montanide and Poly-ICLC Adjuvant on Human Self/Tumor Antigen–Speciﬁc CD4þ

Research Article

Effect of Montanide and Poly-ICLC Adjuvant on HumanSelf/Tumor Antigen–Specific CD4þ T Cells in Phase IOverlapping Long Peptide Vaccine Trial

Takemasa Tsuji1,8, Paul Sabbatini3,6, Achim A. Jungbluth1,4, Erika Ritter1,5, Linda Pan2, Gerd Ritter1,2,Luis Ferran1,7, David Spriggs3, Andres M. Salazar9, and Sacha Gnjatic1,7

AbstractVaccination of patients with ovarian cancer with overlapping long peptides (OLP) from cancer-testis

antigen NY-ESO-1 and poly-ICLC in Montanide-ISA-51 (Montanide) was found to consistently induceintegrated immune responses (antibody, CD4þ, and CD8þ T cells). Using detailed methods, we investigatedthe respective effects of poly-ICLC and Montanide adjuvant on pre- and postvaccine NY-ESO-1–specificCD4þ T cells, because of their central function for induction and maintenance of both antibody and CD8þ Tcells. Polyclonal NY-ESO-1–specific CD4þ T-cell lines were generated from 12 patients using CD154-basedselection of precursors before and after vaccination with (i) OLP alone, (ii) OLP in Montanide, or (iii) OLPand poly-ICLC in Montanide. Kinetics, quantification, fine specificity, avidity, and cytokine-producingpattern were analyzed in depth and compared between vaccine cohorts. Vaccination with OLP alone did notelicit CD4þ T-cell responses; it suppressed high-avidity CD4þ T-cell precursors that recognized naturallyprocessed NY-ESO-1 protein before vaccination. Emulsification of OLP in Montanide was required for theexpansion of high-avidity NY-ESO-1–specific CD4þ T-cell precursors. Poly-ICLC significantly enhancedCD4þ Th1 responses while suppressing the induction of interleukin (IL)-4–producing Th2 and IL-9–producing Th9 cells. In summary, Montanide and poly-ICLC had distinct and cooperative effects forthe induction of NY-ESO-1–specific Th1 cells and integrated immune responses by OLP vaccination. Theseresults support the use of admixing poly-ICLC in Montanide adjuvant to rapidly induce antitumor typeI immune responses by OLP from self/tumor antigens in human cancer vaccines. Cancer Immunol Res; 1(5);1–11. �2013 AACR.

IntroductionMost human cancer vaccine trials target self-antigens that

are overexpressed in malignant cells but have limited expres-sion in normal tissues, such as cancer-testis antigens, oncofetalantigens, and melanosomal antigens (1). Although spontane-ous immune responses can develop against certain self/tumorantigens and lead to antitumor effects in vitro and in vivo,effectorsmediating these immune responses are still regulated

by central and peripheral tolerance (2, 3). Furthermore, cancercells are known to utilize multiple mechanisms to escapeimmunosurveillance (4–6). As a consequence, achieving con-sistent polyclonal and high-avidity antibody, CD8þ and CD4þ

T-cell responses by cancer vaccination has been challenging.In animal models, Toll-like receptor (TLR) signals were

shown to improve the efficacy of cancer vaccines targetingself-antigens (7). Activation of innate immune cells and pro-duction of inflammatory cytokines also play a central role inthe induction of self/tumor antigen-specific immuneresponses (8, 9). On the basis of such experimental evidence,several TLR agonists have been developed for use as vaccineimmunostimulatory adjuvants in human immunotherapy (10).However, in clinical trials, the specific effect of TLR ligands hasrarely been analyzed primarily due to a lack of comparativecontrol groups. Because the expression pattern of TLR isdifferent between mice and humans, the effect of TLR ligandsas adjuvants in humans may differ from experimental animalmodel predictions (11). Therefore, it is critical to understandthe in vivo effect of TLR ligands in humans to further improvethe composition of cancer vaccines.

TLR3 is an endosomalmolecule that recognizes viral double-stranded RNA molecules and plays an important role in anti-viral immunity (12). Synthetic mimic of double-stranded RNA,

Authors' Affiliations: 1Ludwig Institute for Cancer Research Ltd., 2Officeof Clinical Trials Management, Ludwig Institute for Cancer Research Ltd.;Departments of 3Medicine and 4Pathology; 5Ludwig Center for CancerImmunotherapy, Memorial Sloan-Kettering Cancer Center; 6Weill CornellMedical College; 7Tisch Cancer Institute, Icahn School of Medicine atMount Sinai, New York; 8Center for Immunotherapy, Roswell Park CancerInstitute, Buffalo, New York; and 9Oncovir, Washington, District ofColumbia

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

Corresponding Author: Sacha Gnjatic, Tisch Cancer Institute, IcahnSchool of Medicine at Mount Sinai, 1470 Madison Avenue, Box 1128,New York, NY 10029. Phone: 212-824-8438; Fax: 646-537-9577; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-13-0089

�2013 American Association for Cancer Research.

CancerImmunology

Research

www.aacrjournals.org OF1

on April 15, 2021. © 2013 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst September 16, 2013; DOI: 10.1158/2326-6066.CIR-13-0089

http://cancerimmunolres.aacrjournals.org/

polyinosinic-polycytidylic acid (poly-IC) effectively stimulatesTLR3 to induce inflammatory responses. In addition, poly-ICstabilized with polylysine and carboxymethylcellulose (poly-ICLC) can enhance the efficacy of self/tumor antigen-targetingvaccine in mice (13). Poly-ICLC has been tested in humanclinical trials, and demonstrated safety as well as inductionof inflammatory responses (14). However, its effect as a vac-cine adjuvant on the quantity and quality of vaccine-inducedimmune responses has not been characterized in detail.

