Research Article

CD25 Identifies a Subset of CD4þFoxP3� TIL ThatAre Exhausted Yet Prognostically Favorable inHuman Ovarian CancerRonald J.deLeeuw1,2,DavidR.Kroeger1, SaraE. Kost1,2, Pheh-PingChang1, JohnR.Webb1,2,and Brad H. Nelson1,2,3

Abstract

CD25, the alpha subunit of the IL2 receptor, is a canonicalmarker of regulatory T cells (Treg) and hence has been impli-cated in immune suppression in cancer. However, CD25 is alsorequired for optimal expansion and activity of effector T cells inperipheral tissues. Thus, we hypothesized that CD25, in addi-tion to demarcating Tregs, might identify effector T cells incancer. To investigate this possibility, we used multiparameterflow cytometry and IHC to analyze tumor-infiltrating lympho-cytes (TIL) in primary high-grade serous carcinomas, the mostcommon and fatal subtypeofovarian cancer.CD25was expressedprimarily by CD4þ TIL, with negligible expression by CD8þ TIL.In addition to conventional CD25þFoxP3þ Tregs, we identified asubset of CD25þFoxP3� T cells that comprised up to 13% ofCD4þ TIL. In tumors with CD8þ TIL, CD25þFoxP3� T cells

showed a strong positive association with patient survival(HR, 0.56; P ¼ 0.02), which exceeded the negative effect of Tregs(HR, 1.55; P ¼ 0.09). Among CD4þ TIL subsets, CD25þFoxP3�

cells expressed the highest levels of PD-1. Moreover, after in vitrostimulation, they failed to produce common T-helper cytokines(IFNg , TNFa, IL2, IL4, IL10, or IL17A), suggesting that they werefunctionally exhausted. In contrast, the more abundant CD25�

FoxP3� subset of CD4þ TIL expressed low levels of PD-1 andproduced T-helper 1 cytokines, yet conferred no prognostic ben-efit. Thus, CD25 identifies a subset of CD4þFoxP3� TIL that,despite being exhausted at diagnosis, have a strong, positiveassociation with patient survival and warrant consideration aseffector T cells for immunotherapy. Cancer Immunol Res; 3(3); 245–53.�2014 AACR.

IntroductionStudies over the past decade have highlighted the strong

influence of the immune system on the survival of patients withcancer. In particular, CD8þ tumor-infiltrating lymphocytes(CD8þ TIL) are associated with improved survival in virtuallyevery human solid cancer studied, including ovarian cancer (1, 2).Importantly, however, CD8þ TIL do not act in isolation but ratherthrough cooperative interactions with other immune cells (3).Thus, to enhance the beneficial effects of TIL, it is imperative tounderstand how different TIL subsets work together to mediatetumor immunity.

Gooden and colleagues (4) performed a meta-analysis of 52studies investigating the prognostic significance of TIL expressingthe T-cell markers CD3, CD8, CD4, and FoxP3. Intriguingly,CD3þ TIL showed a stronger prognostic effect than CD8þ TIL(pooled HRs of 0.58 and 0.71, respectively), suggesting that other

CD3þ TIL, in particular CD4þ T cells, might contribute to thefavorable prognosis. The prognostic influence of CD4þ TIL hasbeen difficult to assess directly as they include both effector andregulatory subsets (5). Furthermore, CD4 is also expressed bymacrophages in humans, thus complicating the histopathologicscoring (6). Owing to this complexity, CD4 as a standalonemarker has shown no association with patient survival in ovariancancer (7, 8).

CD4þ TIL have been divided into several distinct functionalsubsets based on cytokine secretion patterns and phenotypic mar-kers (5). For example, IFNg-producing CD4þ TIL (Th1 cells) havebeen identified in ovarian cancer (9), and Th1-like gene expressionsignatures have been reported as prognostically favorable in manycancers (1). More recently, IL17-producing CD4þ T cells (Th17cells) have been identified in several cancers (1). The prognosticeffect of Th17 cells appears to depend on tumor site, ranging fromunfavorable in colorectal cancer (10) to favorable in ovarian andgastric cancer (11, 12). CD4þ TIL with a regulatory phenotype(Treg) are alsoprevalent inhumancancers. RegulatoryT cells (Treg)are commonly defined by coexpression of the IL2 receptor (IL2R)alpha subunit (CD25) and the transcription factor FoxP3. Thesecells can inhibit tumor immunity by a variety of mechanisms,including the production of immunosuppressive cytokines, deple-tion of extracellular ATP, and inhibitory cell contacts (13). Accord-ingly, Tregs have been associated with poor prognosis in ovariancancer (8, 14–16) andmanyother cancer types (1, 17). Thus,CD4þ

TIL represent complexmixtures of T-cell subsets with both positiveand negative influences on tumor immunity.

Although IL2 can serve as a growth factor for virtually all T-cellsubsets, Tregs exhibit the most obvious IL2 dependency in vivo.

