TLR Stimulation during T-cell Activation Lowers PD-1 ...€¦ · Research Article TLR Stimulation during T-cell Activation Lowers PD-1 Expression on CD8þ T Cells Christopher D. Zahm1,Viswa

Research Article

TLR Stimulation during T-cell Activation LowersPD-1 Expression on CD8þ T CellsChristopher D. Zahm1, Viswa T. Colluru1, Sean J. McIlwain1,2, Irene M. Ong1,2,3,and Douglas G. McNeel1

Abstract

Expression of T-cell checkpoint receptors can compromiseantitumor immunity. Blockade of these receptors, notablyPD-1 and LAG-3, which become expressed during T-cellactivation with vaccination, can improve antitumor immu-nity. We evaluated whether T-cell checkpoint expressioncould be separated from T-cell activation in the context ofinnate immune stimulation with TLR agonists. We foundthat ligands for TLR1/2, TLR7, and TLR9 led to a decrease inexpression of PD-1 on antigen-activated CD8þ T cells. Theseeffects were mediated by IL12 released by professional

antigen-presenting cells. In two separate tumor models,treatment with antitumor vaccines combined with TLR1/2or TLR7 ligands induced antigen-specific CD8þ T cells withlower PD-1 expression and improved antitumor immunity.These findings highlight the role of innate immune activa-tion during effector T-cell development and suggest that atleast one mechanism by which specific TLR agonistscan be strategically used as vaccine adjuvants is by modu-lating the expression of PD-1 during CD8þ T-cell activation.Cancer Immunol Res; 6(11); 1364–74. �2018 AACR.

IntroductionThe expression of T-cell checkpoint molecules, notably pro-

grammed death 1 (PD-1), lymphocyte activation gene 3 (LAG-3),and T-cell immunoglobulin and mucin-domain containing 3(TIM-3), was first described on T cells specific for viruses andassociated with cells with a reduced functional phenotype (1, 2).They were thus initially described as markers of T-cell exhaustion(3). It has become increasingly recognized, however, that expres-sion of these molecules occurs normally with T-cell activation,and cells expressing these molecules can have functional activity(4). This activation-induced upregulation is also consistent withthe role of T-cell checkpoint molecules as safeguards againstautoimmunity. In the case of PD-1, signalingmediatedbybindingto one of its ligands leads to reduced effector function (5). Thus,the expression of PD-1 sustains peripheral tolerance, as normaltissues express the ligand for PD-1 (PD-L1) to abrogate autoreac-tive toxicity by CD8þ T cells. It has been demonstrated inmultiplemodels that PD-L1 expression by tumors is a mechanism ofimmune evasion and that blocking PD-1 and PD-L1 interactioncan restore T-cell function and enhance antitumor responses (6).The demonstration of antitumor activity using PD-1 or PD-L1blockade alone has led to multiple new FDA approvals over the

last 5 years, underscoring the power of this single mechanism ofimmune regulation.

The notion that T-cell checkpoint receptors are purely markersof T-cell exhaustion has been contested (7). We have found thatT cells activated with a strong antigen signal have increased PD-1expression, and this expression can persist over time (8). CD8þ

T cells stimulated with a lower affinity epitope became activated,but with lower, more transient, PD-1 expression. Furthermore,activated CD8þ T cells with reduced PD-1 expression mediatedgreater antitumor activity in vivo. Similarly, blocking PD-1 signal-ing at the time of activation with vaccination led to greaterantitumor activity (8, 9). We have demonstrated that this mech-anism of resistance is critical for successful human immunother-apy, as blockade of PD-1/PD-L1 interaction at the time of T-cellactivationwith vaccination led to greater antitumor response thanblockade alone in patients with metastatic, castration-resistantprostate cancer (mCRPC; ref. 10). Thesefindings suggest that if theexpression of T-cell checkpoint inhibitors such as PD-1 could bereduced following CD8þ T-cell activation, these cells would havegreater antitumor activity.

Certain bacterial and viral pathogens can elicit robust and rapidimmune responses. This response serves as the basis for usingspecific pathogens as viral delivery vehicles and using bacterialcomponents as vaccine adjuvants. The molecular basis of theserapid "innate" immune responses is partially due to recognitionofpathogenic characteristics by Toll-like receptors (TLR; ref. 11). TLRligands are being increasingly explored as vaccine adjuvants.However, although studies suggest that innate immune stimula-tion at the time of T-cell activation leads to a superior adaptiveeffector and cytotoxic T-cell response, the precise mechanisms forthis have not been elucidated (12).

Other groups have demonstrated that innate immune activa-tion with vaccination can lead to autoreactive immunity. Inaddition to molecular mimicry of pathogens encoding self-antigens, TLR activation is thought to be required to developautoreactive T cells (13). Such findings suggest that TLR stimula-tion might have effects on T-cell function by bypassing normal

1University of Wisconsin Carbone Cancer Center, University of Wisconsin,Madison, Wisconsin. 2Department of Biostatistics and Medical Informatics,University of Wisconsin, Madison, Wisconsin. 3Department of Obstetrics andGynecology, University of Wisconsin, Madison, Wisconsin.

