ox40+ regulatory t cells in cutaneous squamous cell...

Biology of Human Tumors

OX40þRegulatory TCells inCutaneousSquamousCell Carcinoma Suppress Effector T-CellResponses and Associate with MetastaticPotentialChester Lai1,2, Suzannah August1,2, Amel Albibas1, Ramnik Behar1, Shin-Young Cho1,Marta E. Polak1, Jeffrey Theaker3, Amanda S. MacLeod4, Ruth R. French5,Martin J. Glennie5, Aymen Al-Shamkhani5, and Eugene Healy1,2

Abstract

Purpose: Cutaneous squamous cell carcinoma (cSCC) is themost common human cancer withmetastatic potential. Despite Tcells accumulating around cSCCs, these tumors continue to growand persist. To investigate reasons for failure of T cells to mount aprotective response in cSCC, we focused on regulatory T cells(Tregs) as this suppressive population is well represented amongthe infiltrating lymphocytes.

Experimental Design: Flow cytometry was conducted on cSCClymphocytes and in vitro functional assays were performed usingsorted tumoral T cells. Lymphocyte subsets in primary cSCCswerequantified immunohistochemically.

Results: FOXP3þ Tregs were more frequent in cSCCs than inperipheral blood (P < 0.0001, n ¼ 86 tumors). Tumoral Tregssuppressed proliferation of tumoral effector CD4þ (P ¼ 0.005,n ¼ 10 tumors) and CD8þ T cells (P ¼ 0.043, n ¼ 9 tumors) and

inhibited IFNg secretion by tumoral effector T cells (P ¼ 0.0186,n¼ 11 tumors). The costimulatory molecule OX40was expressedpredominantly on tumoral Tregs (P < 0.0001, n¼ 15 tumors) andtriggering OX40 with an agonist anti-OX40 antibody overcamethe suppression exerted by Tregs, leading to increased tumoraleffector CD4þ lymphocyte proliferation (P ¼ 0.0098, n ¼ 10tumors). Tregs and OX40þ lymphocytes were more abundant inprimary cSCCs that metastasized than in primary cSCCs that hadnot metastasized (n ¼ 48 and n ¼ 49 tumors, respectively).

Conclusions: Tregs in cSCCs suppress effector T-cell responsesand are associated with subsequent metastasis, suggesting a keyrole for Tregs in cSCC development and progression. OX40agonism reversed the suppressive effects of Tregs in vitro, suggest-ing that targeting OX40 could benefit the subset of cSCC patientsat high risk of metastasis. Clin Cancer Res; 1–13. �2016 AACR.

IntroductionNon-melanoma skin cancer (NMSC), which comprises cuta-

neous squamous cell carcinomas (cSCC) and basal cell carcino-mas (BCC), is the most common type of cancer and greatlyimpacts on global health, with estimated annual incidences of2.8 million cSCCs and 10 million BCCs worldwide (1). In theUnited States alone, 3.5 million NMSCs arise each year, with

cSCCs accounting for 700,000 of these tumors (2). Skin cancercreates a substantial economic burden and, in 2004, the estimatedcost of skin cancer and precancerous skin lesions to US healthservices was $6.6 million (3). Despite treatment with surgery,cSCC poses problems because of its propensity to metastasize,occurring in 16% to 30% of tumors, which have invaded 6mm indepth or over 2 cm in diameter (4, 5).

cSCCs are keratinocyte-derived neoplasms associated withchronic exposure to ultraviolet radiation, which causes geneticalterations within the epidermis and promotes squamous cellcarcinogenesis and subsequent metastases (6–8). Alterations inimmunity have a key role in regulating cutaneous carcinogenesis,as evidenced by immunosuppression greatly increasing the inci-dence of cSCCs, with these tumors 65- to 250-fold more frequentin organ transplant recipients (9). Indeed, cSCC constitutes anideal tumor model for investigating cancer immunology becausemost cSCCs are surrounded by an immune cell infiltrate, yet thisinfiltrate is generally incapable of mounting a successful antitu-mor response. Tregs have a physiologic function in maintainingimmunologic self-tolerance and homeostasis; however, incertain cancers, increased numbers of tumor-infiltrating Tregs areassociated with poorer clinical outcome, suggesting that Tregssuppress immune responses within the tumoral environment(10–13). As a result of this, there has been considerable interestin developing immunotherapeutic strategies that target Tregs and

1Dermatopharmacology, Clinical and Experimental Sciences, Facultyof Medicine, University of Southampton, Southampton, United King-dom. 2Dermatology, University Hospital Southampton NHS Founda-tion Trust, Southampton, United Kingdom. 3Histopathology, Univer-sity Hospital Southampton NHS Foundation Trust, Southampton,United Kingdom. 4Dermatology, DukeUniversity Medical Center, Dur-ham,NorthCarolina. 5CancerSciences, FacultyofMedicine,Universityof Southampton, Southampton, United Kingdom.

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

Corresponding Author: Eugene Healy, Dermatopharmacology, University ofSouthampton, Sir Henry Wellcome Laboratories, Southampton General Hospi-tal, Southampton, Hampshire SO16 6YD, United Kingdom. Phone: 4423-8079-5232; Fax: 4423-8070-4183; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2614

�2016 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org OF1

Research. on August 8, 2019. © 2016 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 31, 2016; DOI: 10.1158/1078-0432.CCR-15-2614

http://clincancerres.aacrjournals.org/

boost effector immune responses in cancer (14–18). One suchapproach involves the provision of costimulatory signals throughreceptors that belong to the tumor necrosis receptor superfamily,including OX40. Engagement of this receptor has been shown topromote T-cell activation through effects on different subpopula-tions of T cells, for example, by promoting proliferation andsurvival of effector memory T cells following antigenic activation,as well as by suppressing regulatory T-cell activity (16–19). Theseeffects highlight OX40 as a promising target for tumor therapy,and cases of tumor regression have been recently reported in aphase I clinical trial with anti-OX40 agonistic antibody (20).

In this study, we show that cSCCs contain a higher proportionof Tregs than peripheral blood from the same individuals, andthat these tumoral Tregs suppress tumoral effector T-cell prolif-eration and IFNg secretion. In addition, increased expression ofthe costimulatory receptor OX40 was noted on cSCC Tregs andactivation of OX40 enhanced tumoral CD4þ effector T-cellresponses. Furthermore, increased frequencies of Tregs andOX40þ lymphocytes are observed in primary cSCCs that subse-quently metastasize. The results demonstrate that OX40þ Tregs incSCCs are immunosuppressive and are associated with a propen-sity of cSCCs to metastasize.

Materials and MethodsIHC

Ethical approval for the study was provided by the SouthCentral Hampshire B NRES Committee (reference number07/H0504/187). Archived formalin-fixed paraffin-embedded(FFPE) cSCC samples were obtained from University HospitalSouthampton NHS Foundation Trust. IHC was performed asdescribed previously (21), with microwave antigen retrieval per-formed in either 10 mmol/L citrate or 1.6 mmol/L EDTA buffers.Primary antibodies included anti-CD3 (1:200, DAKO), anti-CD4(1:100, DAKO), anti-CD8 (1:20, Abcam), anti-CD25 (1:50,Novocastra), anti-FOXP3 (1:50, Abcam), and anti-OX40 (1:50,BD Biosciences), and biotinylated goat anti-mouse (1:200,DAKO) or swine anti-rabbit (1:400, DAKO) were used as

secondary antibodies. For cell quantification, five representativeimages at �40 magnification were taken from each immunos-tained cSCC section and analyzed using ImageJ software.