1Deeley Research Centre, BC Cancer Agency,Victoria, British Colum-bia, Canada. 2Department of Biochemistry and Microbiology, Univer-sity of Victoria, Victoria, British Columbia, Canada. 3Department ofMedical Genetics, University of British Columbia, Vancouver, BritishColumbia, Canada.

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

Corresponding Author: Brad H. Nelson, BC Cancer Agency, 2410 Lee Avenue,Victoria BCV8R 6V5, Canada. Phone: 250-519-5705; Fax: 250-519-2004; E-mail:bnelson@bccrc.ca

doi: 10.1158/2326-6066.CIR-14-0146

�2014 American Association for Cancer Research.

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This was revealed most strikingly when IL2- and IL2R-deficientmice were first generated. Rather than being immune compro-mised as many had expected, these animals develop a lethal,systemic autoimmune syndrome that is attributable to the lossof Tregs (18). Thus, CD25 serves not only as a marker of Tregsbut also as a component of an essential developmental andhomeostatic signaling pathway for these cells (18). According-ly, several strategies have been developed to deplete Tregs inpatients with cancer by targeting the IL2 pathway. For example,IL2 conjugated to diphtheria toxin has been used to depleteTregs in vivo (19, 20). Tregs can also be depleted with antibodiesto CD25 (21). Although these agents have been shown toreduce the number of circulating Tregs in patients with cancer,and improve responses to tumor-specific vaccines, this hasgenerally not resulted in major antitumor effects. Indeed, inthe settings of transplantation and autoimmunity, these agentshave proven efficacious at inhibiting T-cell responses ratherthan reversing immune suppression (22).

Consistent with this, a closer examination of IL2- or CD25-deficient mice has revealed an important role for IL2 in theexpansion of effector T cells in nonlymphoid tissues such as gutand lung epithelium (23, 24). Moreover, we found in a mousemodel of advanced ovarian cancer that IL2 signaling, thoughdispensable for the initial expansion ofCD8þ T cells, was essentialforCD8þT-cell responses in the tumor environment (25). Indeed,systemic IL2 infusion can have potent antitumor effects as amonotherapy and in the setting of adoptive immunotherapy(26). In ovarian cancer, intraperitoneal IL2 administrationyielded a 25% objective response rate in early-phase clinical trials(27, 28). Thus, the physiologic role of IL2 is highly context-dependent, and a better understanding of its role in the tumormicroenvironment is needed if we are to rationally manipulatethis signaling axis.

Toward this end, we evaluated the phenotype and prognosticsignificance of CD25þ TIL subsets in high-grade serous carcinoma(HGSC), the most common and lethal form of ovarian cancer. Inaccord with prior reports (8, 14–16), CD4þCD25þFoxP3þ Tregswere a prominent component of CD4þ TIL in many patients, andtheir presence trended toward decreased patient survival. Unex-pectedly, we also identified a CD4þCD25þFoxP3� TIL subset thathad a highly exhausted phenotype yet was strongly associatedwith patient survival. Our results reveal a potentially beneficialrole for CD4þCD25þFoxP3� T cells in tumor immunity andprovide new insights into immune modulatory strategies forHGSC and related malignancies.

Materials and MethodsPatient characteristics and biospecimens

All specimens and clinical data were obtained with eitherwritten informed consent or a formal waiver of consent underprotocols approved by the Research Ethics Board of the BritishColumbia Cancer Agency (BCCA) and the University of BritishColumbia (Vancouver, British Columbia, Canada). All patients inthis study were diagnosed with advanced stage HGSC. Survivalanalyseswere performedwith apreviously described retrospectivecohort comprised of 187HGSC cases (Table 1; refs. 7, 29). Briefly,a tissue microarray (TMA) with 0.6-mm cores was constructedfrom formalin-fixed paraffin-embedded tumor samples obtainedat the time of primary surgery from patients seen at the BC CancerAgency from 1984 to 2000 (OvCaRe Ovarian Tumour Bank).

Patients in this cohort were deemed optimally de-bulked, mean-ing they had no macroscopic residual disease.

Flow cytometry studies were performed with viable tumor andblood specimens collected at the time of primary surgery frompreviously untreated patients with HGSC admitted to BCCA from2007 to 2010. Tumor tissue samples were mechanically disag-gregated in RPMI media containing 0.5 mg/mL collagenase Type I(Sigma-Aldrich), 0.5 mg/mL collagenase Type IV (Sigma-Aldrich),0.25 mg/mL hyaluronidase (Sigma-Aldrich), and 0.1 mg/mLDNAse I (Sigma-Aldrich) and incubated for 12 hours at 4�C.Single-cell suspensions were prepared by passing digested tissuethrough a 100-mm filter. Cellular yield and viability were deter-minedbyflow cytometry, and aliquots of 1�107 cellswere frozenin RPMI media containing 50% FBS (Fisher Scientific) and 10%DMSO (Sigma-Aldrich).