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

Corresponding Author: Douglas G. McNeel, University of Wisconsin CarboneCancer Center, 7007 Wisconsin Institutes for Medical Research, 1111 HighlandAvenue, Madison, WI 53705. Phone: 608-265-8131; Fax: 608-265-0614; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-18-0243

�2018 American Association for Cancer Research.

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immune tolerance mechanisms, perhaps via changes to theexpression of T-cell checkpoint molecules. Such a finding mightthen be strategically used to improve the efficacy of antitumorvaccines by using specific TLR agonists as molecular adjuvants.Consequently, we sought to determine if TLR stimulation, at thetime of T-cell activation, leads to changes in CD8þ T-cell check-point expression and whether TLR stimulation might be used toimprove the efficacy of antitumor vaccines. We used two modelsystems, theOT-1model in which transgenic CD8þ T cells expressthe same T-cell receptor, and a nativemodel targeting a dominantepitope derived from the synovial sarcoma X breakpoint 2 (SSX2)tumor antigen. In each model, we activated CD8þ T cells withpeptide ligands in the presence or absence of TLR agonists. Usingin vitro and murine models, we evaluated the effect of TLRstimulation on T-cell activation and antitumor activity. Ourfindings provide insight into the role of TLR stimulation at thetimeof T-cell activation in eliciting an effective adaptive antitumorT-cell response. Our data support the use of defined TLR agonistsas vaccine adjuvants because of their ability to modulate expres-sionof a specific T-cell checkpointmolecule at the timeofCD8þT-cell activation.

Materials and MethodsMice

HLA-A2.01/HLA-DR1-expressing (HHDII-DR1) mice wereobtained fromCharles River Laboratories courtesy of Dr. FrancoisLemonnier (14). OT-1 (Stock No: 003831) and C57BL/6J (B6,Stock No: 000664) were purchased from The Jackson Laboratory.All experiments were conducted under an IACUC-approved pro-tocol conforming to NIH guidelines.

Cell linesE.G7-OVA (CRL-2113) cellswere obtained fromATCC in2015.

E.G7-OVA cells were lentivirally transduced to express PD-L1, aspreviously described (8). E.G7-OVA cells were not used beyondseven passages from original purchase. The A2/sarcoma cell lineexpressing SSX2 (A2/Sarc-SSX2) was generated as previouslydescribed (9). Cell identity and Mycoplasma testing were con-firmed by DDC Medical.

PeptidesPeptides encoding the H2Kb-restricted epitope from chicken

ovalbumin (SIINFEKL, OVA), or variants with lower affinity(SIINTEKL, SIINFEKP), and the dominant HLA-A2–restrictedepitope from SSX2 (FLQGISPKI, ref. 15) were synthesized, andthe purity and identity of each peptide was confirmed (LifeTein,LLC.).

In vitro T-cell stimulationSplenocytes were isolated from OT-1 mice after red blood cell

osmotic lysis (0.15 mol/L NH4Cl, 10 mmol/L KHCO3,0.1 mmol/L EDTA). Splenocytes were cultured at 2 � 106/mL inRPMI 1640 medium supplemented with L-glutamine, 10%fetal calf serum (FCS), 200 U/mL penicillin/streptomycin, 1%sodium-pyruvate, 1% HEPES, 50 mmol/L b-MeOH, and 2 mg/mLof the designated peptide. TLR agonists were purchased fromInvivoGen and added 1 hour before peptides at the followingconcentrations: 300 ng/mL Pam3CSK4, 10 mg/mL Poly(I:C)HMW, 10 mg/mL MPLAs, 3 mg/mL Gardiquimod, 10 mg/mLR848, 5 mmol/L ODN 1826. At the time points indicated, cells

were stained, fixed, and frozen. Cells were thawed, washed, andresuspended in PBS þ 3% FCS þ 1 mmol/L EDTA for flowcytometry. Cells were stained for 30 minutes at 4�C in a 1:4dilution of brilliant stain buffer (BD 563794) in PBSþ 3% FCSþ1 mmol/L EDTA. Intracellular cytokine staining was performedusing standard procedures (8). In order to assess costimulation,blocking antibodies for CD80 (0.6 mg/mL), CD86 (0.25 mg/mL),or OX40L (0.6 mg/mL) were added 1 hour prior to antigen.

RNA preparation and sequencingCells were stimulated in vitro as above, and at the times

indicated CD8þ T cells were isolated via immunomagnetic neg-ative selection (Stemcell Technologies; 19853). RNA was collect-ed as per themanufacturer's instruction (Direct-zol RNAMiniPrepPlus w/ TRI Reagent, Zymo Research), treated with DNAse(TURBO DNA-free Kit, Invitrogen), and stored at �80�C untilanalysis at the UW-Madison Biotechnology Center. Total RNAwas verified via Agilent 2100 BioAnalyzer. Samples were preparedusing the Illumina TruSeq Stranded mRNA kits (Illumina Inc.).For each library, mRNA was purified from 1,000 ng total RNA.Each poly A–enriched sample was fragmented, synthesized intodouble-stranded cDNA, incubatedwith KlenowDNAPolymeraseand DNA fragments, ligated to Illumina adapters, and purified byparamagnetic beads. Adapter ligated DNA was amplified in aLinker Mediated PCR reaction (LM-PCR) for 11 cycles and thenpurified by paramagnetic beads. Libraries were standardized to2 mmol/L. Cluster generation was performed using standardCluster Kit (v3) and the Illumina Cluster Station. Single 100 bpsequencing was performed, using standard SBS chemistry (v4) onan Illumina HiSeq2500 sequencer. Images were analyzed usingthe standard Illumina Pipeline, version 1.8.2.

