T-Cell Trafficking Facilitated by High Endothelial Venules Is Required for Tumor Control After Regulatory T-Cell Depletion

Published OnlineFirst September 7, 2012; DOI: 10.1158/0008-5472.CAN-12-1912

Cancer Microenvironment and Immunology Research

T-Cell Trafﬁcking Facilitated by High Endothelial Venules Is Required for Tumor Control 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

Abstract The 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 that dampens 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 to infiltrate 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 growth rate. In contrast, there was a highly significant association between low tumor growth rate and the extent of T-cell infiltration. 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 lymphoid tissue where they are specialized for lymphocyte recruitment. Thus, our findings suggest that Treg depletion may promote 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, the manipulation of which may refine strategies for more effective cancer immunotherapy. Cancer Res; 72(21); 5473–82. 2012 AACR.

þ Introduction CD8 T-cell response and slower tumor growth. Use of the þ þ þ Several laboratories have shown that CD4 CD25 Foxp3 DTR-Foxp3 transgenic mice has also shown that Treg deple- regulatory T cells (Treg), which normally serve to control tion can prevent development and even limit the progression of fl tumors induced by the carcinogen methylcholanthrene (MCA) autoimmunity and in ammation, also inhibit immune þ responses to tumor antigens (1). Thus, strategies aimed at in a manner that is dependent on CD8 T cells and IFNg (8). boosting antitumor responses, through manipulation of Tregs, The success of these interventions however remains sub- are being intensely investigated. In mice, prophylactic deple- optimal. Treg depletion, even when combined with vaccination of Tregs using CD25-specific antibodies can limit and tion, does not readily result in the elimination of established even prevent tumor development and/or progression (2–4). tumors as tumor rejection is often observed in only a pro- The development of transgenic mice expressing the primate portion of treated mice. Several factors, including the extent of þ fi diphtheria toxin receptor (DTR) on Foxp3 cells has allowed T-cell activation and/or tumor in ltration, may account for specific and complete depletion of Tregs by administration of this failure (6). In this study, we set out to identify factors diphtheria toxin (DT; 5). Using the melanoma cell line B16, it limiting the success of antitumor immune responses. For this purpose, we used the MCA chemical carcinogenesis model in has been shown that selective Treg depletion using anti-CD25 DTR antibodies (6) or DT (7) results in activation of a tumor-specific combination with Foxp3 mice (5), to identify the key features that distinguish progressing tumors from those that are controlled after Treg depletion. Our findings clearly Authors' Affiliations: 1Infection and Immunity, School of Medicine, Henry indicate that T-cell infiltration and not the extent of acti- 2 Wellcome Building, Cardiff University, Cardiff, United Kingdom; and MRC vation, is a critical bottleneck to tumor destruction in Treg- Centre for Immune Regulation, Institute for Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, depleted animals. Moreover, our extensive immunohisto- United Kingdom chemical analyses of MCA-induced tumors from Treg- Note: Supplementary data for this article are available at Cancer Research replete and Treg-depleted mice revealed that control of Online (http://cancerres.aacrjournals.org/). tumor growth, observed in Treg-depleted mice, was determined by development of high endothelial venules (HEV), J.P. Hindley and E. Jones have contributed equally to this work. specialized blood vessels that alter both the composition Corresponding Author: Awen M. Gallimore, Infection and Immunity, fi School of Medicine, Henry Wellcome Building, Cardiff University, Cardiff, and size of the T-cell in ltrate (9, 10). Overall, these data CF144XN, United Kingdom. Phone: 44-2920-687012; Fax: 44-2920- provide a new perspective on the impact of Treg depletion, 687079; E-mail: [email protected] demonstrating that Tregs control immune responses, not doi: 10.1158/0008-5472.CAN-12-1912 only through limiting immune activation, but also through 2012 American Association for Cancer Research. influencing blood vessel differentiation.