Recently, we reported safety data and immune responses inpatientswith ovarian cancerwho received immunizationswithoverlapping long peptides (OLP) from cancer-testis antigenNY-ESO-1 (15). To investigate the immunogenicity of OLP andthe comparative effect of adjuvants, patients received OLPalone, OLP emulsified in Montanide-ISA-51 (Montanide), orOLP and poly-ICLC inMontanide. Cohort 1 received OLP aloneto establish safety. Cohort 2 received OLP emulsified in Mon-tanide as a water-in-oil adjuvant historically used for multipleother NY-ESO-1–based trials. For cohort 3, poly-ICLC wasadded to the emulsion to ascertain whether it could furtherimprove vaccination. From immunomonitoring of NY-ESO-1–specific antibody and T cells, we found that vaccination withOLP alone did not induce measurable humoral or cellularimmune responses, whereas OLP in Montanide induced NY-ESO-1–specific CD4þ T cells but inconsistent or transientantibody and CD8þ T-cell responses. Inclusion of poly-ICLCsignificantly accelerated the induction of NY-ESO-1–specificantibodies with increased titers and polyclonality, acceleratedthe induction of CD4þT-cell responses, and induced persistentCD8þ T-cell responses. In summary, the combination of OLP/poly-ICLC/Montanide induced integrated immune responsesconsisting of OLP-specific CD4þ T cells, CD8þ T cells, andantibody responses in nearly all patients. Importantly, thoughnot a primary endpoint, OLP and poly-ICLC in Montanidesignificantly prolonged progression-free survival in patientswho had NY-ESO-1–expressing tumors as compared withthose who had tumors with no NY-ESO-1 expression. However,due to the limited number of T cells used for standardimmunomonitoring, mechanisms for the activities of Monta-nide and poly-ICLC as adjuvant were not explored in thatoriginal study (15).

We hypothesized that Montanide was important for pro-tecting and slowly releasing the antigen, thereby increasingimmunogenicity, whereas poly-ICLC helped prime antigen-presenting cells (APC) at the injection site and led to betterquality and quantity of effector cells. It has been known thatantigen-specific CD4þ T cells are required for the inductionand maintenance of cognate antigen-specific antibody andCD8þ T-cell responses through direct cell–cell interaction andindirect cytokine production (16, 17). To investigate the effectsof Montanide and poly-ICLC in the differential immunogenic-ity of OLP, we addressed the quantitative and qualitativechanges in vaccine-induced NY-ESO-1–specific CD4þ T cellsdepending on vaccine compositions using an in-depth sensi-tive approach. As we reported previously, upregulation ofCD154 (CD40-ligand) on CD4þT cells restimulatedwith tumorantigen peptides after a single in vitropeptide sensitizationwasused successfully to isolate tumor antigen-specific CD4þ

T cells (18). CD154 upregulation is sufficiently sensitive toallow the isolation of low-frequency tumor antigen–specificCD4þ T-cell precursors even from healthy donors or frompatients before vaccination. By sorting CD154-expressingCD4þ T cells followed by polyclonal expansion, we generatedNY-ESO-1–specific CD4þ T-cell lines before and after OLPvaccination in four representative patients from each vaccinecohort, i.e., OLP alone (cohort 1), OLP inMontanide (cohort 2),or OLP and poly-ICLC in Montanide (cohort 3). Frequency,epitopes, avidity, and cytokine-producing pattern of vaccine-induced CD4þ T cells were compared to characterize therespective effects of Montanide and poly-ICLC.

Materials and MethodsPatients and vaccination

All patients provided written informed consent for thisInstitutional Review Board–approved protocol (trial identifier:NCT00616941). Safety, immune responses, and characteristicsof each patient were reported previously (15). Patients had nodetectable tumor at the start of vaccination. In the presentstudy, 4 patients from each group were selected for detailedanalyses based on availability of samples. From immunohis-tochemical analyses of prevaccination specimens, patientsM01 in cohort 1, M05 in cohort 2, and M21 in cohort 3 hadNY-ESO-1–expressing tumors, whereas tumors from all otherpatients showed no NY-ESO-1 expression except for M20 incohort 3 whose tumor specimen was not available. VaccineOLPs were a mixture of NY-ESO-179–108, NY-ESO-1100–129, NY-ESO-1121–150, and NY-ESO-1142–173 peptides and were manu-factured by Multiple Peptide Systems and Phares. Another setof OLPs (assay OLP) was prepared independently for in vitroexperiments (Bio-Synthesis or GenScript). Patients in cohort 1received 1.0 mg NY-ESO-1 OLP in 0.5 mL diluent; cohort 2received 1.0 mg NY-ESO-1 OLP in 0.5 mL diluent þ 0.5 mLMontanide-ISA-51 VG (total of 1.0 mL); and cohort 3 received1.0 mg NY-ESO-1 OLP in 0.3mL diluentþ 0.7 mL (1.4 mg) poly-ICLC þ 1.0 mL Montanide-ISA-51 VG (total of 2.0 mL). Vac-cines were administered subcutaneously on weeks 1, 4, 7, 10,and 13 with final study assessment on week 16. Samples atthree time points (prevaccination, week 4 or 7, and week 13 or16) were used for each patient based on the availability ofsamples. In cohort 1, only 4 patients were originally enrolledand therefore all patients were analyzed in this study. Forpatient M02 in cohort 1, the vaccine was discontinued afterweek 7 because of disease progression. Therefore, only twotime points (prevaccine and week 7) were analyzed for M02.Other patientswhowere analyzed in the present study receivedall five vaccines. In total, the 12 patients selected in the presentreport represent 60% of patients who completed the originalclinical trial. About 1 � 107 cryopreserved peripheral bloodmononuclear cells (PBMC) were used for the analyses.

Generation of CD4þ T-cell linesCD4þ and CD8þ T cells were isolated by magnetic beads

(Dynal-Invitrogen). CD4�CD8� cells were pulsed overnightwith a 20-mer overlapping peptide pool (assay OLP), exten-sively washed, and irradiated. CD4þ T cells (5 � 105) werestimulated with peptide-pulsed CD4�CD8� cells (1� 106) and

Tsuji et al.

Cancer Immunol Res; 1(5) November 2013 Cancer Immunology ResearchOF2




cultured for 19 to 21 days in the presence of 10 U/mL inter-leukin (IL)-2 (Roche) and 20 ng/mL IL-7 (R&D Systems). Thisexpansion was required to ensure sufficient numbers of anti-gen-specific CD4þ T cells. Remaining CD4þ T cells werepolyclonally expanded using phytohemagglutinin (PHA) in thepresence of IL-2 and IL-7 and were used as APCs (T-APC) in aCD154 expression assay (18, 19). Remaining CD4�CD8� cellswere cultured in supernatant from B95-8 cell line to generateEpstein–Barr virus (EBV)–transformed B-cell lines (EBV-B).NY-ESO-1 peptide-presensitized CD4þ T cells were restimu-latedwith peptide-pulsed and carboxyfluorescein succinimidylester (CFSE)–labeled T-APC for 6 hours in the presence ofphycoerythrin (PE)-labeled anti-CD154 monoclonal antibody(mAb; BD Biosciences) and monensin. CFSE�PEþ cells weresorted by a FACSAria instrument using FACSDiva software (BDBiosciences). Data were analyzed using FlowJo software.Sorted CD4þ T cells were expanded by PHA in the presenceof irradiated allogeneic PBMCs for 20 days. Typically, morethan 2 � 107 NY-ESO-1–specific CD4þ T-cell lines were avail-able after expansion for detailed characterization.

