effects of adjuvant chemoradiotherapy on the frequency and ... · cd4þ t-cell subsets share the...

Cancer Therapy: Clinical

Effects of Adjuvant Chemoradiotherapy on the Frequencyand Function of Regulatory T Cells in Patients with Head andNeck Cancer

Patrick J. Schuler1,2,6, Malgorzata Harasymczuk1,2, Bastian Schilling1,2, Zenichiro Saze1,2,7, Laura Strauss1,2,Stephan Lang6, Jonas T. Johnson1,2,5, and Theresa L. Whiteside1,2,3,4,5

AbstractPurpose: Regulatory T cells (Treg) accumulate in tumor tissues and the peripheral blood of cancer

patients and may persist after therapies. This cross-sectional study examines effects of adjuvant chemor-

adiotherapy (CRT) on Treg numbers and function in head and neck squamous cell carcinoma (HNSCC)

patients.

Experimental Design: The frequency and absolute numbers of CD4þ, ATP-hydrolyzing CD4þCD39þ

and CD8þ T cells, and expression levels of CD39, CD25, TGF-b–associated LAP and GARP on Treg were

measured by flow cytometry in 40 healthy donors (NC) and 71 HNSCC patients [29 untreated with active

disease (AD); 22 treated with surgery; 20 treated with CRT]. All treated subjects had no evident disease

(NED) at the time of phlebotomy. In an additional cohort of 40 subjects with AD (n¼ 15), NED (n¼ 10),

and NC (n ¼ 15), in vitro sensitivity of CD4þ T-cell subsets to cisplatin and activation-induced cell death

(AICD) was tested in Annexin V–binding assays.

Results: CRT decreased the frequency of circulating CD4þ T cells (P < 0.002) but increased that of CD4þ

CD39þ Treg (P � 0.001) compared with untreated or surgery-only patients. Treg frequency remained

elevated for >3 years. CRT increased surface expression of LAP, GARP, and CD39 on Treg. In vitro Treg were

resistant to AICDor cisplatin but conventional CD4þ T cells (Tconv) were not. CRT-induced Treg fromADor

NC subjects upregulated prosurvival proteins whereas Tconv upregulated proapoptotic Bax.

Conclusions: Highly suppressive, cisplatin-resistant Treg increase in frequency and persist after CRT

and could be responsible for suppression of antitumor immune responses and recurrence in HNSCC.

Clin Cancer Res; 19(23); 6585–96. �2013 AACR.

IntroductionStandard of care therapy for patients with head and neck

squamous cell carcinoma (HNSCC) includes radical sur-gery and various regimens of chemoradiotherapy (CRT;ref. 1). However, survival rates remain at less than 50% inHNSCCbecause of disease recurrence and the developmentof distant metastases (2). It has been suggested that CRTeliminates subsets of immune cells, thus reducing antitu-mor effector functions and contributing to disease recur-

rence (3). For this reason, current interest has been focusedon the influence of CRT on the host immune system and onalterations in various hematopoietic cell populations thatoccur duringCRT.We andothers have reported thatCD4þTcells are especially sensitive to CRT, and that their absolutenumber decreases after treatment (3, 4). In contrast, thefrequency of regulatory T cells (Treg) was reported to beincreased after chemotherapy in some studies (5), and waslinked to poor prognosis and shorter patient survival (6). Inour earlier studies, we observed an increased frequencyand elevated suppressor function of Treg in the peripheralblood of HNSCC patients and at the tumor sites. Further-more, the Treg frequency was found to be elevated in theblood of patients treated with adjuvant CRT (7). Thesefindings suggesting that in contrast to conventionalCD4þ T cells (Tconv), Treg seemed to be resistant to CRTprovided a rationale for amoredetailed examinationof Tregattributes in HNSCC patients treated with adjuvant CRT.

Recently, human Treg have been reported to expresssurface CD39, an ectonucleotidase, which is a componentof the adenosine pathway (8–10) and is considered as areliable marker for isolation of human Treg (11). Wereported that the CD4þCD39þ T-cell subset consists of 2

Authors' Affiliations: 1University of Pittsburgh Cancer Institute; 2Univer-sity of Pittsburgh School of Medicine; Departments of 3Pathology, 4Immu-nology, and 5Otolaryngology, Pittsburgh, Pennsylvania; 6Department ofOtolaryngology, University of Essen, Germany; and 7Department of Sur-gery, Fukushima Medical University, Fukushima, Japan

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

Corresponding Author: Theresa L. Whiteside, University of PittsburghCancer Institute, Research Pavilion at the Hillman Cancer Center, 5117Centre Avenue, Suite 1.32, Pittsburgh, PA 15213-1863. Phone: 412-624-0096; Fax: 412-624-0264; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-0900

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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on July 31, 2020. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 4, 2013; DOI: 10.1158/1078-0432.CCR-13-0900

http://clincancerres.aacrjournals.org/

ATP-hydrolyzing subsets: CD25þFOXP3þ cells, which sup-press cytokine expression and proliferation of autologousCD4þ Tconv; and CD25negFOXP3neg cells, which are notsuppressive in CFSE-based assays, but are capable of medi-ating suppression via the adenosine pathway (12). These 2CD4þ T-cell subsets share the ability to hydrolyze exoge-nous (e)ATP to ADP and AMP (12, 13). Effects of CRT onthese Treg subsets are unknown, and we considered thepossibility that their functions and interactions with eachother or with CD4þ Tconv are influenced by oncologicaltherapies.