RNA-seq data analysisIllumina sequencing reads were adapter and quality trimmed

using the Skewer trimming program. Quality reads were subse-quently aligned to the annotated reference genome using theSTAR aligner (16). Quantification of expression for each gene wascalculated by RSEM (17), the expected read counts from RSEMwere filtered for low/empty values and used for differential gene-expression analysis using DESeq2 (18). All remaining genes fromthe pairwise comparisons were analyzed using Gene Set Enrich-ment Analysis (GSEA; refs. 19, 20) to identify pathways [based onGO terms (c5.all.v6) from the Molecular Signatures Database(MSigDB; ref. 21] as well as custom gene sets involved in CD8þ T-cell activation.GSEAwas performedby calculating a ranked vectoras log2 fold change. Analysis was performed using R 3.3.1 andBioconductor 2.32.0 (22). Venn diagrams were generated usingthe VennDiagrampackage. Data (accession numberGSE110144)are publicly available at (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE110144).

In vitro coculture experimentsAPC subsets were enriched from splenocytes using PE-labeled

antibodies specific for either CD19 or CD11c (Stemcell Technol-ogies). CD8þ T cells were isolated as above. Primary dendritic cells(DC) were harvested as previously described (23). Each APCsubset, and a subset of T cells with no APCs present, were culturedat 2�106 cells/mL inPBS in the presence of 2mg/mLpeptide,withor without TLR agonists for 1 hour. Cells were rinsed and trans-ferred to freshmedium. Na€�ve T cells were added at a 1:1 ratio andincubated for 3 days, after which CD8þ T cells were analyzed by

TLR Stimulation and PD-1 Expression

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flow cytometry. To assess the effects of secreted factors, cells wereincubated as described for 4 hours, rinsed, and incubated for 24hours in fresh media. These conditioned media were then addedto a well containing untreated APCs and OT-1 T cells, isolated asabove, and incubated for an additional 24 hours. Where indicat-ed, 1.0 ng/mL recombinant IL12 (R&D Systems; 419-ML-010)was added in place of the TLR agonists or 0.06 mg/mL blockingantibody (R&DSystems, AF-419-SP)was added in addition to theagonists. Where indicated, IL12 concentrations in culture super-natant were detected by ELISA according to the manufacturer'srecommendations (R&D Systems, kit M1270).

Adoptive transfer and immunization of miceOT-1 splenocytes were harvested, and CD8þ T cells were

isolated as described. OT-1 T cells (2 � 106) were adoptivelytransferred into 6- to 10-week-old female B6 mice intraperitone-ally. The following day, mice were immunized subcutaneouslywith 100 mg SIINFEKL (OVA) peptide in PBS. Alternatively, HHD-II-DR1 mice were immunized directly with 100 mg of the dom-inant HLA-A2–restricted epitope from SSX2 (FLQGISPKI) in PBS.TLR agonists were coinjected with vaccine: 20 mg/mousePam3CSK4, 100 mg/mouse Gardiquimod, 50 mg/mouse ODN1826, or vehicle. Mice were euthanized at the times indicated.Spleens were collected, processed as described, and T-cell popula-tions were analyzed by flow cytometry. Tetramers specific forFLQGISPKI (NIH Tetramer Core Facility, Emory University,Atlanta, GA) were used as previously described (15). Data col-lected on different days were normalized using rainbow beads(Spherotech; RFP-30-5A).

Ovalbumin tumor treatment studiesOvalbumin-expressing E.G7PD-L1high cells (106)were injected

subcutaneously into 6- to 10-week-old female B6 mice. Whentumors were palpable and similarly sized (�0.2 cm3), 2 � 106

na€�ve OT-1 CD8þ T cells were adoptively transferred to eachmouse as described above. The following day, mice were immu-nized subcutaneously with 100 mg of an individual peptide withor without TLR agonists as described. In mice receiving PD-1blocking antibody, 100 mg of antibody (clone G4) was injectedintraperitoneally on the day following vaccination. Tumor vol-ume was measured using calipers and calculated in cubic cen-timeters according to the formula: (p/6) � (long axis) � (shortaxis)2. Tumors obtained at necropsy were digested in collagenaseand DNAse I for 1 to 2 hours at 37�C, and passed through a100-mm screen and analyzed by flow cytometry.

SSX2 tumor treatment studiesSix- to 8-week-old HHDII-DR1 mice were inoculated with

105 A2/Sarc-SSX2 cells administered subcutaneously and thenimmunized intradermally with DNA vaccines weekly beginning1 day after tumor implantation (9). TLR agonists or vehicle werecoadministered with the vaccine intradermally, and tumorvolumes were measured as described above.