Flow cytometryTumor-cell suspensions were thawed at 37�C followed by

washing and a 2-hour rest at 37�C in RPMI media (Life Technol-ogies) containing 10% FBS (Fisher Scientific), L-glutamine (LifeTechnologies), sodium pyruvate (Life Technologies), b-mercap-toethanol (Sigma-Aldrich), and HEPES (Life Technologies). Cellswere stained with fluorochrome-conjugated monoclonal antibo-dies to the following cell surface markers: CCR4, CCR6, CD3,CD4, CD8, CD25, CD127, CD357 (GITR), CTLA-4, CXCR3,CXCR5, LAG-3, OX40, PD-1, and TIM-3 (Supplementary TableS1). To analyze cytokine production, bulk tumor preparationswere stimulated with phorbol myristate acetate (PMA; 50 ng/mL;Sigma-Aldrich) and ionomycin (1 mmol/L; Sigma-Aldrich) in thepresence of GolgiStop (BD Biosciences) for 3 hours at 37�C. Cellswere then fixed and permeabilizedwith Perm/Fix solution (eBios-ciences) and stained with fluorochrome-conjugated monoclonalantibodies to IFNg , TNFa, IL2, IL4, and IL17 (SupplementaryTable S1). Analysis of FoxP3 and Helios was performed simulta-neously with intracellular cytokine staining. For analysis of T-bet,GATA-3, and RORgt, unstimulated bulk tumor preparations werestained using the Transcription Factor Buffer Set (BD Biosciences)according to the manufacturer's protocol. To generate positivecontrols for T-bet and GATA-3 staining, Th1 and Th2 lines wereproduced by stimulating healthy donor peripheral blood mono-nuclear cells (PBMC) for 5 days with anti-CD3 and anti-CD28 inthe presence of IL2 (30 U/mL) þ IL12 (20 ng/mL) or IL4 (10 mg/mL), respectively. Flow cytometry was performed using eight-channel Influx or FACSCalibur instruments (BDBiosciences), anddata were analyzed with FlowJo software v10.0.7 (TreeStar Inc.).

IHCMulticolor IHC was performed as previously described (30).

Briefly, TMAs were stained with pan-cytokeratin alone or thefollowing antibody combinations: (i) CD25, CD8, and FoxP3and (ii) CD3, CD8, and FoxP3 (Supplementary Table S1). Allslides were deparaffinized, treated with Diva Decloaker in aDecloaking Chamber for antigen retrieval, and then blocked withPeroxidased-1 and Background Sniper. All staining was per-formed at room temperature for one hour for the primary anti-bodies and 30 minutes for secondary amplification. For the pan-cytokeratin stain, the primary signal was amplified using theMACH-2 Mouse-AP polymer Kit and visualized with Warp Red(10minutes). The first antibody combination utilized anti-CD25(clone 4C9) and anti-CD8a (clone SP16) antibodies, theMACH-2

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polymer kit Double Stain 1, and the chromogens Warp Red andBetazoid DAB, respectively. Slides were denatured at 50�C for 45minutes to remove the primary and secondary antibodies (31).The anti-CD25 antibody (clone 4C9) was reapplied (to boost thesignal) together with the anti-FoxP3 antibody (clone SP97),followed by the secondary polymer Kit MACH-2 Double Stain1 and development with Warp Red and Vina Green, respectively.For the second antibody combination, we used the primaryantibodies anti-CD3 (clone SP7) and anti-CD8a (clone C8/144B), the secondary polymer kit MACH-2 Double stain 2, andWarp Red and Betazoid DAB, respectively. After denaturation, thesecond round of staining used anti-FoxP3 antibody (clone SP97),the secondary polymer MACH-2 Double Stain 2, and Vina Green.Where indicated, slides were counterstained with hematoxylin.Other than antibodies (Supplementary Table S1), all IHC reagentsand equipment were obtained from Biocare Medical.

Image analysis and scoringImages were captured using an Olympus BX53 microscope

equipped with a motorized stage (Quorum Technologies Inc.)and the Nuance multispectral imaging system (CRI). Pan-cyto-keratin–stained TMA cores were captured as a single image at200� magnification. For cores stained with the CD8/CD25/FoxP3 and CD3/CD8/FoxP3 combinations, each chromogen wasscanned at its optimal wavelength and 400� magnification.Images were then combined electronically using MetaMorphsoftware (Quorum Technologies Inc.). To aid the identificationof intraepithelial TIL (as opposed to stromal lymphocytes), cyto-keratin-positive tumor regions were overlaid on serial sectionsstained for TIL markers (Supplementary Fig. S1). To enumerateTIL, multispectral images were deconvoluted into individualcomponents using Nuance software. Using Metamorph software(Quorum Technologies Inc.), images containing three chromo-gens were re-created and used to visually count cells with theindicated phenotypes.

Cases were considered positive for a given TIL subset if at leastone cell was present per 0.6-mm core. The density of eachintraepithelial TIL subset was calculated by normalizingcell counts by epithelial area as defined by positive cytokeratinstaining on a neighboring tissue section. The mean number ofCD25�FoxP3� TIL was inferred for the cohort by subtractingthe mean number of CD8�CD25þFoxP3� T cells seen with theCD25/CD8/FoxP3 IHC combination from the mean numberof CD3þCD8�FoxP3� T cells seen with the CD3/CD8/FoxP3combination.