In this cross-sectional study, we prospectively examinethe frequency and functions of these 2 Treg subsets incohorts of HNSCC patients who were either untreated ortreated with surgery alone or CRT. The objective was toevaluate short-term and long-term effects of CRT on theseTreg subsets relative to CD4þ Tconv or CD8þ Tconv and toconsider the mechanisms responsible for persistently ele-vated frequency of Treg after CRT and its potential impacton disease recurrence.

Materials and MethodsPatients and normal donors

Peripheral venous blood samples obtained from 20 nor-mal donors (NC) and 71 HNSCC patients were used forphenotyping studies. An additional cohort of 25 patientsand 15 NC donated peripheral blood for in vitro experi-ments with cisplatin. All subjects signed an informed con-sent approved by the Institutional Review Board of theUniversity of Pittsburgh (IRB #991206). The first patientcohort included 23 females and48maleswith amean age of59.3� 10.1 years (range: 31–78 years). The 29 patients with

active disease (AD) were untreated at the time of blooddraws; 22 patients were treated with surgery alone (SRG);and 20 patients had received adjuvant CRT 14 � 9 months(mean � SD) before the phlebotomy for this study. Alltreated patients were no evident disease (NED) at the timeof blood draws. Chemotherapy was platinum-based andconsisted of cisplatin or carboplatin. Panitunumab andpaclitaxel each were added in one case, respectively. Allpatients received radiotherapy, which ranged from 44 to 70Gy.Clinicopathologic anddemographic data for the patientcohorts are listed in Table 1. The age-matched NC cohortincluded 9 females and 31males with a mean age of 51� 6years (range 39–69 years).

The cohort of 25 patients who donated blood for the invitro sensitivity/resistance studies included 17 males and 8females with amean age of 60 years (range 23–82 years); 10patients had AD (7 of 10 had primary untreated tumors and3 of 10 had a recurrent disease); all 15 NED patientsunderwent surgery andwere treatedwithCRT. Their therapywas terminated from 3 weeks to 12 months before phle-botomy for this study.

Collection of peripheral blood mononuclear cellsBlood samples (20 mL) were drawn into heparinized

tubes, and plasma was collected after centrifugation at lowspeed. The cell pellets were resuspended in 40 mL PBS andcentrifuged on Ficoll-Hypaque gradients (GE HealthcareBioscience). Peripheral blood mononuclear cells (PBMC)were recovered, washed in AIM-V medium (Invitrogen),counted in a trypan blue dye, and immediately used forexperiments. A fraction of harvested cells was cryopreservedfor cytokine expression assays by flow cytometry.

Flow cytometry reagentsThe following antihumanmonoclonal antibodies (mAb)

were used for staining: anti-CD19-ECD, anti-CD4-PC5,anti-CD8-PE, anti-HLA-DR-ECD (all Beckman Coulter);anti-CD73-PE, anti-Bcl-2-FITC, anti-Bcl-2-PE, anti-Bcl-xL-FITC, and anti-Bax-FITC (all BD Pharmingen); anti-CD39-FITC, anti-CD38-FITC, FOXP3-FITC (ClonePCH101), LAP-PE, GARP-APC (all eBioscience), and anti-CD25-PE (Mil-tenyi). Isotype controls were included in all assays andserved as negative controls for surface aswell as intracellularstaining. All Abs were pretitered using activated as well asnonactivated PBMC to determine the optimal stainingdilution for each.

Surface and intracellular stainingFreshly isolated cells to be used for flow cytometry were

incubated with mAbs specific for each surface marker in 50mL PBS for 30minutes at room temperature in the dark andwashed with PBS before acquisition for surface markerdetection. For intracellular staining of prosurvival BcL-2and BcL-xL proteins or proapoptotic Bax, cells were firstincubated with relevant mAbs specific for surface markers.After washing, cells were fixed with 4% (v/v) paraformal-dehyde in PBS for 20minutes at room temperature, washedonce with PBS, and permeabilized with PBS containing

Translational RelevanceDespite radical surgery and various regimens of

chemoradiotherapy (CRT), survival rates remain lowerthan 50% for head and neck squamous cell carcinoma(HNSCC). The patients initially responsive to therapydevelop recurrent disease, second primaries, or metas-tases. Current interest has been focused on effects ofCRT on the host immune system. In this cross-sec-tional study, absolute numbers, frequency, and func-tions of CD4þCD39þ regulatory T cells (Treg) weremonitored in patients with HNSCC and were com-pared in cohorts of patients who were either untreatedor treated with surgery-only or adjuvant CRT. The datashow that in HNSCC patients, CRT increases thefrequency of highly suppressive Treg that are resistantto activation-induced cell death and to cisplatin.Mechanisms responsible for this resistance includeenhanced expression of prosurvival proteins BcL-2/BcL-xL in Treg. The persistence of Treg in the patients’circulation long after CRT is finished could be respon-sible for post-CRT immunosuppression, leading tocancer recurrence in HNSCC patients.

