T-Cell Trafﬁcking Facilitated by High Endothelial Venules ... · Gene expression analysis Samples were cut in 10 mm sections from OCT embedded tissue and RNA extracted using TRIzol

Microenvironment and Immunology

T-Cell Trafficking Facilitated by High Endothelial Venules IsRequired for TumorControl after Regulatory T-Cell Depletion

James P. Hindley1, Emma Jones1, Kathryn Smart1, Hayley Bridgeman1, Sarah N. Lauder1, Beatrice Ondondo1,Scott Cutting1, Kristin Ladell1, Katherine K. Wynn1, David Withers2, David A. Price1, Ann Ager1,Andrew J. Godkin1, and Awen M. Gallimore1

AbstractThe evolution of immune blockades in tumors limits successful antitumor immunity, but the mechanisms

underlying this process are not fully understood. Depletion of regulatory T cells (Treg), a T-cell subset thatdampens excessive inflammatory and autoreactive responses, can allow activation of tumor-specific T cells.However, cancer immunotherapy studies have shown that a persistent failure of activated lymphocytes toinfiltrate tumors remains a fundamental problem. In evaluating this issue, we found that despite an increase in T-cell activation and proliferation following Treg depletion, there was no significant association with tumor growthrate. In contrast, therewas a highly significant association between low tumor growth rate and the extent of T-cellinfiltration. Further analyses revealed a total concordance between low tumor growth rate, high T-cell infiltration,and the presence of high endothelial venules (HEV). HEV are blood vessels normally found in secondary lymphoidtissue where they are specialized for lymphocyte recruitment. Thus, our findings suggest that Treg depletionmaypromote HEV neogenesis, facilitating increased lymphocyte infiltration and destruction of the tumor tissue.These findings are important as they point to a hitherto unidentified role of Tregs, themanipulation of whichmayrefine strategies for more effective cancer immunotherapy. Cancer Res; 72(21); 5473–82. �2012 AACR.

IntroductionSeveral laboratories have shown that CD4þ CD25þ Foxp3þ

regulatory T cells (Treg), which normally serve to controlautoimmunity and inflammation, also inhibit immuneresponses to tumor antigens (1). Thus, strategies aimed atboosting antitumor responses, throughmanipulation of Tregs,are being intensely investigated. In mice, prophylactic deple-tion of Tregs using CD25-specific antibodies can limit andeven prevent tumor development and/or progression (2–4).The development of transgenic mice expressing the primatediphtheria toxin receptor (DTR) on Foxp3þ cells has allowedspecific and complete depletion of Tregs by administration ofdiphtheria toxin (DT; 5). Using the melanoma cell line B16, ithas been shown that selective Treg depletion using anti-CD25antibodies (6) or DT (7) results in activation of a tumor-specific

CD8þ T-cell response and slower tumor growth. Use of theDTR-Foxp3 transgenic mice has also shown that Treg deple-tion canprevent development and even limit the progression oftumors induced by the carcinogen methylcholanthrene (MCA)in a manner that is dependent on CD8þ T cells and IFNg (8).

The success of these interventions however remains sub-optimal. Treg depletion, even when combined with vaccina-tion, does not readily result in the elimination of establishedtumors as tumor rejection is often observed in only a pro-portion of treated mice. Several factors, including the extent ofT-cell activation and/or tumor infiltration, may account forthis failure (6). In this study, we set out to identify factorslimiting the success of antitumor immune responses. For thispurpose, we used the MCA chemical carcinogenesis model incombination with Foxp3DTR mice (5), to identify the keyfeatures that distinguish progressing tumors from thosethat are controlled after Treg depletion. Our findings clearlyindicate that T-cell infiltration and not the extent of acti-vation, is a critical bottleneck to tumor destruction in Treg-depleted animals. Moreover, our extensive immunohisto-chemical analyses of MCA-induced tumors from Treg-replete and Treg-depleted mice revealed that control oftumor growth, observed in Treg-depleted mice, was deter-mined by development of high endothelial venules (HEV),specialized blood vessels that alter both the compositionand size of the T-cell infiltrate (9, 10). Overall, these dataprovide a new perspective on the impact of Treg depletion,demonstrating that Tregs control immune responses, notonly through limiting immune activation, but also throughinfluencing blood vessel differentiation.

