Human Cancer Biology

Immune Suppression in Premalignant RespiratoryPapillomas: Enriched Functional CD4þFoxp3þ RegulatoryT Cells and PD-1/PD-L1/L2 Expression

Lynda J. Hatam1,4, James A. DeVoti1, David W. Rosenthal1,2,4,5, Fung Lam1, Allan L. Abramson1,3,5,Bettie M. Steinberg1,2,3,5, and Vincent R. Bonagura1,2,4,5

AbstractPurpose: Respiratory papillomas, caused by human papillomaviruses types 6 and 11 (HPV6/11), are

premalignant lesions with potential formalignant conversion. The cytokine and chemokinemicromilieu of

papillomas is TH2-likewith amarked absence of IFN-g expression. To illuminatewhypatientswith recurrent

respiratory papillomatosis (RRP) fail to effectively control their disease, we further investigated the

suppressive cellular microenvironment in papillomas.

Experimental Design: CD4þCD25þCD127low/�Foxp3þ regulatory T cells (Treg) and

CD4þCD25�CD127low/�Foxp3� T cells within papillomas were characterized and isolated. Their suppres-

sor function was measured by inhibition of peripheral blood mononuclear cell (PBMC) proliferation.

Expression of PD-1, CD69, and Helios was identified on these T cells. PD-L1, PD-L2, CCL17, and CCL22

mRNA was also identified in papillomas by quantitative PCR.

Results: Functional Tregs were markedly enriched in papillomas and strongly inhibited anti-CD3 and

anti-CD28 antibody activated PBMC proliferation. The natural Treg marker Helios was reduced on Tregs

from papillomas, indicating that the majority of Tregs in papillomas are adaptive. The majority of the

papilloma-derived CD4þ T cells expressed the CD4þCD25�CD127low/�Foxp3�PD1þCD69þ phenotype

and failed to suppress PBMCproliferation, suggesting that they are chronically activated and exhausted. The

Treg-attracting chemokine CCL22was equally expressed by all laryngeal tissues examined.However, CCL17

was robustly expressed by papillomas compared with unaffected laryngeal tissues from RRP patients and

individuals without RRP. PD-L1 was elevated in papillomas compared with control laryngeal tissues.

Conclusions: Papilloma CD4þ T cells are enriched with functional Tregs, and the adaptive Helios� Treg

fractionwas increased within the TH2-like papillomamicromilieu. CD4þCD25�CD127low/�Foxp3� T-cells

failed to suppress PBMC proliferation and may be exhausted. The PD-1/PDL-1 pathway may represent an

additional immunosuppressive mechanism that contributes to defective HPV6/11 clearance in RRP. Clin

Cancer Res; 18(7); 1925–35. �2012 AACR.

IntroductionRecurrent respiratory papillomatosis (RRP) is character-

ized by the relentless recurrence of laryngeal papillomas

that are induced by human papillomavirus (HPV) types 6and 11. Laryngeal papillomas are premalignant lesionswithpotential for malignant conversion. This rare disease has anestimated incidence in the United States of 4.3/105 inchildren less than 14 years old, and of 1.8/105 in adults.The estimated cost of surgical treatment for RRP in theUnited States is greater than 100 million USD per year (1).

The strategic location of these lesions in the larynx andupper airway causes significantmorbidity and, on occasion,mortality. Patients with severe disease can require morethan 150 general surgeries under general anesthesia tomaintain a patent airway. The interval between surgicalinterventions can vary between patients ranging from 3weeks to several years. Laryngeal disease can spread to thelower respiratory tract, and on occasion RRP can progress tomalignancy (1, 2).

The persistent recurrence of HPV6/11 infection in theselesions highlights the failure of RRP patients to generate

Authors' Affiliations: 1The Feinstein Institute for Medical Research,2Elmezzi Graduate School for Molecular Medicine, North Shore-LIJ HealthSystem, Manhasset; 3Department of Otolaryngology, North Shore-LIJHealth System, New Hyde Park; 4Division of Allergy and Immunology,Department of Pediatrics, Steven and Alexandra CohenChildren'sMedicalCenter of New York, North Shore-LIJ Health System, Great Neck; and5Hofstra North Shore-LIJ School of Medicine, Hempstead, New York

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

Corresponding Author: Vincent R. Bonagura, The Feinstein Institute forMedical Research, 350 Community Drive, Room 1239, Manhasset, NY11030. Phone: 516-562-1185; Fax: 516-562-1077; E-mail: Bonagura@lij.edu

doi: 10.1158/1078-0432.CCR-11-2941

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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effective HPV-specific, innate and/or adaptive immuneresponses, although they show normal immune homeo-stasis, and respond normally to other infectious microbes(1). Thus, a better understanding of the HPV-specific, T-celldysfunction in RRP that supports chronic HPV infection oflaryngeal keratinocytes could provide a rationale to developnonsurgical strategies that would correct this defect.