Immunofluorescence/confocal microscopyTissue from freshly excised cSCC was obtained from patients

during surgery at the Dermatology Department, University Hos-pital Southampton NHS Foundation Trust (Southampton, Unit-ed Kingdom). The tissue samples (n ¼ 15 tumors) were snapfrozen in liquid nitrogen, embedded in OCT medium, cryosec-tioned, and immunostained as described previously (22). Prima-ry antibodies included anti-CD3 (1:200, DAKO), anti-CD4(1:50, Abcam), anti-CD8 (1:20, Invitrogen), anti-FOXP3 (1:20,eBioscience), anti-cytokeratin 16 (1:20, Thermo Scientific), anti-cytokeratin 17 (1:20,Dako), anti CD31 (1:200, eBioscience), anti-CLA (1:200, Biolegend), anti-E-selectin (1:20, R&D Systems), andanti-OX40 (1:200, BD Biosciences). Fluorophore-conjugated sec-ondary antibodies comprised Alexa Fluor 488 goat anti-mouseIgG1a, Alexa Fluor 488 goat anti-rat IgM, Alexa Fluor 555 goatanti-rabbit IgG, Alexa Fluor 555 goat anti-rat IgG, and Alexa Fluor633 goat anti-mouse IgG2a (all from Invitrogen). Tissue sectionswere counterstained with DAPI (Sigma) before beingmounted inMowiol 4-88 (Harco) and imaged using a Zeiss Axioskop 2fluorescence microscope or a Leica SP5 confocal microscope.

Lymphocyte isolation/flow cytometryFollowing formal surgical excision of the cSCC (n ¼ 93

tumors), part of the fresh tumor sample was taken and cut intosmall pieces, then enzymatically digested with 1 mg/mL collage-nase I-A (Sigma) and10mg/mLDNAse I (Sigma) inRPMImedium(Gibco) at 37�C for 1.5 hours. The resulting suspension waspassed through 70-mm cell strainers (BD Biosciences) and cen-trifuged in OptiPrep (Axis-Shield). Peripheral blood mononucle-ar cells (PBMC) were isolated from venous blood from the cSCCpatients by centrifugation with Lymphoprep (Axis-Shield).The following fluorophore-conjugated antibodies were used forflow cytometry: anti-CD3 (APC-Cy7, Biolegend), anti-CD4(PerCP-Cy5.5 or FITC, Biolegend), anti-CD8 (PE-Cy7, Biole-gend), anti-CD25 (PE, Invitrogen), anti-CD127 (Brilliant Violet421, Biolegend), anti-FOXP3 (APC, eBioscience), anti-CD45RO(PerCP-Cy5.5, Biolegend), anti-Helios (PerCP-Cy5.5, Biolegend),anti-CLA (Brilliant Violet 421, BD Biosciences), anti-CCR4(PerCP-Cy5.5, Biolegend or FITC, R&D Systems), and anti-OX40(PE, eBioscience). For cell surface staining, cells were incubatedwith the antibodies in the dark for 30minutes at 4�C in PBSþ 1%BSA þ 10% FBS. For intracellular markers, a FOXP3 stainingbuffer set (eBioscience) was used to fix and permeabilize cellsbefore staining (23, 24). An aqua LIVE/DEAD Viability stain(Invitrogen) was added before flow cytometry using a BDFACSAria.

Generation of anti-human OX40 mAbsFor the production of anti-hOX40mAb, mice were immunized

with hOX40-hFc fusion protein and 293F cells transfected withhOX40 (primary fusion protein in complete Freund adjuvant,subcutaneously; secondary transfected cells in incomplete, sub-cutaneously; final boost fusion protein, intravenously). Spleencells from the immunized mice were fused with NS-1 cells usingconventional hybridoma technology. Plates were screened byELISA using hOX40-hFC fusion protein and positive wells testedfor binding to hOX40-transfected 293F cells. Agonist mAb were

Translational Relevance

Cutaneous squamous cell carcinoma (cSCC) is a highlyprevalent cancer that is capable of metastasizing despite treat-ment by surgical excision. Alterations in immunity greatlyinfluence cancer development, especially cSCC development.As cSCCs are heavily infiltrated with T cells, cSCCs form anattractive model to study cancer T-cell immunology. In thisstudy, increased Treg frequencies were identified in cSCCscompared with peripheral blood. Moreover, tumoral Tregssuppressed tumoral effector T-cell responses in vitro, indicatinga role for Tregs in preventing effector T-cell response againstthis tumor. Significantly more Tregs were observed in primarycSCCs that had metastasized than in primary cSCCs that hadnot metastasized, suggesting that tumoral Tregs may influenceclinical outcome in this tumor. In addition, an agonistic anti-OX40 antibody enhanced tumoral CD4þ effector T-cellresponses in vitro, supporting a rationale for investigatingOX40 activation as a potential immunotherapeutic strategyfor cSCC.

Lai et al.

Clin Cancer Res; 2016 Clinical Cancer ResearchOF2




identified by their ability to enhance T-cell proliferation usingcultured PBMCs obtained from healthy volunteers that werestimulated with suboptimal concentrations of anti-CD3 mAb.

Functional assaysTumoral CD3þCD4þCD25highCD127low Tregs and CD3þ

CD4þCD25low or CD3þCD8þ effector T cells were sorted by FACSusing the `purity' setting. Ninety-six–well U-bottomed plateswereused for coculture assays and all functional experiments wereperformed using triplicate wells. Tumoral effector T cells (2,500per well) were cocultured with/without tumoral Tregs (1,250 perwell) in the presence of irradiated (47 Gy) autologous PBMCs(25,000 per well), stimulated with 1 mg/mL phytohemagglutinin(PHA, Sigma), or 1 mg/mL soluble anti-CD3 (eBioscience). Afterculture for 48 hours, tritiated thymidine-based lymphocyte pro-liferation assayswere performed as described previously (25); thismethod of measuring cell proliferation was used because thelimited numbers of lymphocytes isolated from the tumor samplesmeant that carboxyfluorescein diacetate succinimidyl ester(CFSE) dilution experiments were not feasible. IFNg enzyme-linked immunospot (ELISPOT) assays were conducted asdescribed previously (26) with cocultures of tumoral Tregs, effec-tor T cells, and CD3þ cell–depleted irradiated autologous PBMCsstimulated as above. For experiments with anti-OX40 antibody,tritiated thymidine uptake and IFNg ELISPOT assays were per-formed with tumoral CD4þ T cells cultured with/without anti-OX40 (5 mg/mL) or mouse IgG1 (Biolegend) and stimulated asabove in the presence of accessory cells.

Statistical analysisStatistical analysis was conducted using GraphPad Prism soft-

ware. For functional assays, paired Wilcoxon rank tests were usedto assess significance, and for IHC and flow cytometric quantifi-cation of normally distributed data, paired t tests or one-wayANOVA with Tukey test for multiple comparisons were used.