Statistical analysisAll statistical analyses were performed using GraphPad Prism

6.0. Survival analysis was performed using Kaplan–Meier plots

and log-rank tests. Pearson correlations were used to assessassociations between TIL subsets. The c2 analysis was used toassess the association of TIL subsets withMHC class II. P values ofless than 0.05 were considered statistically significant.

ResultsCD25 and FoxP3 define four subsets of CD4þ TIL

To assess which populations of TIL express CD25, we per-formed multiparameter flow cytometry on disaggregated tumorsamples from 12 patients with HGSC. CD25 was expressed by asmall but significant proportion of CD3þ TIL (mean, 12%; range,3%–20%) and was not found on any non–T (CD3�) cells. Themajority of CD25þ T cells were CD4þCD8� (mean, 84%; range,78%–91%), and a small proportionwereCD4�CD8þ (mean, 3%;range, 1%–7%; Fig. 1A and D). The remaining CD25þ cells wereCD4�CD8� or CD4þCD8þ. Among the CD4þ TIL, CD25 wasexpressed by an average of 20% of cells (range, 4%–28%; Fig. 1BandD). To investigatewhether thesewere Tregs, we stained for thetranscription factor FoxP3. Consistent with prior reports (8, 14–16), cells with a canonical Treg phenotype (CD25þFoxP3þ)constituted a significant fraction of CD4þ TIL (mean, 13%; range,3%–25%) and are hereafter referred to as Tregs. Notably, asubstantial fraction of CD25þ TIL did not express FoxP3 (mean,7%; range, 2%–13%; Fig. 1C and D). The remaining CD4þ TILwere CD25�FoxP3þ (mean, 7%) or CD25�FoxP3� (mean,74%; Fig. 1C and D). Among CD8þ TIL, only a small percentageexpressed CD25 (mean, <1%), FoxP3 (mean, 2%), or both(mean, <1%; data not shown). Similar T-cell subsets were foundin peripheral blood samples from healthy donors except that, inline with the findings of others (32), CD4þCD25þFoxP3� T cellswere rare and CD4þCD25�FoxP3þ T cells were absent (data notshown).

Prognostic significance of TIL subsetsTo investigate the prognostic significance of TIL subsets, a 187-

case HGSC TMA was stained with antibodies to CD8, CD25, andFoxP3 (Fig. 2A). Because CD4 is also expressed by macrophages(6), it is difficult to score CD4þ TIL directly. Instead, we assumedthat anyCD25þ and/or FoxP3þ cell that didnot expressCD8was aCD4þ T cell, an assumption that was justified by the flow cyto-metry results shown in Fig. 1. With this staining combination,we could directly visualize all CD8þ TIL subsets, as well as theCD25þFoxP3�, CD25þFoxP3þ, and CD25�FoxP3þ subsets ofCD4þ TIL. To infer the number of CD4þCD25�FoxP3� TIL, weemployed a second IHC combination involving antibodiesto CD3, CD8, and FoxP3. We determined the number of CD4þ

FoxP3� cells, which appeared as CD3þCD8�FoxP3� cells. Fromthis, we subtracted the number ofCD25þFoxP3� cells determinedfrom the first staining combination. This yielded an estimate ofthe number of CD4þCD25�FoxP3� cells. Our detection andscoring approach proved valid, as the results obtained by multi-color IHC and flow cytometry were concordant (Fig. 1D and E).We stained an adjacent section of the TMA for cytokeratin tounequivocally identify tumor epithelium versus stroma; for allsubsequent analyses, we focused on intraepithelial TIL, as thesehave the greatest prognostic significance (2).

A large proportion of cases (74%) scored positive for CD8þ TIL(Fig. 2B), and consistent with the flow cytometry data, only asmall number of CD8þ TIL expressed CD25 and/or FoxP3 (datanot shown). Over 90%of cases with CD8þ TIL also had CD4þ TIL

Table 1. Clinical characteristics of the retrospective HGSC cohort

N ¼ 187 Univariate P

Disease-specific survival (y), median 6.6Age (y), median (range) 60.0 (37.6–85.9) 0.16FIGO stageI 47 (25%) <0.0001II 76 (41%)III 64 (34%)

GradeG2 52 (28%) 0.55G3 135 (72%)

CD25 Identifies a Prognostically Favorable CD4þ TIL Subset

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(defined as CD3þCD8� cells); conversely, almost all cases withCD4þ TIL also hadCD8þ TIL (Fig. 2B). Thus, CD8þ andCD4þ TILwere strongly associated with one another. Tregs (defined asCD8�CD25þFoxP3þ cells) were the most prevalent subset ofCD4þ TIL, being present in 67% of cases (Fig. 2B). The CD25þ

FoxP3� andCD25�FoxP3þ subsets were present in 55% and 43%of cases, respectively (Fig. 2B). Thus, the different subsets of CD4þ

TIL were usually found together, although there were exceptions.All subsets of CD4þ TIL showed a strong association with MHCclass II expression by tumor cells (determined in a previous studyof this cohort; ref. 7; P < 0.01 for all subsets).