Previously we described RRP as a TH2-like disease that ischaracterized by an imbalance in TH1-/TH2-like cytokineand chemokine expression (1–3). We identified a correla-tion between RRP disease severity and the expression ofselect class II MHC genes that likely regulate this imbalance(1–4) and predispose HPV6/11-infected individuals todevelop RRP (1, 4). Recently, we described differences inthe repertoire of killer cell immunoglobulin-like receptor(KIR) gene haplotypes in RRP as compared with controls(1). In addition, we also identified natural killer cell dys-function in RRP (1) and differential expression of TH2-like

polarizing immune response genes in papillomas com-pared with autologous unaffected laryngeal tissue (1, 5).

The objective of this study was to further characterize therepertoire of T-cell subpopulations within tissue-infiltratinglymphocytes (TIL) from respiratory papillomas that sup-port chronic HPV6/11 infection in these lesions.

In this article, we provide evidence for the first timethat there is an enrichment of functional CD3þCD4þ-CD25þFoxp3þCD127low/� regulatory T cells (Treg; ref. 6)that infiltrate HPV6/11 induced, respiratory papillomas.We also identified the two chemokines (CCL17 andCCL22) that recruit Tregs into tissues where they areexpressed (7). In addition, we show that the majority ofCD4þFoxp3� TILs (non-Tregs) in papillomas express asurface phenotype that is characteristic of exhaustedT cells (PD1þCD69þCD25�CD127�; refs. 8–10), suggest-ing that this major T-cell fraction is chronically activated,quiescent, and unable to produce effector cytokinesin response to these HPVs. Furthermore, we provideevidence that the PD-1/PD-L1 pathway (11) is likely activein RRP, given that papilloma-derived TILs express PD-1strongly, and that papilloma tissue robustly expresses PD-L1, compared with control-derived laryngeal tissues. Tak-en together, these mechanisms may contribute to theimmunosuppressive microenvironment in papillomasthat prevent HPV-specific, effector TH1-like T-cell functionin RRP.

Materials and MethodsSubjects and index of disease severity

Thirty-seven randomly selected patients with RRP withvarying disease severity were studied. Before enrollment, allpatients signed informed consent that was approved by theInstitutional Review Board at the North Shore-LIJ HealthSystem. Table 1 shows the demographics of all patients withRRP included in this study.

Heparinized blood and biopsies of autologous respira-tory papillomaswere obtained during surgical intervention.HPV typing was carried out as previously described in detail(3, 12). Disease severity is defined by a composite score thatrepresents the extent of disease and is inversely proportionalto the frequency of recurrence (1). The composite score wasused to define the overall severity for an individual patient.Three or more surgeries in the previous 12 months with a

Translational RelevanceRespiratory papillomas induced by human papilloma-

viruses 6 and 11 are premaligant lesions with potentialformalignant conversion and cause significantmorbidityand, on occasion, mortality. Standard therapy for recur-rent respiratory papillomatosis (RRP) is surgical extirpa-tion, which significantly impacts on lifestyle (>150 sur-geries per lifetime) and medical costs (>100 million USdollars yearly). The cytokine–chemokine micromilieu ofpapillomas is TH2-like with markedly reduced IFN-g .We further investigated the microenvironment in papil-lomas toward ultimately developing a nonsurgical,medical treatment for RRP. Our studies show for the firsttime that papillomas contain an enrichment of functionalCD4þ regulatory T cells (Treg), express Treg/TH2-liketropic chemokines (CCL17, CCL22), and contain T cellsthat are CD127 negative and express CD69 and PD-1, aphenotype of chronically activated and exhausted T cells.PD-L1, an inducer of effector T-cell dysfunction, is alsoexpressed in papillomas. These studies provide potentialnew targets for future medical therapies that can reversethe immunosuppressive micromilieu in papillomas byrestoring effective TH1-like anti-HPV responses.

Table 1. RRP patient demographics

Ethnicityb

Patients All Severity score Male gender Juvenile onset W B A

Mild/moderate 20 0.004–0.048 13 (65) 7 (35) 16 3 1Severea 17 0.061–0.685 11 (65) 7 (41) 16 1 0

NOTE: Values in parenthesis are expressed in percentage.aIncludes 3 patients with tracheal involvement.bW, White/Caucasian; B, Black/African-American; A, Asian.

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numeric score of 0.06 or more, or the extension of diseaseinto the trachea, was defined as severe. A severity score ofless than 0.06 was defined as mild/moderate disease (1).Severity scores for these study patients ranged from0.004 to0.685. Seventeen patients had mild/moderate RRP (score0.004–0.048), and 17 had severe disease (score 0.061–0.685).

Isolation of PBMCs and CD4þ T regulatory cells fromrespiratory papillomas and peripheral bloodPeripheral blood mononuclear cells (PBMC) were iso-

lated by Ficoll-Paque Plus gradient centrifugation (GEHealthcare). These PBMCs were stained for surface andintracellular markers, or used as responders in the in vitrosuppression assay described below. TILs were isolated fromfresh papilloma biopsies by mechanical dissociation, andthe resulting cell suspension was passed through a 70-micron mesh filter. Tregs were isolated from TILs using aCD4þCD25þ Regulatory T-cell Isolation Kit (Miltenyi Bio-tec), as recommended by the manufacturer.In addition to the isolation of cells by bead purification,

effector memory CD3þCD4þCD127low/�CD25� T cells(13)were isolated by cell sorting on afluorescence-activatedcell sorting (FACS) Aria IIu flow cytometer. Minced papil-loma tissue was filtered, washed, resuspended in sortingbuffer (0.25% bovine serum albumin/PBS with 1 mMEDTA) and surface stained with antibodies to CD3-PacificBlue, CD4-FITC, CD127-PerCP-Cy5.5, and CD25-PE.Aliquots of the sort purified T-cell subpopulation werecoculturedwith autologous PBMCs in the suppression assaydescribed below.