ResultsQuantification of Tregs in cSCCs

cSCCs are infiltrated by immune cells, with IHC conducted onarchived FFPE cSCCs showing accumulationofCD3þT cells in thestroma around islands of tumor keratinocytes (i.e., peritumoral),whereas CD3þ T cells within tumor cell nests (i.e., intratumoral)were found less frequently (Fig. 1A and B and SupplementaryFig. S1A and S1B). In addition to CD4þ T cells and CD8þ T cellswithin the tumoral infiltrate (i.e., combination of peritumoraland intratumoral infiltrate), frequent numbers of CD25þ andFOXP3þ Treg populations were also present (Fig. 1C and D andSupplementary Fig. S1C and S1D). Quantification of the T-cellsubsets that comprised the tumoral immune infiltrate in 24 cSCCsdemonstrated that CD3þ, CD4þ, and CD8þ T cells accounted for40.5% � 10.3%, 36.6% � 7.1%, and 22.0% � 7.5% of theinfiltrate, respectively, whereas FOXP3þ Tregs comprised 11.0%� 4.4% of the immune infiltrate, indicating a Treg to CD4þ T-cellratio of approximately 1:3 and a Treg: CD8þ T-cell ratio of 1:2(Fig. 1C). To determine whether there were differences in Tregnumbers between precancerous and cancerous skin lesions, IHCwas performed which demonstrated that actinic keratoses (AK)contained similar FOXP3þ Treg frequencies to cSCCs (24.1% �14.2% of AK immune infiltrate vs. 29.2% � 19.4% of cSCCimmune infiltrate, n¼ 44 AKs, n¼ 79 cSCCs, P¼ 0.131, Fig. 1D).

Using anti-cytokeratin 16 to delineate the cSCC tumor islands,immunofluorescence microscopy showed the presence ofFOXP3þ Tregs and CD3þ T cells in the stroma surrounding thecSCCs with fewer of these cells within the tumor islands (Fig. 1Eand F). Tregs exert suppressive function via contact-dependentmechanisms (27), although noncontact mechanisms may alsoplay a role in some instances (28). Confocal microscopy of cSCCsindicated close approximation of tumoral Tregs (identified bynuclear FOXP3 and cell membrane CD4) and CD4þFOXP3�

T cells and, separately, CD8þ T cells (Fig. 1G), suggesting con-tact-dependent interactions between Tregs and effector T cells inthis tumor. Flow cytometry of tumoral immunocytes from freshlyexcised cSCCs demonstrated that cSCCs contained lower CD4þ

T-cell frequencies than peripheral blood of the same individuals(55.7%� 18.7% vs. 70.9%� 17.8%of the live CD3þ lymphocytepopulation, respectively, P¼ 0.0211, n¼ 17 tumors, Fig. 1H andI). In contrast, Tregs were more numerous in cSCCs than thecorresponding peripheral blood and nonlesional skin (19.8% �8.6% vs. 5.5%� 4.1% and 7.3%� 4.1%of the CD4þ population,respectively expressed FOXP3, P < 0.0001 for both comparisons,n ¼ 86 tumors, Fig. 1J and K), indicating that in cSCC, there is anaccumulation of tumoral Tregs, potentially contributing to theprotumorigenic microenvironment. Tregs were also significantlymore frequent in BCC than in corresponding peripheral blood(16.7%�7.7%vs. 2.8%�2.2%ofCD4þpopulation for BCCandblood, respectively, P ¼ 0.0045 n ¼ 6, Fig. 1L). Although Tregsseemedmore frequent in keratoacanthoma (KA, a skin lesion thatclinically resembles cSCC but is known to regress spontaneously)than in corresponding blood (11.2% � 7.5% vs. 4.4%� 3.3% ofCD4þ population for KA and blood, respectively, n ¼ 6, Fig. 1L),there were significantly overall fewer Tregs in KA than in cSCC(P ¼ 0.0321, Supplementary Fig. S1E).

Tregs in cSCCs arememory T cells that express the skin homingmarker CLA

Many of the T cells within normal skin are effector memoryT cells that express CD45RO (29). In the current study, themajority of FOXP3þ Tregs (87.8% � 11.7%) and CD4þFOXP3�

T cells (85.7%� 7.3%) in cSCCs were CD45ROþ (n¼ 8 tumors),whereas 77.6%� 16.7% of FOXP3þ Tregs and 52.7%� 19.3% ofCD4þFOXP3� T cells in peripheral blood were CD45ROþ (P ¼0.0171 and P¼ 0.0013, respectively, for FOXP3þ Tregs and CD4þ

FOXP3� T cells comparison between cSCCs and peripheral bloodfrom same individuals; Fig. 2A–C). The T-cell marker Helios maydenote thymically derived natural Tregs (24) but can also beupregulated on activated T cells (30). Flow cytometry establishedthat Helios was expressed at higher levels in tumoral FOXP3þ

Tregs (47.2% � 7.9%, n ¼ 9 tumors) than peripheral bloodFOXP3þ Tregs (26.7% � 8.5%, P ¼ 0.0022) and tumoral non-regulatory CD4þFOXP3� T cells (9.6%� 5.8%, P < 0.0001) fromthe same subjects, (Fig. 2D and E).

Clark and colleagues (29) have reported that T cells, includingTregs, from cSCCs lack expression of the skin homing addressins,cutaneous lymphocyte antigen (CLA), and C-C chemokine recep-tor 4 (CCR4). We observed that CLA was expressed by 78.8% �13.0% of the FOXP3þ Tregs population in cSCCs, which washigher than the proportion of FOXP3þ Tregs (40.0% � 13.7%)expressing CLA in peripheral blood (P < 0.0001, n ¼ 19tumors; Fig. 2F and G). The latter is similar to that reported byBooth and colleagues in blood (23). Likewise, greater numbers ofCD4þFOXP3� T cells (58.2% � 15.1%) in cSCC expressed CLA

OX40þ Tregs in Cutaneous Squamous Cell Carcinoma

www.aacrjournals.org Clin Cancer Res; 2016 OF3




Lai et al.





than those in peripheral blood (P < 0.0001, n¼ 19 tumors, Fig. 2Fand H). Furthermore, experiments performed in a separate insti-tution on a different set of cSCCs (n ¼ 13) identified CLApositivity in 64.7% � 31.2% of tumoral CD3þ T cells (Supple-mentary Fig. S2A and S2B). CCR4 expression was detected on44.1% � 32.7% of FOXP3þ Tregs in cSCCs, although this waslower than the number of FOXP3þ Tregs expressing CCR4 inperipheral blood (72.7% � 17.8%; P ¼ 0.0139, n ¼ 17tumors; Fig. 2F and I). Equal numbers of tumoral CD4þFOXP3�

T cells (25.4% � 21.4%) and peripheral blood CD4þFOXP3� Tcells (27.6% � 19.8%) expressed CCR4 (Fig. 2J). Further experi-mentswere performed to confirm the expression of CLA andCCR4in cSCC Tregs; CCR4 was detected on cSCC Tregs at similar levelswith an alternate anti-CCR4 antibody and immunofluorescence/confocal microscopy using a different anti-CLA antibody showedCLAþ cells in cSCCs (Fig. 3A) and that the tumoral FOXP3þ cellsexpressed CLA (Fig. 3B and Supplementary Fig. S2C).

CLA permits lymphocyte trafficking to the skin through itsbinding to E-selectin on cutaneous endothelial cells (29). Immu-nofluorescence microscopy was performed using cytokeratin(CK) 16 or 17 costaining that allows the cSCC tumor cells to bevisualized. Blood vessels (highlighted by CD31þ endothelialcells) were detected in the peritumoral areas of cSCCs with noevidence of intratumoral vessels (Fig. 3C and Supplementary Fig.S3A and S3B), and that most of the peritumoral vasculatureexpressed E-selectin (Fig. 3D and Supplementary Fig. S4), suggest-ing that CLAþ Tregs can readily be directed to the site of the tumorfrom the blood via interaction with E-selectin on these endothe-lial cells.