Analyses of NY-ESO-1–specific CD4þ T cellsNY-ESO-1–specific cytokine production was evaluated by

ELISA. Expanded NY-ESO-1–specific CD4þ T-cell lines werestimulated by peptide- or recombinant NY-ESO-1 protein-pulsed autologous EBV-B cells for 16 to 20 hours. Culturesupernatant was harvested and stored at�20�C until the mea-surement. All ELISA kits were purchased from eBioscience. Insome experiments, cytokine productionwas confirmedby intra-cellular cytokine staining. NY-ESO-1–specific CD4þ T-cell lineswere cocultured for 6 hours with peptide-pulsed and CFSE-labeledEBV-B cells in the presence ofmonensin. Cellswerefixedand permeabilized using the BD Cytofix/Cytoperm Kit (BDBiosciences) according to the manufacturer's instructions andstained by fluorochrome-conjugated anticytokine mAbs. Allanticytokine mAbs were purchased fromBDBiosciences exceptfor PE-labeled anti-IL-4 mAb from eBioscience.

Statistical analysesData were analyzed using Prism 5 (GraphPad Software, Inc.)

by unpaired two-tailed t test. P values less than 0.05 wereconsidered significant. Responses for NY-ESO-1 protein–specificCD4þ T-cell lines were considered significant if they were >0.5ng/mL granulocyte macrophage colony—stimulating factor(GM-CSF) and if they were >2� the level of GM-CSF withunpulsed control targets.

ResultsDetection of NY-ESO-1–specific CD4þ T cells by CD154expression assayWe previously reported that tumor antigen–specific CD4þ

T cells with different functions such as IFN-g–producing Th1and IL-4–producing Th2 are sensitively detected by theexpression of CD154, also known as CD40-ligand, which istransiently expressed on antigen-specific CD4þ T cells afterantigenic stimulation (18, 20). To assess NY-ESO-1–specificcellular immune response, CD4þ T cells isolated fromPBMCs of OLP-vaccinated patients were presensitized oncewith an assay NY-ESO-1 peptide pool (assay OLP) and after a

20-day culture period, CD154-expressing cells were enumer-ated after restimulation with peptide-pulsed or -unpulsedtarget cells by flow cytometry. In preliminary analyses, wefound that in vitro expansion of antigen-specific CD4þ Tcells by presensitization was required to consistently detectCD154-expressing cells (data not shown). As shown in Fig.1A, CD154 was significantly upregulated on CD4þ T cellsafter peptide restimulation not only at week 7 or 13 aftervaccination, but also in some prestudy samples, even thoughthe 3 patients shown in Fig. 1A neither had detectable IFN-g–producing T cells at baseline by ELISpot assays (data notshown) nor NY-ESO-1 expression in their resectedtumor. Figure 1B summarizes the frequency of NY-ESO-1–specific CD4þ T cells by CD154 expression assays in allpatients tested. As expected, before vaccination, baselineseropositive patient M01 showed the highest frequency ofCD154-expressing CD4þ T cells, whereas the baseline sero-negative patients showed variable frequencies (Fig. 1B). Thefrequency of CD154-expressing cells significantly increasedin most patients after vaccinations with OLP in Montanidein the presence or absence of poly-ICLC. In contrast, OLPalone vaccination induced no significant increase in 2 of 4patients. It is clear that vaccination with OLP and poly-ICLCin Montanide (cohort 3: M18-M21) significantly acceleratesthe induction of CD4þ T cells compared with OLP alone orOLP in Montanide vaccination, resulting in a higher fre-quency of CD154-expressing cells at week 4 or 7 (Fig. 1C).

Although CD154-based detection following presensitizationwas a very sensitive assay that allowed the detection of low-frequency NY-ESO-1–specific CD4þ T cells as na€�ve precursorsfrom all samples tested, the number of antigen-specific cellswas not sufficient for functional characterization such ascytokine production. To enable further analyses, CD154-expressing CD4þ T cells were isolated by flow-cytometric cellsorting, and polyclonally expanded with PHA. After expansionfor 14 to 20 days, at least 1 � 107 polyclonal CD4þ T-cell linesbecame available from all samples except for the prevaccinesample from M19, which showed the lowest frequency ofCD154-expressing cells after restimulation (0.2% increase com-pared with unstimulated CD4þ T cells).

Epitopes recognized by vaccine-induced NY-ESO-1–specific CD4þ T cells

Because most NY-ESO-1 epitopes that were previouslyreported to be recognized by CD4þ T cells (21) were includedin the present vaccine OLP, the vaccine is able to inducemultiple epitope-specific CD4þT cells. The number and regionof epitopes recognized by NY-ESO-1–specific CD4þ T cellsbefore and after vaccination were determined by testingreactivity of NY-ESO-1–specific CD4þ T-cell lines againstindividual assay OLP (22). Peptide recognition was determinedby measuring GM-CSF because GM-CSF was reported to beproduced by multiple CD4þ T-cell subsets including Th1 andTh2 cells (23). Figure 2 summarizes GM-CSF production fromNY-ESO-1–specific CD4þ T-cell lines from samples at week 13or 16 except for the sample from M02 at week 7. In mostpatients, the NY-ESO-1119–143 peptide, which was reportedto bind to multiple MHC class II molecules (24), was the

Role of Montanide and Poly-ICLC on Tumor Vaccine CD4 T Cells

www.aacrjournals.org Cancer Immunol Res; 1(5) November 2013 OF3




most strongly recognized. In addition, the NY-ESO-181–100peptide was recognized by all CD4þ T cells induced aftervaccination with either OLP in Montanide or OLP and poly-ICLC inMontanide. TheseNY-ESO-1119–143 andNY-ESO-181–100peptides align with the hydrophobic regions of the NY-ESO-1protein (Fig. 2, red line; ref. 25). All 20-mer assay peptides invaccinated OLP region were recognized by at least one CD4þ

