Adjuvants That Improve the Ratio of Antigen-Speci Effector ... · design of more effective cancer...

Microenvironment and Immunology

Adjuvants That Improve the Ratio of Antigen-SpecificEffector to Regulatory T Cells Enhance Tumor Immunity

Rachel Perret, Sophie R. Sierro, Natalia K. Botelho, St�ephanie Corgnac, Alena Donda, and Pedro Romero

AbstractAntitumor immunity is strongly influenced by the balance of tumor antigen-specific effector T cells (Teff) and

regulatory T cells (Treg). However, the impact that vaccine adjuvants have in regulating the balance of antigen-specific T-cell populations is not well understood. We found that antigen-specific Tregs were induced followingsubcutaneous vaccination with either OVA or melanoma-derived peptides, with a restricted expansion of Teffs.Addition of the adjuvants CpG-ODN or Poly(I:C) preferentially amplified Teffs over Tregs, dramatically increasingthe antigen-specific Teff:Treg ratios and inducing polyfunctional effector cells. In contrast, two other adjuvants,imiquimod and Quil A saponin, favored an expansion of antigen-specific Tregs and failed to increase Teff:Tregratios. Following therapeutic vaccination of tumor-bearing mice, high ratios of tumor-specific Teffs:Tregs indraining lymph nodes were associated with enhanced CD8þ T-cell infiltration at the tumor site and a durablerejection of tumors. Vaccine formulations of peptideþCpG-ODN or Poly(I:C) induced selective production ofproinflammatory type I cytokines early after vaccination. This environment promoted CD8þ and CD4þ Teffexpansion over that of antigen-specific Tregs, tipping the Teff to Treg balance to favor effector cells. Our findingsadvance understanding of the influence of different adjuvants on T-cell populations, facilitating the rationaldesign of more effective cancer vaccines. Cancer Res; 73(22); 6597–608. �2013 AACR.

IntroductionThe development of therapeutic cancer vaccines is a chal-

lenging task, and generating clinically relevant, potent, andpolyfunctional T-cell responses to tumor/self-antigens hasproven difficult. Therapeutic peptide vaccines stimulate anti-tumor immune responses in patients with advanced melano-ma (1, 2), but clinical benefits have not lived up to theexpectations (3, 4). For therapeutic vaccines to be effective,they must circumvent regulatory mechanisms that limit theactivation and expansion of CD8þ andCD4þTcells. RegulatoryT cells (Treg) play a major role in the control of antitumorimmunity (5–7) and existing cancer vaccines activate andexpand Tregs, resulting in suppression of antitumor responses(8–10). Thus, the need to enhance immunogenicity of peptidevaccines is paramount. Adjuvants provide a means to improvethe generation of potent and durable T-cell immunity by

cancer vaccines (11). A greater understanding of the role ofadjuvants in modulating T-cell responses and in particularTregs is therefore urgently needed. The rational developmentof new adjuvant formulations to augment T-cell responses iskey in cancer vaccine development. Adjuvants that stronglystimulate T-cell responses are still not readily available and themajor clinically licensed adjuvants Alum and incompleteFreund's adjuvant (IFA) primarily promote antibody responsesbut are poor at inducing the cytotoxic CD8þ T-cell responsesneeded in the case of cancer (12).

Members of the Toll-like receptor (TLR)-ligand class ofadjuvants, including CpG-ODN (TLR-9), LPS (TLR-4), andPam3Cys (TLR-2), induce antigen-presenting cell (APC) mat-uration and production of inflammatory cytokines, favoringtype I effector T cell (Teff) responses and restricting Tregexpansion (13–15). Conflicting data show that TLR agonistsCpG-ODN, LPS, Zymozan (TLR-6), Poly(I:C) [TLR3], Imiqui-mod and R-848 (TLR-7/8) can expand both Teffs and Tregsleading to suppression of effector responses (16–18). In thestudies mentioned above, CD8þ T-cell responses were com-pared with those of polyclonal Tregs. Very few studies haveinvestigated the effect of adjuvants on antigen-specific Tregs.An ex vivo study from patients with colorectal carcinomaidentified shared antigen-specificities between tumor-specificTeffs and Tregs. These Tregs were shown to suppress prolif-eration of Teffs in an antigen-specific manner when culturedwith tumor peptide-loaded dendritic cells (19). In a clinicalstudy of patients with melanoma immunized with NY-ESO-1protein in ISCOMATRIX, vaccination increased the frequencyof NY-ESO-1–specific Tregs in peripheral blood mononuclearcells (PBMC) and tumor tissue (20). We have shown that

Authors' Affiliation: Ludwig Center for Cancer Research, University ofLausanne, Lausanne, Switzerland

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for S.R. Sierro: Service of Immunology and Allergy,Department of Medicine, Lausanne University Hospital, Lausanne,Switzerland.

Corresponding Author: Rachel Perret, Clinical Research Division, FredHutchinson Cancer Research Center, D3-100, 1100 Fairview Avenue N.,Seattle, WA 98109. Phone: 206-667-5932; Fax: 206-667-7983; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-0875

�2013 American Association for Cancer Research.