cSCC Tregs suppress tumoral effector T-cell responsesIn vitro coculture experiments with Tregs and effector T cells

were performed to investigate cSCCTreg function. cSCCTregs andeffector T cells were cocultured in a 1:2 ratio based on their relativefrequencies observed in the prior IHC quantification experiments(Fig. 1C). Tumoral Tregs were identified by expression of CD3,CD4, high levels of CD25, and low levels of CD127 and isolatedusing FACS (Fig. 4A). Sorted tumoral CD4þ effector T cellsidentified as CD3þCD4þCD25low and CD8þ effector T cells wereCD3þCD8þ (Fig. 4A). After sorting, a sample of the cells werefixed and permeabilized for analysis of FOXP3 expression, con-firming that most of the sorted CD3þCD4þCD25highCD127low

cellswere Tregs (Fig. 4B and Supplementary Fig. S5A). In addition,IFNg was produced by <4% of tumoral CD3þCD4þCD25highC-D127low cells following PMA and ionomycin stimulation, sug-gesting that this CD3þCD4þCD25highCD127low population wasminimally contaminated by effector T cells (Fig. 4C). Tritiatedthymidine-based lymphocyte proliferation assays showed that

tumoral CD3þCD4þCD25highCD127low Tregs were able to sup-press PHA-induced proliferation of tumoral CD3þCD4þCD25low

effector T cells (median suppression 41.7%, n ¼ 10 tumors, Fig.4D) and, to a lesser extent, CD3þCD8þ effector T cells (mediansuppression 12.6%, P ¼ 0.043, n ¼ 9 tumors, Fig. 4E). TumoralTregs also suppressed proliferation of anti-CD3 stimulatedtumoral CD4þ effector T cells (median suppression 46.2%,n¼ 4 tumors; Supplementary Fig. S5B) and CD8þ T cells (mediansuppression 40.2%, n ¼ 4 tumors; Supplementary Fig. S5C). Inaddition, ELISPOT assays demonstrated that tumoral Tregsreduced effector T-cell IFNg secretion in response to PHA (medianinhibition 24.2%, P ¼ 0.0186, n ¼ 11 tumors, Fig. 4F). Theseresults indicate that tumoral Tregs from cSCCs can suppresstumoral effector T-cell function, and may therefore contribute toan immunosuppressive milieu that prevents immune-mediateddestruction of the tumor.

OX40 is expressed by cSCC Tregs and OX40 agonism enhancestumoral CD4þ T-cell function

As the costimulatory receptor OX40 is expressed on effectorand regulatory T cells and can augment T-cell receptor signaling(15–19), we next investigated whether OX40 was present ontumoral lymphocytes in cSCC. Immunofluorescence microscopydemonstrated the presence of OX40 predominantly on tumoralFOXP3þ Tregs (Fig. 5A). Flow cytometry confirmed FOXP3þ Tregsin cSCC expressedOX40 (39.3%� 13.6%of FOXP3þ Tregs), withsignificantly more tumoral Tregs expressing OX40 than CD4þ

FOXP3� T cells and CD8þ T cells in cSCCs, and FOXP3þ Tregs,CD4þFOXP3� T cells and CD8þ T cells in peripheral blood(P < 0.0001 for all comparisons, n ¼ 15 tumors, Fig. 5B and Cand Supplementary Fig. S5D). To assess whether OX40 agonismattenuates the suppressive effects of Tregs in cSCC,we assessed theproliferation of tumoral CD4þ T cells from cSCCs in the presenceof an agonistic anti-OX40 mAb. The addition of anti-OX40, butnot an isotype control mAb, led to enhancement of PHA-inducedCD4þ T-cell proliferation (median increase in proliferation 45%,P ¼ 0.0098, n ¼ 10 tumors, Fig. 5D); proliferation of CD4þ

CD25highCD127low Tregs was not increased by anti-OX40 whenculturedwith PHA in the presence of accessory cells alone [isotypecontrol ¼ 108.5 cpm (IQR 68.0–129.5 cpm), anti-OX40 ¼ 107cpm (IQR 73.3–135.5 cpm), n ¼ 4 tumors; SupplementaryFig. S5D). Subsequently, tumoral CD4þCD25low effector T-cellproliferation was measured following culture with PHA � anti-OX40 in the absence or presence of tumoral CD4þCD25highC-D127low Tregs. In cultures containing tumoral CD4þCD25low Tcells without Tregs, median cell proliferation increased by 5.3%with the addition of anti-OX40 compared with isotype control,whereas in cultures containing tumoral CD4þCD25low T cells and

Figure 1.Tregs accumulate in cSCC and cluster with CD4þ and CD8þ T cells predominantly in the peritumoral stroma. IHC of cSCC demonstrating CD3þ T cells in (A)intratumoral and (B) peritumoral areas. C, immunohistochemical quantification of T-cell subsets in 24 cSCCs. D, no differences in frequencies of infiltratingFOXP3þ cells were observed between actinic keratoses (AKs, n ¼ 44) and cSCCs (n ¼ 79). Immunofluorescence staining demonstrating (E) CD3þ T cells and (F)FOXP3þ Tregs in cSCC. Cytokeratin (CK) 16 staining highlights tumor keratinocytes. G, confocalmicroscopy of CD4þ T cells, CD8þ T cells, and CD4þFOXP3þ T cells incSCC, boxes highlight examples of CD4þFOXP3þ Tregs (red membrane, green nucleus) in close contact with CD8þ T cells (blue membrane, gray nucleus)and CD4þFOXP3� T cells (red membrane, gray nucleus). H, flow cytometric plots of CD3þ gated lymphocytes isolated from peripheral blood and matched cSCCdemonstrating CD4 and FOXP3 expression. I, aggregate data showing fewer CD4þ T cells as a proportion of the CD3þ population in cSCCs compared withblood (n ¼ 17). J, CD3þCD4þ gated lymphocyte populations in blood and corresponding cSCC plotted for FOXP3 and CD25 expression. K, data from32 cSCCs demonstrating higher FOXP3þ Treg frequencies as a percentage of the CD4þ T-cell population in cSCC than blood. L, FOXP3þ Treg frequencies inkeratoacanthoma (KA), basal cell carcinoma (BCC), and corresponding blood. In images A, B, E, F, and G, scale bars¼ 50 mm, dashed lines indicate tumor outlines.In graphs C and D, dots ¼ mean cell frequencies in five separate 40� fields for each lesion, horizontal bars ¼ mean values for all lesions. In graphs I–L,horizontal bars ¼ means.






Tregs, the improvement in effector T-cell function with the addi-tion of anti-OX40 was more apparent (median increase in cellproliferation ¼ 252.4% compared with isotype control, P ¼

0.0313, n ¼ 5 tumors; Fig. 5E and Supplementary Fig. S5E).Similar results were also observed when anti-CD3 was used asa stimulus instead of PHA, with anti-OX40 increasing tumoral

Figure 2.Phenotypic characterization of cSCC Tregs. A, FACS plots showing CD45RO expression on CD3þCD4þ gated lymphocyte populations from peripheral bloodand cSCC. CD45RO positivity as a percentage of CD4þFOXP3þ Treg (B) and CD4þFOXP3� (C) T-cell populations from blood and cSCC (n¼ 8), showing that thesepopulations in cSCCs aremainly CD45ROþmemory T cells. D, representative histograms demonstratingHelios expression in CD3þCD4þFOXP3þ gated lymphocytesfromperipheral blood and corresponding cSCC. Gray shaded areas represent isotype control. E, percentage of tumoral CD4þFOXP3þTregs, tumoral CD4þFOXP3�Tcells, and peripheral blood CD4þFOXP3þ Tregs that are Heliosþ (n ¼ 9). F, representative plots demonstrating CLA and CCR4 expression in CD3þ and CD3þ

CD4þFOXP3þ gated lymphocytes from peripheral blood and cSCC from the same subject. Graphs showing aggregate data for CLA expression in peripheral bloodand tumoral CD3þCD4þFOXP3þ Tregs (G) and CD3þCD4þFOXP3� T cells (n ¼ 19; H), and CCR4 expression in peripheral blood and tumoral CD3þCD4þFOXP3þ

Tregs (I) and CD3þCD4þFOXP3� T cells (n ¼ 19; J). Horizontal bars in graphs B, C, E, and G–J, means.