Schuler et al.

Clin Cancer Res; 19(23) December 1, 2013 Clinical Cancer Research6586




0.5%bovine serumalbumin (BSA) and0.1%(v/v) saponin.Next, pretitered antibodies specific for BcL-1, BcL-xL, or Baxwere added and incubated with the cells for 30 minutes atroom temperature. Cells were further washed twice withPBS containing 0.5% BSA and 0.2% saponin, resuspendedin FACS flow solution and analyzed by flow cytometry.Expression of intracellular FOXP3 was evaluated using astaining kit available from eBioscience as previouslydescribed (14).

Flow cytometryFlow cytometry was performed using an EPICS XL-MCL

or Gallios flow cytometer equipped with Expo32 software(BeckmanCoulter). The acquisition and analysis gates wererestricted to the live cells based on forward and side scatterproperties of the cells. At least 1� 105 events were acquiredfor analysis and, where applicable, gates were restricted tothe CD4þ, CD4þCD39þ, or CD8þ T-cell subsets. The cellfrequency and mean fluorescence intensity (MFI) weredetermined. Absolute numbers of CD4þ T cells were deter-mined using the whole blood counts; absolute numbers ofTreg were calculated by multiplying the Treg frequencyvalues by the absolute number of CD4þ T cells.

Isolation of TregCD4þ T cells were isolated from buffy coats of NC by

negative magnetic bead separation according to the man-ufacturer’s protocol (AutoMACS, Miltenyi). CD4þCD39þ

T cells were then isolated by magnetic immunobeads aspreviously described (11). Briefly, CD4þ T cells were incu-bated with anti-CD39 biotinylated Ab and anti-biotin mag-netic beads before isolation by AutoMACS. The purity ofisolated cell populations was >85%.

Ex vivo activation of T cellsPBMC obtained from NC or patients with HNSCC were

incubated in anti-CD3/antiCD28 Abs-coated wells in flat-bottom 96-well plates in the presence of interleukin (IL)-2 (150 IU/mL) for 48 hours at 37�C in the atmosphere of5% CO2 in air. In some experiments, PBMC were pre-treated with physiologic doses of cisplatin determined asdescribed by van den Bongard and colleagues (15) for 4hours before further incubation with activating Abs andIL-2.

Detection of Annexin V-binding (ANXVþ) to activatedT cells

PBMC immediately after their isolation; PBMC after exvivo activation as described earlier; or PBMC preincubatedwith cisplatin � activating Abs and IL-2 were analyzed forANXV binding to Treg or Tconv by flow cytometry. Sampleswere stained with FITC-conjugated or PE-conjugatedANXV (Molecular Probes), according to instructions pro-vided by the manufacturer. Flow cytometry was per-formed with gates restricted to CD3þCD4þCD25high andCD3þCD4þCD25neg T-cell subsets.

Table 1. Clinicopathologic characteristics of patients studied for the Treg frequency

Control Untreated Treated with surgery alonea Treated with CRT

N (female/male) 40 (9/31) 29 (6/23) 22 (6/16) 20 (2/18)Age (�SD)b range (y) 51 � 6 (39–69) 58 � 9 (39–74) 60 � 10 (38–78) 55 � 12 (31–76)Stage (n)c

T1 5 10 3T2 10 5 3T3 6 2 7T4 8 5 7

Nodal status (n)c

N0 13 20 7N1 5 1 1N2 11 1 12

Location (n)Tongue 10 7 3Oral cavity 8 4 7Pharynx 4 1 5Larynx 7 8 4Other 0 2 1

Days since treatment (range)d 321 � 389 (8–1680) 408 � 279 (1–975)

aTwo patients had received adjuvant radiation without chemotherapy.bAge at diagnosis.cThe patients' TNM status was based on clinical/pathological diagnoses. In the CRT cohort, diagnostic biopsies were performed in 12of 20 patients.dThe patients who donated blood for the Treg studies were NED at the time of phlebotomy.

CD4þCD39þ Treg and CRT

www.aacrjournals.org Clin Cancer Res; 19(23) December 1, 2013 6587




In vitro sensitivity of T-cell subsets to cisplatinCD4þCD39þ and CD4þCD39neg T-cell subsets were iso-

lated from PBMC of NC using magnetic beads as describedearlier. These T-cell subsets were then incubated with cis-platin (3–5 mg/mL) in the presence of Staphylococcal Pro-tein B (SEB, 1 mg/mL; Sigma-Aldrich) or activating Abs andIL-2 in RPMI media. On day 2 and 5, cells were harvestedand stained for Annexin-FITC, 7AAD (Invitrogen), andCD4-PE (Beckman-Coulter) for 20 minutes on ice as pre-viously described (16).