Authors' Affiliations: 1Infection and Immunity, School of Medicine, HenryWellcome Building, Cardiff University, Cardiff, United Kingdom; and 2MRCCentre for Immune Regulation, Institute for Biomedical Research, Collegeof Medical and Dental Sciences, University of Birmingham, Birmingham,United Kingdom

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J.P. Hindley and E. Jones have contributed equally to this work.

Corresponding Author: Awen M. Gallimore, Infection and Immunity,School of Medicine, Henry Wellcome Building, Cardiff University, Cardiff,CF144XN, United Kingdom. Phone: 44-2920-687012; Fax: 44-2920-687079; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-1912

�2012 American Association for Cancer Research.

CancerResearch

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Materials and MethodsMice

We are grateful to Professor Alexander Rudensky for sup-plying Foxp3DTR mice. These were backcrossed with C57BL/6mice for 5 or more generations and housed in accordance withUK Home Office regulations.

Tumor induction, DT administration, and tumormonitoring

Foxp3DTRmice were injected subcutaneously in the left hindleg with 400 mg of 3-MCA (Sigma-Aldrich) in 100 mL of olive oilunder general anesthetic as described previously (11). Miceweremonitored for tumor development weekly up to 2monthsand daily thereafter. Tumor-bearing mice were culled beforetheir tumors reached 1.7 cm in diameter, typically 80 to 150days after injection or if tumors caused discomfort. DT (Sigma)diluted in PBS was administered intraperitoneally (i.p.) everyother day after tumor detection.

Tumor growth rate (k, days�1) was determined using a stat-istical software package Prism 5 (GraphPad) with the followingequation for exponential growth: Y ¼ Y0 � exp(k � X). Tumordiameter (X, mm) was measured every 2 days using calipers. Onaverage, measurements were taken for 12 days for PBS-treatedmice and 17 days for DT-treated mice. Therefore, on average 6to 8 measurements were used to calculate tumor growth rate.

Flow cytometrySingle-cell suspensions of spleens, inguinal lymph nodes,

and tumors were prepared. The inguinal lymph node from thetumor (left) side was taken as the tumor-draining lymph nodeand the contralateral inguinal lymph node as a nondraininglymph node.

For analysis of intracellular cytokines by flow cytometry,single-cell suspensions were stimulated with 20 ng/ml phor-bol myristate acetate (Sigma-Aldrich) and 1 mg/mL ionomycin(Sigma-Aldrich). Cells were incubated at 37�C for a total of 4hours. After 1 hour, 1 mL/mL of GolgiStop (containing mon-ensin; Becton Dickinson) was added to each well.

To identify dead cells, a fixable dead cell staining kit (LIVE/DEAD Aqua; Invitrogen) was used. Cells were washed twice inPBS and 3 to 6 mL of diluted (1:10; in PBS) dead cell stain wasadded to the cell pellet. Cells were stained at room temperaturefor 15 minutes in the dark then washed twice in fluorescence-activated cell sorting (FACS) buffer (PBS containing 5 mMEDTA and 2% fetal calf serum). For surface-marker staining,fluorescently labeled mAbs: anti-CD4 Pacific Blue (BectonDickinson), anti-CD8 PerCP-Cy5.5 (eBioscience), anti-CD25PE (eBioscience) were used. Then cells were fixed, permeabi-lized, and stained for intracellular antigens using the Foxp3Fix/Perm buffer set (eBioscience) and the following fluores-cently labeled mAbs: anti-Foxp3 PE-Cy7 (eBioscience), anti-Ki67 Alexa Fluor 647 (Becton Dickinson), anti-IFNg APC(eBioscience), anti-IL2 PE (eBioscience), anti-granzyme BAlexaFluor 647 (eBioscience), and anti-CD107a PE (eBioscience).For flow cytometric analysis, samples were acquired using aFACS Canto II flow cytometer (Becton Dickinson) and wereanalyzed using FACSDiva software (Becton Dickinson).