Lai et al.





effector CD4þ T-cell proliferation from 5474 to 6572 cpm(20.1%) when Tregs were absent, and from 2,906 to 5,263 cpm(81.1%), when Tregs were present, n¼ 4 tumors (SupplementaryFig. S5F). Furthermore, the increase in IFNg spot production bytumoral CD4þ effector T cells with the addition of anti-OX40 wasmore apparent in the presence of tumoral Tregs (53.5with isotypevs. 93.7 with anti-OX40) than in the absence of tumoral Tregs(114.2 with isotype to 131.5 with anti-OX40, Fig. 5F). Taken incombination with the fact that OX40 was found mainly on Tregsin cSCCs, these results suggest that OX40 agonism reduces Treg-mediated suppression of tumoral CD4þ T-cell responses.

Increased Treg frequencies are associated with primary cSCCsthat metastasize

Todeterminewhether Tregs in cSCCs are associatedwithpoorerclinical outcome, Tregs were quantified in archived FFPE primarycSCCs that hadmetastasized and in cSCCs that were knownnot tohave metastasized after 5 years. As expected, histologic datademonstrated differences in known prognostic factors betweenthemetastatic and nonmetastatic groups, with primarymetastaticcSCCs being larger, invading deeper, and being more poorlydifferentiated than nonmetastatic cSCCs (Fig. 6A). Nevertheless,there were significantly higher frequencies of FOXP3þ Tregs inprimary cSCCs that metastasized than those that did not metas-tasize (49.3% � 13.8% vs. 23.5% � 11.0% of immune infiltrate,respectively, P < 0.0001, n ¼ 29 and 26 tumors, respectively)despite similar frequencies of CD3þ T cells in both groups (Fig.6B–D), suggesting that increased Treg numbers in primary cSCCsmay influence the development of subsequent metastasis. OX40-expressing cells were also quantified in FFPE primary cSCCs, withincreased percentages of tumoral immunocytes expressing OX40observed in primary metastatic cSCCs (17.0% � 10.7% ofimmune infiltrate, n ¼ 48 tumors) than primary nonmetastaticcSCCs (11.7% � 6.9% of immune infiltrate, n ¼ 49 tumors, P ¼0.0041, Fig. 6B and E).

DiscussionFactors that predispose to skin cancer development in humans

include light skin pigmentation secondary to genetic polymorph-isms in genes such asMC1R, UV radiation exposure, which causesDNA damage/gene mutation in relevant skin cells and altered/suppressed immunity (6–8, 21, 31). cSCCs have one of thehighest mutational burdens compared with other tumor types(6–8) and high frequencies of UV-inducedmutations could resultin neoantigens that can be recognized by T cells (32). Indeed, theimportance of the immune system in cSCC development isexemplified by the markedly increased prevalence of this tumortype in immunosuppressed individuals (9).However, the fact thatcSCCs are frequently accompanied by a substantial immuneinfiltrate in most individuals (including nonimmunosuppressedsubjects who constitute the majority of cSCC cases) highlighted aneed to characterize this infiltrate from a functional perspective.

On thebasis of the viewpoint that local cutaneous immunity playsa key role in preventing cSCCdevelopment and that dysregulationof this local immunity may allow the expansion of neoplastickeratinocytes to go unchecked, we investigated the Treg popula-tion within cSCCs. Our findings demonstrate that the cSCCtumoral immune infiltrate contains higher Treg frequencies thanperipheral blood, which is in keeping with studies indicating thatTregs may accumulate in various tumors via several mechanismsand with reports that exposure of skin to UV is conducive to thegeneration of Tregs (33). Functionally immunosuppressive Tregshave been reported in human cancer previously (10, 34–36) andthis current study confirms that cSCC tumoral Tregs have thecapacity to suppress tumoral effector T-cell function in vitro,therefore highlighting Tregs as a mechanism by which cSCCs canavoid destruction by the skin immune system. In addition, higherTreg frequencies were found to be associated with primary cSCCsthat had metastasized rather than primary cSCCs that had notmetastasized, indicating that Tregs may play a role in allowingcSCC to metastasize and thus are of potential prognosticimportance.

This study showed no difference in Treg numbers betweenactinic keratoses and cSCC, as reported previously (37), suggest-ing that Tregs accumulate during a precursor lesion stage, that is,actinic keratoses, rather than being a determinant of progressionfrom actinic keratosis to cSCC. However, we found an associationbetween higher Treg frequencies in primary cSCCs and subse-quent metastasis, in keeping with studies in other cancer typeswhere Tregs have been shown to promote metastasis and influ-ence clinical outcome (10–13, 38–40). Related to this, althoughwe found an association between the histologic differentiationstatus of cSCC and Treg infiltration, which was similar to a recentstudy (41), the association did not remain after excluding cSCCswith known clinical outcome (i.e., had metastasized or notmetastasized at 5 years; Supplementary Fig. S6), suggesting amore robust association was present between Treg infiltrationand metastasis than that with cSCC differentiation status in ourstudy.

Although Gelb and colleagues reported that lymphocyte-infil-trating cSCC express CLA (42), a more recent investigationrecorded that T cells infiltrating cSCCs are noncutaneous centralmemory T cells, which lack expressionofCLAandCCR4 (29).Ourphenotypic characterization of tumoral Tregs in cSCC showedthat most expressed CD45RO and CLA, indicating a skin residentmemory phenotype (which is the predominant T-cell phenotypein skin) in cSCCs. We confirmed the expression of CLA on cSCCTregs using both flow cytometry and immunofluorescencemicroscopy with two different anti-CLA antibodies (from BDBiosciences and Biolegend). However, our results demonstratea high degree of variability in CLA and CCR4 expression betweentumors, suggesting heterogeneous T-cell responses in cSCC, pos-sibly accounting for the differences between previous studies. Inthe context of the E-selectin expression by the majority of theperitumoral blood vessels, our findings signify that Tregs are

Figure 3.cSCCTregs express the skin homingmarker CLA. A, immunofluorescencemicroscopyof cSCC cryosections showingCLAþ tumoral immunocytes; cytokeratin (CK) 16highlights tumor keratinocytes. B, immunofluorescence microscopy demonstrating CLAþ FOXP3þ Tregs in cSCC. C, CD31þ staining highlighting peritumoralblood vessels in cSCC stroma, cytokeratin (CK) 17 positivity indicates tumor keratinocytes. D, sequential cSCC sections showing e-selectin expression in theperitumoral vasculature (highlighted by CD31 straining) between cytokeratin (CK) 16-positive tumor islands. Arrows in the right hand boxes, which depicthigher power images of the merged images, indicate the same blood vessels in sequential sections. Dashed lines represent the outlines of the tumor in A to D,scale bars ¼ 50 mm.

Lai et al.





generally recruited to cSCCs via CLA on the Treg surface interact-ingwithE-selectinon the tumoral vasculature. This is akin to T-cellrecruitment in inflammatory skin conditions including psoriasis(43), atopic dermatitis (44), and contact dermatitis (45) aswell asin other cutaneous neoplasms such as melanoma (42).