T-cell line, which supports using overlapping peptides span-ning an immunogenic (hydrophobic) region to simultane-ously inducemultiple epitope-specific CD4þ T cells in patientswith different HLAs. Unexpectedly, NY-ESO-1–specific CD4þ

T-cell lines from 3 patients (M01, M05, and M21) showedrelatively weak but significant (�1 ng/mL GM-CSF) re-activity against a region not included in the vaccine OLP(NY-ESO-151–70 and/or NY-ESO-161–80). These nonvaccinatedregion-specific reactivities were not observed before vacci-nation and at week 7 for patients M05 and M21, whereasbaseline seropositive patient M01 showed strong preexistingimmunity against NY-ESO-161–80 (data not shown). The poten-tial mechanism is epitope-spreading, i.e., vaccine-inducedT cells destroyed NY-ESO-1–expressing cancer cells, andNY-ESO-1 protein was cross-presented to CD4þ T cells.Although this unexpected reactivity was restricted to 3 pati-ents (M01, M05, and M21) who had NY-ESO-1–expressingcancer cells, the patients had no detectable tumor during theentire vaccination period.

Recognition of naturally processed NY-ESO-1 protein byOLP-induced NY-ESO-1–specific CD4þ T cells

Because vaccination with peptides may induce peptide-reactive T cells that are unable to respond to naturally pro-cessed antigens, it is important to test the recognition ofnaturally processed NY-ESO-1 protein by peptide vaccine-induced T cells. Significant NY-ESO-1 protein recognition wasdetectable before vaccination in 5 of 12 patients, indicatingthe presence of high-avidity T-cell precursors in PBMC (Fig.3A). Interestingly, NY-ESO-1 protein-recognizing CD4þ

T-cell precursors in patients M02 and M03, who receivedOLP-alone vaccination, rapidly became undetectable aftervaccination. In addition, NY-ESO-1 protein-recognizingCD4þ T cells were not detected before and after vaccinationwith OLP alone in M04. Patient M01, who showed sponta-neous NY-ESO-1–specific antibody before vaccination, hadhigh-frequency preexisting NY-ESO-1–specific CD4þ T cellsthat efficiently recognized NY-ESO-1 protein before vacci-nation. In this patient, the NY-ESO-1 protein-recognizingcapability of CD4þ T cells continued to be detectable fol-lowing vaccination, suggesting that in vivo–primed NY-ESO-1–specific T cells are expanded by OLP-alone vaccination. Insharp contrast, independent of the presence of poly-ICLCadjuvant, OLP emulsified in Montanide consistently inducedNY-ESO-1 protein-recognizing CD4þ T cells, even in patientswho did not have detectable protein-recognizing CD4þ

Figure 1. Detection of NY-ESO-1–specific CD4þ T cells by CD154expression before and aftervaccination with OLP with orwithout adjuvant. CD4þ T cellswere isolated from PBMC andstimulated with NY-ESO-1 assayOLP. After about 20 days, cellswere restimulated with assay OLP-pulsed autologous T-APC, andCD154 expression was evaluatedby flow cytometry. A, CD154expression on CD4þ T cells in atypical patient in each vaccinationgroup. B, induction of NY-ESO-1–specific CD4þ T cells in each timepoint in patients vaccinated withOLP alone (open symbols anddotted lines), OLP in Montanide(gray symbols and thin lines), orOLP and poly-ICLC in Montanide(closed symbols and thick lines).M01 was a baseline seropositivepatient. C, comparison of thefrequency of NY-ESO-1–specificCD4þ T cells at week 4 or 7. Barsindicate mean � SD. �, P ¼ 0.024by Student t test.

Tsuji et al.





T-cell precursors before vaccination. All 8 patients whoreceived Montanide-containing OLP injections had signifi-cant increases in CD4þ T-cell responses to NY-ESO-1 proteinafter vaccination compared with baseline (even patientM17, whose responses were weaker at week 13 comparedwith week 7, but still positive compared with the absence ofsignificant response at baseline).To assess differences in the functional avidity of NY-ESO-1–

specific vaccine-induced CD4þ T cells, reactivity against titrat-ed amounts of individual 20-mer assay OLP was determined.To facilitate the comparison of apparent avidity for each pep-tide, an EC50 value was interpolated from titration curves asthe peptide concentration required to induce 50% of theGM-CSF produced from saturating amounts of peptide (10mmol/L). As shown in Fig. 3B for M05 after vaccination, therewas a wide range of avidities elicited by each peptide withinthe polyclonal CD4þ T-cell line. Consistent with a decrease inNY-ESO-1 protein recognition after vaccination with OLPalone, apparent avidities of CD4þ T-cell lines generated fromcohort 1 patients were low (EC50 > 100 nmol/L) except forpatient M01, who had preexisting responses (Fig. 3C and

Supplementary Fig. S1A). In contrast, NY-ESO-1–specific vac-cine-induced CD4þ T-cell lines from cohort 2 (OLP in Mon-tanide) and cohort 3 patients (OLP and poly-ICLC in Mon-tanide) recognized at least 1 peptide with high avidity (EC50 <100 nmol/L; Fig. 3C). Consistent with the change in proteinrecognition, the avidity generally decreased after OLP-alonevaccination but increased after OLP vaccination in the pres-ence of Montanide (Supplementary Fig. S1A–S1C). Althoughmultiple HLA-binding epitopes may be included within the20-mer peptides tested, and knowing that minimal concentra-tion of 20-mer peptide triggering T-cell recognition is only arough estimation of the avidity of T cells for minimal epitopes,vaccine-induced CD4þ T cells with high EC50 apparent aviditywere only observed in patients receiving OLPs emulsified inMontanide (median of 3 peptides with high-avidity recogni-tion in cohorts 2 and 3 vs. 0 peptide in cohort 1; Fig. 3C).