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therapeutic vaccination of patients with melanoma withMelan-A peptide plus CpG-ODN inMontanide results in robustexpansion of Melan-A–specific CD8þ T cells within PBMCswith a concomitant decrease in Melan-A–specific Tregs (1,2, 21). Importantly, although we observed a decrease in vac-cine-specific Tregs, the total polyclonal Treg populationremained unchanged. These results suggest that antigen-spe-cific Tregs are regulated differently than polyclonal Tregsfollowing adjuvanted-peptide vaccination, and that adjuvantchoice may be important in selectively controlling the specificTreg response.

We therefore set out to extend these clinical observationsusing mouse models of peptide vaccination to dissect the roleof vaccine formulations in shaping the antitumor immuneresponse. We developed models that allowed us to comparetumor-specific CD8, CD4, and Treg responses to peptidevaccination in various adjuvant formulations. Here, we showthat vaccines containing TLR-9 ligand CpG-ODN or TLR-3ligand Poly(I:C) preferentially induce strong proliferation ofantigen-specific Teffs, while minimizing antigen-specific Tregexpansion. High Teff:Treg ratios were linked to strong proin-flammatory cytokine production in the lymph nodes early afterimmunization and resulted in polyfunctional CTLs withenhanced tumor infiltration and protective function.

Materials and MethodsMice

Mouse strains were maintained at the University of Lau-sanne (UNIL; Lausanne, Switzerland) SPF Unit. C57BL/6,CD45.1 congenic (B6.SJL-PtprcaPep3b/BoyJArc), OT-I mice,and OT-II mice were obtained from Harlan Laboratories (22,23). Pmel and Trp-1 mice were obtained from The JacksonLaboratory (24, 25). Foxp3-eGFP reportermicewere purchasedfrom EMMA (EM:01945; ref. 26). Foxp3-eGFP mice werecrossed to TCR-transgenics to create OT-IIxFoxp3-eGFP(referred to as OT-II) and Trp-1xFoxp3-eGFP (referred to asTrp-1). Age- and sex-matched mice between 6 and 14 weeks ofage were used for all experiments. This study was approved bythe local Veterinary Authority and performed in accordancewith Swiss ethical guidelines.

Cell linesThe B16.OVA melanoma cell line was obtained from B.

Huard (University Medical Center, Geneva, Switzerland;ref. 27). The B16 and EG7 lymphoma cell lines were obtainedfrom the American Type Culture Collection (CRL-6475 andCRL-2113; refs. 28, 29). Tumor cell lines were maintained incomplete Iscove'sModifiedDulbecco'sMedium (IMDM)medi-um supplemented with G418, at 1 mg/mL for B16.OVA and 0.4mg/mL for EG7.OVA.

Adoptive cell transfersAntigen-specific CD8þ and CD4þ T cells (CD45.2) were

isolated from spleens of TCR-transgenic mice. The frequencyof transgenic T cells was determined by flow cytometry. OT-Iand OT-II cells were labeled with Va2 and Vb5.1/5.2 antibo-dies, Trp-1 cells with CD4 and Vb14 antibodies, and Pmel cellswith H-2Db/hgp10025-33 tetramers. Na€�ve CD45.1 recipient

mice received 1 � 105 or 1 � 106 OT-I cells and 3 � 106 or1 � 106 OT-II cells in 200 mL of Dulbecco's Modified EagleMedium (DMEM) intravenously, as indicated. Alternatively,mice received 1� 105 Pmel and 1� 105 Trp-1 cells in 200 mL ofDMEM intravenously.

ImmunizationsMice were immunized with 10 mg OVA257-264 and 10 mg

OVA323-339 peptides or with 10 mg hgp10025-33 and 10 mg Trp-1106-130 peptides in 100 mL PBS subcutaneously at the base ofthe tail. Peptides were injected alone or in combination withthe following adjuvants: 50 mg CpG-ODN 1826 (CpG),Pam3CSK4 (Pam3Cys), HMW Poly(I:C), imiquimod, and QuilA or 5 mg LPS and Flagellin from Salmonella typhimurium, all�emulsification in 50 mL IFA (30, 31). Peptides were manufac-tured by the Protein and Peptide Chemistry Facility (PPCF) oftheUNIL. Adjuvantswere sourced from InvivoGen except CpG-ODN (Coley Pharmaceuticals) and theQuil A saponinmix fromQuillaja saponaria (generously provided by Brenntag NordicA/S). IFA was purchased from Sigma–Aldrich, Inc.

Flow cytometryDraining lymphnodes (inguinal) and spleenswere harvested

on day 7 after immunization. Cell suspensions were incubatedwith appropriate concentrations of antibody in PBS containing2% FBS. The anti-FcgRII monoclonal antibody 2.4G2 was usedto inhibit nonspecific antibody binding. Antibodies wereobtained from BD Pharmingen and eBioscience or grownin-house fromB-cell hybridomas. Samples were acquired usingLSR-II and FACSCanto flow cytometers (Becton-Dickinson)and analyzed using FlowJo software (TreeStar). Lymphocyteswere gated on the basis of forward scatter and side scatterproperties and LIVE/DEADAquaCell Stain (Life Technologies)was used to exclude dead cells.