ImmunohistochemistryParaffin sections. Tumors were collected from

Foxp3DTR mice and fixed in neutral buffered formalin andembedded in paraffin. Sections 5 mm in thickness, were cutand mounted on slides, dewaxed in xylene and hydratedusing graded alcohols to tap water. After conducting antigenretrieval, by microwaving in 10 mmol/L Tris, 1 mmol/LEDTA buffer pH9 for 8 minutes, sections were cooled for30 minutes and then equilibrated in PBS. Endogenous per-oxidase activity was quenched with Peroxidase Suppressor(Thermo Scientific) for 15 minutes and nonspecific antibodybinding was blocked by incubating sections with 2.5% nor-mal horse serum (VectorLabs) in PBS for 30 minutes. Sec-tions were stained overnight at 4�C with the followingprimary antibodies diluted in 1% bovine serum albumin(BSA) in PBS: rat antiperipheral node addressin (PNAd)antibody (clone MECA-79; Biolegend) and rat anti-Mucosaladdressin cell adhesion molecule-1 (MAdCAM-1) antibody(clone MECA-367; Biolegend). Primary antibodies weredetected with anti-rat Immpress (VectorLabs) for 30 min-utes and visualized with impact 3,30-diaminobenzidine (Vec-torLabs). Sections were counterstained with haematoxylin,dehydrated in an ethanol series, and mounted in distyrene,plasticizer, xylene. Photomicrographs were taken using aNIKON microscope and digital camera.

Frozen sections. Tumors and lymph nodes were collectedfrom Foxp3DTR mice, embedded in optimum cutting temper-ature compound (OCT; RA Lamb), and snap frozen in liquidnitrogen. Sections, 5 mm in thickness, were fixed for 10minutesin ice-cold acetone and left to dry at room temperature. Slideswere washed in PBS and blocked with Avidin/Biotin BlockingKit (VectorLabs) and then 2.5% normal horse serum (Vector-Labs) in PBS for 30minutes. Sections were stained overnight at4�Cwith the following primary antibodies diluted in 1% BSA inPBS: rat anti-PNAd antibody (clone MECA-79; Biolegend),Alexa Fluor 488 rat anti-MAdCAM-1 antibody (clone MECA-367; BioLegend), biotin rat anti-PNAd antibody (clone MECA-79; BioLegend), rat anti-CD4 (clone RM4–5; eBioscience), ratanti-CD8 (clone 53–6.7; eBioscience), fluorescein isothiocya-nate rat anti-CD31 (clone 390; eBioscience), biotin rat anti-CD45R/B220 (clone RA3–6B2; eBioscience), rabbit anti-CD3(DAKO), and rat anti-CD35 (clone 8C12; BD Pharmingen).Primary antibodies were detected with Alexa Fluor 488 goatanti-rat, Alexa Fluor 488 goat anti-rabbit, and streptavidin-Alexa Fluor 555 (all from Invitrogen Life Sciences). Whensections were double-stained with 2 unconjugated rat primaryantibodies, the first rat primary antibody (anti-CD4 or -CD8)was detected with Alexa Fluor 488 anti-rat then fixed in 1%paraformaldehyde for 10 minutes before using a biotinylatedsecond rat primary antibody (anti-PNAd), which was detectedby streptavidin-Alexa Fluor 555. All sections were counter-stained with TOTO-3 iodide (Invitrogen) before fixing with 1%paraformaldehyde for 10 minutes and mounting with Vecta-shield, containing 4', 6-diamidino-2-phenylindole (Vectorlabs).Images were collected with a Zeiss LSM5 Pascal confocalmicroscope. Images recorded in the far-red channel werepseudocolored blue. Images were assembled in Adobe Photo-shop software.

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Gene expression analysisSamples were cut in 10 mm sections from OCT embedded

tissue and RNA extracted using TRIzol reagent (Invitrogen).Gene expression profiling was carried out using MouseRef-8v2.0 whole genome expression bead chip (Illumina) as recom-mended by the manufacturer. Probe intensity values werecorrected by background subtraction in GenomeStudio soft-ware and subsequently log-2 and baseline (median) trans-formed using Genespring software (Agilent) before analysisof genes.

Statistical analysesAll statistical analyses were conducted using Prism 5

(GraphPad). Unless stated otherwise, data groups were com-pared using an unpaired, nonparametric Mann–Whitney testand displayed as median. Where stated, data groups werecompared by an unpaired t test following confirmation thatthe set was normally distributed using the D'Agostino andPearson omnibus normality test. In this instance themeanwasgraphed. Correlation analyses were conducted using the Pear-son method and Pearson correlation coefficients (r2) aredisplayed.