There has been considerable interest in developing cancertreatments that use monoclonal antibodies, which act on T-cellcostimulatory pathways to augment antitumor immunity (15–20). Indeed, effector T-cell responses can be modulated throughengaging costimulatory receptors that can attenuate Treg-suppres-sive ability (15–19, 46). In vitro studies of human peripheral

blood T cells have demonstrated that anti-OX40 mAbs can beagonistic and can enhance the resistance of effector T cells tosuppression and reduce the suppressive activity of Tregs (19). Inaddition, anti-OX40 mAbs could stimulate antitumor immunityby preferential depletion of Tregs, as shown in preclinical studies(17, 18). Furthermore, a phase I clinical trial using an anti-OX40agonisticmAb has shown an acceptable safety/toxicity profile andsome evidence of tumor regression in cases of melanoma, renalcancer, urethral squamous cell carcinoma, prostate cancer andcholangiocarcinoma (20). In our study, the tumoral Tregs werethe T-cell subset that expressed OX40 at the highest frequencies,

Figure 4.cSCCTregs suppress PHA-induced tumoral effector T-cell responses in vitro. A, representative flowcytometry gating strategy for isolating cSCCTregs and effector Tcells. Highlighted box in the left FACS plot shows the CD3þCD4þ population, and subgating of this population is displayed in the right FACS plot, where the sortedCD25highCD127low Treg population is shown in the highlighted area. Effector T cells were CD3þCD4þCD25low or CD3þCD8þ. B, the majority of sorted CD3þ

CD4þCD25highCD127low cells expressed FOXP3, consistent with their Treg status. C, sorted CD3þCD4þCD25highCD127low cells were stimulated with PMA andionomycin for 5 hours and intracellular flow cytometry was performed for FOXP3 and IFNg . D–F, tumoral effector T cells were cocultured in the presence ofautologous irradiated PBMCs and 1 mg/mL PHA with/without the addition of tumoral Tregs. Tritiated thymidine uptake was used to assess tumoral CD4þ T-cellproliferation, n ¼ 10 tumors (C), tumoral CD8þ T-cell proliferation, n ¼ 9 tumors (D), and ELISPOT assays to determine tumoral effector T-cell IFNg secretion (E),n ¼ 11 tumors. In C–E, dots, median values for each tumor from triplicate well experiments; horizontal bars, median values for all tumors.






consistent with observations from studies on murine tumorswhich used anti-OX40 antibodies to deplete intratumoral Tregsand enhance antitumor immunity (16–18), suggesting a similarstrategy could be beneficial in cSCC. OX40 has been demonstrat-ed on CD4þ tumor-infiltrating lymphocytes in melanoma andhead and neck cancer (47), and whereas a recent study hasdocumented the presence of OX40-expressing lymphocytes incSCC (48), our work highlights that this molecule is predomi-nantly expressed by Tregs in this cancer. Furthermore, our studyshows that higher percentages of tumor-infiltrating lymphocytesexpress OX40 in primary metastasizing cSCCs compared with

primary tumors that had not metastasized. Although this con-trasts with some other cancers in which OX40 expression corre-lates with improved prognosis (49–51),muchhigher proportionsof tumoral Tregs expressedOX40 than the tumoral CD4þFOXP3�

and CD8þ T cells in our cSCC population. Furthermore, usingan in vitro culture model, enhanced tumoral CD4þ T-cellresponses were observed with addition of an OX40 agonist,suggesting that the use of an OX40 agonist approach in vivo mayhave potential benefits in patients with cSCCs at high risk ofmetastasizing after surgical excision. Admittedly, although OX40agonism had little effect on enhancing proliferation of tumoral

Figure 5.OX40 is expressed by Tregs in cSCC and in vitro OX40 activation enhances tumoral CD4þ T-cell proliferation. A, immunofluorescence microscopy showingOX40 on tumoral FOXP3þ Tregs. Dashed lines indicate the outline of the cSCC tumor islands, scale bars, 50 mm. B, flow cytometric plots of CD3þCD4þ gatedlymphocytes from cSCC and corresponding peripheral blood showing OX40 and FOXP3 expression. C, FACS quantification of OX40 expression in tumoralTregs, tumoral effector T cells, and peripheral blood Tregs, n¼ 15 tumors; horizontal bars, means. D, tumoral CD4þ T cells isolated by flow cytometry from 10 freshcSCCs were cultured with 1 mg/mL PHA þ autologous irradiated CD3þ cell depleted irradiated PBMCs � agonistic anti-OX40 antibody (5 mg/mL). Proliferationwas assessed by tritiated thymidine uptake; dots, median values from triplicate wells for each tumor; horizontal bar, median value from all tumors. E, tritiatedthymidine uptake assays were performed where tumoral CD4þ effector T cells were cultured� tumoral Tregs� agonistic anti-OX40 antibody. Median values fromtriplicate wells are shown and represented as normalized to 100% of CD4 Teff þ Treg þ iso, n ¼ 5 tumors. F, IFNg ELISPOT assays, showing that the effect of theagonistic OX40 antibody is more apparent when tumoral Tregs are present in culture. Dots/circles, median values for each tumor with circles for isotypecontrol, squares for anti-OX40, horizontal bar ¼ median for all tumors. Error bars ¼ IQR; n ¼ 4 tumors.

Lai et al.





CD4þ effector T cells and IFNg production in the absence of Tregs,it is unclear whether part of the effect of OX40 agonism could bemediated by the presence of OX40þFOXP3� cells in the sortedCD3þCD4þCD25highCD127low population (Supplementary Fig.S5D). This is because, due to FOXP3 being an intracellular stain, it

was not possible to isolate the FOXP3� fraction of the CD3þ

CD4þCD25highCD127low population from human cSCCs andperform functional studies using this subset alone. Neither is itclear whether the effect of OX40 agonism is mediated by OX40þ

CD4þ effector T cells in overcoming or influencing Treg

Figure 6.Higher numbers of tumoral Tregs are associated with subsequent development of metastasis from primary cSCCs. IHC was conducted on archived FFPE sections ofprimary cSCCs that had not metastasized at 5 years postexcision (primary nonmetastatic) and primary cSCCs which had metastasized (primary metastatic)postexcision. A, table showing histologic details of primary nonmetastatic and primary metastatic cSCCs. B, representative images of tumoral CD3þ T cells,FOXP3þ Tregs, and OX40þ immunocytes in primary nonmetastatic and primary metastatic cSCCs, scale bars ¼ 50 mm. Quantification of tumoral CD3þ T cells (C),FOXP3þTregs (D) in 26primary nonmetastatic, and 29primarymetastatic cSCCs, andOX40þ cells (E) in 49primary nonmetastatic and48primarymetastatic cSCCs.Dots on the graphs indicate mean values for each tumor from five high-power fields; horizontal bars represent mean values for each group.






suppression; however, our results indicate that the effect of OX40agonism in boosting the effector T-cell response is only seen in thepresence of the CD3þCD4þCD25highCD127low Treg population.

In conclusion, this study demonstrates that cSCCs contain anabundance of Tregs that can suppress tumoral effector T-cellfunction and that activation of the costimulatory receptor OX40enhances tumoral T-cell responses. Primary cSCCs that metasta-size are associated with higher Treg frequencies, therefore pro-viding evidence that Tregs play a key role in the pathogenesis ofcSCC.