In vitro restimulation and intracellular cytokine stainingCell suspensions were incubated in complete DMEM con-

taining 1 mmol/L of specific MHC-I and MHC-II peptides for 4hours in the presence of CD107a-specific antibodies. Of note, 1mmol/L Golgiplug and Golgistop were added after 1 hour ofincubation. Cells were harvested, surface labeled, fixed, andpermeabilized using the Fix/Perm Kit. Intracellular cytokineswere detected using anti-IFN-g and anti-interleukin (IL)-2antibodies. All reagents were purchased from BD Biosciencesand fixation and staining was performed according to themanufacturer's specifications.

In vivo cytotoxicity assayCytotoxicity was measured using the VITAL assay (32).

Briefly, C57BL/6 splenocytes were left untreated and labeledwith 10 mmol/L CellTracker Orange (CTO; Molecular Probes),or incubated for 2 hours with 10 or 100 nmol/L OVA or hgp100peptide, and then labeled with 0.02 or 0.2 mmol/L carboxy-fluorescein diacetate succinimidyl ester (CFSE) (MolecularProbes), respectively. Labeled cells were mixed at equal ratios,and approximately 2 � 106 cells of each population wereinjected intravenously. At 6 or 24 hours after target celladministration, blood was collected for flow cytometric

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analysis. Percentage specific killing ¼ 100 � [100 � (expnumber CFSEþ cells/exp number CTOþ cells)/(control num-ber CFSEþ cells/control number CTOþ cells)].

Tumor-infiltrating lymphocyte analysisMice that had received OT-I and OT-II T cells were chal-

lenged the next day with 2 � 105 B16.OVA tumor cells subcu-taneously in the left flank. One week later, once tumors werepalpable, mice were immunized as described above. After 7days, draining lymph nodes (dLN) and tumors were excisedand tumors were weighed and digested in collagenase I andDNase I (Roche). CD45þ cells were purified by positive selec-tion using magnetic cell separation (MACS) beads and theAutoMACS automatic cell separator (Miltenyi Biotech).

Tumor protectionProphylactic setting. Mice received OT-I andOT-II T cells

intravenously and were immunized the next day with theindicated OVA vaccine formulations. A total of 2 � 105 B16.OVA melanoma cells were injected subcutaneously in the leftflank 1 week later, tumor growth was monitored every 2 to 3days as described previously (33). Ten days after tumor chal-lenge, mice received a vaccine boost proximal to the tumor.Therapeutic setting. Mice received OT-I and OT-II T cells

intravenously and were challenged the next day with 5 � 106

EG7.OVA tumor cells subcutaneously in the left flank, andexamined every 2 to 3 days to monitor tumor growth. Ten dayslater, mice with well-established tumors were immunized asabove.Self-antigen model. Mice received Pmel and Trp-1 cells

intravenously and were immunized the next day with theindicated hgp100/Trp-1 vaccine formulations and boosted 1week later. A total of 1 � 105 B16.F10 melanoma cells wereinjected subcutaneously at the time of boosting.

Cytokine multiplex assay and IFN-b ELISAC57BL/6 mice were immunized subcutaneously at the base

of the tail with peptides � adjuvants as described above. ThedLNs (inguinal) were harvested 12 or 24 hours later. Totallymph node cell suspensions were incubated at 37�C in IMDMsupplemented with 5% FBS. Supernatant samples were col-lected and frozen at 1 hour for analysis of type I IFN and at 1, 6,and 12 hours for cytokine multiplex analysis. Cytokine pro-ductionwasmeasuredwith themouse IFN-b ELISAKit and 10-plex Luminex panel [granulocyte macrophage colony–stimu-lating factor (GM-CSF), IFN-g , IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10,IL-12p40/p70 and TNF-a], using the Luminex 200 System withxPONENT Software (all from Life Technologies) and EpochELISA plate-reader (BioTek).

Statistical calculationsStatistical differences between groups were calculated using

the ANOVA and Dunnett multiple comparison tests, compar-ing all groups to the peptide alone group. Differences insurvival were calculated using the log-rank test, comparingall groups to the untreated control. The two-way ANOVA andBonferroni posttests, matched by adjuvant group, were usedfor cytokine multiplex analysis. Error bars indicate SEM. All

tests were performed using GraphPad Prism software. (�, P <0.05; ��, P < 0.01; ��, P < 0.001).