As expected, CD8þ TIL were strongly associated with disease-specific survival (HR, 0.33; P < 0.0001; not shown) and progres-sion-free survival (HR, 0.38; P < 0.0001; Fig. 3A). The strongprognostic effect of CD8þ TIL can confound the analysis of closelyassociated TIL subsets, such as Tregs (30). Therefore, to determinethe prognostic significance of CD4þ TIL subsets, we restricted our

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Figure 1.CD25 and FoxP3 define four subsets of CD4þ TIL. Multiparameter flowcytometry was performed on bulk disaggregated tumor samples from 12patients with HGSC, and data for one representative case are shown. A,CD25þ TIL predominantly consisted of CD3þCD4þ T cells. B, approximately20% of CD3þCD4þ TIL expressed CD25. C, within the population of CD3þ

CD4þ TIL, expression of CD25 and FoxP3 defined four subsets, indicated bythe quadrants. D, average proportions of the four CD4þ TIL subsets (� range)for the 12 cases analyzed by flow cytometry. E, average proportions of thefour CD4þTIL subsets (� SEM) for 181 evaluable cores of the TMA analyzed bymulticolor IHC. For flow cytometry experiments in plots A to D, events weregated on live cells with forward and side scatter characteristics oflymphocytes. The numbers shown indicate the percentage of events fallingwithin an indicated gate.

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Figure 2.Analysis of TIL subsets by multicolor IHC and spectral image analysis. A 187-case TMA was triple stained with antibodies to CD8, CD25, and FoxP3 andcounterstained with hematoxylin. A, brightfield (upper left plot) and pseudo-colored images from a representative tumor core showing examples of fourTIL subsets: (a) CD8�CD25þFoxP3þ, (b) CD8þCD25�FoxP3�, (c) CD8�

CD25�FoxP3þ, and (d) CD8�CD25þFoxP3�. Magnification, �400. B, Venndiagram showing the distribution of the five indicated TIL subsets, withnumbers indicating the percentage of cases scoring positive for each TILsubset.

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analysis to cases that were positive for CD8þ TIL. As expected,cases with a higher than median ratio of Tregs to CD8þ TILtrended toward decreased survival (HR, 1.55; P ¼ 0.09; Fig.3B). In contrast, a higher than median ratio of CD25þFoxP3�

TIL to CD8þ TIL was strongly associated with survival (HR,0.56; P ¼ 0.02; Fig. 3C). The prognostic effect of CD25þ

FoxP3� TIL was even more pronounced in cases with lowerlevels of Tregs (HR, 0.29; P ¼ 0.003; Fig. 3D). No otherCD4þ TIL subsets showed an association with survival(Fig. 3E and F). Thus, the CD25þFoxP3� subset was uniqueamong CD4þ TIL in showing a positive association withpatient survival.

CD25þFoxp3� T cells are phenotypically distinct fromother TILsubsets

To better understand the prognostic effect of CD25þFoxP3�

TIL, we used multicolor flow cytometry to assess their activationstatus and cytokine production profile in comparison with otherCD4þ TIL subsets (n¼ 6 cases; Fig. 4A). CD25þFoxP3� TIL had anactivated phenotype as evidenced by high CD69 and low CCR7expression (Supplementary Fig. S2B). Similar to Tregs, CD25þ

FoxP3� TIL expressed low levels of CD127 andmoderate levels ofGITR (Supplementary Fig. S2C). However, they expressed lowerlevels of CTLA-4 and OX40 than did Tregs and were negative forHelios (Fig. 4B). These data suggested that CD25þFoxP3� cellsmight be Th1 cells, which are widely reported among TIL (1).However, CD25þFoxP3� T cells expressed only low levels of theTh1 transcription factor T-bet (Supplementary Fig. S3B) and failedto produce any of the hallmark Th1 cytokines IFNg , TNFa, or IL2after in vitro stimulation with PMA and ionomycin (Fig. 4C).

Indeed, Th1 cytokines were only produced by the CD25�FoxP3�

subset (Fig. 4C). None of the CD4þ TIL subsets produced IL4 orIL17A (Supplementary Fig. S2D), and the expression of transcrip-tion factors GATA-3 (Th2) and RORgt (Th17) was restricted tominor subsets of CD4þCD25�FoxP3� cells (Supplementary Fig.S3D and S3F, and data not shown). All four CD4þ TIL subsetsexpressed CXCR3 (Supplementary Fig. S3C), which is typical ofT cells in inflammatory environments.