Statistical analysesComparison of group means was performed using GraphPad

Prism software, v5.01. Analysis of variance (ANOVA; one- or two-sided depending on the nature of the data) statistical modelingwas used to analyze data sets, followed by the Bonferroni mul-tiple-comparison post hoc procedure to compare individual

groupmeans. For all comparisons, P values� 0.05 (or 0.01whereindicated) were considered statistically significant.

ResultsTLR stimulation at the time of CD8þ T-cell activation in vitrolowers expression of PD-1

We have previously found that immunization with peptideswith high MHC-I affinity, or DNA immunization encoding epi-topes with highMHC-I affinity, led to increased expression of PD-1 on cognate CD8þ T cells (8, 9). Others have reported thatimmunization with a TLR9 agonist led to decreased expression ofPD-1 on antigen-specific CD8þ T cells (24, 25). These observa-tions suggest that innate signals at the time of T-cell activationmight affect the expression of checkpoint molecules on activatedCD8þ T cells. To evaluate this directly, we used the well-characterized ovalbumin model to question whether TLR stimu-lation could similarly affect expression of different T-cell check-points. OT-1 splenocytes were stimulated in vitro with SIINFEKLpeptide alone or in the presence of different TLR ligands: TLR 1/2(Pam3CSK4), TLR 3 (Poly I:C), TLR 4 (MPLAs), TLR 7 (Gardi-quimod), TLR 7/8 (R848), or TLR 9 (ODN1826). OT-1 cellsstimulated with OVA peptide (SIINFEKL) were activated, asmeasured by 4-1BB expression, and activation was not affectedover 4 days by the presence of different TLR ligands (Fig. 1).Similarly, LAG-3 expression increased by day 3, but was notaffected by the different TLR ligands. However, PD-1 expressionwas reduced in the presence of TLR1/2, TLR7, TLR7/8, and TLR9ligands. This reduction in PD-1 expression in the presence of theseTLR ligands was also observed following activation with loweraffinity epitopes (Supplementary Fig. S1; ref. 8). Effects on T-cellcheckpoint expression appeared to be limited to PD-1, as we didnot detect decreases in expression of LAG-3, TIM-3, CTLA-4,VISTA, CD244, TIGIT, or CD160 following activation of OT-1cells in the presence of TLR1/2, TLR7, or TLR9 ligands (Supple-mentary Fig. S2). Moreover, OT-1 T cells activated in the presenceof peptide and TLR ligands continued to exhibit a Th-1 bias interms of cytokine production (Fig. 2A and B).

TLR1/2 or TLR7 ligands induce expression of genes associatedwith effector T-cell function

To determine whether CD8þ T cells activated in the presence ofTLR ligands were functional, or whether the decrease in PD-1 wasdue to decreased activation, and to determine whether the effectsof TLR1/2 and TLR7 activation were redundant, OT-1 splenocyteswere activated in the presence of SIINFEKL peptide alone or withTLR1/2 (Pam) or TLR7 (Gardiquimod) ligands. CD8þ OT-1T cells were then purified and evaluated for gene-expressionchanges by mRNA sequencing (RNA-seq). OT-1 CD8þ T cellsactivated in the presence of either TLR1/2 (Pam3CSK4) or TLR7(Gardiquimod) ligands showed similar but distinct changes inenrichedGO-term expressionwhen comparedwith cells activatedby peptide antigen alone (Fig. 3A). Both TLR ligands increasedexpression of genes associated with Th1-biased effector function(IFNg , TNFa, granzymes A–C, perforin, and T-Bet), reducedEOMES expression, and reduced expression of genes associatedwith T-cell regulation (including PD-1 and IL10) when comparedwith cells stimulated with peptide alone (Fig. 3B). Hence, thereduced PD-1 expression we observed corresponded to increasedexpression of molecules associated with Th1-biased effectorCD8þ T cells.

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Changes in PD-1 expression following TLR stimulation drivenby professional APC

TLRs are expressed by both T cells and professional APCs. Todetermine if the effects on T-cell checkpoint expression weremediated by direct effects on T cells or APCs, purified OT-1CD8þ T cells were activated with SIINFEKL peptide alone or inthe presence of TLR1/2 or TLR7 agonist, or with purifiedDCs or B cells pretreated with TLR agonists and peptide.After 24 hours, cells were evaluated for the expression of 4-1BB, PD-1, and LAG-3. Cells were activated in the presence orabsence of professional APCs, as measured by 4-1BB expression(Fig. 4A). The expression of LAG-3 was not affected by the APC

type or the presence of TLR ligand. PD-1 expression requiredpresentation by professional APCs and was reduced in thepresence of either TLR ligand. Blockade of CD80, CD86, orOX40L did not affect PD-1 expression, suggesting that APCsignals other than these costimulatory molecules were required(Supplementary Fig. S3). To determine if these APC signalswere due to secreted factors, conditioned medium from DCsincubated with or without TLR ligands was used in place of theTLR agonist during T-cell stimulation. OT-1 CD8þ T cellsactivated by DCs in the presence of TLR-conditioned mediashowed a reduction in PD-1 similar to that observed with TLRagonist treatment directly, suggesting the decrease of PD-1

Figure 2.