ResultsActivation of tumor-infiltrating lymphocytes after Tregdepletion and impact on tumor progressionPreviously we have found that Foxp3þ Tregs are signifi-

cantly enriched in all MCA-induced fibrosarcomas (2, 11).Tumors were induced using the chemical carcinogen MCAin transgenic mice expressing the primate DTR on Foxp3þ

cells (Foxp3DTR mice; ref. 5). Optimal Treg depletion inFoxp3DTR mice bearing MCA-induced tumors was deter-mined by injecting increasing doses of DT (0.1 – 5 mg/kg)every other day. Successful depletion of Foxp3þ T cells wasobtained in all mice after injecting 5 mg/kg DT (<0.5% CD4þ

cells expressing Foxp3 remain; Supplementary Fig. S1 andref. 5); concomitant activation of CD4þ FoxP3� and CD8þ Tcells was confirmed by examining expression of Foxp3,CD25, and Ki67 (Supplementary Fig. S2 and ref. 5). A highlysignificant reduction in tumor growth rate was observed inDT-treated (Treg–; n ¼ 43) compared with control-treated(Tregþ; n ¼ 24) mice (P ¼ 0.0008; Fig. 1) and tumorregression was observed in 12% (n ¼ 5) of mice (tumorgrowth rate <0; Fig. 1). However, despite the overall reduc-tion in tumor growth rate, no difference in growth rate wasobserved in many of the treated mice.As T cells have been shown to control MCA-induced tumors

following Treg depletion (7), we postulated that control oftumor growth would correlate with the extent of T-cell acti-vation following depletion of Tregs. Indeed, analysis by flowcytometry revealed a highly significant increase in the per-centage of both CD8þ and CD4þ T cells in the spleen andtumors of Treg-depleted compared with control mice (Sup-plementary Fig. S3). However, when we examined CD4þ andCD8þ tumor-infiltrating lymphocytes (TIL) for expression ofthe activation markers CD25 and Ki67 and also for functionalmarkers, IFNg , IL-2, granzyme-B, and CD107a, we found no

significant correlation between tumor growth rate and anyof these markers (Fig. 2 and Supplementary Fig. S4), indi-cating that following Treg-depletion, the extent of CD8þ orCD4þ T-cell activation is not a critical bottleneck to suc-cessful control of tumor growth. We also evaluated whethertumor size at the start of treatment correlated with treat-ment success. The data, shown in Fig. 3, clearly indicate thatthis is not the case, as the impact of Treg depletion on tumorgrowth kinetics was not influenced by the size of the tumorat the start of treatment.

Tumor control is associated with accumulation of TILsNext, we addressed whether the extent of T-cell infiltration

following Treg depletion was associated with control of tumorgrowth. To enumerate tumor-infiltrating T cells, we conductedextensive immunohistochemical analyses of 14 Treg– and 8control, Tregþ tumors with representative examples of CD8-and CD4-specific staining shown in Fig. 4A and B. A significantincrease in the number of CD8þ cells (P ¼ 0.0140) and anoverall increase in the number of CD4þ cells (P ¼ 0.0818) wasobserved in the tumors of Treg– versus control Tregþ mice(Fig. 4C and D). However, the accumulation of tumor infiltrat-ing T cells was not universal among treated mice, as many ofthese did not differ significantly from the control group (Fig. 4Cand D). Indeed, the Treg– mice could clearly be split into 2subgroups: those with less than 100 (TILlo) and those withgreater than 100 (TILhi) CD4þ or CD8þ cells per high-powerfield (Fig. 4A–D). The correlation between the level of CD8þ

andCD4þ cell infiltration and tumor growth rate in Treg–micewas striking (P ¼ 0.0238 and P ¼ 0.0047, respectively; Fig. 4Eand F) whereas no correlation was observed in control Tregþmice (Fig. 4G and H). Although we do not rule out a role for Tcells in controlling tumors in any Tregþ tumor (particularlythose with lower growth rates), these data indicate thatfollowing Treg-depletion, T-cell infiltration determines suc-cessful control of tumor growth.

Figure 1. Depletion of Foxp3þ Tregs reduces tumor growth rate. Tumorbearing Foxp3DTR mice received either DT (n ¼ 43) or control treatment(n ¼ 24). Tumors were measured every other day during treatment andtumor growth rates (k, days–1) were calculated. ��, P < 0.001; Mann–Whitney test. Data from at least 3 independent experiments.