Antibody staining and flow cytometric analysesPBMCs and TILs were washed in PBS containing 2% FBS

and 0.1% sodium azide and surface stained for Tregs usingthe following combinations of directly conjugated anti-bodies: anti-CD4 PerCP, anti-CD25 FITC, and anti-CD127PE (BD Biosciences). After washing, cells were stainedfor intracellular FoxP3 (anti-FoxP3-APC, clone PCH101,eBioscience) using the manufacturer’s recommended pro-cedure. Stained and washed cell suspensions were resus-pended in 1% formaldehyde. Additional specimens werestained for surface expression with anti-PD-1 APC or PE-Cy7, anti-PD-L1 PE, anti-CD69 PE, (BD Biosciences), orintracellular anti-Helios PE, (Biolegend).Samples were analyzed on a FACSCalibur or FACSCanto

II flow cytometer (BD Biosciences). Single fluorochromestained cells or antibody capture beads were used to correctfor spectral overlap. FlowJo version 7.2.5 (TreeStar) wasused for compensation and data analysis. For determina-tion of Treg frequencies, CD4þ cells with low side scatterwere identified and then a forward versus side scatter gateapplied to isolate small lymphocytes and facilitate theexclusion of cellular debris and nonviable cells. Positivefluorescence staining was established using isotype andfluorescence minus one controls. Only biopsies that pro-ducedmore than1�103CD4þ events ondata analysiswere

included in the study of Treg frequency. Studies evaluatingPD-1, PD-L1, and Helios expression were stained as aboveand analyzed on the FACSCanto II using forward scatterarea versus forward scatter height gating to remove cellaggregates and Live/Dead Fixable Aqua Dead Cell Stain Kit(Invitrogen) to exclude nonviable cells.

Treg function measured by suppression of PBMCproliferation

Tregs isolated from the papillomas of 3 patients weretested for their ability to suppress autologous PBMC pro-liferation using a functional assay done in vitro. In short,5 � 103 PBMCs were cocultured with Tregs enriched frompapilloma TILs or with CD4þ cells isolated from auto-logous PBMCs and activated by 2 mg/mL of anti-CD3 and1 mg/mL anti-CD28 (Pharmingen) monoclonal anti-bodies. Cell suspensions were incubated for 6 days in96-well, round bottom plates in RPMI supplemented with10% bovine serum. One mCi of 3H-thymidine was addedto each well for the last 18 hours of incubation. Plates wereharvested as previously described (14). The proliferativeresponses were expressed as mean 3H-thymidine incorpo-rated counts per minute (CPM) of triplicate wells minusbackground.

mRNA expression of CCL17, CCL22, and programmedcell death ligands, PD-L1 and PD-L2

To determine whether papillomas expressed CCL17and CCL22, known to be chemoattractants for Tregs (7),or expressed PD-L1 (CD274) and/or PD-L2 (CD273), theligands for PD-1 (CD279; ref. 11), we determined therelative expression of these genes by quantitative real-time reverse transcriptase PCR (qRT-PCR). Relative levelsof mRNA from papillomas (PAP, n ¼ 9), clinically normallaryngeal tissues from RRP patients (normal adjacent,NA, n ¼ 11), and controls without RRP (true normal, TN,n ¼ 6) were determined. Briefly, total RNA was isolatedusing nucleic acid affinity spin columns (Qiagen) togetherwith DNase-1 digestion. I-Script One-step RT-PCR forProbes (BioRad) was done on an Applied Biosystems7900HT thermocycler with gene-specific, intron-spanning,primers and either the appropriate FAM-labeled probefrom theUniversal Probe Library (Roche) or a FAM/TAMRAcustom TaqMan probe (Supplementary Table). The house-keeping gene glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was used to normalize the input RNA. mRNAexpressionofCCL17, normalized toGAPDH, was calculatedrelative to the mean NA tissue, using the DDCT method (5).Similarly, mRNA expression of PD-L1 and PD-L2, normal-ized toGAPDH, was calculated relative to TN PD-L1 expres-sion. The expression efficiency of the PD-L1 and PD-L2probe/primer sets were linearly efficient among the rangeof cycles measured in this assay, allowing for cross-primerrelative expression calculations. Data from each group hadsimilar SDs, confirmed by the method of Bartlett, and wereGaussian distributed, confirmed by the method of Kolmo-gorov and Smirnov. Therefore ANOVA was used to analyzethe 3 groups (TN, NA, and PAP), and a Turkey–Kramer

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multiple comparison test and 2-tailed unpaired t tests weredone to compare between 2 groups.

Statistical analysisAll statistical analysis employed a 2-tailed, unpaired t test

done by InStat (GraphPad). When the data was not nor-mally distributed, it was log-transformed, and if the SDwasdifferent between groups, a Welch correction was applied.