StatisticsThe data are presented as mean values� SD. For samples

with nonparametric distribution, P-values were calculatedby Kruskal–Wallis and two-tailed exact Wilcoxon–Mann–Whitney tests. Correlations were calculated by the Spear-man test. P-values < 0.05 and R2 values > 0.5 were consid-ered to be significant.

ResultsFrequency and absolute numbers of T-cell subsets

The frequency and absolute numbers of CD4þ andCD8þ

T cells in the peripheral blood specimens obtained fromHNSCC patients who were not yet treated (i.e., with ADbefore surgery) as well as patients treatedwith surgery alone(SRG) or with CRT were compared (Fig. 1). The frequencyand absolute numbers of both CD4þ andCD8þ T cells weresignificantly decreased in the blood of patients with AD aswell as those treated with surgery alone relative to NCvalues. In the cohort of CRT patients, percentages and

absolute numbers of CD4þ T cells were dramaticallydecreased (P < 0.002 for both; Fig. 1A). However, thefrequency of CD8þ T cells increased following CRT in thispatient cohort relative to NC values, although the absolutenumber of CD8þ T cells significantly decreased (P < 0.001)as shown in Fig. 1B. The data suggest that although CRTadversely affects both T-cell subsets, it has especially detri-mental effects on the CD4þ T cells. CD8þ T cells seem to beless sensitive to CRT or they recover faster than CD4þ T cellsafter CRT.

Frequency and absolute numbers of CD4þCD39þ TregBecause CD4þ T cells contain Treg, we were especially

interested in therapy-induced changes in the CD4þ T-cellsubset. We have previously shown that in humans, func-tional CD4þ Treg are CD39þCD25þ, express FOXP3 andhydrolyze (e)ATP to 50AMP (12). Within the CD4þ T-cellcompartment, the frequency of these cells was found to beincreased (P < 0.01) in the AD and SRG patient cohortsrelative to that seen in NC (Fig. 1C). However, the mostdramatic increase in the frequency, although not the abso-lute number (Fig. 1C), of Treg was seen in the CRT patients(P<0.001). These results suggest that in the shrinkingCD4þ

T-cell compartment, CD4þCD39þCD25þ Treg are relative-ly resistant to CRT as compared with CD4þ Tconv. Theirfrequency increases whereas that of CD4þ T cells declinesfollowingCRT.Wehave also calculated theCD8þ/Treg ratiofor all patient cohorts, because this ratio has beenoften usedin evaluating changes in the immune profile in situ and inthe peripheral circulation of cancer patients treated with

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Figure 1. The frequency and absolute numbers of CD4þ and CD8þ T lymphocyte subsets in the peripheral blood of HNSCC patients. Box plots showmedian,25 and 75 percentiles, and the highest and lowest values. NC, normal control (n¼ 40); AD, untreated active disease (n¼ 29); SRG, treated with surgery alone(n ¼ 22); CRT, treated with adjuvant chemoradiotherapy (n ¼ 20). �, P < 0.05; ��, P < 0.01.

Schuler et al.





various therapies (6, 17). As shown in Fig. 1D, the CD8þ/Treg ratio was lower in all patient groups than in NC, but itwasmost prominently decreased in the CRT patient cohort,which is consistent with the significant increase in thefrequency of Treg after CRT and the resistance of these cellsto CRT as described earlier. Here, the significantly decreasedCD8þ/Treg ratio after therapy suggests that Treg are moreresistant to CRT than CD8þ T cells. Interestingly, the fre-quency of CD19þ B cells was also increased after CRT(Supplementary Fig. S1), an indication that B cells mightalso be resistant to CRT.A decline in the size of the CD4þ T-cell compartment

following oncological therapies was further evaluated,and Fig. 2A shows that it is accompanied by increasingpercentages of CD4þCD39þ Treg in representativepatients selected from each of the cohorts. Importantly,CD4þCD39þCD25þ Treg seem as a distinct, well-definedpopulation among CD4þ T cells, which can be easilydistinguished from CD4þCD39negCD25þ Tconv by flow

cytometry. For patients in the SRG and CRT cohorts, thefrequency and absolute numbers of total circulatingCD4þ T cells negatively correlate with the percentage ofCD4þCD39þCD25þ Treg at P < 0.001 and P < 0.002,respectively (Fig. 2B). We have previously shown thatbased on expression of surface markers and functionalcharacteristics, human Treg can be divided into (i) strong-ly suppressive CD4þCD39þCD25þ T cells that areFOXP3þ and (ii) CD4þCD39þCD25neg T cells that donot express FOXP3 and mediate little suppression (12).Within the Treg subpopulation, a strong positive rela-tionship exists between these 2 subsets in NC (12) and, asshown in Fig. 2C, this relationship is not altered inuntreated patients (AD) or in patients with NED whowere treated with SRG or CRT. The 2 Treg subsets trackeach other and the frequency of both was found to beequally increased in all patient cohorts compared withNC (Fig. 2C). As expected, the frequency of CD4þ