Given that CD25þFoxP3� T cells did not express canonical Thcytokines, we investigated the possibility that they might repre-sent other less common CD4þ T-cell phenotypes. CD4þ cytolyticT cells have been described in cancer (33); however, similar toother non-Tregs, very few CD25þFoxP3� T cells expressed thecytolytic markers TIA-1, granzyme B, or perforin (SupplementaryFig. S2E). Furthermore, CD25þFoxP3� TIL did not expressCXCR5, a marker of T follicular helper cells (SupplementaryFig. S2F).

On the basis of their lack of discernible functional attributes, wehypothesized that CD25þFoxP3� TIL might be in a suppressed orexhausted state. In accord with this, we found that CD25þFoxP3�

T cells expressed very high levels of the exhaustion marker PD-1(Fig. 4D). Indeed, the level of PD-1 expressed by CD25þFoxP3�

TILwas on average 3.1-fold higher than that by Tregs, and 6.6-foldhigher than that by CD25�FoxP3� cells. In addition, CD25þ

FoxP3� TIL expressed the exhaustion markers LAG-3 (Supple-mentary Fig. S2G) and TIM-3, the latter being found on cells withthe highest PD-1 levels (Fig. 4D). Thus, despite being prognos-tically favorable, CD25þFoxP3� TIL exhibited an exhausted phe-notype based on the expression of these markers and deficientcytokine production.

BA C

FEDCD25+ TIL combinations

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Low (n = 68)

HR: 1.03P = 0.73

Figure 3.Prognostic significance of TIL subsets. Kaplan–Meier plots showing the association between TIL subsets and progression-free survival. A, density of intraepithelialCD8þ TIL. B, ratio of CD25þFoxP3þ to CD8þ TIL. C, ratio of CD25�FoxP3þ to CD8þ TIL. D, ratio of CD25þFoxP3� to CD8þ TIL. E, ratio of CD4þ (i.e., CD3þCD8�)to CD8þ TIL. F, cases were stratified into two groups: above median CD25þFoxP3:CD8þ ratio and below median CD25þFoxP3þ:CD8þ ratio (n ¼ 26) versusbelow median CD25þFoxP3:CD8þ ratio and above median CD25þFoxP3þ:CD8þ ratio (n ¼ 26). In each plot, log-rank tests were used to determine P values,and HRs are shown for each analysis. For plots B to F, analyses were restricted to cases that were positive for CD8þ TIL.

CD25 Identifies a Prognostically Favorable CD4þ TIL Subset

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DiscussionUsingmultiparameter flow cytometry and IHC,we have shown

that CD25 and FoxP3 delineate four subsets of CD4þ TIL withdistinct functional and prognostic attributes. Consistent withprior reports (8, 14–16), CD25þFoxP3þ Tregs constituted asubstantial proportion of CD4þ TIL, expressed canonical Tregmarkers (Helios, CTLA-4, andOX40), and showed a trend towarddecreased patient survival. CD25�FoxP3� T cells constituted thelargest subset of CD4þ TIL and were the sole source of Th1cytokines; however, despite their abundance and functional com-petence, these cells conferred no apparent prognostic benefit.Instead, the CD25þFoxP3� subset showed a strong associationwith patient survival, despite expressing very high levels of PD-1and failing to produce cytokines. This paradoxical relationshipbetween functional status and prognosis might be explained bythe fact that PD-1 is amarker of tumor-reactive TIL (34, 35). Thus,the exhausted state of CD25þFoxP3� TIL observed here mightreflect ongoing recognition of tumor antigens. Although specu-lative, this state of exhaustion might subsequently be relieved bythe cytoreductive and immune stimulatory effects of surgery andchemotherapy (36), ultimately leading to increased patient sur-vival. Thus, CD25þFoxP3� TIL warrant further investigation fortheir contribution to spontaneous tumor immunity as well astheir potential to serve as effector cells for immunotherapy.

CD25þFoxP3� T cells have been largely overlooked in priorstudies of CD4þ TIL, likely because of their low abundance andfailure to make cytokines typical of Th1, Th2, Th17, or Tregs.Adding to their inconspicuous nature, these cells are very rare inPBMCs and hence would not be obvious in blood-based immuneanalyses. Nonetheless, cells with this phenotype are evident inpublished data of CD4þ TIL in ovarian (14, 37, 38) and other

tumors (39). Moreover, their prognostic significance has beendirectly assessed in one prior study of HGSC. Preston and col-leagues (8) used multicolor IHC to enumerate CD4þ

CD25þFoxP3� TIL in patients withHGSCwho had demonstratedlong (>18 months) versus short survival. Although these cellswere equally abundant in the two groups, the ratio of CD8þ toCD4þCD25þFoxP3� TILwasmodestly higher in the long survivalgroup (0.65 vs. 0.46), leading the authors to propose that CD25þ

FoxP3� TIL play an inhibitory role in tumor immunity. However,this interpretation could potentially reflect a confounding effect ofCD8þ TIL. Specifically, cases exhibiting a low ratio of CD8þ toCD25þFoxP3� TIL were undoubtedly enriched for tumors withfew or no CD8þ TIL, which itself is a poor prognostic factor (2).We attempted to correct for this effect by restricting our survivalanalysis to cases that were positive for CD8þ TIL, thereby allowingus to assess any additional prognostic effects contributed by thedifferent CD4þ TIL subsets. The validity of this approach wassupported by our finding, with few exceptions, that CD25þ