Cytokine-producing pattern of vaccine-induced CD4þ

T cellsTo investigate the effect of Montanide and poly-ICLC on the

differentiation and polarization of CD4þ T cells, cytokines in

Figure 2. Peptide specificity ofNY-ESO-1–specific CD4þ T-celllines after vaccination withoverlapping peptides. CD154-expressing NY-ESO-1–specificCD4þ T cells after stimulation withNY-ESO-1 OLP were isolated andexpanded. CD4þ T-cell lines werestimulated for 16 to 20 hours withautologous EBV-B cells pulsedwith a NY-ESO-1 peptide, and theGM-CSF level in the culturesupernatant was measured byELISA. Bars indicate GM-CSFproduction at week 13 or 16 exceptfor M02 at week 7. A red lineindicates predicted hydrophobicitydetermined from the NY-ESO-1protein sequence. Location ofNY-ESO-1 assay and vaccine OLPis shown by lines with amino acidnumbers.






the supernatant of CD4þ T-cell lines stimulated with the assayOLP pool were evaluated by ELISA. The amount of GM-CSF,which is produced by multiple CD4þ T-cell subsets, wassignificantly different between CD4þ T-cell lines, most likelyreflecting the difference in the purity of NY-ESO-1–specificCD4þ T cells in each CD4þ T-cell line (Fig. 4A). The differencein purity of NY-ESO-1–specific CD4þ T cells made it difficult tocompare absolute cytokine levels associated with CD4þ T-celldifferentiation. To compensate for the different percentages ofNY-ESO-1–specific CD4þ T cells within T-cell lines, cytokineproduction was normalized against GM-CSF (Fig. 4B). Figure 5compares the normalized cytokine production by vaccine-induced NY-ESO-1–specific CD4þ T-cell lines following vacci-nation. There was a trend for more IFN-g production fromCD4þ T cells induced by OLP and poly-ICLC in Montanide ascompared with those induced by OLP in Montanide, althoughthe difference was not statistically significant because of thelarge variations within groups. Moreover, poly-ICLC adjuvantsignificantly reduced the production of IL-4 and IL-13, cyto-kines produced by Th2 cells. It appeared that emulsification in

Montanide increased IL-4 and IL-13 production as comparedwith OLP alone, indicating a Th2-differentiating effect ofMontanide. The IFN-g/IL-4 ratio, as an indication of theTh1/Th2 ratio, was increased by adding poly-ICLC adjuvantto Montanide. Differentiation to IL-10–producing Th2 or Tr1cells was also suppressed by poly-ICLC. Recently, various CD4þ

T-cell subsets, other than Th1 and Th2 cells, were identified inbothmice and humans, including IL-17–producing Th17, IL-9–producing Th9, and TGF-b–producing regulatory T cells. Asshown in Fig. 4B, significant NY-ESO-1–specific IL-17 produc-tion was detected only in patient M21 before vaccination (IL-17/GM-CSF ¼ 0.31) but it disappeared after vaccination,whereas all other CD4þ T-cell lines showed low IL-17/GM-CSF ratio (below 0.02), indicating that the present vaccinecompositions did not induce Th17 cells. In addition, TGF-bproduction was low (below 1,000 pg/mL) and transient. Inter-estingly, significant IL-9 production was detected in all NY-ESO-1–specific CD4þ T cells induced after vaccination withOLP in Montanide and it showed a gradual increase followingvaccinations (Fig. 4B). IL-9–producing CD4þ T cells were also

Figure 3. Avidity of vaccine-inducedNY-ESO-1–specific CD4þ T cells.A, recognition of naturallyprocessed NY-ESO-1 proteinby CD4þ T-cell lines from samplesbefore and after vaccination. NY-ESO-1–specific CD4þ T-cell lineswere stimulated for 20 hours withautologous EBV-B cells pulsedwith 20 mg/mL recombinant NY-ESO-1protein. GM-CSF level in thesupernatant was measured byELISA. B, determination ofapparent avidity of CD4þ T-celllines fromM05 atweek 13. TheNY-ESO-1–specific CD4þ T-cell linewas stimulated with EBV-B cellspulsed overnight with the indicatedconcentrations of respective NY-ESO-1 assay peptide for 20 hours.GM-CSF levels in the supernatantweremeasured by ELISA. GM-CSFlevels normalized to thoseproduced against 10 mmol/Lpeptide are shown. EC50 wasdefined as the peptideconcentration able to induce 50%of GM-CSF levels elicited by 10mmol/L peptide and was calculatedby interpolation of a fitting curve. C,comparison of apparent avidity.EC50 values for all recognizedepitopes at week 13 or 16 exceptfor week 7 for M02 are shown, andresponses were considered ashigh avidity for EC50 < 102 nmol/L(above gray area), correlating withthe ability to recognize naturallyprocessed NY-ESO-1 protein.

Tsuji et al.





detectable by intracellular cytokine staining (Fig. 6). Thestaining demonstrated the presence of both IL-9 single posi-tive cells and IL-9 and IL-4 coproducing cells. In contrast,IFN-g–producing cells rarely coproduced IL-9. Vaccine-in-duced differentiation to IL-9–producing cells was completelyinhibited by inclusion of poly-ICLC in the adjuvant (Figs. 5and 6). In contrast, no significant difference in cytokine pro-duction was found between the three cohorts before vacci-nation, except for the IFN-g/IL-4 ratio that was slightly higherin cohort 1 compared with cohort 2 (Supplementary Fig. S2).This suggested that differences in cytokine profiles postvac-

cination reflected the effect of vaccine adjuvants, and notpreexisting variability in precursors.

DiscussionMontanide and poly-ICLC were previously found to coop-

eratively enhance the induction of both humoral and cellularresponses to NY-ESO-1 OLP vaccination, resulting in an inte-grated NY-ESO-1–specific antibody, CD4þ and CD8þ T-cellimmune response in nearly all patients (15). In this study, weused a sensitive CD154 expression–based assay to characterizevaccine-induced CD4þT-cell responses in detail and assess the

Figure 4. Cytokine production ofNY-ESO-1–specific CD4þ T-celllines obtained before and aftervaccination. A, GM-CSFproduction from NY-ESO-1–specific CD4þ T-cell lines aftercoculture with autologous EBV-Bcells pulsed (filled) or unpulsed(open) with an NY-ESO-1 assayOLP pool. B, normalized cytokineproduction. Measured cytokinelevels were normalized against theGM-CSF level.