The effect of TLR1/2, TLR7, and TLR9 agonists is greatest on PD-1 following T-cell activation. OT-1 splenocytes were stimulated with either the SIINFEKL (OVA)peptide or a nonspecific peptide control (NS), alone or in combination with the designated TLR agonists, for 3 days. The expression of PD-1 and intracellularcytokines, as a percentage of total CD8þ T cells, were assessed by flow cytometry (A). Representative flow cytometry plots showing IFNg and TNFaexpression with each stimulation condition are shown in B. � , P < 0.01, with each comparison made to stimulation with OVA peptide alone. Beads ¼ stimulationof OT-1 cells with beads coated with anti-CD3 and anti-CD28. Results are from one experiment, with samples assessed in triplicate, and are representative oftwo similar experiments.

Figure 1.

TLR stimulation at the time of T-cell activation in vitro affects expression of PD-1. Splenocytes were prepared from the spleens of OT-1 mice and stimulated in vitrowith the high-affinity SIINFEKL peptide in the presence or absence of specific TLR agonists [TLR 1/2 (Pam3CSK4), TLR 3 (Poly I:C), TLR 4 (MPLAs), TLR 7(Gardiquimod), TLR 7/8 (R848), or TLR 9 (ODN1826)]. Shown are the mean fluorescence intensity (MFI) of 4-1BB, PD-1, and LAG-3 on CD8þ T cells collecteddaily for 4 days. � , P < 0.01, with each comparison made to stimulation with SIINFEKL peptide alone, and color corresponding to the agent compared. Resultsare from one experiment, with samples assessed in triplicate, and are representative of four similar, independent experiments.






expression was due to release of a secreted factor by TLR-stimulated DCs (Fig. 4B).

Changes in PD-1 expression following TLR stimulation can bemediated by IL12

CpG treatment of mice leads to CD8þ T cells with lowerexpression of PD-1, an effect dependent on IL12 signaling(26). Given this, and the Th1 effector bias of T cells identifiedin our gene-expression studies, we tested whether treatment ofAPCs with TLR ligands elicited production of IL12. Treatment ofDCswith TLR1/2 and TLR7 agonists led to the greatest secretion ofIL12 (Fig. 5A). No significant secretion of IL12 was detectedfollowing treatment with TLR3 or TLR4 agonists. Moreover,OT-1 cells activated with peptide and DCs in the presence ofIL12 had lower PD-1 expression, and the reduction of PD-1

expression with TLR stimulation was decreased in the presenceof IL12 blockade (Fig. 5B). Finally, when beads coated with anti-CD3 and anti-CD28 were used in place of APCs, IL12 alonereduced PD-1 expression (Fig. 5C). Together, these findingsdemonstrate that TLR stimulation leads to secretion of IL12 fromDCs, and IL12 acting directly on T cells can lead to reduced PD-1expression during T-cell activation.

TLR stimulation at the time of T-cell activation reducedexpression of PD-1 in vivo

To determine whether TLR stimulation in vivo at the time of T-cell activation with vaccination elicited a similar reduction in PD-1,OT-1 cells were adoptively transferred to na€�ve C57BL/6mice asabove, and immunized once with OVA peptide (or nonspecificpeptide) 1 day later in the presence or absence of a TLR 1/2, TLR7,

Figure 3.

CD8þ T cells activated in the presence of TLR1/2 or TLR7 ligands exhibit a gene-expression profile consistent with enhanced effector function: OT-1 splenocyteswere activated for 24 or 72 hours in the presence of SIINFEKL peptide (OVA) alone, or with TLR1/2 or 7 ligands. CD8þ OT-1 T cells were then purified andevaluated for gene-expression changes by RNA-seq. All samples were evaluated in six replicates from a single experiment. A, Venn diagrams showing the numberof enriched GO-term expression with significantly increased (up) or decreased (down) expression following treatment with TLR1/2 (TLR2) or TLR7 relative toSIINFEKL treatment alone. Results are from GSEA with an FDR of <0.25. B, Gene-expression changes for CD8þ T cells stimulated with OVA in the presenceof TLR agonists versus CD8þ T cells stimulated with OVA alone (green ¼ increased expression, red ¼ reduced expression).

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or TLR9 ligand. Splenocytes were collected up to 14 days afterimmunization and evaluated for expression of 4-1BB, PD-1, andLAG-3. 4-1BB and LAG-3 expression increased after immuniza-tion, and their expression was not affected by concurrent deliveryof one of the TLR agonists (Fig. 6A). PD-1 expression, however,was significantly reducedwithin days of vaccinationwhen vaccinewas codelivered with a TLR ligand. Similar results were foundfollowing immunization of HLA-A2 transgenic mice with a sep-arate high-affinity HLA-A2–restricted peptide derived from theSSX2 tumor antigen (8) in the presence or absence of TLRstimulation (Fig. 6B).