FoxP3� TIL were found only in cases with CD8þ TIL (Fig. 2B).With this correction, a strong positive prognostic effect of CD25þ

FoxP3þ TIL was revealed.To explain this positive effect, we propose that CD25þFoxP3�

TIL might be tumor-reactive T-helper cells that have becomefunctionally impaired or exhausted due to chronic antigen expo-sure. Indeed, their appearance on flow cytometric plots initiallysuggested to us that they might represent a CD25hi "shoulder" ofthe Th1-like CD25�FoxP3� subset. To address this, we assessedthe prognostic significance of CD3þCD8�FoxP3� TIL, a groupingthat included both the CD25�FoxP3� and CD25þFoxP3� sub-sets. CD3þCD8�FoxP3� TIL showed no prognostic significance,indicating that the positive effect of CD25þFoxP3� TIL was lostwhen combined with CD25�FoxP3� TIL (Fig. 3F). Although an

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Figure 4.Phenotype and functional characteristics of CD4þ TIL subsets. Multiparameter flow cytometry was used to assess expression of the indicated activation anddifferentiationmarkers and cytokines for four CD4þ T-cell subsets defined by CD25 and FoxP3 expression. A, contour plot showing four CD4þ TIL subsets and colorscheme used in other plots: CD25þFoxP3� (red), CD25þFoxP3þ (orange), CD25�FoxP3� (blue), CD25�FoxP3þ (green). B, expression of T-cell activation anddifferentiation markers by the four TIL subsets. C, cytokine production after 3 hours of stimulation with PMA and ionomycin. D, expression of exhaustion markers(note that the right plot shows only the CD25þFoxP3� and CD25�FoxP3� subsets). Data are shown for one representative case from a total of six.

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indirect assessment, this indicates that CD25�FoxP3� TIL havenegligible (or possibly even negative) prognostic significance andthat CD25þFoxP3� TIL make a unique positive contribution totumor immunity.

At first glance, the above findings may appear inconsistent withprior studies reporting that TIL with Th1, Th17, or other cytokinepatterns are prognostically favorable in ovarian (40) and othercancers (1). However, many of these prior studies were based onthe analysis of bulk tumor samples for expression of Th1-associ-ated genes, such as IFNg , TNFa, T-bet, IRF-1, and STAT4 (10, 40).Notably, these genes are also expressedbyCD8þ T cells andnaturalkiller cells; therefore, gene expression analysis of bulk tumor tissuedoes not reveal the prognostic influence of CD4þ Th1 cells per se.Using a flow cytometric approach, Kryczek and colleagues showedthat production of IFNg , IL2, TNFa, and IL17 by CD4þ TIL wasrestricted to CD25�, FoxP3�, and PD-1lo cells and that IL17 levelsin ascites fluid were associated with survival (37). In accord withtheir results, we found that cytokine production was restricted tothe CD25�/FoxP3�/PD-1lo subset; however, we failed to detectIL17 production by any CD4þ TIL subset. This might reflect a lowabundance of Th17 cells in ovarian cancer (as others have alsoreported; refs. 38, 41), or inadequate sensitivity of our flow cyto-metric assay for IL17 production. Finally, two studies have usedMHC class II tetramers specific for the tumor antigen NY-ESO-1 toisolate CD4þ T cells from peripheral blood (42) or tumors (9) ofpatients withovarian cancer.NY-ESO-1–specific T cellswere foundto produce Th1-associated cytokines after in vitro stimulation.However, the Th1 competence of these T cells may reflect the factthat theywere derived fromperipheral blood,which has negligibleCD25þFoxP3� T cells, or expanded in vitrobyphytohemagglutininstimulation, which can reverse T-cell exhaustion. Our observationthat CD25þFoxP3� TIL express CXCR3 and low levels of T-betsuggests they might represent exhausted Th1 cells. Indeed, othershave recently reported that T-bet expression is diminished indysfunctional effector cells (43). Alternatively, CD25þFoxP3� TILcould represent early activated Th1 cells that have not yet acquiredrobust T-bet expression or effector cytokine production, althoughone would not expect such cells to express multiple exhaustionmarkers. In summary, our data appear consistent with previousreports of Th1-likeTIL in cancer buthighlight the exhausted state ofthese cells at diagnosis.