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Figure 2. Changes in the CD4þ T-cell compartment after oncological therapies. A, decreased total CD4þ T-cell frequency is accompanied by increasedfrequency of CD4þCD39þ Treg. Density plots show data from representative HNSCC subjects selected from each study group. Gates are set on thelymphocyte window (left) and on the CD4þ T-cell subset (right). B, negative associations between percentages (left) or absolute numbers (right) of totalCD4þT cells and the percentages ofCD4þCD39þCD25þTreg in the circulation ofHNSCCpatients treatedwith SRGorCRT.C, a positive correlation betweenthe frequency of CD4þCD39þCD25þ Treg and CD4þCD39þCD25neg cells in the peripheral circulation. D, a positive correlation between the frequencyof CD4þCD25high and CD4þCD39þCD25þ Treg subsets in the peripheral circulation of NC as well as all HNSCC patients.






CD4þCD25high T cells in the peripheral blood of HNSCCpatients, indicating that these 2 subsets of Treg closelyoverlap (Fig. 2D).

Functional characteristics of Treg in patients treatedwith CRT

Suppressor cell assays based on proliferation inhibitionrequire large cell numbers and, therefore, they are not usefulfor monitoring of patient specimens, which are often lim-ited in volume. For this reason, we used flow cytometry–based assays to measure expression of functionally relevantproteins, such as the latency-associated peptide (LAP) andthe glycoprotein A repetitions predominant (GARP),known to be expressed on the surface of cells that produceand release TGF-b (18). Because Treg utilize TGF-b as asuppressive mechanism (19), and LAP as well as GARP areestablished surrogate markers for TGF-b (20, 21), we eval-uated expression of LAP and GARP on the surface of CD4þ

CD39þ Treg, as surrogate markers for Treg suppressoractivity in all patient cohorts (Fig. 3A and B). We havepreviously reported that suppressive CD4þCD39þFOXP3þ

Treg expressed LAP and GARP, whereas CD4þ

CD39þFOXP3neg T cells did not (12). The highest percen-tages of LAPþ or GARPþ Treg were seen in CRT-treatedpatients. (Fig. 3C). Also, expression of these markers washighly and significantly upregulated on Treg in the CRTpatient cohort (Fig. 3A andB). In addition, theMFI of CD39was upregulated on Treg in all theHNSCCgroups relative tothat in NC, and the highest MFI for CD39 was observed inTreg of patients in the CRT cohort (Fig. 3D). These datasuggest that Treg of the CRT-treated patients have a betterability to hydrolyze exogenous ATP to 50-AMP than Treg ofpatients in the other cohorts. We have previously shownthat CD39 expression levels on the cell surface positivelycorrelate with enzymatic hydrolysis of exogenous ATP byTreg (12).

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Schuler et al.





Persistent upregulation of the Treg frequency inCRT-treated patientsBlood specimens were obtained from NED patients trea-

ted with CRT at various times after the end of therapy (n ¼20). The average time period elapsed between the end oftreatment and phlebotomy for this study was 14 � 9months (mean � SD). The increased Treg frequencyobserved in the CRT-treated patients was independent ofthe time of blood draws after the end of therapy (Fig. 4A andB). This suggests that the increase in the Treg frequencyrepresents a long-lasting effect in these patients. A subset ofHNSCC patients (N ¼ 22) treated with surgery alone alsoshowed an increase in the Treg frequency shortly aftersurgery (1–6 months). However, in patients tested 6 to15 months after surgery, the Treg frequency was normal,suggesting that in contrast to CRT, surgery was not associ-ated with persistent elevation of Treg (Fig. 4). Importantly,in some of the NED patients who were treated with CRT,the frequency of Treg was elevated even after 3 years afterend of therapy. In 3 NED patients treated with CRT, serial

blood specimens were obtained at 6, 8, and 12months aftertermination of therapy. The Treg frequency remained ele-vated, ranging between 12 and 16%, in these patients’blood, further suggesting that a stable and persistentincrease in Treg characterizes this patient cohort. Figure 4shows that the percentages of total CD4þ T cells remainedwithin the normal range in patients treated with surgery,whereas CRT-treated patients had low percentages of CD4þ

T cells months after therapy.

Clinical significance of persistently high TregThe cohort of 20 CRT patients was followed for 37 � 13

months (mean � SD from the end of treatment) to deter-mine whether anyone experienced disease recurrence. Dur-ing this period of time, 4 patients recurred and 3 othersdeveloped second primary HNSCC (35%). Two of thesecond primary tumors occurred at 25 months and thethird at 45 months after CRT ended, and all 3 patients hadelevated percentages of Treg (6–19%) in the peripheralcirculation. Within the CRT cohort, there was no significant

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Figure 4. Persistence of Treg in the circulation of NED patients after therapy and in vitro resistance of Treg to cisplatin. A, percentages of CD4þCD39þCD25þ