Disclosure of Potential Conflicts of InterestA. Al-Shamkhani reports receiving commercial research grants from Celldex

Therapeutics. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: C. Lai, S. August, A.S. MacLeod, M.J. Glennie,A. Al-Shamkhani, E. HealyDevelopment of methodology: C. Lai, S. August, M.E. Polak, E. HealyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Lai, S. August, R. Behar, S.-Y. Cho, J. Theaker,A.S. MacLeod, R.R. French, A. Al-Shamkhani, E. HealyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Lai, S. August, A. Albibas, R. Behar, S.-Y. Cho,M.E. Polak, J. Theaker, A.S. MacLeod, R.R. French, A. Al-Shamkhani, E. HealyWriting, review, and/or revision of the manuscript: C. Lai, S. August,A. Albibas, R. Behar, S.-Y. Cho, J. Theaker, A.S. MacLeod, R.R. French, M.J.Glennie, A. Al-Shamkhani, E. HealyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Lai, S. August, S.-Y. Cho, E. Healy

Study supervision: A. Al-Shamkhani, E. HealyOther (doing some lab work): A. AlbibasOther (Carried out part of the research): R. Behar

AcknowledgmentsThe authors thank Richard Jewell and Carolann McGuire in Dermatophar-

macology, University of Southampton; Ian Mockeridge and Dima Taraban inCancer Sciences, University of Southampton; Susan Wilson and JonWard fromthe Histochemistry Research Unit, University of Southampton; and DaveJohnston from the Biomedical Imaging Unit, University of Southampton fortheir technical support. The authors also thank Trevor Bryant and Scott Harris,Medical Statistics, University of Southampton, for providing statistical adviceand the administrative and nursing staff, including Margaret Wheeler andAmanda Clowes, in the Dermatology Department, University Hospital South-ampton NHS Foundation Trust for assistance with procurement of samplesfrom patients.

Grant SupportC. Lai was supported by a Wellcome Trust Research Training Fellowship.

Support was also provided from National Institute for Health Research Aca-demic Clinical Fellowships (to C. Lai, S. August, and S.-Y. Cho), the Ministry ofHigher Education and Scientific Research – Libya (to A. Albibas), and CancerResearch UK (to R.R. French, A. Al-Shamkhani, M.J. Glennie). A.S. MacLeodwas supported by a NIH K08 award (5K08 AR063729) and by the Skin DiseaseResearch Center at Duke University.

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.

ReceivedOctober 27, 2015; revisedMarch 14, 2016; acceptedMarch18, 2016;published OnlineFirst March 31, 2016.

References1. Lucas R, McMichael T, Smith W, Armstrong B. Solar ultraviolet radiation:

Global burden of disease from solar ultraviolet radiation. Geneva, Switzer-land:WorldHealthOrganization Public Health and the Environment; 2006.

2. Rogers HW,WeinstockMA,Harris AR, HinckleyMR, Feldman SR, FleischerAB, et al. Incidence estimate of nonmelanoma skin cancer in the UnitedStates, 2006. Arch Dermatol 2010;146:283–7.

3. Bickers DR, LimHW, Margolis D, Weinstock MA, Goodman C, Faulkner E,et al. The burden of skin diseases: 2004 a joint project of the AmericanAcademy of Dermatology Association and the Society for InvestigativeDermatology. J Am Acad Dermatol 2006;55:490–500.

4. Alam M, Ratner D. Cutaneous squamous-cell carcinoma. N Engl J Med2001;344:975–83.

5. Brantsch KD, Meisner C, Schonfisch B, Trilling B, Wehner-Caroli J, RockenM, et al. Analysis of risk factors determining prognosis of cutaneoussquamous-cell carcinoma: a prospective study. Lancet Oncol 2008;9:713–20.

6. Durinck S, Ho C, Wang NJ, Liao W, Jakkula LR, Collisson EA, et al.Temporal dissection of tumorigenesis in primary cancers. Cancer Discov2011;1:137–43.

7. Li YY,HannaGJ, Laga AC,Haddad RI, Lorch JH,HammermanPS.Genomicanalysis ofmetastatic cutaneous squamous cell carcinoma. Clin Cancer Res2015;21:1447–56.

8. Pickering CR, Zhou JH, Lee JJ, Drummond JA, Peng SA, Saade RE, et al.Mutational landscape of aggressive cutaneous squamous cell carcinoma.Clin Cancer Res 2014;20:6582–92.

9. Euvrard S, Kanitakis J, Claudy A. Skin cancers after organ transplantation.N Engl J Med 2003;348:1681–91.

10. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, et al. Specificrecruitment of regulatory T cells in ovarian carcinoma fosters immuneprivilege and predicts reduced survival. Nat Med 2004;10:942–9.

11. Deng L, Zhang H, Luan Y, Zhang J, Xing Q, Dong S, et al. Accumulation offoxp3þ T regulatory cells in draining lymph nodes correlates with diseaseprogression and immune suppression in colorectal cancer patients.Clin Cancer Res 2010;16:4105–12.

12. Bates GJ, Fox SB, Han C, Leek RD, Garcia JF, Harris AL, et al. Quantificationof regulatory T cells enables the identification of high-risk breast cancerpatients and those at risk of late relapse. J Clin Oncol 2006;24:5373–80.

13. Ichihara F, Kono K, Takahashi A, Kawaida H, Sugai H, Fujii H. Increasedpopulations of regulatory T cells in peripheral blood and tumor-infiltratinglymphocytes in patients with gastric and esophageal cancers. Clin CancerRes 2003;9:4404–8.

14. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F,et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med2013;210:1695–710.

15. Taraban VY, Rowley TF, O'Brien L, Chan HT, Haswell LE, Green MH, et al.Expression and costimulatory effects of the TNF receptor superfamily mem-bers CD134 (OX40) and CD137 (4-1BB), and their role in the generation ofanti-tumor immune responses. Eur J Immunol 2002;32:3617–27.

16. Piconese S, Valzasina B, ColomboMP.OX40 triggering blocks suppressionby regulatory T cells and facilitates tumor rejection. J Exp Med 2008;205:825–39.

17. Marabelle A, Kohrt H, Sagiv-Barfi I, Ajami B, Axtell RC, Zhou G, et al.Depleting tumor-specific Tregs at a single site eradicates disseminatedtumors. J Clin Invest 2013;123:2447–63.

18. Bulliard Y, Jolicoeur R, Zhang J, Dranoff G, Wilson NS, Brogdon JL. OX40engagement depletes intratumoral Tregs via activating FcgRs, leading toantitumor efficacy. Immunol Cell Biol 2014;92:475–80.

19. Voo KS, Bover L, Harline ML, Vien LT, Facchinetti V, Arima K, et al.Antibodies targeting human OX40 expand effector T cells and blockinducible and natural regulatory T cell function. J Immunol 2013;191:3641–50.

20. Curti BD, Kovacsovics-Bankowski M, Morris N, Walker E, Chisholm L,Floyd K, et al. OX40 is a potent immune-stimulating target in late-stagecancer patients. Cancer Res 2013;73:7189–98.

21. Robinson S, Dixon S, August S, Diffey B, Wakamatsu K, Ito S, et al.Protection against UVR involves MC1R-mediated non-pigmentary andpigmentary mechanisms in vivo. J Invest Dermatol 2010;130:1904–13.


Lai et al.




22. Pickard C, Louafi F, McGuire C, Lowings K, Kumar P, Cooper H, et al. Thecutaneous biochemical redox barrier: a component of the innate immunedefenses against sensitization by highly reactive environmental xenobio-tics. J Immunol 2009;183:7576–84.

23. Booth NJ, McQuaid AJ, Sobande T, Kissane S, Agius E, Jackson SE, et al.Different proliferative potential and migratory characteristics of humanCD4þ regulatory T cells that express either CD45RA or CD45RO.J Immunol 2010;184:4317–26.

24. Thornton AM, Korty PE, Tran DQ,Wohlfert EA,Murray PE, Belkaid Y, et al.Expression of Helios, an Ikaros transcription factor family member, differ-entiates thymic-derived from peripherally induced Foxp3þ T regulatorycells. J Immunol 2010;184:3433–41.