ResultsAntigen-specific Teff and Treg responses to peptidevaccination can be specifically modulated by adjuvantchoice

To perform an in-depth investigation of the vaccine-medi-ated regulation of antigen-specific T-cell populations, micewere given an adoptive transfer of OT-I and OT-II T cells andthen immunized with OVA257-264 and OVA323-339 peptides �adjuvants. After 7 days, antigen-specific Teffs and Tregs wereexamined by flow cytometry in inguinal dLNs and spleen. Weinitially vaccinated mice with OVA peptide alone, OVAþCpG�IFA to determine whether this system could faithfully repro-duce the effect observed in the blood of patients with mela-noma vaccinated with MelanAþCpGþIFA (21). This wasindeed the case, with CpG �IFA significantly increasing theOVA-specific Teff:Treg ratio in vaccine-dLNs and spleen (Sup-plementary Fig. S1A and S1B). Polyclonal T cells did notundergo the same regulation in response to adjuvanted vac-cines, as evidenced by stable Teff:Treg ratios (SupplementaryFig. S1C).

We next set out to determine whether the antigen-specificTeff:Treg ratios were similarly regulated by other adjuvants.Antigen-specific T cells were identified using the gating strat-egy shown in Fig. 1A. We observed that antigen-specific CD8þ

T cells uniformly upregulated the activation marker CD44(Supplementary Fig. S2A) in response to all vaccine formula-tions. However, there was substantial variation in the absolutenumbers of OT-I T cells following immunization, withOVAþCpG and OVAþPoly(I:C) inducing significantly greaterexpansion than other vaccine formulations (Fig. 1B). Antigen-specific CD4þTeffs decreased expression of the restingmarkerCD62L (Supplementary Fig. S2B) and expanded to a similarextent in response to all adjuvants except imiquimod and QuilA (Fig. 1C). Absolute OT-II Tregs generally increased followingvaccination regardless of the adjuvant (Fig. 1D). All adjuvants,with the notable exception of Quil A, reduced the proportion ofTregs among OT-II cells (Fig. 1E). Ratios of OVA-specific Teffsto Tregs were calculated from absolute Teff and Treg counts.OVAþCpG and OVAþPoly(I:C) significantly enhanced anti-gen-specific CD8þ Teff:Treg ratios compared with mice vac-cinated with OVA alone (Fig. 1F). CpG, Pam3Cys, and Flagellinenhanced antigen-specific CD4þ Teff:Treg ratios (Fig. 1G). IFAincreased the immune response to some of the pepti-deþadjuvant vaccines but did not significantly impact anti-gen-specific Teff:Treg ratios (Supplementary Fig. S3). CpG,Poly(I:C), imiquimod, and Quil A (from which QS21 is derived)were selected for further analysis in in vivo models of tumorprotection due to their contrasting immunologic effects andparticular clinical relevance for cancer vaccination.

Adjuvants promoting high Teff:Treg ratios enhancelymphocyte infiltration into B16.OVA tumors and delaytumor growth

The immune response to peptide vaccination in a tumorsetting was examined by immunizing mice bearing palpable

Adjuvants Modulate Antitumor Teff:Treg Ratios

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Figure 1. CpG and Poly(I:C) induced the highest OVA-specific Teff:Treg ratios among a panel of adjuvants. CD45.1 mice received 1 � 106 OT-I and 3 � 106

OT-II T cells intravenously. One day later, mice were immunized subcutaneously with OVA257-264 and OVA323-339 peptides alone or in combinationwith Pam3Cys, Poly(I:C), LPS, flagellin (FLA), imiquimod (Imi), CpG, or Quil A. Draining lymph nodes were harvested 7 days later and antigen-specificT cells were analyzed. A, gating strategy for identifying antigen-specific T-cell populations. B, absolute number of OT-I effector cells. C, absolute number ofOT-II effector cells. D and E, absolute number (D) and frequency (E) of OT-II Tregs. F, ratios of OT-I Teffs:OT-II Tregs. G, ratios of OT-II Teffs:Tregs. Combineddata from three independent experiments comprising a total of 3 to 9 mice/group are shown.

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B16.OVA melanomas. One week following vaccination, micewere sacrificed and tumor size as well as tumor-infiltratinglymphocytes was examined. The degree of OT-I tumor infil-tration was significantly greater in mice immunized withOVAþCpG and OVAþPoly(I:C) compared with other groups(Fig. 2A, left) and was inversely related to tumor size (Fig. 2A,right). OT-II Teffs and Tregs infiltrated tumors poorly in all ofthe experimental groups (Fig. 2B). Because of low numbers ofinfiltrating OT-II T cells, ratios of antigen-specific Teffs:Tregscould not be reliably calculated at the tumor site. Accumula-tion of OT-I, OT-II Teffs, and OT-II Tregs in vaccine-dLNs oftumor-bearing mice resembled that of tumor-free mice (Sup-plementary Fig. S4). Lymph node Teff:Treg ratios correlatedwith the degree of antigen-specific CD8þ T-cell infiltration andearly tumor control (Fig. 2C vs. Fig. 2A). To investigate long-lasting tumor protection, mice were immunized 1 week beforetumor challenge, followed by a booster immunization 10 daysafter, and tumor growth was monitored over time. Micevaccinated with OVAþCpGwere completely protected againsttumor development, andmice vaccinated with OVAþPoly(I:C)developed tumors much more slowly than controls, leading toa significant improvement in survival time. Tumors developedand grew rapidly in allmice immunizedwithOVAþImiquimodor OVAþQuil A (Fig. 2D). Cytotoxic function of OT-I cells wastested in vivo 1week after vaccination in the absence of tumors.The highest killing of specific targets was seen in theOVAþCpG and Poly(I:C) groups, supporting the role of anti-gen-specific CTL in mediating tumor protection when presentat high ratios relative to antigen-specific Tregs (Fig. 2E).