Immunization in the presence of TLR1/2 or TLR7 ligandsimproved antitumor immunity

Todeterminewhether the reduced expression of PD-1 onT cellsactivated in the presence of TLR stimulation could mediategreater antitumor response, ovalbumin- and PD-L1–expressing

E.G7 cells were implanted in C57BL/6 mice and permitted togrow until palpable (14 days). OT-1 T cells were then adoptivelytransferred and the following day mice were immunizedwith SIINFEKL (OVA) peptide alone or with TLR1/2 or TLR7agonist.Other groups ofmice received immunizationwith 100mganti–PD-1 delivered the day following. In this model, PD-1blockade alone had limited antitumor efficacy in the absence ofT-cell activation, as we have previously reported (8). Immuniza-tion with OVA peptide in the presence of TLR1/2 or TLR7 agonistproduced a greater antitumor effect than with peptide alone(Fig. 7A). The combination of vaccine with TLR agonists was atleast as effective as combination with PD-1 blockade. This anti-tumor effect was not observed with TLR agonist treatment alone(Supplementary Fig. S4). As shown in Fig. 7B, immunizationwithTLR1/2 (Pam3CSK4) or TLR 7 (Gardiquimod) agonists elicited agreater number of CD8þ tumor-infiltrating lymphocytes (TIL).These CD8þ TILs had similar expression of 4-1BB, but reduced

Figure 4.

Changes in PD-1 expression following TLR stimulation are mediated by factors secreted from professional APC. A, Purified OT-1 T cells (T), B cells (B), or DC wereactivated with SIINFEKL peptide (OVA) or with a nonspecific peptide (NS) and in the presence of TLR1/2 (TLR1/2) agonist, TLR7 agonist (TLR7), or media alone.After 24 hours, cells were washed and cocultured with na€�ve purified OT-1 CD8þ T cells for an additional 24 hours and then assessed for the expression ofthe indicated surface receptors (4-1BB, PD-1, and LAG3) by flow cytometry. Shown is the MFI for each marker on CD8þ T cells, with samples assessed in triplicate.B, Purified DCs were stimulated in the presence of TLR1/2 agonist, TLR7 agonist, or media alone. After 24 hours, media were collected and used for cultureof purified OT-1 CD8þ T cells with na€�ve DCs in the presence of OVA or NS peptide. After 24 hours, CD8þ T cells were assessed for the expression of theindicated surface receptors (4-1BB, PD-1, and LAG3) by flow cytometry. Shown is the mean and standard error of the MFI for each marker, with samplesassessed in triplicate. Results shown are from one experiment, and are representative of two similar experiments. � , P < 0.01.






expression of PD-1 (Fig. 7C). Similar findings were observed in aseparate tumor model using a DNA vaccine. We have previouslyreported that aDNAvaccine encoding the SSX2antigen expressingepitopes with high affinity for HLA-A2 (SSX2 opt) elicited anti-gen-specific CD8þ T cells expressing higher PD-1 and an inferiorantitumor response (9). HLA-A2–expressing transgenicmicewereimplanted with SSX2-expressing tumor cells. After 2 days, micewere immunized weekly with control vector (pTVG4) or DNAencoding high-affinity HLA-A2 epitopes (pTVG-SSX2 opt) in thepresence or absence of Pam3CSK4 (TLR1/2) or Gardiquimod(TLR7) agonist. Although TLR1/2 or TLR7 agonists alone (withvector DNA) had modest effects on tumor growth, in combina-tion with antigen-specific DNA vaccine, they significantlyincreased antitumor efficacy (Fig. 7D).

DiscussionIn this article, we investigated whether expression of PD-1, and

other T-cell immune-checkpoint molecules, at the time of T-cell

activation can be reduced in the context of innate immunestimulation using TLR ligands.We found that specific TLR ligands,notably ligands for TLR1/2, TLR7, and TLR9, led to lower expres-sion of PD-1 on activated CD8þ T cells, with less effect on othercheckpoint molecules. This was mediated by professional anti-gen-presenting cells, but not through the direct interaction withCD80, CD86, or OX40L on APCs. Rather, IL12 release from APCsfollowing TLR stimulation was found to occur, and IL12 alonecould reduce expression of PD-1 on CD8þ T cells during activa-tion. In vivo use of TLR ligands at the time of T-cell activation withvaccines led to superior antitumor immunity in two differentmurine tumor systems. TLR agonists have been previouslyexplored as vaccine adjuvants. Our findings demonstrate that atleast onemechanismbywhich they can act as adjuvants is by theireffects on PD-1 expression on vaccine-elicited CD8þ T cells. Ourresults also show that PD-1 expression can be separated fromT-cell activation, a finding that has implications for T-cell thera-pies for cancer. Finally, gene-expression results suggested that TLRstimulation may modulate development of effector and memory

Figure 5.