ResultsThe frequency of Foxp3þCD127low/� Tregs areincreased in CD4þ T cells from papillomas comparedwith blood of patients with RRP

The flow cytometric strategy for identifying Tregs withinTILs from a respiratory papilloma, and from the blood ofthe same patient, is shown in Fig. 1. CD4þ Tregs wereidentified by their expression of Foxp3þCD127low/�,CD25þCD127low/�, or the coexpression of both Foxp3 andCD25 on CD4þ T cells (Fig. 1A–E). The fraction of Tregsidentified by these surface markers in TILs from this pap-illoma was 3-fold greater than in the corresponding CD4þ

T-cell fraction from the autologous PBMCs (Fig. 1C and D,top vs. bottom rows). Some Foxp3þCD127low/� CD4þ Tcells in the papilloma were negative, or showed reducedexpression of CD25, in contrast to the strong CD25 coex-pression by the majority of Foxp3þCD127low/�CD4þ cellsin PBMCs (Fig. 1E).

Figure 2A shows the composite results of 17 patientswith RRP. Foxp3þ CD127low/� Tregs were increased inCD4þ TILs obtained from respiratory papillomas (25.0%� 8.0% of CD4þ T cells), compared with CD4þ T cellsfrom autologous PBMCs (7.3% � 1.8% of CD4þ T cells,P < 0.0001). Similar frequencies were observed for the

CD25þCD127low/� subset of CD4þ cells in the TILs com-pared with PBMCs (19.5% � 6.7% and 7.8% � 1.8%,respectively, P < 0.0001). Tregs were increased in thetissue compared with blood of all patients studied, asshown by a tissue-to-blood ratio that ranged from 1.7 to6.9 (mean � SD ¼ 3.5 � 1.4). The frequency of Foxp3þ

Tregs in CD4þ TILs from papillomas obtained from 5patients with mild/moderate disease versus 12 patientswith severe disease (27% � 11% vs. 24% � 7%, respec-tively) did not predict disease severity. In addition, theFoxp3þ CD127low/� CD4þ cell fraction with reducedexpression of CD25 (Fig. 2B) was prominent in TILs, butonly a minor population in blood (46.7% � 13.9%and 13.4% � 5.4% of Foxp3þ CD4þ T cells, respectively(P < 0.0001), consistent with that reported in the blood ofcontrols without RRP (15, 16).

The ratio of natural versus adaptive Tregs is altered inpapillomas

The transcription factor Helios has recently beenreported to distinguish nTregs from extrathymicallyderived adaptive Tregs (aTregs) and is expressed on70% (on average) of Foxp3þ Tregs in human controls(17). Therefore we analyzed both the TILs and PBMCs of6 patients for Helios expression on CD3þCD4þ Foxp3þ

cells. The frequency of Tregs with bright Helios expressionwas significantly reduced in papilloma TILs comparedwith blood (31.2% � 18.51% TILs, vs. 66.1% � 6.5%blood, P < 0.005; Fig. 2C and D). Concomitantly, therewas a significant increase in the Helioslow/� Tregs sub-population in papilloma TILs, suggesting that within thepapilloma microenvironment, there are more aTregs thanin the blood.

Figure 1. Flow cytometric identification of Tregs in papilloma TILs and peripheral blood. PBMCs and papilloma tissue were stained and analyzed for surfaceexpression of CD4, CD25, CD127, and intracellular Foxp3. A representative staining of PBMCs (top row) and TILs (bottom row) from a patient withRRP is shown. A, the ungated events (all cells) from these specimens; B, forward and side scatter characteristics of CD4þ gated cells; C–E, CD25, CD127,and Foxp3 expression of CD4þ lymphocytes. A gate is drawn around CD4þ cells which are CD25þCD127low/� (C) and the Foxp3þCD127low/� (D); E, thecorrelation of CD25þ and Foxp3þ on CD4þ T cells. The percentages of CD4þ cells are indicated for each subset.

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Tregs isolated from papilloma TILs function bysuppressing PBMC proliferationThe functional capacity of Tregs isolated from TILs was

determined using an in vitro micro coculture suppressionassay (Fig. 2E). CD25þCD4þ cells, enriched by magneticbead selection from the papillomas of patients with RRP,were cocultured with anti-CD3 and anti-CD28 activated,autologous PBMCs. There was marked suppression ofPBMC proliferation (66% � 11.8%) when autologousTregs, enriched from TILs, were added. In contrast, whenautologous, additional blood-derived CD4þ T cells wereadded back as a control to similarly activated PBMCs,proliferation was enhanced. The mean � SD suppressionratios were calculated by dividing the CPM of PBMCsalone (black bars), by the CPM of PBMCs with added TILs(light grey bars), or by the CPM of PBMCs with addedautologous CD4 cells (grey bars; Fig. 3E). Using theseratios, the TILs markedly suppressed PBMC proliferation

(�3.3 �1.3), whereas the autologous CD4 cells did not(1.5 � 0.4; P ¼ 0.0033).