Treg in the peripheral circulation of HNSCC patients who are NED after CRT. The elevated Treg frequency in patients was independent of the timeperiod elapsed between the last treatment and phlebotomy for this study. B, in HNSCC patients treated with SRG alone, the Treg frequency wasincreased shortly after treatment but later seemed to normalize. C, percentages of total CD4þ T cells were persistently decreased in CRT-treated patients.A–B, the solid line represents the mean Treg (3.2%) or CD4þ T-cell (42%) frequency established for NC, and the dotted lines show � 1 SD of the mean.D, isolatedCD4þCD39þ andCD4þCD39neg T cells obtained from4different NCwere incubated in the presence of physiological concentrations of cisplatin for48 hours. At the cisplatin concentration of 3 mg/mL, the increase in frequency of ANXVþ cells was greater (P < 0.03) among CD4þCD39neg Tconv thanamong CD4þCD39þ Treg.






difference in the Treg frequency (P¼ 0.18) between patientswho recurred or developed new primary tumors (12� 4%)and those who did not recur (9� 5%; means� SD). In thesimilarly followed surgery-only cohort, 4 of 22 patientsexperienced recurrence (18%), and there were no secondprimary cancers observed. There was also no significantdifference in the Treg frequency (P > 0.2) between patientswho had recurrence and those who did not.

In vitro sensitivity of CD4þ T-cell subsets to cisplatinor AICD

Our data suggest that the high frequency of Treg seen inthe blood of HNSCC patients treated with CRT could bebecause of resistance of these cells induced by therapy. Totest the hypothesis that the long-term Treg survival afterCRT is a characteristic of these cells, sensitivity/resistanceto cisplatin of CD4þCD39þ Treg freshly isolated from thePBMCofNCandactivated ex vivo (i.e., incubated eitherwithSEB or anti-CD3/CD28 Abs and IL-2) was tested in ANXV-binding assays. CD4þCD39þ Treg and CD4þCD39neg Tconvwere incubatedwith physiologic concentrations of cisplatinas previously described by van den Bongard and colleagues(15). The CD4þCD39þ Treg subset was found to be moreresistant (P < 0.03) to cisplatin than CD4þCD39neg Tconv(Fig. 4D).

Using PBMC obtained from NC and HNSCC patientstreated or not treated with CRT, we studied resistance/sensitivity of Treg to activation-induced cell death (AICD)following in vitro activation of PBMC with anti-CD3/CD28Abs and IL-2 (150 IU/mL). After 48-hour incubation, weused flow cytometry to measure ANXV binding to CD4þ

CD24high versus CD4þCD25neg T-cell subsets and to com-pare their resistance/sensitivity of these cells to AICD orcisplatin. Figure 5A shows that after ex vivo activation, onlyCD4þCD25high Treg of patients treated with CRT wereresistant to activation-induced cell death, as evidenced bythe lack of ANXV binding (left), whereas activated Treg ofuntreated patients or of NC were highly sensitive to AICD.In contrast, no difference in ANX binding to CD4þCD25neg

Tconv was seen among the 3 cohorts: only 20–40% of Tconvbound ANXV (right). Furthermore, as shown in Fig. 5B,preincubation with cisplatin (4 hours) followed by PBMCactivation for 48 hours induced resistance of CD4þCD25high

Treg obtained from NC or untreated HNSCC patients toAICD (left), and this resistance was also evident in Treg ofpatients treated with CRT. In contrast, pretreatment withcisplatin induced greater sensitivity to AICD in CD4þ

CD25neg Tconv of NC as well as HNSCC patients, as nearlyall of these cells became ANXVþ as shown in Fig. 5B (right).

Differential effects of cisplatin on Treg versus CD4þ

TconvTo investigate the mechanisms responsible for resistance

of Treg in CRT-treated patients to AICD, expression ofprosurvival Bcl-2/Bcl-xL and of proapoptotic Bax was com-pared in Treg versus Tconv preincubated with or withoutcisplatin before in vitro activation (Fig. 5C). Cisplatin upre-gulated expression of the prosurvival proteins in Treg,

whereas it significantly downregulated prosurvival proteinexpression in Tconv (Fig. 5C, right) Also, the pretreatmentwith cisplatin decreased expressionofBax inTreg,whereas itdramatically upregulated expression of this proapoptoticprotein in Tconv. These CD4þCD25þ Treg in the blood ofHNSCC patients treated with CRT (POST) upregulatedexpression levels of survival proteins relative to those inPRE Treg (P < 0.001) and were highly resistant to in vitrocisplatin, perhaps because of cumulative direct or indirect invivo effects of CRT. In contrast, CD4þCD25þ Treg in theblood of NC incubated in vitro with cisplatin for 4 hours(Fig. 4D) showed a relatively small, albeit statisticallysignificant, increase in resistance versus that observed inCD4þCD25neg Tconv.