Changes in PD-1 expression following TLR stimulationcan be mediated by IL12: A, Purified DCs werestimulated in the presence of TLR1/2 agonist(Pam3CSK4), TLR3 agonist (HMW PolyI:C), TLR4agonist (MPLA), TLR7 agonist (Gardiquimod), TLR 7/8 agonist (R848, resiquimod), TLR9 agonist (ODN1826), or media only (no TLR) for 24 hours. Culturesupernatant was then evaluated for the presence ofIL12 p70 by ELISA. � , significant (P < 0.05) IL12 releasecompared with media alone. B, Purified DCs werestimulated in the presence of TLR1/2 agonist, TLR7agonist, or media alone. After 24 hours, media werecollected and used for culture of purified OT-1 CD8þ

T cells with na€�ve DCs in the presence of SIINFEKLpeptide (OVA) and, where indicated, 1.0 ng/mLrecombinant IL12 or 0.06 mg/mL anti-IL12 blockingantibody. After 24 hours, CD8þ T cells were assessedfor the expression of the indicated surface receptors(4-1BB or PD-1) by flow cytometry. Shown is meanand standard error of the MFI for each marker, withsamples assessed in triplicate, and are representativeof two independent experiments. � , P < 0.01. C,Purified OT-1 CD8þ T cells were stimulated withanti-CD3– and anti-CD28–coated latex beads and inthe presence or absence of recombinant IL12, orleft untreated (NS). After 24 hours, cells wereassessed for the expression of Th1 cytokines or PD-1by flow cytometry. Results shown are from oneexperiment and are representative of two similarexperiments.

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function in CD8þ T cells, leading to more effective antitumorresponses.

Many investigators have evaluated TLR agonists as vaccineadjuvants. Indeed, CpG-richDNAanddouble-strandedRNAwerefound to promote Th1-biased immune responses and were sug-gested to be part of the mechanism of plasmid DNA immuniza-tion (27). This effect was mediated by activation of TLR9. TLR9agonists continue to be explored as antitumor therapies alone orin combination with other therapies in clinical trials (28). TLR9-deficient mice have impaired priming of CD8þ T cells (29).Similarly, TLR9 activation using CpG-rich oligonucleotides hadan adjuvant effect promoting Th1-biased immunity, augmentedTh1 memory responses, and induced protective immunity wheninjected into tumors (30–32). TLR9 agonists can stimulate anti-gen-presenting cell function and promote the lytic activity of NKcells (33). The specific effects of TLR9 agonists on CD8þ T cellshave not been entirely elucidated, although TLR9 activation couldlead to a decrease in PD-1 expression on activated CD8þ T cellsin vitro (27).Ourfindings support and expandupon thesefindingswith other TLR agonists and further demonstrate that changes inT-cell checkpoint molecule expression are specific for PD-1 com-pared with other immunomodulatory molecules expressed withT-cell activation.

The effect of TLR1/2 activation on T-cell checkpoint expressionhas not been specifically explored, although TLR2 activation hasbeen evaluated as a means of circumventing T-cell exhaustion(34): with na€�ve T cells stimulated with anti-CD3 and anti-CD28,stimulation in the presence of the TLR1/2 agonist Pam2Cys led topersistent expression of Th1 cytokines and decreased expression

of PD-1 and LAG-3. These results are consistent with our findingthat TLR1/2 activation at the time of T-cell activation led to adecrease in PD-1 expression; however, we did not observe changesin LAG-3 expression.

TLR7 agonists have been explored as antitumor treatmentsalone, and as vaccine adjuvants. In particular, imiquimod is atopical TLR7 agonist approved for the treatment of basal cellcarcinoma (35). Many studies have used imiquimod and otherTLR7 agonists as vaccine, and specifically antitumor vaccine,adjuvants (36, 37). Although TLR7 activation has been demon-strated to increase the activity of tumor-specific CD8þ T cells, thismechanism of action has not been explored. However, consistentwith our findings, TLR7 knockoutmice infected with lymphocyticchoriomeningitis virus (LCMV) developed LCMV-specific CD8þ

T cells that expressed higher PD-1 than similarly treated wild-typemice (38).

Our results provide evidence that PD-1 expression can beseparated fromCD8þT-cell activation. In the context of antitumorimmunity, this can have important consequences, because wehave demonstrated that even a short-term increase in PD-1expression following T-cell activation by vaccination can impairantitumor immunity (8). We demonstrate here that using specificTLR stimulation to decrease PD-1 expression following CD8þ

T-cell activation with vaccines can lead to antitumor responses atleast as effective as those following use of vaccines combinedwithPD-1 blockade directly. Our results further suggest that PD-1expression on CD8þ T cells was affected at the time of activationwith TLR stimulation, and this was not altered in tumor-bearingmice; however, this needs to be confirmed in other tumor systems.

Figure 6.

TLR stimulation at the time of CD8þ T-cell activation reduces the expression of PD-1 in vivo. A,OT-1 T cells were adoptively transferred to na€�ve C57BL/6 mice and 1day later immunized once with 100 mg OVA peptide (or nonspecific peptide, NS) in the presence or absence of TLR 1/2, TLR7, or TLR9 ligands. CD8þ T cells werecollected from the spleens of mice up to 14 days after immunization and evaluated for expression of 4-1BB, PD-1, and LAG-3 by flow cytometry. Shownare the MFI of 4-1BB, PD-1, and LAG-3 on OT-1 CD8þ T cells collected. � , P < 0.01, with each comparison made to peptide alone, and color corresponding tothe treatment group compared. B, HLA-A2 transgenic (HHD-II-DR1) mice were immunized once with 100 mg FLQGISPKI (SSX2) peptide in the presence or absenceof TLR ligands, as above. Splenocytes were collected from groups up to 14 days after immunization, and the expression of 4-1BB, PD-1, and LAG-3 was evaluatedon CD3þCD4� CD8þSSX2 tetramerþ T cells by flow cytometry. Results are from one experiment, with N ¼ 5 mice/group, and are representative of twosimilar experiments.