effect of adjuvants on these responses. We generated 12 pre-vaccine and 23 postvaccine NY-ESO-1–specific CD4þ T-celllines from 4 representative patients of each cohort (corre-sponding to 60% of patients who completed the originalstudy). Consistent with our previous report, in which IFN-g-ELISpot assays were used as the primary assay to detectNY-ESO-1–specific CD4þ T cells (15), CD154 expression–baseddetection indicated enhancement of OLP vaccine-inducedCD4þ T-cell responses by Montanide. Inclusion of poly-ICLCsignificantly accelerated the induction of CD4þ T cells.Increased expression of costimulatory molecules on APC byTLR3 signaling and induction of a type I IFN milieu couldexplain the accelerated induction of CD4þ T-cell responses(26). Consequently, CD4þ T-cell frequency at week 13 or 16after four to five injections did not increase from that atweek 7 after two injections, indicating the response reacheda plateau early after vaccination, whereas rapidly inducedCD4þ T cells were maintained throughout vaccination.Because of the high sensitivity of the assay, we were able todetect and isolate NY-ESO-1–specific CD4þ T cells in mostsamples before vaccination, even from patients who did notshow responses to the vaccine by IFN-g ELISpot assays. Afterpolyclonal expansion of isolated NY-ESO-1–specific CD4þ

T cells, they were characterized for peptide specificity,avidity, and cytokine-producing profiles. It is possible thatthese characteristics are affected by in vitro culture, whichcontains IL-2 and IL-7 that could skew repertoires and function

of T cells. This same cytokine cocktail was also used inour original article, following T-cell stimulation with CD4�

CD8� APC pooled from pre- and postvaccine to minimizeAPC-related qualitative differences. In both previous andcurrent studies, we found statistically significant differencesin vaccine-induced CD4þ T-cell responses depending on adju-vant used, suggesting that our experimental protocol did notintroduce important in vitro distortions and was still able tosignificantly distinguish characteristics and functions ofCD4þ; T cells established in vivo by vaccine components.Likely, the differences in CD4þ T-cell frequency and qualityreflect the effect of adjuvants on APC and their capacity toprime effectors. Further studies on activation and cytokine-producing profiles for APC subsets in the various cohorts arewarranted.

As we reported previously, circulating NY-ESO-1–specificCD4þ T-cell precursors capable of recognizing naturallyprocessed NY-ESO-1 protein were detected in many pati-ents before vaccination although at very low frequency (6).Interestingly, these high-avidity precursors became unde-tectable after vaccination with OLP alone, strongly suggest-ing that OLP should be administered in a proper carriersystem such as Montanide. Preferential expansion of low-avidity CD4þ T cells by OLP-alone vaccination is unlikelybecause total NY-ESO-1 peptide-reactive CD4þ T-cell fre-quency did not significantly increase following OLP-alonevaccination. These results indicated that OLP-alone

Figure 5. Comparison of cytokineproduction. Normalized cytokineproduction from vaccine-inducedNY-ESO-1–specific CD4þ T cells isshown for statistical consideration.The shape of symbols indicates asingle patient, as shown in Fig. 1B.Open and closed symbols indicateCD4þT-cell lines atweek 4 or 7 andweek 13 or 16, respectively. Barsindicate mean � SD. �, P < 0.05;��, P < 0.01; and ��, P < 0.001by Student t test.

Tsuji et al.





vaccination selectively deleted or anergized high-avidityprecursors. A similar observation was made in patientsvaccinated with MAGE-A3 protein alone in comparison withpatients vaccinated with MAGE-A3 in the adjuvant systemAS02B, indicating that this phenomenon is not limited topeptide vaccination (18). Interestingly, patients who re-ceived MAGE-A3 protein alone did not respond to boostervaccinations with MAGE-A3þAS02B adjuvant, indicatingthat deletion or persistent anergy of high-avidity CD4þ

T cells led to long-term tolerance (27, 28). In contrast, OLPin Montanide with or without poly-ICLC increased theprotein-recognizing capability of NY-ESO-1–specific CD4þ

T cells. Taken together, emulsification in Montanide seemsessential to expand high-avidity CD4þ T cells by OLP vac-cine, possibly due to involvement of local inflammation,facilitated antigen uptake, or slow release of antigens. Theuse of Montanide was recently called into question becauseit was found to act as a trap for T cells after short peptidevaccination, reducing circulating high-avidity effectors byattracting cells to the vaccine site itself instead of the tumor(29). Yet, this trapping effect in Montanide depots was notobserved with long peptide vaccination (30). Aside frompeptide length, species difference, i.e., humans and mice,may contribute to the favorable effect of Montanide

observed in the current study. In mice, it was shown thatlong peptides are selectively presented by professionalAPCs (31). However, we found that in humans, many celltypes including activated T and B cells efficiently processand present long peptides on both HLA class I and IIin vitro (32). Although it is possible that presentation ofOLP by nonprofessional APCs contributes to the deletionor unresponsiveness of high-avidity CD4þ T cells in theabsence of Montanide, the emulsification of OLP seems toovercome this limitation, possibly by routing antigen pre-sentation to professional APCs for adequate priming of na€�veT cells (33).

Characterization of a cytokine-producing pattern of OLPvaccine-induced CD4þ T cells revealed a significant effect ofMontanide and poly-ICLC on CD4þ T-cell differentiation.As known for incomplete Freund's adjuvant in mice, Mon-tanide seemed to promote Th2 differentiation of vaccine-induced NY-ESO-1–specific CD4þ T cells (34). Inclusion ofpoly-ICLC with Montanide significantly suppressed Th2-associated cytokines such as IL-4, IL-13, and IL-10, whichresulted in Th1 over Th2 polarization of vaccine-inducedNY-ESO-1–specific CD4þ T cells. It has been proposed thatTh1 immunity is more favorable for antitumor responses(35). In addition, differentiation to IL-9–producing Th9 cellswas completely suppressed by poly-ICLC. In vitro cultureexperiments have shown that IL-9 production is regulatedby IL-4 and TGF-b in mice and humans (36, 37). Therefore,Th9 suppression after vaccination with OLP and poly-ICLCin Montanide may be due to the strong suppressing effect ofpoly-ICLC on IL-4 production. Th9 cells have only recentlybeen characterized as a novel CD4þ T-cell subset, and theirin vivo differentiation, especially in humans, is largely un-known (36, 37). Our results suggest that similar to in vitrocultures, in vivo Th9 differentiation is regulated by Th2-derived cytokine(s), presumably IL-4. As reported forin vitro–differentiated Th9 cells, in patients vaccinated withOLP in Montanide, IL-9 production can be found in CD4þ

T cells whether or not they produce IL-4. However, IL-9production is rarely found in IFN-g–producing cells. Recent-ly, an antitumor effect of Th9 cells was demonstrated inmouse models in vivo via direct induction of tumor apopto-sis and indirect mechanism through mast cells (38). Becausethe NY-ESO-1–specific IL-9–producing cells were only about1% of total NY-ESO-1–specific CD4þ T cells (Fig. 6), the roleof tumor antigen-specific Th9 cells in vivo in humans wouldrequire a new vaccine strategy that preferentially inducesantigen-specific Th9 cells, but not Th2 cells.