HEV Neogenesis Facilitates Tumor Rejection

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HEV neogenesis in Treg-depleted, but not Treg-repletetumors

We hypothesized that those tumors that are controlled afterdepleting Tregs would differ from those that continue toprogress with respect to mechanisms governing T-cell traf-ficking. Thus, tumors were examined by immunohistochem-istry for markers associated with HEVs, specialized postcapil-lary venules found in secondary lymphoid tissues that facilitateextravasation of naive and central memory T cells from bloodto lymphoid organs (9). For this purpose, tumors were stainedusing antibody clone MECA-79, which recognizes PNAd, a

carbohydrate epitope expressed on a variety of L-selectinligands that is specifically expressed by HEV (12). No PNAdstaining was observed in any of the tumors recovered fromTregþ control animals (Fig. 5A, n ¼ 14). However, PNAdstaining was identified in 50% of tumors recovered fromanimals depleted of Treg (Fig. 5B–E, n ¼ 14). The cuboidalmorphology of the cells (arrowhead) expressing PNAd inthe tumors was consistent with HEV morphology (Fig. 5D).Furthermore, PNAd expression often colocalized with MAd-CAM-1, an HEV-associated molecule, normally expressed onimmature HEV, adult mucosal lymphoid tissue, and lamina

Figure 2. Reduced tumor growth rate following depletion of Foxp3þ Tregs does not correlate with the extent of T-cell activation. The proportion oftumor infiltrating CD8þ T cells expressing CD25, Ki67, the cytokines IFNg and IL-2, and the functional markers granzyme B and CD107a werecorrelated with tumor growth rate (A–F). Statistical significance was evaluated using the Pearson correlation method. Data from at least 3 independentexperiments. ns, not statistically significant.

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propria (Fig. 5F). To determine whether PNAd was expressedon endothelial cells, we costained tumor sections with theendothelial cell marker CD31 (PECAM-1), and PNAd.Although many CD31þ cells do not express either MAd-CAM-1 or PNAd, our data clearly show coexpression of CD31on all MAdCAM-1 or PNAdþ cells (Fig. 5G). Collectively,these data indicate the presence of HEV within tumor tissue.Under nonpathologic conditions, HEVs are confined tosecondary lymphoid tissues. HEV develop as an integralpart of the stromal component of lymph nodes duringlymphoid neogenesis in embryonic life. Lymphotoxins a(LTa) and b (LTb) are required for the development of allHEV-containing lymph nodes in mice and the lymphoidchemokines, CCL21, CCL19, and CXCL13 segregate incom-ing lymphocytes into discrete T- and B-cell containing areas(9, 13–15). To determine whether the same mechanismscontribute to HEV neogenesis in tumors, expression of thesemolecules, as well as the PNAd scaffold protein GlyCAM-1,was compared in tumors containing HEVs (Treg–, HEVþ)and in tumors with no detectable HEVs (Tregþ, HEV– andTreg–, HEV–). Expression of each molecule was increased inthe HEVþ tumors (Fig. 6A) indicating that mechanismsfacilitating development of HEV-containing lymph nodesalso exist during HEV neogenesis in tumors (reviewed inref. 9). HEV neogenesis has also been reported under con-ditions of chronic inflammation and in autoimmune lesions,where their presence is often associated with the develop-ment of tertiary lymphoid organs (TLO; refs. 9, 16, 17). Theevents that govern HEV neogenesis and TLO development atthese sites are undefined. However, TLOs, such as secondarylymph nodes are characterized by discrete T- and B-cellareas with a follicular dendritic cell (FDC) network formingthe center of the B-cell area. We therefore conducted exten-sive immunohistochemical staining for T and B cells andFDCs but found that in contrast to the organization found in

lymph nodes, T and B cells were intermingled throughoutthe tumor mass and there was no FDC network (Fig. 6B andC). Furthermore, CD4þCD3� IL7Rþ,RORgþ lymphoid tissueinducer (LTi) cells, which are required for secondary lymphnode development were not detected in HEVþ tumors(Supplementary Fig. S5; 18–20). Collectively, these dataindicate that although HEV neognesis is accompanied byexpression of the lymphoid chemokines CCL19, CCL21, andCXCL13, it takes place without the need for LTi cells or forlymphoid neo-organogenesis. It is however possible thatHEV development and expression of lymphoid chemokinesrepresent the early stages of this process, preceding devel-opment of TLO.