25. Cooper A, Robinson SJ, Pickard C, Jackson CL, Friedmann PS, Healy E.Alpha-melanocyte-stimulating hormone suppresses antigen-induced lym-phocyte proliferation in humans independently of melanocortin 1 recep-tor gene status. J Immunol 2005;175:4806–13.

26. Newell L, PolakME, Perera J,OwenC, BoydP, PickardC, et al. Sensitizationvia healthy skin programs Th2 responses in individuals with atopicdermatitis. J Invest Dermatol 2013;133:2372–80.

27. Gondek DC, Lu LF, Quezada SA, Sakaguchi S, Noelle RJ. Cutting edge:contact-mediated suppression by CD4þCD25þ regulatory cells involves agranzyme B-dependent, perforin-independent mechanism. J Immunol2005;174:1783–6.

28. Delgoffe GM,Woo SR, Turnis ME, Gravano DM, Guy C, Overacre AE, et al.Stability and function of regulatory T cells ismaintained by a neuropilin-1-semaphorin-4a axis. Nature 2013;501:252–6.

29. Clark RA, Huang SJ, Murphy GF, Mollet IG, Hijnen D,MuthukuruM, et al.Human squamous cell carcinomas evade the immune response by down-regulation of vascular E-selectin and recruitment of regulatory T cells. J ExpMed 2008;205:2221–34.

30. Gottschalk RA, Corse E, Allison JP. Expression of Helios in peripherallyinduced Foxp3þ regulatory T cells. J Immunol 2012;188:976–80.

31. Healy E, Flannagan N, Ray A, Todd C, Jackson IJ, Matthews JN, et al.Melanocortin-1-receptor gene and sun sensitivity in individuals withoutred hair. Lancet 2000;355:1072–3.

32. LinnemannC, vanBuurenMM,Bies L, Verdegaal EM, Schotte R, Calis JJ, et al.High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4þ T cells in human melanoma. Nat Med 2015;21:81–5.

33. Yamazaki S, Nishioka A, Kasuya S, Ohkura N, Hemmi H, Kaisho T, et al.Homeostasis of thymus-derived Foxp3þ regulatory T cells is controlled byultraviolet B exposure in the skin. J Immunol 2014;193:5488–97.

34. Gobert M, Treilleux I, Bendriss-Vermare N, Bachelot T, Goddard-Leon S,Arfi V, et al. Regulatory T cells recruited through CCL22/CCR4 are selec-tively activated in lymphoid infiltrates surrounding primary breast tumorsand lead to an adverse clinical outcome. Cancer Res 2009;69:2000–9.

35. Jie HB, Gildener-Leapman N, Li J, Srivastava RM, Gibson SP,Whiteside TL,et al. Intratumoral regulatory T cells upregulate immunosuppressivemolecules in head and neck cancer patients. Br J Cancer 2013;109:2629–35.

36. Pedroza-Gonzalez A, Verhoef C, Ijzermans JN, Peppelenbosch MP, Kwek-keboom J, Verheij J, et al. Activated tumor-infiltrating CD4þ regulatory

T cells restrain antitumor immunity in patients with primary or metastaticliver cancer. Hepatology 2013;57:183–94.

37. Muhleisen B, Petrov I, Gachter T, Kurrer M, Scharer L, Dummer R, et al.Progression of cutaneous squamous cell carcinoma in immunosuppressedpatients is associated with reduced CD123þ and FOXP3þ cells in theperineoplastic inflammatory infiltrate. Histopathology 2009;55:67–76.

38. LeeHE, ParkDJ, KimWH,KimHH, LeeHS.High FOXP3þ regulatory T-celldensity in the sentinel lymph node is associated with downstream non-sentinel lymph-node metastasis in gastric cancer. Br J Cancer 2011;105:413–9.

39. TanW, ZhangW, Strasner A, Grivennikov S, Cheng JQ, Hoffman RM, et al.Tumour-infiltrating regulatory T cells stimulate mammary cancer metas-tasis through RANKL-RANK signalling. Nature 2011;470:548–53.

40. deLeeuw RJ, Kost SE, Kakal JA, Nelson BH. The prognostic value of FoxP3þtumor-infiltrating lymphocytes in cancer: a critical review of the literature.Clin Cancer Res 2012;18:3022–9.

41. Azzimonti B, Zavattaro E, Provasi M, Vidali M, Conca A, Catalano E, et al.Intense Foxp3þ CD25þ regulatory T-cell infiltration is associated withhigh-grade cutaneous squamous cell carcinoma and counterbalanced byCD8þ/Foxp3þ CD25þ ratio. Br J Dermatol 2015;172:64–73.

42. Gelb AB, Smoller BR, Warnke RA, Picker LJ. Lymphocytes infiltratingprimary cutaneous neoplasms selectively express the cutaneous lympho-cyte-associated antigen (CLA). Am J Pathol 1993;142:1556–64.

43. Lowes MA, Suarez-Farinas M, Krueger JG. Immunology of psoriasis. AnnuRev Immunol 2014;32:227–55.

44. Islam SA, Luster AD. T cell homing to epithelial barriers in allergic disease.Nat Med 2012;18:705–15.

45. Cavani A, Nasorri F, Ottaviani C, Sebastiani S, De Pita O, Girolomoni G.Human CD25þ regulatory T cells maintain immune tolerance to nickel inhealthy, nonallergic individuals. J Immunol 2003;171:5760–8.

46. Taraban VY, Slebioda TJ, Willoughby JE, Buchan SL, James S, Sheth B, et al.Sustained TL1A expression modulates effector and regulatory T-cellresponses and drives intestinal goblet cell hyperplasia. Mucosal Immunol2011;4:186–96.

47. Vetto JT, Lum S,Morris A, SicotteM,Davis J, LemonM, et al. Presence of theT-cell activation marker OX-40 on tumor infiltrating lymphocytes anddraining lymph node cells from patients with melanoma and head andneck cancers. Am J Surg 1997;174:258–65.

48. Feldmeyer L, Ching G, Vin H, Ma W, Bansal V, Chitsazzadeh V, et al.Differential T-cell subset representation in cutaneous squamous cell car-cinoma arising in immunosuppressed vs. Immunocompetent Individuals.Exp Dermatol. 2016;25:245–7.

49. Sarff M, Edwards D, Dhungel B, Wegmann KW, Corless C, Weinberg AD,et al. OX40 (CD134) expression in sentinel lymph nodes correlates withprognostic features of primary melanomas. Am J Surg 2008;195:621–5.

50. Petty JK, He K, Corless CL, Vetto JT, Weinberg AD. Survival in humancolorectal cancer correlates with expression of the T-cell costimulatorymolecule OX-40 (CD134). Am J Surg 2002;183:512–8.

51. Ladanyi A, Somlai B, Gilde K, Fejos Z, Gaudi I, Timar J. T-cell activationmarker expression on tumor-infiltrating lymphocytes as prognostic factorin cutaneous malignant melanoma. Clin Cancer Res 2004;10:521–30.






Published OnlineFirst March 31, 2016.Clin Cancer Res Chester Lai, Suzannah August, Amel Albibas, et al. Associate with Metastatic PotentialCarcinoma Suppress Effector T-Cell Responses and

Regulatory T Cells in Cutaneous Squamous Cell+OX40

Updated version

10.1158/1078-0432.CCR-15-2614doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2016/03/31/1078-0432.CCR-15-2614.DC1Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/early/2016/06/15/1078-0432.CCR-15-2614To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-15-2614

http://clincancerres.aacrjournals.org/content/suppl/2016/03/31/1078-0432.CCR-15-2614.DC1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2016/06/15/1078-0432.CCR-15-2614


ox40+ regulatory t cells in cutaneous squamous cell...

Documents