CD4þ T cells help the induction of antibody production bysecreting cytokines and activating B cells. From the immu-nomonitoring of humoral immune responses, it was foundthat poly-ICLC significantly accelerated and enhancedhumoral immune responses, probably by modulating andenhancing CD4þ T-cell responses. In addition, CD8þ T-cellinduction was higher and more sustained if poly-ICLC waspresent in the vaccine formulation; this observation maybe explained by the interplay with CD4þ T cells and ade-quately activated APCs at priming. As both antigen-specificCD4þ and CD8þ T-cell populations could be detected after

Figure 6. Intracellular cytokine staining of NY-ESO-1–specific CD4þ

T-cell lines. CD4þ T cells were cocultured for 6 hours with autologousCFSE-labeled EBV-B cells pulsed with or without an NY-ESO-1 assayOLP pool in the presence of monensin. After fixation andpermeabilization, cells were stained with fluorochrome-labeled mAbsagainst IFN-g , IL-4, and IL-9. Staining patterns of CD4þ T cellsstimulated with peptide-pulsed EBV-B cells are shown. Numbers ineach quadrant indicate the percentages of cytokine-producing cells.Parenthesized numbers indicate the background cytokineproduction after coculture with unpulsed EBV-B cells.






a single vaccine injection in many patients receiving OLPand poly-ICLC in Montanide (15), a direct role of adjuvant onCD8þ T-cell responses may need to be explored in futurestudies. Nevertheless, the current study identified priming ofhigh-avidity polyclonal Th1 responses with poly-ICLC andMontanide adjuvants as critical in achieving integratedimmune response by helping the induction and maintenanceof antibody production and CD8þ T-cell responses. Ourstudy did not evaluate poly-ICLC as adjuvant without Mon-tanide, and clinical trials to address this question are ongo-ing. These and future studies testing the effect of checkpointblockade on vaccine-induced responses should be evaluatedusing the detailed quantitative and qualitative analysesdescribed here.

Disclosure of Potential Conflicts of InterestT. Tsuji, G. Ritter, and S. Gnjatic have ownership interest (including patents)

and are coinventors on primary affiliation assigned NY-ESO-1 patents. A.M.Salazar is employed as CEO of Oncovir and has ownership interest (includingpatents) in the same.

Authors' ContributionsConception and design: T. Tsuji, L. Pan, G. Ritter, D. Spriggs, A.M. Salazar,S. GnjaticDevelopment of methodology: T. TsujiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Tsuji, P. Sabbatini, A.A. Jungbluth, E. Ritter,G. Ritter, L. Ferran, D. SpriggsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):T. Tsuji, A.A. Jungbluth, E. Ritter, L. Ferran, S. GnjaticWriting, review, and/or revision of the manuscript: T. Tsuji, P. Sabbatini,A.A. Jungbluth, L. Pan, G. Ritter, A.M. Salazar, S. GnjaticAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Pan, D. SpriggsStudy supervision: L. Pan, G. Ritter, S. Gnjatic

Grant SupportThis study was sponsored and funded by the Ludwig Institute for Cancer

Researchwith a supplemental grant from theCancer Research Institute as part ofthe Cancer Vaccine Collaborative.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 5, 2013; revised August 28, 2013; accepted September 7, 2013;published OnlineFirst September 16, 2013.

References1. Cheever MA, Allison JP, Ferris AS, Finn OJ, Hastings BM, Hecht TT,

et al. The prioritization of cancer antigens: a national cancer institutepilot project for the acceleration of translational research. Clin CancerRes 2009;15:5323–37.

2. Gotter J, Brors B, HergenhahnM, Kyewski B. Medullary epithelial cellsof the human thymus express a highly diverse selection of tissue-specific genes colocalized in chromosomal clusters. J Exp Med2004;199:155–66.

3. Tsuji T,Matsuzaki J, Ritter E, Miliotto A, Ritter G, Odunsi K, et al. Split Tcell tolerance against a self/tumor antigen: spontaneous CD4þ but notCD8þ T cell responses against p53 in cancer patients and healthydonors. PLoS ONE 2011;6:e23651.

4. Whiteside TL. The tumor microenvironment and its role in promotingtumor growth. Oncogene 2008;27:5904–12.

5. Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. IntJ Cancer 2010;127:759–67.

6. Nishikawa H, Jager E, Ritter G, Old LJ, Gnjatic S. CD4þ CD25þ

regulatory T cells control the induction of antigen-specificCD4þhelperT cell responses in cancer patients. Blood 2005;106:1008–11.

7. Yang Y, Huang CT, Huang X, Pardoll DM. Persistent Toll-like receptorsignals are required for reversal of regulatory T cell-mediated CD8tolerance. Nat Immunol 2004;5:508–15.

8. Kaisho T, Akira S. Toll-like receptors as adjuvant receptors. BiochimBiophys Acta 2002;1589:1–13.

9. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4þ

CD25þ T cell-mediated suppression by dendritic cells. Science2003;299:1033–6.

10. Duthie MS, Windish HP, Fox CB, Reed SG. Use of defined TLRligands as adjuvants within human vaccines. Immunol Rev 2011;239:178–96.

11. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptiveimmune responses. Nat Immunol 2004;5:987–95.

12. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition ofdouble-stranded RNA and activation of NF-kappaB by Toll-like recep-tor 3. Nature 2001;413:732–8.

13. Zhu X, Nishimura F, Sasaki K, Fujita M, Dusak JE, Eguchi J, et al. Tolllike receptor-3 ligand poly-ICLC promotes the efficacy of peripheralvaccinations with tumor antigen-derived peptide epitopes in murineCNS tumor models. J Transl Med 2007;5:10.

14. Caskey M, Lefebvre F, Filali-Mouhim A, Cameron MJ, Goulet JP,Haddad EK, et al. Synthetic double-stranded RNA induces innateimmune responses similar to a live viral vaccine in humans. J ExpMed 2011;208:2357–66.

15. Sabbatini P, Tsuji T, Ferran L, Ritter E, Sedrak C, Tuballes K, et al.Phase I Trial of Overlapping long peptides from a tumor self-antigenand poly-ICLC shows rapid induction of integrated immune responsein ovarian cancer patients. Clin Cancer Res 2012;18:6497–508.