Although CD25 is a well-established marker of Tregs, to ourknowledge, this is the first report of CD25 expression by T cellswith an exhausted phenotype. This observation may lead tofurther insights into the signaling state of CD25þFoxP3� TIL, asthe mechanisms of transcriptional regulation of the CD25 geneare relatively well characterized. The CD25 promoter has bothpositive and negative regulatory elements, which bind multiplefactors, including NFAT, NF-kB, STAT5, and SMAD3/4 (44).Transient CD25 expression is induced on virtually all T cells byactivation of the T-cell receptor (TCR) and the downstreamNFATand NF-kB pathways (44). CD25 can also be induced by a varietyof cytokines, including IL1, IL2, IL7, IL12, IL15, TNFa, and TGFb(44). In particular, IL2 induces CD25 expression via STAT5 (44)and in combination with extensive antigen stimulation inducesexpression of Blimp-1, which also upregulates CD25 expression(18). Finally, CD25 is expressed at high, constitutive levels byTregs through a combination of TCR signaling, IL2-inducedSTAT5 activation, and constitutive expression of FoxP3 (18). Inthe case of CD25þFoxP3� TIL, this FoxP3-dependent mechanismwould not apply. Rather, CD25 might be induced by antigen

stimulation in combination with IL2, a cytokine for which theywould be competitive consumers based on their CD25hi phe-notype. Another candidate is TGFb, which can induce CD25(44) and is present in most epithelial tumors. In this regard, it isnoteworthy that CD25þFoxP3� cells expressed high levels ofPD-1, which is also induced by TCR stimulation in combina-tion with TGFb (45). One paradoxical aspect of the phenotypeof CD25þFoxP3� cells is that they somehow maintain CD25expression despite high levels of PD-1, which normally bluntsTCR signaling through SHP-2–mediated dephosphorylation ofZAP70 (46). This suggests that CD25 might be relatively insen-sitive to PD-1–mediated inhibition, as described for other geneproducts (47).

The findings reported here have implications for the devel-opment of effective immunotherapies for HGSC and relatedcancers. First, our data indicate that strategies to deplete Tregsby targeting CD25 might have the detrimental effect of alsoeliminating CD25þFoxP3� T cells. Indeed, in renal transplan-tation and autoimmunity, the net effect of anti-CD25 antibodytherapy is immune suppression not activation (22). Second,our findings might help to explain the positive clinical effects ofIL2 in ovarian cancer, where a 25% response rate was achievedwith intraperitoneal administration of IL2 as a monotherapy(26). Third, our data have implications for checkpoint blockadestrategies. Because many CD25þFoxP3� TIL express high levelsof CTLA-4 (Fig. 4B), treatment with antibodies to CTLA-4 mightinadvertently deplete this TIL subset (48). On the other hand,our data support further investigation of PD-1 blockade inHGSC, given that CD25þFoxP3� TIL expressed the highestlevels of PD-1 compared with all other CD4þ TIL subsets (Fig.4D). That said, our data suggest that PD-1 might not be the onlyinhibitory mechanism affecting CD25þFoxP3� TIL, because thestimulatory agents we used (PMA and ionomycin) are expectedto bypass PD-1/SHP-2–mediated inhibition, yet failed toinduce cytokine production by these cells (Fig. 4C). Finally,our data reveal OX40 as a promising target for immune mod-ulation, because this molecule was highly expressed by Tregsbut not the other three CD4þ TIL subsets (Fig. 4B). Antibodiesto OX40 can directly stimulate effector T cells (39, 49), and theycan also enhance antitumor immunity by depleting OX40þ

Tregs (50). In summary, our study highlights the IL2, PD-1, andOX40 pathways as potential immunotherapeutic targets todifferentially enhance the positive effects of CD25þFoxP3� TILover the inhibitory effects of Tregs.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R.J. deLeeuw, D.R. Kroeger, S.E. Kost, B.H. NelsonDevelopment of methodology: R.J. deLeeuw, D.R. Kroeger, S.E. Kost, P.-P.ChangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.J. deLeeuw, D.R. Kroeger, S.E. Kost, P.-P. ChangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):R.J. deLeeuw,D.R. Kroeger, P.-P. Chang, J.R.Webb, B.H. NelsonWriting, review, and/or revisionof themanuscript:R.J. deLeeuw,D.R. Kroeger,S.E. Kost, P.-P. Chang, J.R. Webb, B.H. NelsonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.J. deLeeuw, B.H. NelsonStudy supervision: B.H. NelsonOther (designed and carried out experiments): P.-P. Chang

CD25 Identifies a Prognostically Favorable CD4þ TIL Subset

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AcknowledgmentsThe authors thank the donors and clinicians who provided specimens for

research, and Peter Watson and Katy Milne for technical advice.

Grant SupportThis study was supported by the Canadian Institutes of Health Research

(MOP97897 and a fellowship to R.J. deLeeuw), U.S. Department of Defense

(OC110435), BCCancer Foundation, and the BCCancer Agency Tumour TissueRepository, a member of the Canadian Tumour Repository Network.

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.

ReceivedAugust 7, 2014; revisedNovember 23, 2014; acceptedNovember 25,2014; published OnlineFirst December 5, 2014.

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2015;3:245-253. Published OnlineFirst December 5, 2014.Cancer Immunol Res   Ronald J. deLeeuw, David R. Kroeger, Sara E. Kost, et al.   Yet Prognostically Favorable in Human Ovarian Cancer

TIL That Are Exhausted−FoxP3+CD25 Identifies a Subset of CD4

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