DiscussionEffects of oncological therapies on lymphocyte subsets

havebeenof considerable interest, because of thepossibilitythat therapy-induced changes in immune-cell homeostasismight interfere with antitumor activity. In this respect,in vivo studies in mice indicate that radiation and chemor-adiation exert strong effects on the host immune system,including Treg (22). The limited data that are available forpatients with cancer suggest that CRT reduces the number ofcirculating lymphocytes (23). In HNSCC patients, CD4þ Thelper cells were previously reported to be particularlysensitive to CRT, as they were found to be significantlydepleted relative to other T-cell subsets (3, 24). Recently,attention has focused on Treg, a subset of CD4þ T cellsresponsible for mediating suppression of antitumorimmune responses (25). The presence and accumulationsof Treg at tumor sites and in the peripheral circulation ofcancer patients, including HNSCC patients, have beenwidely reported (6, 7, 26). These high Treg frequencies havebeen negatively or positively correlated with disease out-come in various malignancies (6, 27, 28). Studies alsoindicate that the Treg frequency tends to increase afteroncologic therapies (23), introducing a possibility thatTreg are more resistant to CRT than CD4þ Tconv. Thisprospective cross-sectional study in a large cohort ofHNSCC patients was performed to examine this possibilityand to provide insights into potential mechanisms respon-sible for Treg resistance to oncological therapies.

Confirming our previous observations, we show that incomparison to NC, untreated HNSCC patients or thosetreated with surgery or surgery plus CRT have reducedpercentages and absolute numbers of CD4þ T cells butincreased percentages of CD4þCD39þ Treg. As the CD4þ

T cell compartment shrinks in disease, the proportion butnot absolute number of circulating CD4þCD39þ Tregincreases. This contraction was especially magnified in theCRT patient cohort, implying that Treg, unlike CD4þ Tconv,are resistant to CRT. Treg resistance to radiation and drugshas been reported (29, 30).However, the exactmechanismsresponsible for Treg resistance to CRT remain unclear. Thisresistance might reflect the slow proliferation rate of Tregin the periphery or existence of protective mechanisms, for

Schuler et al.





example, multidrug transporter pumps, and the enhancedcapability for DNA repair (31). A short exposure of Treg tocisplatin before activation seems toprotect them fromAICDand is mediated by elevated expression levels of survival

proteins, Bcl-2 and Bcl-xL, and lower levels of proapoptoticBax than those observed in Tconv (Fig. 5). Still anotherpossibility is that toll-like receptors (TLR) present on Treg(32) and signaling in response to ligands released by

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Figure 5. In vitro resistance of CD4þCD25high Treg to cisplatin and upregulated expression of the prosurvival proteins in Treg treated with cisplatin. A, ANXVbinding to Treg (CD4þCD25high) (left) or Tconv (CD4þCD25neg) (right) in the in vitro activated PBMC obtained from NC (n ¼ 15), untreated HNSCC patients(pre; n ¼ 10) or HNSCC patients treated with CRT (post; n ¼ 15). PBMC were incubated with anti-CD3/CD28 Abs and 150 IU/mL of IL-2 for 48 hoursand studied for the evidence of AICD (ANXVbinding) by flowcytometry, gating on Treg and Tconv cells. Only Treg obtained from theCRT-treated patients (post)were resistant toAICD.B,ANXVbinding to Treg (CD4þCD25high) (left) or Tconv (CD4þCD25neg) (right) inPBMC,whichwerepretreated in vitrowith cisplatin for 4hours before incubation of the cells in the presence of anti-CD3/CD28 Abs and IL-2 for 48 hours. Only Treg became resistant to AICD as determined bymeasuring ANXV binding. C, expression of Bcl-2, Bcl-xL, and Bax are shown as MFI � SD in CD4þCD25high Treg and CD4þCD25neg Tconv. PBMC wereobtained from the blood ofNC, untreated, or CRT-treatedpatients andwere preincubated or notwith cisplatin and then activated asdescribed earlier in A. Thesolid lines (PRE to POST panels) show increased expression levels of Bcl-2 and Bcl-xL in POST Treg as comparedwith those in PRE Treg that were incubatedwith in vitro cisplatin. �, P < 0.001; ��, P < 0.005.






radiation-induced tissue damage activate the PI3K/Akt sur-vival pathway, protecting Treg from effects of CRT. SuchTLR-mediated chemoresistance has been previously des-cribed for tumor cells (33–35). Others report that plati-num-based chemotherapies decrease immune suppressionacting not on Treg but on dendritic cells (DC) or tumorcells, reducing expression of the T-cell inhibitory moleculePD-L2 on these cells and upregulating recognition of tu-mor targets by T cells (36).

Here, we advance the hypothesis that CRT induces tissuechanges that have indirect effects on Treg, favoring theirsurvival and suppressor functions. For example, increasedTGF-b levels in irradiated tissues could promote Treg dif-ferentiation (12, 22). The persistently enlarged post-CRTTreg compartment includes cells with increased immuno-suppressive capabilities (e.g., upregulated LAP, GARP, andCD39). These CRT-induced Treg producemore 50-AMP andADO, acquire and maintain the ‘killer phenotype’ associ-ated with expression of Fas, FasL, and GrB/perforin, aspreviously reported by us (37), and upregulate prosurvivalproteins. We show here that activated CD4þCD39þ Tregare more resistant to cisplatin than CD4þ Tconv and thatCD4þCD25high Treg obtained from HNSCC patients withNED after CRT become resistant to AICD, whereas CD4þ

CD25neg Tconv remain sensitive (Fig. 5). Together, our datasupport the hypothesis that Treg induced in the CRT envi-ronment acquire new characteristics that make them intolong-surviving and potentially more dangerous immune-regulatory cells.