Therapeutic vaccination with OVAþCpG or OVAþPoly(I:C) induces the rejection of established tumorsWe next wanted to determine whether high Teff:Treg–

inducing vaccines could induce the rejection of establishedtumors in a therapeutic setting. Mice with well-established 10-day EG7 tumors were vaccinated with OVA peptides � adju-vant. Tumor growth was substantially delayed in theOVAþCpG and OVAþPoly(I:C) vaccinated groups comparedwith controls. Three and 2 of 5 mice in these respective groupscompletely rejected their tumors (Fig. 3A and B). The overallsurvival of these two groups of mice was consequentlyenhanced (Fig. 3B, right), with a significant proportion of themice remaining tumor-free for at least 50 days. On the otherhand, the tumor growth rate in the OVA alone, OVAþQuil A,and OVAþImiquimod groups was similar to that of theuntreated controls (Fig. 3A).

Poly(I:C) and CpG enhance the tumor/self-antigen–specific Teff:Treg balanceTo ascertain whether the adjuvant effects on Teff:Treg ratios

were translatable to a self-/tumor-antigen system, we repeatedour experiments in a gp100 and Trp-1 melanoma antigenmodel. We transferred CD8þ Pmel and CD4þ Trp-1 T cellsinto CD45.1þ recipient mice and vaccinated themwith hgp100and Trp-1 peptides. A week later antigen-specific T-cellresponses were measured in dLNs and spleen. Vaccinationwith hgp100/Trp-1þPoly(I:C) or CpG induced significantlygreater expansion of Pmel T cells than peptide alone (Fig.

4A, left). Effector potential was assessed by restimulatingsplenocytes with specific peptides for 4 hours. A substantialproportion of Pmel Teffs in all adjuvanted vaccinationgroups acquired at least one effector characteristic: produc-ing IL-2, IFN-g , or releasing cytotoxic granules via external-ization of CD107a (Supplementary Fig. S5A). Polyfunctionaleffectors, simultaneously producing IL-2, IFN-g , and degra-nulating, were most frequent following vaccination with self-peptidesþPoly(I:C) or CpG (Fig. 4A, right). This corre-sponded with the greatly enhanced Teff:Treg ratios observedin the hgp100/Trp-1þPoly(I:C) or CpG groups (Fig. 4D). Trp-1 Teff expansion was significantly enhanced in response toimmunization with self-peptidesþPoly(I:C), CpG, or Quil A(Fig. 4B, left). Similar proportions of Trp-1 Teffs in allvaccinated groups displayed at least one effector function(Supplementary Fig. S5B), whereas significantly higher fre-quencies of Trp-1 Teffs in the hgp100/Trp-1þPoly(I:C) orCpG groups simultaneously produced IFN-g and IL-2 (Fig.4B, right). Self-peptide immunization with Poly(I:C), CpG, orimiquimod reduced the number of Tregs among Trp-1 Tcells, whereas Quil A increased their number (Fig. 4C). Theratio of Trp-1 Teffs:Tregs was significantly increased forgroups receiving Poly(I:C) and CpG, but not for Quil A orimiquimod (Fig. 4E). Importantly, the higher antigen-specificTeff:Treg ratios observed with hgp100/Trp-1þPoly(I:C) orCpG immunization resulted in significantly greater in vivokilling of hgp100-pulsed targets (Fig. 4F). Altogether, thesedata corroborate the results obtained in the OVA model.

Adjuvants promoting high Teff:Treg ratios conferprotection against tumor growth in a self-/tumor-antigen system

We next set out to determine whether the polyfunctionalTeffs generated by vaccination with tumor/self-peptide andCpG or Poly(I:C) could confer protection against B16 tumorchallenge. Mice received Pmel and Trp-1 T cells and wereimmunized with gp100þTrp-1 peptides � adjuvant 1 weekbefore tumor challenge, followed by a booster immunizationon the day of tumor graft. Mice vaccinated with hgp100/Trp-1þCpG or Poly(I:C) developed tumors much more slowlythan controls (Fig. 5A), leading to a significant improvementin survival time (Fig. 5B). Depigmentation was observed inseveral groups of vaccinated mice and was most prevalent inthe peptideþCpG and Poly(I:C) groups, correlating withenhanced survival (Fig. 5C) and confirming that vaccinationwith high Teff:Treg–inducing adjuvants in a natural tumor-antigen setting induces a functional and protective immuneresponse.