16. Pardoll DM, Topalian SL. The role of CD4þ T cell responses inantitumor immunity. Curr Opin Immunol 1998;10:588–94.

17. Bevan MJ. Helping the CD8(þ) T-cell response. Nat Rev Immunol2004;4:595–602.

18. Tsuji T, Altorki NK, Ritter G, Old LJ, Gnjatic S. Characterization ofpreexisting MAGE-A3-specific CD4þ T cells in cancer patientsand healthy individuals and their activation by protein vaccination.J Immunol 2009;183:4800–8.

19. Atanackovic D, Matsuo M, Ritter E, Mazzara G, Ritter G, Jager E, et al.Monitoring CD4þ T cell responses against viral and tumor antigensusing T cells as novel target APC. J Immunol Methods 2003;278:57–66.

20. Chattopadhyay PK, Yu J, Roederer M. A live-cell assay to detectantigen-specific CD4þ T cells with diverse cytokine profiles. Nat Med2005;11:1113–7.

21. van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B.Peptide database: T cell-defined tumor antigens. Cancer Immun2013;13:15.

22. Tsuji T, Matsuzaki J, Kelly MP, Ramakrishna V, Vitale L, He LZ, et al.Antibody-targetedNY-ESO-1 tomannose receptor orDEC-205 in vitroelicits dual humanCD8þandCD4þT cell responseswith broad antigenspecificity. J Immunol 2011;186:1218–27.

23. Cousins DJ, Lee TH, Staynov DZ. Cytokine coexpression duringhuman Th1/Th2 cell differentiation: direct evidence for coordinatedexpression of Th2 cytokines. J Immunol 2002;169:2498–506.

24. Zarour HM, Maillere B, Brusic V, Coval K, Williams E, Pouvelle-Moratille S, et al. NY-ESO-1 119-143 is a promiscuous majorhistocompatibility complex class II T-helper epitope recognized byTh1- and Th2-type tumor-reactive CD4þ T cells. Cancer Res 2002;62:213–8.

25. Sweet RM, Eisenberg D. Correlation of sequence hydrophobicitiesmeasures similarity in three-dimensional protein structure. J Mol Biol1983;171:479–88.

26. de Jong EC, Vieira PL, Kalinski P, Schuitemaker JH, Tanaka Y,Wierenga EA, et al. Microbial compounds selectively induce Th1cell-promoting or Th2 cell-promoting dendritic cells in vitro withdiverse th cell-polarizing signals. J Immunol 2002;168:1704–9.

27. Atanackovic D, Altorki NK, Stockert E, Williamson B, JungbluthAA, Ritter E, et al. Vaccine-induced CD4þ T cell responses to

Tsuji et al.





MAGE-3 protein in lung cancer patients. J Immunol 2004;172:3289–96.

28. Atanackovic D, Altorki NK, Cao Y, Ritter E, Ferrara CA, Ritter G, et al.Booster vaccination of cancer patients with MAGE-A3 protein revealslong-term immunological memory or tolerance depending on priming.Proc Natl Acad Sci U S A 2008;105:1650–5.

29. Hailemichael Y, Dai Z, Jaffarzad N, Ye Y, Medina MA, Huang XF, et al.Persistent antigen at vaccination sites induces tumor-specific CD8þ

T cell sequestration, dysfunction and deletion. Nat Med 2013;19:465–72.

30. Gnjatic S, Bhardwaj N. Antigen depots: T cell traps? Nat Med 2013;19:397–8.

31. Bijker MS, van den Eeden SJ, Franken KL, Melief CJ, van der Burg SH,OffringaR.Superior inductionof anti-tumorCTL immunity by extendedpeptide vaccines involves prolonged, DC-focused antigen presenta-tion. Eur J Immunol 2008;38:1033–42.

32. Gnjatic S, Atanackovic D, Matsuo M, Jager E, Lee SY, Valmori D,et al. Cross-presentation of HLA class I epitopes from exogenous

NY-ESO-1 polypeptides by nonprofessional APCs. J Immunol2003;170:1191–6.

33. Banchereau J, Steinman RM. Dendritic cells and the control of immu-nity. Nature 1998;392:245–52.

34. Yip HC, Karulin AY, Tary-Lehmann M, Hesse MD, Radeke H, HeegerPS, et al. Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response. J Immunol1999;162:3942–9.

35. Ikeda H, Chamoto K, Tsuji T, Suzuki Y, Wakita D, Takeshima T, et al.The critical role of type-1 innate and acquired immunity in tumorimmunotherapy. Cancer Sci 2004;95:697–703.

36. Noelle RJ, Nowak EC. Cellular sources and immune functions ofinterleukin-9. Nat Rev Immunol 2010;10:683–7.

37. Perumal NB, Kaplan MH. Regulating Il9 transcription in T helper cells.Trends Immunol 2011;32:146–50.

38. Purwar R, Schlapbach C, Xiao S, Kang HS, Elyaman W, Jiang X, et al.Robust tumor immunity to melanoma mediated by interleukin-9-pro-ducing T cells. Nat Med 2012;18:1248–53.






Published OnlineFirst September 16, 2013.Cancer Immunol Res Takemasa Tsuji, Paul Sabbatini, Achim A. Jungbluth, et al. Overlapping Long Peptide Vaccine Trial

T Cells in Phase I+Specific CD4−Self/Tumor Antigen Effect of Montanide and Poly-ICLC Adjuvant on Human

Updated version

10.1158/2326-6066.CIR-13-0089doi:

Access the most recent version of this article at:

Material

Supplementary

.DC1

http://cancerimmunolres.aacrjournals.org/content/suppl/2013/09/17/2326-6066.CIR-13-0089Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerimmunolres.aacrjournals.org/content/early/2013/10/09/2326-6066.CIR-13-0089To request permission to re-use all or part of this article, use this link



http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-13-0089

http://cancerimmunolres.aacrjournals.org/content/suppl/2013/09/17/2326-6066.CIR-13-0089.DC1

http://cancerimmunolres.aacrjournals.org/content/suppl/2013/09/17/2326-6066.CIR-13-0089.DC1

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/content/early/2013/10/09/2326-6066.CIR-13-0089


Documents

Self/Tumor Antigen Speci c CD4þ T Cells in Phase I ... · 10/9/2013 · Research Article Effect of Montanide and Poly-ICLC Adjuvant on Human Self/Tumor Antigen–Speciﬁc CD4þ