Treg-attracting chemokine CCL17 is differentiallyexpressed by papillomas

To determine whether the increase of Tregs in papil-lomas was the result of the expression of chemokinesknown to attract Tregs into tissues (18), we comparedCCL17 and CCL22 mRNA expression in 9 papillomas(PAP), 11 clinically normal laryngeal tissues from RRPpatients (NA), and 6 control laryngeal tissues (TN) frompatients without RRP (Fig. 3). All 3 types of tissuesexpressed equivalent amounts of CCL22 mRNA (data notshown). In contrast, the expression of CCL17 mRNA wasupregulated in papillomas, compared with both types ofcontrol laryngeal tissues (Fig. 3). Thus, Tregs may berecruited to papillomas by CCR4 receptor engagement ofan increasing gradient of CCL17 that is differentially

Figure 2. Characterization of suppressive Tregs isolated from the blood and tissue of patients with RRP. A, the frequency of Foxp3þCD127low/� andCD25þCD127low/� cells in the CD4þ T-cell populations from papilloma tissues (TILs; black bars, 25.0% � 8.0% and 19.5% � 6.7%, respectively), andautologous blood (grey bars, 7.3%� 1.8%and 7.8%� 1.8%, respectively) was determined by flow cytometry. Both populationswere significantly increasedin the tissue when compared with autologous blood (n¼ 17, P < 0.0001, unpaired t test with Welch correction). B, percent of CD4þFoxp3þCD127low/� Tregswith reduced CD25 levels was 46.9%� 13.5% in papilloma tissues (TILs) and 13.4%� 5.5% in the blood of patients with RRP (n¼ 17, P < 0.0001, unpaired ttest with Welch correction). C and D, Helios expression on CD4þ T cells isolated from laryngeal papilloma tissue and autologous blood. C, the compositeresults of 6 patients with RRP. Bright expression of Helios was detected on 66.1% � 6.5% of Tregs isolated from the blood and on 31.2% � 18.5% ofpapilloma-derived Tregs, P < 0.005, 2-tailed t test. D, representative example of Helios expression on CD4þFoxp3þCD127low/� T cells. High expression ofHelios was detected on 61.47% and 38.93% of Tregs isolated from blood and tissue, respectively. E, Tregs enriched from papilloma-derived TILs suppressPBMC proliferation. PBMCs (black bars) were cultured alone or with Tregs isolated from papilloma tissue (light grey bars), or with autologous CD4þ cellsisolated from PBMCs (grey bars). Suppression of PBMCs proliferation by Tregs was observed in all 3 patients studied (76%, 53%, and 69%, respectively,light grey bars), whereas control PBMC cultures with added autologous CD4þ T cells showed an anticipated increase in proliferation (grey bars).

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expressed by papillomas, together with normal constitu-tive robust expression of CCL22.

CD4þ "non-Tregs" in TILs from papillomas showreduced CD127 expression and lack suppressorfunction

We observed that the majority of the CD4þ T cells in TILsfrom papillomas were CD4þFoxp3�CD25� (non-Tregs)and had low or absent expression of CD127 (49.2% �7.4%; Fig. 4). In comparison, the majority of CD4þ non-Tregs in thebloodwereCD127þ, andonlyaminorsubgroupof these cells lacked CD127 (22.2% � 5.4%, P < 0.001).CD127 is constitutively expressed on naive T cells, isdownregulated upon T-cell activation, and then reex-pressed on a subset of effector T cells that ultimatelydifferentiate into memory T cells (19). The absence ofCD127 expression on T cells has been associated with T-cell exhaustion in patients with chronic infection ortumors (8, 19), whereas absent CD127, Foxp3, and CD25expression is also a characteristic of effector Tr1-like cells(20, 21). Therefore we asked whether this major T-cellsubpopulation in TILs (CD4þCD25low/�CD127low/�)could also suppress PBMC proliferation. This purified

Figure 3. Differential CCL-17 mRNA expression in laryngeal tissues.Total mRNA from laryngeal tissue obtained from true normaltissues (TN), clinically normal adjacent laryngeal tissue (NA) frompatients with RRP, and from respiratory papillomas (PAP) werecompared using real-time RT-PCR, and expressed as a relativemRNA concentration (DDCt), after being normalized to ahousekeeping gene (GAPDH), and also standardized to NA tissue.Papilloma (PAP) CCL-17 mRNA expression was increased betweengroups (P < 0.02, ANOVA) and between NA and PAP (P ¼ 0.01,t test).

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population in TILs (non-Tregs) havereduced expression of CD127.Papilloma tissues and PBMCswere stained for CD4, CD25,CD127, and Foxp3 and analyzedby flowcytometry. A representativeanalysis of the expression of Foxp3and CD127 on the CD4þ cells frompapilloma tissue (A) and blood (B)from a patient with RRP is shown.Foxp3� "non-Tregs" arerepresented in the lower 2quadrants. C, the CD127expression of CD4þ "non-Tregs"(lower 2 quadrants) in papillomatissue (dashed line) and autologousblood (solid line). D, frequency ofCD127low/� cells within theCD4þFoxp3� "non-Tregs" fromthe blood (22.2% � 5.4%) and thetissue (49.2% � 7.4%) of 17patients (P � 0.0001, 2-tailed ttest). Expression of CD127 greaterthan that of Foxp3þ cells wasconsidered positive.