High Teff:Treg promoting adjuvants induce earlyproduction of type I cytokines

There is abundant evidence that certain TLR ligandsinduce maturation of APCs and proinflammatory cytokineproduction, differentially polarizing T cells during priming.Thus, dLNs were harvested 12 or 24 hours after immuniza-tion and cultured in the absence of further stimulation toassess the cytokine milieu produced by APC. Significantlevels of IFN-b were detected in supernatants from dLNs






Figure 2. Increased infiltration ofOT-I cells at the tumor site andreduction in tumor growth correlatewith the Teff:Treg ratios in thedLNs.Mice receivedOT-I andOT-IIT cells intravenously as in Fig. 1 andwere challenged with B16.OVAtumor cells subcutaneously in theleft flank. Seven days later, micewere immunized with OVA257-264

and OVA323-339 peptides �adjuvant. Tumors and dLNs wereexcised 7 days later andlymphocyte populations wereanalyzed by flow cytometry (A–C).Alternatively,micewere vaccinated1 week before tumor challenge andboosted 10 days after tumorestablishment. Tumor growth wasmonitored over time (D). To assessin vivo killing, splenocytes wereloaded with OVA-peptide andinjected intravenously 1 week aftervaccination in the absence oftumors. Surviving target cellfrequencies were detected in blood6 hours later (E). A, absolutenumber of OT-I effector cells permilligram of tumor tissue (left) andtotal tumor weight in milligrams(right). B, number of OT-II effectorcells (left) andOT-II Tregs (right) permilligram of tumor tissue. C, ratiosof OT-I Teffs:OT-II Tregs (left) andOT-II Teffs:OT-II Tregs (right) in thetumor/vaccine dLNs. D, meantumor sizes (left) and Kaplan–Meiersurvival curves (right). E, specifickilling of 10 nmol/L peptide-pulsedtargets at 6 hours. A, B, C, and E,the data for groups of 3 mice in oneof two independent experiments.Data in D are from groups of 5 micerepresenting one of fourindependent experiments.

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extracted 12 hours after vaccination and incubated in vitrofor 1 hour (Fig. 6A). Inflammatory mediators IL-12, IFN-g ,and IL-6 were detected in 1-, 6-, and 12-hour culture super-natants of dLNs from mice vaccinated with OVAþCpG orOVAþPoly(I:C) 12 hours earlier (Fig. 6B, left). OVAþQuil Aand OVAþImiquimod induced less or no IL-12, IFN-g , andIL-6 at the same time points. Lymph nodes extracted frommice vaccinated with OVAþCpG 24 hours earlier continuedto produce a small but significant amount of IL-12 detectedin the culture supernatant, whereas the levels of the otheranalytes returned to baseline in all groups (Fig. 6B, right).Variable levels of TNF-a and IL-2 production were detectedbut no significant difference was apparent between groups(Supplementary Fig. S6). No production of GM-CSF, IL-1b,IL-4, or IL-10 was detected (data not shown).

DiscussionIn this study, we performed a comprehensive analysis of a

panel of adjuvants to better characterize their antigen-specificeffects on antitumor immune responses following peptide

vaccination. Our dual T-cell adoptive transfer models wereideal tools to closely examine the relationship between vac-cine-induced antigen-specific Teff and Treg populations,which are extremely rare in a physiologic setting. We wereable to determine a hierarchy among the different adjuvantstestedwith regards to their ability tomodulate antigen-specificTeff and Treg responses. Similar results were obtained in theovalbumin and melanoma peptide models, highlighting therobustness of this experimental system. It is also noteworthythat our results in bothmousemodels tested recapitulated ourobservations in patients with melanoma vaccinated withMelan-A peptide and CpG-ODN (21).

We found that immunization with peptide and adjuvantsCpG-ODN and Poly(I:C) preferentially promoted the expansionof antigen-specific CD8þ and CD4þ Teffs over that of antigen-specific Tregs, resulting in increased Teff:Treg ratios. In addi-tion, these adjuvants endowed antigen-specific effector cellswith polyfunctional effector capacity. In contrast, we foundthat Quil A and imiquimodmaintained or even decreased Teff:Treg ratios due to a greater accumulation of antigen-specific

Figure 3. Therapeutic vaccination with adjuvants that induce high antigen-specific Teff:Treg ratios confers durable rejection of established tumors. Micereceived 1 � 105 OT-I and 1 � 106 OT-II T cells intravenously. Three days later, mice were challenged with EG7 tumor cells subcutaneously After10 days, once tumors were well established, mice were immunized as above, and tumor growth was monitored over time. A, growth curves of EG7tumors are shown for each individual mouse in the different groups. B, mean tumor sizes (left) and survival times (right) are shown for groups of 5 mice in oneof two independent experiments.