HEV neogenesis promotes T-cell infiltration and tumorcontrol

HEV neogenesis has been reported in the context ofchronic inflammation and autoimmune disease in bothmouse models and human disease (9, 21, 22). Consistentwith their ability to facilitate increased lymphocyte extrav-asation, HEV are found in regions of extensive lymphocyticinfiltrate. Thus, we compared the number of tumor-infil-trating T cells in HEVþ and HEV– tumors. A clear associ-ation was observed between presence of HEV within tumortissue and a high number of CD8þ T cells (Fig. 7A; P ¼0.0034). When we compared the presence of HEV with tumorgrowth rate, HEVþ tumors showed significantly reducedtumor growth rate compared with HEV– tumors (Fig. 7B;P ¼ 0.0082). Interestingly, no correlation was observedbetween the extent of CD31 staining and numbers ofCD8þ T cells, indicating that the development of HEV andnot blood vessel development per se, is central to theincrease in T-cell infiltration and control of tumor growth(Fig. 7C). In support of the premise that tumor HEV allowthe infiltration of T cells into the tissue, we frequently foundT cells attached to or extravasating through the luminal wallof the vessel (Fig. 7D and E, arrowheads). Moreover, whenexpression of CD3, CCR7, L-Selectin, and the transcriptionfactor, KLF2 were assessed and compared between HEV–and HEVþ tumors, there was a clear increase in the expres-sion of these molecules in the HEVþ tumors (Fig. 7F).Thus, these data suggest that development of HEV intumors, allows infiltration of L-selectinhiCCR7þ T cells,which includes both central memory and naive T cells. Insupport of this, we have observed clonal expansion withintumor-infiltrating T cells, suggesting that an antigen-drivenresponse is generated intratumorally (Supplementary Fig.S6). Overall, these data strongly support the premise thatfollowing Treg depletion, the formation of HEV shapes thesize and composition of the T-cell infiltrate resulting intumor control.

DiscussionSelective depletion of Foxp3þ Tregs can result in immune-

mediated control of tumors. We examined the distinguishingfeatures of controlled versus progressing tumors followingtherapeutic Treg depletion in mice bearing palpable

Figure 3. Reduced tumor growth rate following depletion of Foxp3þ Tregsdoes not correlate with tumor size at start of treatment. Mice weremonitored for tumor development weekly and injected with DT typcially80 to 150 days after MCA injection. Tumor size was evaluated at the startof treatment and measured every 2 days thereafter as described inMaterials and Methods. Statistical significance was evaluated using thePearson correlation method. Data from at least 3 independentexperiments. ns, not statistically significant.






carcinogen-induced tumors. Previously, using the tumorcell line, B16, it was reported that although Treg-depletionpromoted systemic T-cell activation (as also shownabove), control of tumor growth was limited by failure ofthe T cells to infiltrate the tumor (6). In this and previousstudies, it was shown that sensitizing the tumor stromathrough irradiation or inflammation, induced vascularremodeling and upregulation of adhesion molecules facili-tating T-cell infiltration into tumors (6, 23). The data pre-sented herein identify, for the first time, a previously unsus-pected effect of Treg-depletion in facilitating neogenesisof HEV, which enable T-cell recruitment into the tumor

from blood. The significance of this effect is striking: HEVare not observed unless Tregs are depleted and there is anabsolute concordance between presence of HEV, high num-bers of tumor-infiltrating T lymphocytes, and control oftumor growth.

As yet, we have not identified themechanisms underpinningHEV neogenesis in tumors. Under nonpathologic conditions,HEV development is confined to secondary lymphoid tissue.Ligation of the LTb receptor (LTbR) on stromal-organiser cellsby LTab expressing LTi cells represents a key event in thedevelopment of lymph nodes (18, 19). Although LTbR signalingis required to maintain fully differentiated HEV (24, 25),

Figure 4. Tumor growth rate isassociated with the extent of CD8þ

and CD4þ T-cell infiltration. Tumorsections were stained with anti-CD8 or -CD4 antibodies (green)and counterstained with TOTO-3(blue). Representative images fromcontrol and Treg-depleted micewith low or high T-cell counts areshown (A andB). CD8þ andCD4þTcells from Treg-depleted (n ¼ 14)and control mice (n ¼ 8) werecompared (C and D) and correlatedwith individual tumor growth rates(E and F, DT-treated mice; G and Hcontrol untreated mice). ns, notstatistically significant.

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mechanisms underpinningHEV formation in lymphnodes andwhether they are related to those driving stromal cell differ-entiation remain incompletely defined. HEV neogenesis within

chronically inflamed tissue was first reported around 15 yearsago and more recently in tumors (26–28). As in lymph nodes,the presence of HEV is almost always associated with thedevelopment of a stromal cell network that organizes infil-trating lymphocytes into discrete T- and B-cell areas in so-called TLO (29–31). However, themechanisms underlying HEVneogenesis and how it relates to the development of thestromal cell network of TLO are poorly defined. In the caseof the HEVþ tumors described in this study, although we havefound no evidence of LTi cells or TLOs, expression of inflam-matory cytokines and lymphoid chemokines is increased.Collectively these data imply that HEV neogenesis in tumorsoccurs through mechanisms that promote inflammation (inthis case, Treg depletion) and deviate from those operating insecondary lymph nodes, possibly in terms of the cellular sourceof LTab.