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cell fraction from TILs was cocultured with anti-CD3 andanti-CD28 antibody activated autologous PBMCs todetermine whether they could suppress PBMC prolifera-tion. CD4þCD25low/�CD127low/� T cells from papillo-ma-derived TILs from 2 different RRP patients did notsuppress PBMC proliferation (data not shown). However,in a parallel experiment, CD4þCD25low/�CD127low/�

T cells from the blood of a control without RRP showed40% suppression of autologous PBMC proliferation.Thus, CD4þCD25low/�CD127low/� T cells in control bloodcontain a subpopulation of functional Tr1-like effectorcells, as described (6, 20, 21), whereas T cells with thisphenotype in TILs from papillomas do not contain suffi-cient Tr1-like cells to show suppression in vitro.

PD-1 and CD69 are highly expressed on TILs frompapillomasTo determine whether the PD-1/PD-L1/L2 pathway was

relevant in the immunosuppressive micromilieu present inpapillomas, TILs from7patientswere stainedwith anti–PD-1 antibodies and analyzed by flow cytometry. PD-1 wasexpressed on 85%� 7% of the CD3þ T cells and on 92%�4% of CD4þ T cells from these papillomas (Fig. 5A). Inaddition, the intensity of expression of PD-1 on TILs wasmuch greater than that observed on T cells from the blood.The majority of TILs also expressed CD69, another markerof activation (9). These results suggested that the CD4þ

effector T cells within papilloma TILs are chronically acti-vated as continuous engagement of the TCR is necessary formaintaining a high PD-1 expression (9). The majority ofTILs did not express CD25 and therefore they may be in anexhausted state with reduced capacity to proliferate andproduce cytokines, as has been shown for CD8þPD-1þ Tcells during chronic infection (10). Thus, in addition to thedecreased CD127 expression that was observed on themajority of CD4þ TILs, the papilloma microenvironmentalso contributes to effector dysfunction by upregulating PD-1 expression on T cells.

PD-L1 and PD-L2 are expressed by papillomasTo determine whether the engagement of PD-1 that is

highly expressed on the majority of CD3þ TILs couldrepresent a mechanism that induces T-cell exhaustion inpapillomas, we analyzed PD-L1 and PD-L2mRNA extractedfrom papilloma tissue, clinically normal laryngeal tissue,and control laryngeal tissues from individuals without RRP.PD-L1 mRNA in papillomas (PAP, n ¼ 9) was relativelyexpressed 145 times more than laryngeal tissues from indi-viduals without RRP (TN, n ¼ 6) and 93 times more thanclinically normal laryngeal tissues fromRRP patients (NA, n¼ 11; P < 0.0001 ANOVA and P < 0.0001 unpaired 2-tailed ttest for TN vs. NA, and TN vs. PAP; Fig. 5B). In contrast, all 3types of laryngeal tissues expressed PD-L2 mRNA equiva-lently and at relative expression levels of 15 to 32 times thatof PD-L1 in TNs. Thus, PD-1 ligation on CD3þ TILs by PD-L1 expressed by papillomas may represent an additionalinhibitory pathway that blocks effector T-cell function inthese lesions as previously reported in other systems (8).

DiscussionThere has been intense interest in the role of Tregs and

their respective subpopulations in bothmurine and humandisease. Increased Treg numbers and function have been

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Figure 5. Papilloma TILs have high expression of CD69, PD-1, and PD-L1.A, TILs and autologous PBMCs were stained with antibodies to CD3,CD4, and CD69 (row 1), or CD3, CD4, CD127, Foxp3, and PD-1 (rows 2and 3). Representative samples from 2 patients with RRP (row 1, patient1; rows 2–3, patient 2) are shown. Gating was done using CD3þ T cells(rows 1 and 2) and CD3þ4þ T-cells (row 3). B, total mRNA normallaryngeal tissue from individualswithout RRP (TN; n¼6), clinically normaladjacent laryngeal tissue from patients with RRP (NA; n ¼ 11), and fromrespiratory papillomas (PAP; n ¼ 9) were compared using real-time RT-PCR. Relative mRNA expression (DDCt), was normalized to GAPDHmRNA, a housekeeping gene, and standardized to PD-L1 TN. PD-L1wasupregulated in both NA and PAP compared with TN (P < 0.0001, ANOVAand post hoc t tests). There was no significant difference in PD-L2mRNAlevels between the 3 different laryngeal tissues.

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linked to chronic bacterial, parasitic, and viral (HIV, HCV,and EBV) infection, whereas a low frequency and/orreduced Treg function have been linked to the resolutionof acute infection and the reestablishment of tumor immu-nity in various cancers, as recently reviewed (22). Mostrelevant to our studies are recent descriptions of Tregs inchronic HPV infection of the human cervix, specificallycervical intraepithelial neoplasia and cervical cancer causedby HPV16 and 18. These studies showed that the frequencyof Tregs increased with HPV-induced cervical disease(23, 24).

In this article, we provide evidence for the first time thatthere is an increased frequency of functional Foxp3þ

CD127low/� Tregs within the total CD4þ TIL populationin HPV6/11-induced respiratory papillomas. The frequen-cy of these Tregs within respiratory papillomas did notpredict disease severity, unlike that reported in cervicalneoplasia (23, 24). Thus, an increased frequency of func-tional Tregs likely contributes to the chronicity of HPV-induced RRP, but may not greatly influence the severity ofthis disease.