Tregs, whereas effector cell expansion and acquisition ofpolyfunctionality were reduced. Polyfunctionality is a definingfeature of long-lived T cells, and correlates with increaseddisease protection in both human antiviral vaccination andmurine tumor immunotherapy studies (34–36). Furthermore, arecent report demonstrated that human papillomavirus E7peptideþCpG-ODN or Poly(I:C) vaccine formulations expand-ed multi-cytokine–producing CD8þ effector memory T cells,the presence of which predicted therapeutic efficacy againstcancer in mice (37). Our results are consistent with thesestudies and provide the additional dimension of control ofantigen-specific Treg expansion by CpG-ODN or Poly(I:C)adjuvants.

Previous studies have linked total CD8þ T cell:CD4þ Tregratios to both natural tumor progression (5, 38) and cancerimmunotherapy outcomes in mice and humans (39, 40). How-ever, little information exists on the role of antigen-specificTregs on disease outcome, or how this population might beregulated by immunotherapy. Data are emerging on theTeff and antigen-specific Treg relationships in autoimmunityand transplantation. Immunotherapies that enhance antigen-specific Treg expansion and suppressive function delayedmulti-cytokine–producing Teff activation, resulting in diseasecontrol (41, 42). To date, the relationship between antigen-specific Teffs and Tregs has not been examined in detail incancer, although some in vitro and observational reports exist

Figure 4. Tumor/self-antigen–specific Teff:Treg balance and effector function are most strongly enhanced by vaccination with peptide and Poly(I:C) or CpG.CD45.1 mice received 1 � 105 Pmel and 1 � 105 Trp-1 T cells intravenously. One day later, mice were immunized subcutaneously with hgp10025-33and Trp-1106-130 peptides � adjuvant. Spleens were harvested 7 days later and lymphocytes analyzed (A–E). To assess in vivo killing, splenocyteswere loaded with hgp100-peptide and injected intravenously 1 week after vaccination and surviving target cell frequencies were detected in blood24 hours later (F). A, absolute number of Pmel effector cells (CD8þCD45.2þ, left) and frequency of Pmel cells producing IFN-g , IL-2, and CD107a(right). B, absolute number of Trp-1 effector cells (CD4þCD45.2þFoxp3�, left) and frequency of Trp-1 cells producing IFN-g and IL-2 (right). C, absolutenumber of Trp-1 Tregs (CD4þCD45.2þFoxp3þ). D and E, ratio of Pmel Teffs:Trp-1 Tregs (left; D) and ratio of Trp-1 Teffs:Trp-1 Tregs (right; E). F, specific killingof 10 and 100 nmol/L peptide-pulsed targets at 24 hours. Graphs show data from groups of 3 mice in one of two independent experiments.

Perret et al.





(19–21). Our study extends the knowledge base gained fromprevious investigations by comparing a large panel of adju-vants for their ability to modulate the vaccine-specific Teff:Treg balance. Our results indicate that each adjuvant uniquelymodulates antigen-specific Teff:Treg ratios, which in turncorrelate with tumor control. Interestingly, we found that incontrast to antigen-specific responses, polyclonal Teff:Tregratios are not affected by vaccination with specific peptide inadjuvant (Supplementary Fig. S1C). Thus, the behavior ofpolyclonal T-cell populations cannot be used to predict theresponses of antigen-specific cells. This fact should be consid-ered in rational vaccine design, which should focus on con-trolling the balance of Teffs versus Tregs with defined antigenspecificities to create the most effective vaccines.We found that the ratios of antigen-specific Teffs:Tregs in

tumor-dLNs were similar to those observed in vaccine-dLNs oftumor-free mice. High Teff:Treg ratios induced by CpG-ODNand Poly(I:C) in the lymphoid tissues correlated with increased

OT-I T-cell activation, cytotoxic activity, and tumor infiltra-tion, leading to significantly higher protection against tumorgrowth. Surprisingly, antigen-specific Tregs infiltrated mela-nomas very poorly and are therefore unlikely to play a majorrole at the tumor site. This is in sharp contrast to the profoundinfiltration of polyclonal Tregs into many tumors, includingmelanomas, which has been observed by us (data not shown)and others (43, 44). That antigen-specific T cells should play animportant role in the lymph nodes rather than at the diseasesite is consistent with reports that antigen-engagement byTregs is important for initial priming, allowing fine-regulationof the priming of effector cells of the same specificity (45). Onceactivated, Tregs can suppress nonspecifically, thus eliminatingthe need for antigen-specific Tregs at disease sites (45, 46).These findings outline the relevance of antigen-specific Teff:Treg ratios in the development of antitumor immunity. Mea-suring this balance in the periphery may consequently providea more accurate method of predicting vaccine efficacy (39, 47).

Figure 5. Vaccination with tumor/self-peptides and Poly(I:C) or CpG confers protection against tumor challenge. Mice received Pmel and Trp-1 T cellsintravenously as in Fig. 4 and were vaccinated 1 day later with hgp10025-33 and Trp-1106-130 peptides � adjuvant. Seven days later, mice werechallenged with B16 tumor cells subcutaneously and at the same time received a second vaccine dose. Tumor growth and survival were monitored overtime. A, growth curves of B16 tumors are shown for individual mice in the different groups. B, survival following tumor challenge. C, vaccine-induceddepigmentation (left axis) vs. survival (right axis) at day 20. Graphs show data for groups of 5 mice in one of two independent experiments.