An unexplained aspect of our findings is that HEVdevelopment, although tightly linked to Treg depletion,is only observed in around half of Treg-depleted mice.Several explanations may account for this finding, anexploration of which may offer vital clues to defining themechanisms underpinning HEV neogenesis in tumors.First, the nature of the immune response stimulated fol-lowing Treg depletion may affect whether HEV develop ornot. Perhaps, following Treg depletion, T cells and/or Bcells in some, but not all mice, produce important HEVinducing factors (e.g., LTab). Furthermore, the tumormay specifically impinge on the type of immune responseinduced following Treg depletion. Tumors that retainimmunogenicity, perhaps as a result of Treg-induced im-munosuppression, may activate tumor-specific T and Bcells, which, as described above, drive HEV development.On the other hand, tumors that have escaped the attentionof the immune system through the loss of strong antigens(immunoediting), may fail, even after Treg depletion, toinduce a sufficiently robust, HEV-promoting immuneresponse. Indeed, Matsushita and colleagues recentlyshowed that the outgrowth of MCA-induced tumors couldresult from T-cell dependent selection of tumors lackingstrong antigens (32). Alternatively, tumor-intrinsic featuresmay influence HEV development in a stochastic fashion.For instance, tumors may produce factors that eitheractively inhibit or facilitate HEV development. Gaining anunderstanding of these complex issues will uncover themechanisms underpinning HEV neogenesis in tumors andfacilitate a better understanding of whether a givenimmune response can promote tumor immunity throughblood vessel differentiation.

The importance of understanding how HEV can develop inperipheral sites has clear implications for cancer immunother-apy. Recently, in a study of more than 300 human tumors,Martinet and colleagues discovered a highly significant asso-ciation between the presence of HEV and the frequency oftumor-infiltrating T cells (26). Moreover, when a cohort ofaround 150 breast cancer patients were investigated, the samestudy reported a significant correlation between HEV and afavorable prognosis. These data support a crucial role for HEVneogenesis in tumor rejection. Our study highlights one avenue

Figure 5. Staining of HEV associated molecules PNAd andMAdCAM-1 intumors from Treg depleted mice. Paraffin-embedded sections, fromTreg-replete (Tregþ) andTreg-depleted (Treg–) tumors,were stainedwithanti-PNAd antibodies (brown) and counterstained with haematoxylin(blue; A–D). Representative low-power (A and B) and high-power (C andD) fields of view are shown. Cells stained with anti-PNAd antibodiesdisplay a cuboidal morphology typical of HEV (arrowhead). Frozensections from Tregþ (n¼ 6) and Treg– (n¼ 14) tumors were stained withanti-PNAd antibodies and levels of PNAd expressionwere determined byquantitating the percentage of PNAd-positive pixels per area of tumor (E).Frozen sections, from controlled tumors, were stained with anti-PNAd(red) and anti-MAdCAM-1 (green) antibodies (F) and anti-PNAd (red) andanti-CD31(green) antibodies (G). Cells were counterstained with TOTO-3(blue). Data from at least 2 independent experiments.






toward achieving this goal, namely the depletion of Tregs. Itwould be extremely interesting therefore to examine whetherin large cohorts of human tumors such as those used in theabove studies, there is an inverse association between Tregsand the presence of HEV.

Although the precise mechanism of HEV induction inour study remains to be elucidated, the significant increasein expression of LTb in HEVþ tumors and the previouslydescribed role for LTab in maintaining fully functionalHEV, support an important role for this cytokine. A studyby Schrama and colleagues showed that administration ofan LTa fusion protein into B16 melanoma-bearing miceresulted in HEV neogenesis, which subsequently promotedentry of naive T cells that were primed intratumorally andwhich contributed to tumor destruction (10). Similarly, Yuand colleagues reported the same effect in mice inoculatedwith LIGHT-expressing tumors; this effect was attributedto the ability of LIGHT, such as LTa to facilitate generationof lymphoid structures, which enabled entry of naive T cells