In preclinical models, radiotherapy has been reported toeither interfere with antitumor immune responses by theelimination of immune effector cells (38) or promote theseresponses by enhancing the processing and presentation oftumor antigens by APC (39, 40). These opposing radiother-apy-induced effects are related to alterations in tissues andcells, which seem to be dependent on the dose and type ofradiotherapy (38–40). For example, the ATP concentrationis increased in damaged irradiated tissues (22), providingtherapy-induced Treg with an opportunity to produce 50-AMP and ADO. These factors acting via the adenosinereceptors potentially influence survival and activity of Treg.Because isolated CD4þ25þ T cells contain significantlyhigher levels of mRNA coding for A1R, A2AR, and A3R thanCD4þCD25neg T cells (our unpublished data), we surmisethat autocrine and paracrine signaling via these receptorshas profound effects on functions and survival of CRT-induced Treg. Our experiments with CD4þCD39þ orCD4þCD25high Treg subsets suggest that environmentallygenerated signals, as exemplified by TGF-b, ATP, or ADO,regulate functions of CRT-induced Treg and might contrib-ute to their resistance and survival after CRT. Thus, resis-tance of these Treg to cisplatin or AICD might not be theirintrinsic characteristic but rather reflects an environmental-ly regulated requirement for their activity to regulateimmune homeostasis disturbed by therapy that alters theimmune cell numbers and balance. To what extent thisinduced resistance to drugs such as cisplatin is related toimmune escape mechanisms, which are responsible for the

promotion of tumor growth in cancer patients, remains tobe determined. Our data suggest that human Treg are ableto utilize various survivalmechanisms toprotect themselvesfrom exogenous stress signals. Treg survival seems to becritically important for the host, emphasizing the signi-ficant role these T cells play in health and disease.

The observed elevations in the Treg frequency and func-tions in patients with active (pretherapy) and inactive(posttherapy) disease were independent of the time elapsedbetween the treatment and blood draws. In fact, the Tregfrequency remained elevated for weeks and months inmany, although not all, HNSCC patients who were NEDafter CRT. The suppressive potential of Treg is known to berelated to the Treg/Tconv ratio. The functional consequenceof an increase in the proportion of Treg after CRT is that thisratio changes in favor of Treg. These Treg acquire strongersuppressor function after CRT and express more TGF-b–associated LAP and GARP as well as ATP-hydrolyzingCD39 on their surface. Our data are provocative, as theysuggest that persistent elevations in the CD4þCD39þ Tregfrequency induced in the blood of cancer patients by poten-tially curative therapies, such as surgery and adjuvant CRT,result in suppression of antitumor immune effector cellsand thus promote disease recurrence or the development ofsecondary cancers. The fact that in our study, 3 of the CRT-treated patients with elevated Treg percentages developedsecond primary cancers within 2 to 4 years of therapysuggests that the imbalance in immune regulation mightpromote disease progression.

It has also been suggested that CRT induces a state ofchronic inflammation in tissues, which has been linked tocancer development (41). The presence of iTreg in thissetting could be beneficial to the host by normalizing T-cell responses and maintaining immune responses in bal-ance. The role of Treg that accumulate after CRT in thepromotion or inhibition of tumor progression is currentlyunclear. Prospective serial monitoring will be necessary todetermine the biologic and clinical consequences ofincreased Treg frequency in HNSCC patients treated withCRT.

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

Authors' ContributionsConception anddesign: P.J. Schuler, L. Strauss, J.T. Johnson, T.L.WhitesideDevelopment of methodology: M. Harasymczuk, L. StraussAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Harasymczuk, B. Schilling, Z. Saze, J.T.JohnsonAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): P.J. Schuler, B. Schilling, L. Strauss,S. Lang, T.L. WhitesideWriting, review, and/or revision of the manuscript: P.J. Schuler,B. Schilling, L. Strauss, J.T. Johnson, T.L. WhitesideAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): P.J. Schuler, T.L. WhitesideStudy supervision: P.J. Schuler, T.L. Whiteside

AcknowledgmentsThis study was supported in part by NIH grant PO1 CA109688 to T.L.

Whiteside and by the Pittsburgh-Essen-Partnership Program to P.J. Schuler.

Schuler et al.





The services of the Immunologic Monitoring and Cellular Products Labo-ratory (IMCL) and the FlowCytometry Laboratory were supported in part bythe NCI CCSG 5P30 CA047904.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 5, 2013; revised August 22, 2013; accepted September 12,2013; published OnlineFirst October 4, 2013.

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2013;19:6585-6596. Published OnlineFirst October 4, 2013.Clin Cancer Res Patrick J. Schuler, Malgorzata Harasymczuk, Bastian Schilling, et al. CancerFunction of Regulatory T Cells in Patients with Head and Neck Effects of Adjuvant Chemoradiotherapy on the Frequency and

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