The mechanisms involved in the control of effector versusTreg induction, whether polyclonal or antigen-specific, are notcompletely understood.However, thematuration state of APCsand the resulting proinflammatory cytokine environment atthe site of T-cell priming is known to be important (48–50).There is ample evidence that vaccine adjuvants, and TLRagonists in particular, can mature dendritic cells, inducingexpression of costimulatory molecules such as CD40 and

modifying their T cell priming potential (51). Type I IFNs arenecessary for the maturation of dendritic cells and the gener-ation of CTL and Th1 responses. Vaccines containing Poly(I:C),CpG or the TLR-7/8 ligand R-848 have been shown to induceIFN-a/b-dependent production of type I cytokines (48, 49).Dendritic cells are also matured by stimulation with the TLRligands LPS andCpG. They upregulateMHC-II, CD80 andCD86expression and produce the inflammatory cytokines IL-12,IFN-g and IL-6, which promote effector CTL and Th1 devel-opment as well as directly inhibiting Tregs (50, 52, 53). Wefound that a dendritic cell maturing/type I polarizing cytokinemilieu consisting of IFN-b, IL-12, IFN-g and IL-6was induced indLNs early after vaccination with peptideþCpG or Poly(I:C).These adjuvants induced the highest antigen-specific Teff:Tregratios and the best antitumor outcomes, providing clues aboutthemechanism behind the adjuvant effects on vaccine-specificT cell balance. Antibody-blockade of type I IFNs, and/or IL-12,or experiments in KO mouse strains would be necessary toproduce definitive evidence for the role of these cytokines, butthe concordance of our results with the large body of existingdata provide strong support for this preliminary mechanisticexplanation.

In conclusion, we have shown that not all adjuvants areequal in their ability to modulate vaccine-specific immuneresponses. Our data support the use of the adjuvants CpG-ODN and Poly(I:C) in peptide vaccines containing both CD4and CD8 epitopes, to enhance the activation of polyfunc-tional Teffs and avoid Treg expansion. This study alsohighlights the importance of studying antigen-specific Teffand Treg responses in the context of peptide vaccination, asthese cannot necessarily be extrapolated from an evaluationof total polyclonal responses. Finally, we reveal a correlationbetween the choice of adjuvant in a peptide vaccine formu-lation, the antigen-specific CD8þ and CD4þ Teff:Treg ratios,the size and quality of the resulting Teff response, and thedegree of tumor protection induced. This suggests thatantigen-specific Teff:Treg ratios are a useful measure ofvaccination outcome and have the potential to be a valuablepredictive biomarker of objective clinical responses in can-cer immunotherapy.

Disclosure of Potential Conflicts of InterestP. Romero is a consultant/advisory board member of Immatics Biotechno-

logies, DC Prime, Matwin, and Center for Human Immunology, Pasteur Institute(Paris, France). No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: R. Perret, P. RomeroDevelopment of methodology: R. PerretAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Perret, N.K. Botelho, S. CorgnacAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Perret, S. Sierro, P. RomeroWriting, review, and/or revision of the manuscript: R. Perret, S. Sierro,A. Donda, P. RomeroStudy supervision: P. Romero

AcknowledgmentsThe authors thank Aur�elie Hanoteau for assistance with experiments and the

staff of the SPFmouse facility of the University of Lausanne for animal husbandryand care.

Figure 6. CpG and Poly(I:C) induce strong type I polarization early afterimmunization with peptide and adjuvant. Mice were immunizedsubcutaneously with OVA257-264 and OVA323-339 peptides � adjuvant.dLNs were collected 12 or 24 hours later and homogenized andincubated at 37�C. Supernatants were collected from the lymph nodecultures after 1, 6, and 12 hours and analyzed by ELISA and Luminex. A,IFN-b productionmeasured by ELISA 1 hour after lymph node extraction.B, cytokineproductionmeasuredbyLuminex after lymphnodeextractionand in vitro incubation of cell suspensions. Graphs show data fromthree samples per group in one of three independent experiments. Dottedlines show the detection limit.

Perret et al.





Grant SupportThis work was supported by grants from the New Zealand Foundation

for Research Science and Technology and the Emma Muschamp Foun-dation (R. Perret) and from the Swiss National Science Foundation(310030-130812 and CRSII3_141879) and the Medic Foundation (P.Romero).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 2, 2013; revised July 25, 2013; accepted August 21, 2013;published OnlineFirst September 18, 2013.

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2013;73:6597-6608. Published OnlineFirst September 18, 2013.Cancer Res Rachel Perret, Sophie R. Sierro, Natalia K. Botelho, et al. Regulatory T Cells Enhance Tumor ImmunityAdjuvants That Improve the Ratio of Antigen-Specific Effector to

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