into the tumor (33). In both cases, this resulted in efficientpriming of tumor-specific T cells, presumably because ofthe high probability of these T cells meeting their cognateantigen within the tumor mass. We suggest that depletingTregs can lead to HEV neogenesis; a process that is criticalin determining the composition and size of the T-cellinfiltrate thus permitting tumor destruction. Although themechanisms of HEV induction and the influence of Tregson this process are poorly understood, it is clear that abetter understanding of these mechanisms will presentnew opportunities for more effective antitumor therapy.Furthermore, HEV neogenesis may play deleterious rolein autoimmunity and chronic inflammation (9). Our find-ings may therefore point to a hitherto unidentified mech-anism through which a defect in Treg activity drivesautoimmune disease and chronic inflammation by allowingHEV induction, hence sustained immune cell infiltrationand destruction of the affected tissue. Harnessing thisactivity of Tregs therefore has implications for both cancer

Figure 6. HEV neogenesis isassociated with expression oflymphoid chemokines but notdevelopment of TLO. The relativeexpression of various HEV-associated geneswere determinedand compared between Tregþ,HEV–Treg–, HEV–, and Treg–,HEVþ tumors (A). Frozen sections,from lymph nodes (B) and Treg-depleted (Treg–), HEVþ tumors (C),were stained with antibodiesspecific for B and T cells (red andgreen; anti-B220 and anti-CD3),FDCs (green; anti-CD35) or T cellsandPNAd (green and red; anti-CD3and anti-PNAd). Cells werecounterstainedwith TOTO-3 (blue).Scale bar, 20 mm.Data fromat least2 independent experiments.

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immunotherapy and the treatment of chronic inflamma-tory diseases.

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

Authors' ContributionsConception and design: J.P. Hindley, E. Jones, B. Ondondo, A. GallimoreDevelopment of methodology: J.P. Hindley, E. Jones, B. Ondondo, K. Ladell, D.Price, A. AgerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.P. Hindley, E. Jones, S.N. Lauder, S. Cutting, K. Ladell,K. Wynn, D. Withers, D. Price, A. Gallimore

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.P. Hindley, E. Jones, S. Cutting, K. Ladell, D. Price, A.J. Godkin, A. GallimoreWriting, review, and/or revision of themanuscript: J.P. Hindley, E. Jones, K.Ladell, D. Price, A. Ager, A.J. Godkin, A. GallimoreAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.P. Hindley, E. Jones, K. Smart, H.Bridgeman, A. GallimoreStudy supervision: J.P Hindley, E. Jones, A. Gallimore

AcknowledgmentsThe authors are grateful to the staff at Cardiff University JBIOS for their

continued support.

Figure 7. Increased T-cell infiltrationand reduced tumor growth rate inHEVþ Treg-depleted tumors. Thenumber of tumor infiltrating CD8þ

cells (A) and the tumor growthrates (B) of TregþHEV–, n¼ 8, Treg–HEV–, n¼ 6, and Treg–HEVþ, n¼ 7,tumors were compared. ��, P < 0.01;Mann–Whitney test. Frozen sectionswere stained with CD31- and CD8-specific antibodies. Levels of CD31expression were determined byquantitating the percentage ofCD31-positive pixels per area oftumor and compared with numbersof CD8þ T cells (C). Frozen sections,from HEVþ tumors, were stainedwith anti-PNAd (red) and anti-CD8(green) antibodies (D) or anti-PNAd(red) and anti-CD4 (green) antibodies(E). Cells were counterstained withTOTO-3 (blue). CD8þ and CD4þ cellsare seen attached to andextravasating through the vessel wall(arrowheads; D and E). Geneexpression analysis of T-cellassociated genes was comparedbetween tumor samples (F). Datafrom at least 2 independentexperiments. ns, not statisticallysignificant.






Grant SupportThis work was supported by a project grant from the Medical Research

Council (G0801190) and a University Award (A. Gallimore) from the WellcomeTrust (086983/Z/08/Z).

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 May 16, 2012; revised August 22, 2012; accepted August 24, 2012;published OnlineFirst September 7, 2012.

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2012;72:5473-5482. Published OnlineFirst September 7, 2012.Cancer Res James P. Hindley, Emma Jones, Kathryn Smart, et al. Required for Tumor Control after Regulatory T-Cell DepletionT-Cell Trafficking Facilitated by High Endothelial Venules Is

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T-Cell Trafﬁcking Facilitated by High Endothelial Venules ... · Gene expression analysis Samples were cut in 10 mm sections from OCT embedded tissue and RNA extracted using TRIzol