The increased frequency of Tregs has been shown tocontribute to the suppression of infiltrating cytotoxic T cellsin human cancerous lesions allowing these tumors toescape immunosurveillance (7). Several types of CD4þ

Tregs have been described, including CD4þ natural Tregs(nTregs) that express the transcription factor Forkhead boxP3 (Foxp3) and the high-affinity interleukin (IL)-2 receptora-subunit (CD25), but fail to express the a-subunit of thehigh-affinity receptor for IL-7 (CD127). They are pheno-typically defined as CD4þ CD25þFoxp3þCD127low/� Tcells (6). nTregs develop within the thymus and exert theirsuppressive activity by several mechanisms as recentlyreviewed (25). Adaptive Tregs (aTregs) share the samephenotype as nTregs (26); however they may also showreduced or absent CD25 and Foxp3, as described in Tr1-likeCD4þ T cells (6, 20, 21). aTregs are generated in theperiphery from naive TH0-like T cells following antigenexposure and a TH2-like cytokine micromilieu, and theysuppress T-cell function by their production of immuno-suppressive cytokines such as IL-10 and TGFb (26).

Tregs may accumulate within papillomas either by inter-nal proliferation, as reported in other tissues (26), or bytheir continued ingress from the blood because they expressCCR4, and papillomas express the CCR4 ligands CCL17and CCL22, that would enable the retention of Tregs inthese tissues (7, 18). Although a detailed analysis of Tregtraffickingwas not the focus of our studies, it is interesting tospeculate from our data that the robust and comparableexpression of CCL22 by papillomas, clinically normalautologous laryngeal tissues, and laryngeal tissues fromcontrols without RRP is important in regulating upperairway homeostasis by the recruitment of anti-inflamma-tory Tregs into the airway (18). In contrast, recent observa-tions in breast and ovarian cancer showed that CCL22 wasdifferentially expressed by tumors compared with normaltissue (27, 28). Similar observations were also described inthenasalmucosaof patientswith allergic rhinitis, compared

with nonatopic controls (29). These differences suggest thatthe effect of CCL22 is site specific and may elicit beneficial(airway patency), or harmful outcomes (malignancy), byattracting Tregs and/or TH2-like T cells into different tissues.

Interestingly, we found CCL17 to be differentiallyexpressed by papillomas in RRP, compared with both,autologous laryngeal tissue and control laryngeal tissuefrom patients without RRP (Fig. 3). These results suggestthat active HPV infection in the larynx induces CCL17expression and likely supports the infiltration of bothTH2-like T cells (1–4) and Tregs into papillomas.

There are several mechanisms in respiratory papillomasthat could induce the generation and/or expansion of Tregs.We previously showed that IL-10 and TGFb are expressed inpapilloma lesions (1). The involvement of these cytokinesin generating, activating, and expanding Tregs from naiveCD4þ T cells that differentiate into Foxp3 expressing aTregfollowing engagement of their T-cell receptors has beendocumented (7, 30). Of interest, recent studies have shownthat prostaglandin E2 (PGE2) can induce Foxp3 expressionand enhance the suppressive activity of CD4þCD25þ Tregsand that these responses could be reversed by COX-2inhibition (31, 32). Respiratory papilloma and their sur-rounding uninfected laryngeal tissues from patients withRRP have elevated COX-2 expression (33). Thus, the laryn-geal micromilieu in RRP is a rich source of PGE2 that canfurther support Treg function.

Our studies also showed that there are a substantialnumber of Tregs in papilloma CD4þ TILs that have reducedor negative CD25 expression. These Tregs have beenreported to function similarly to their CD25high Treg coun-terparts, although less efficiently (6). In studies using FoxP3-GFP knock in mice, the expression of Foxp3-GFP correlatedwith suppressor activity irrespective of CD25 expression(34). It has also been suggested that CD4þFoxp3þCD25�

Tregs can show functional plasticity and can loose Foxp3expression, whereas CD4þFoxp3þCD25high T cells repre-sent a more stable Foxp3þ Treg population (35, 36). StableFoxp3 expression depends on the DNA methylation statusof the Treg-specific determining region of the Foxp3 gene(37). Demethylation of the Foxp3 locus and high expres-sion of the Ikaros transcription factor Helios have recentlybeen reported to be a specific "signature" of thymic-derivednTregs (37, 38). From our studies, the CD4þ Foxp3þ

CD127low/� T cells in papilloma TILs include bothHelioshigh, nTregs, and Helioslow/�, aTregs (Fig. 2C andD). Importantly, the frequency of Helioshigh Tregs in papil-lomas was lower than in the blood of the same RRP patient(Fig. 2C and D). This suggests that the papilloma microen-vironment is conducive to de novo generation of aTregs fromTH0-like T cells. Novel therapies aimed at destabilization ofFoxp3 expression on Tregs in papillomas may benefit RRPpatients by blocking Treg function and thereby restoringeffective TH1-like responses in these premalignant lesions.