Our studies focused on antigen-specific CD8þ T cells, and did notaddress PD-1 expression onCD4þ T cells. Insights into the impactof TLR or IL12 stimulation on activated antigen-specific CD4þ

T cells will benefit from further research.Our gene-expression analysis suggests that TLR1/2 and TLR7

stimulation activates similar T-cell transcriptional profiles; hence,their effects on PD-1 expression may be redundant. Our studiesdemonstrate that this reduction of PD-1 expression can be medi-

ated by IL12; however, other soluble factors released by APCsmayalso play a role. Although certain TLR agonists (notably TLR 7, butnot TLR3 or TLR4) can lead to IL12 production by DC, ourfindings here link IL2 production by DCs to expression of PD-1 on activated T cells (39–41). Our findings are consistent withobservations in which TLR9 activation led to decreased PD-1 onCD8þ T cells that was abrogated in IL12 receptor knockout mice(26). Likewise, treatment ofmice with IL12 led to increased CD8þ

Figure 7.

Immunization in the presence of TLR1/2 or TLR7 ligands, codelivered as vaccine adjuvants, elicits greater antitumor immunity in vivo: A,Ovalbumin-expressing E.G7cells were implanted in C57BL/6 mice and permitted to grow until tumors were palpable (�14 days). OT-1 T cells were then adoptively transferred and mice wereimmunized subcutaneously the following day with SIINFEKL (OVA) peptide alone, or in combination with TLR1/2 or TLR7 agonist, or with 100 mg anti–PD-1delivered the day following immunization. Shown are the tumor growth curves (median� standard error, n¼ 5 animals per group). Results are from one experimentand are representative of two independent experiments.B, Tumors obtained at necropsywere evaluated for the percentage of infiltratingCD8þT cells amongCD45þ

T cells. C, Tumor-infiltrating CD8þ T cells were evaluated for 4-1BB and PD-1 expression by flow cytometry. D, HLA-A2–expressing transgenic mice (HHD-II-DR1)were implanted with SSX-2-expressing tumor cells. After 2 days mice were immunized intradermally weekly with control vector (pTVG4) or DNA encodingSSX2 protein encoding epitopes with high affinity for HLA-A2 (pTVG-SSX2 opt) and delivered alone or codelivered with TLR1/2 or TLR7 agonist. Shown are thegrowth curves for each group (n ¼ 6 animals per group). � , P < 0.05. Results are representative of two independent experiments with 12 total mice per group.

Zahm et al.





T-cell effector function characterized by increased expression ofIFNg , T-bet, but lower expression of IL10 and EOMES (42),consistent with our gene-expression studies.

Our findings demonstrate that specific TLR stimulation canaffect the transcriptional profile of CD8þ effector T cells, poten-tially favoring the establishment of effector memory cells withhigh expression of T-bet and lower expression of EOMES. Theeffect of treatment on the establishment of T-cell memory will beevaluated in future studies. Nevertheless, our findings suggest thatTLR agonists might be used, alone or with other cytokines, toincrease the antitumor efficacy of CD8þ effector memory cellswith vaccination. Moreover, given the findings that TLR stimula-tion can lead to reduced expression of molecules associated withT-cell regulation, such as PD-1 and IL10, future studies shouldexplore whether combinations of TLR ligands, or TLR stimulationwith checkpoint blockade, can further improve CD8þ T-celleffector function and antitumor efficacy.

Disclosure of Potential Conflicts of InterestD.G.McNeel has ownership interest (including stock, patents, etc.) in and is a

consultant/advisory board member for Madison Vaccines, Inc. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: C.D. Zahm, V.T. Colluru, D.G. McNeelDevelopment of methodology: C.D. Zahm, V.T. Colluru, D.G. McNeelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.D. Zahm, V.T. ColluruAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.D. Zahm, S.J. McIlwain, I.M. Ong, D.G. McNeelWriting, review, and/or revision of the manuscript: C.D. Zahm, V.T. Colluru,I.M. Ong, D.G. McNeelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.D. Zahm, S.J. McIlwainStudy supervision: D.G. McNeel

AcknowledgmentsWe thank the UW Biotechnology Center and UWCCC Flow Cytometry

Core staff for support and technical assistance. This work was supported bythe Department of Defense Prostate Cancer Research ProgramW81XWH-15-1-0492 and NIH R01 CA219154 and T32 CA157322.

The costs of publication of this articlewere defrayed inpart 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 11, 2018; revised June 20, 2018; accepted August 30, 2018;published first September 10, 2018.

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2018;6:1364-1374. Published OnlineFirst September 10, 2018.Cancer Immunol Res Christopher D. Zahm, Viswa T. Colluru, Sean J. McIlwain, et al.

T Cells+on CD8TLR Stimulation during T-cell Activation Lowers PD-1 Expression

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TLR Stimulation during T-cell Activation Lowers PD-1 ...€¦ · Research Article TLR Stimulation during T-cell Activation Lowers PD-1 Expression on CD8þ T Cells Christopher D. Zahm1,Viswa