Our studies also showed that a large population ofCD4þFoxp3� cells (non-Tregs) in papilloma TILs lacked,or had reduced CD127 and CD25 expression. Expression ofIL-7 and IL-2 receptors regulate naive and memory T-cell

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homeostasis, proliferation, and differentiation (39). Thefunction of CD3þCD4þCD25�CD127�Foxp3� T cells isvaried. This population can include Tr1-like T cells andpreviously activated, but exhausted, effector memory T cells(6, 8, 10, 20, 21). The expansionof this population has beenshown to be an index of dysfunction in CD4þ cells fromHIV-infected individuals (13) and was linked to enhanced,tumor-specific, CD4þ T-cell apoptosis (19). TheCD3þCD4þCD25�CD127� fraction isolated from papillo-ma TIL did not suppress autologous PBMC proliferation. Inaddition, in a single experiment, this TIL fraction failed toexpress IL-10, TGF-b, or IFN-g mRNA after anti-CD3 andanti-CD28 antibody stimulation (data not shown). Thus,we suspect that this TIL subpopulation contains chronicallyactivated T cells that express CD69þ and PD-1high andrepresent dysfunctional effector T cells, as described in otherdiseases (9, 19).Our observations that CD3þ T cells isolated from

papillomas expressed PD-1 brightly (Fig. 5A), but notPD-L1, identified by flow cytometry (data not shown),and that papillomas express both PD-1 ligands (PD-L1and PD-L2; Fig. 5B), suggest that the PD-1/PD-L1/L2pathway may also contribute to the immunosuppressivemilieu in papillomas. PD-L1 is expressed on a widevariety of tissues and tumors, and both PD-L1 and PD-L2 are expressed on professional antigen-presenting cells(11). High levels of PD-L1 expressed by tumor cells havebeen correlated with an unfavorable prognosis in cancer(40). PD-1 is a major regulator of CD8þ T-cell exhaustionand viral control in chronic infection and tumor progres-sion, and it has become an important potential thera-peutic target in these diseases (41, 42). However, thePD-1/PD-1L pathway in CD4þ effector T-cell and Tregfunction is more controversial, and the mechanisms thatregulate exhaustion in PD1þCD4þ and PD1þCD8þ T cellsmay differ between the these 2 cell types in adaptivecellular immunity (9, 19, 43–45). In addition, conflictingreports of the role of PD-1 on CD4þ Treg functionsuggested that PD-1–PD-L1 ligation on CD4þ Tregs sup-ported Treg stability and expansion (40, 46–48), whereasothers suggested that this pathway inhibited Treg expan-sion and suppressive function (49, 50). Further studiesare needed to determine how nTreg or aTreg stability,expansion, and function, are modulated by interactionsbetween PD-1 and its ligands in RRP and in varioustumors at different anatomic sites.

In our studies, CD4þ non-Treg T cells from HPV6/11-induced papillomas showed upregulated PD-1 expressionalong with expression or absence of other markers ofchronic activation and terminal differentiation(CD25�CD127�CD69þ). Continued interaction withPD-1 ligands within papillomas may therefore block effec-tor T-cell function and/or induce their apoptosis. As aconsequence, these T cells within papillomas are likelyunable to expand into polyfunctional T helper cells thatcould control HPV6/11 infection.

In summary, we show that the overwhelming majorityof CD4þ cells in papillomas are either actively involved inimmune suppression or are chronically stimulated andare likely exhausted effector T cells that fail to respond toHPV/tumor antigen exposure. Better understanding ofPD-1 expressing TILs, and the identification of the anti-gen(s) that induce T-cell exhaustion in papillomas, couldhelp develop novel strategies to reverse T-cell exhaustionand restore TH1-like function in RRP. Clinical trials usingPD-1/PD-L1 blockade in cancer and infectious diseasesshould be monitored closely for their applicability inRRP. Additional characterization of the interactive cycleof cells and antigens within papillomas that polarizeimmune responses toward HPV6/11 tolerance may pro-vide the rationale to develop novel immune-based ther-apies that can restore effective T-cell clearance of theseviruses.

Disclosure of Potential Conflicts of InterestV.R. Bonagura has received honoraria as consultant to Baxter and is a

consultant and is on the advisory board of CSL-Behring. A.L. Abramson andB.M. Steinberg have received commercial research grant from Pfizer, Inc. Theother authors disclosed no potential conflicts of interest.

AcknowledgmentsThe authors thank Virginia Mullooly, R.N. for coordinating the col-

lection of patient samples, the residents from the Department of Oto-laryngology, and the anesthesiologists who obtained blood from RRPpatients.

Grant SupportThis work was supported by a grant from the National Institute of Dental

& Craniofacial Research/NIH, grant R0 1DE017227 (to V.R. Bonagura).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 1, 2011; revised January 30, 2012; accepted February1, 2012; published OnlineFirst February 9, 2012.

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2012;18:1925-1935. Published OnlineFirst February 9, 2012.Clin Cancer Res   Lynda J. Hatam, James A. DeVoti, David W. Rosenthal, et al.   PD-1/PD-L1/L2 Expression

Regulatory T Cells and+Foxp3+Enriched Functional CD4Immune Suppression in Premalignant Respiratory Papillomas:

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