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Materials and Methods Immunohistochemistry Paraffin sections. Mice Tumors were collected from DTR fi We are grateful to Professor Alexander Rudensky for sup- Foxp3 mice and xed in neutral buffered formalin and fi plying Foxp3DTR mice. These were backcrossed with C57BL/6 embedded in paraf n. Sections 5 mm in thickness, were cut mice for 5 or more generations and housed in accordance with and mounted on slides, dewaxed in xylene and hydrated UK Home Office regulations. using graded alcohols to tap water. After conducting antigen retrieval, by microwaving in 10 mmol/L Tris, 1 mmol/L EDTA buffer pH9 for 8 minutes, sections were cooled for Tumor induction, DT administration, and tumor 30 minutes and then equilibrated in PBS. Endogenous per- monitoring oxidase activity was quenched with Peroxidase Suppressor DTR Foxp3 mice were injected subcutaneously in the left hind (Thermo Scientific) for 15 minutes and nonspecificantibody leg with 400 mg of 3-MCA (Sigma-Aldrich) in 100 mL of olive oil binding was blocked by incubating sections with 2.5% nor- under general anesthetic as described previously (11). Mice mal horse serum (VectorLabs) in PBS for 30 minutes. Sec- were monitored for tumor development weekly up to 2 months tions were stained overnight at 4C with the following and daily thereafter. Tumor-bearing mice were culled before primary antibodies diluted in 1% bovine serum albumin their tumors reached 1.7 cm in diameter, typically 80 to 150 (BSA) in PBS: rat antiperipheral node addressin (PNAd) days after injection or if tumors caused discomfort. DT (Sigma) antibody (clone MECA-79; Biolegend) and rat anti-Mucosal diluted in PBS was administered intraperitoneally (i.p.) every addressin cell adhesion molecule-1 (MAdCAM-1) antibody other day after tumor detection. (clone MECA-367; Biolegend). Primary antibodies were 1 Tumor growth rate (k, days ) was determined using a stat- detected with anti-rat Immpress (VectorLabs) for 30 min- istical software package Prism 5 (GraphPad) with the following utes and visualized with impact 3,30-diaminobenzidine (Vec- ¼ equation for exponential growth: Y Y0 exp(k X). Tumor torLabs). Sections were counterstained with haematoxylin, diameter (X, mm) was measured every 2 days using calipers. On dehydrated in an ethanol series, and mounted in distyrene, average, measurements were taken for 12 days for PBS-treated plasticizer, xylene. Photomicrographs were taken using a mice and 17 days for DT-treated mice. Therefore, on average 6 NIKON microscope and digital camera. to 8 measurements were used to calculate tumor growth rate. Frozen sections. Tumors and lymph nodes were collected from Foxp3DTR mice, embedded in optimum cutting temper- Flow cytometry ature compound (OCT; RA Lamb), and snap frozen in liquid Single-cell suspensions of spleens, inguinal lymph nodes, nitrogen. Sections, 5 mm in thickness, were fixed for 10 minutes and tumors were prepared. The inguinal lymph node from the in ice-cold acetone and left to dry at room temperature. Slides tumor (left) side was taken as the tumor-draining lymph node were washed in PBS and blocked with Avidin/Biotin Blocking and the contralateral inguinal lymph node as a nondraining Kit (VectorLabs) and then 2.5% normal horse serum (Vector- lymph node. Labs) in PBS for 30 minutes. Sections were stained overnight at For analysis of intracellular cytokines by flow cytometry, 4C with the following primary antibodies diluted in 1% BSA in single-cell suspensions were stimulated with 20 ng/ml phor- PBS: rat anti-PNAd antibody (clone MECA-79; Biolegend), bol myristate acetate (Sigma-Aldrich) and 1 mg/mL ionomycin Alexa Fluor 488 rat anti-MAdCAM-1 antibody (clone MECA- (Sigma-Aldrich). Cells were incubated at 37 C for a total of 4 367; BioLegend), biotin rat anti-PNAd antibody (clone MECA- hours. After 1 hour, 1 mL/mL of GolgiStop (containing mon- 79; BioLegend), rat anti-CD4 (clone RM4–5; eBioscience), rat ensin; Becton Dickinson) was added to each well. anti-CD8 (clone 53–6.7; eBioscience), fluorescein isothiocya- To identify dead cells, a fixable dead cell staining kit (LIVE/ nate rat anti-CD31 (clone 390; eBioscience), biotin rat anti- DEAD Aqua; Invitrogen) was used. Cells were washed twice in CD45R/B220 (clone RA3–6B2; eBioscience), rabbit anti-CD3 PBS and 3 to 6 mL of diluted (1:10; in PBS) dead cell stain was (DAKO), and rat anti-CD35 (clone 8C12; BD Pharmingen). added to the cell pellet. Cells were stained at room temperature Primary antibodies were detected with Alexa Fluor 488 goat for 15 minutes in the dark then washed twice in fluorescence- anti-rat, Alexa Fluor 488 goat anti-rabbit, and streptavidin- activated cell sorting (FACS) buffer (PBS containing 5 mM Alexa Fluor 555 (all from Invitrogen Life Sciences). When EDTA and 2% fetal calf serum). For surface-marker staining, sections were double-stained with 2 unconjugated rat primary fluorescently labeled mAbs: anti-CD4 Pacific Blue (Becton antibodies, the first rat primary antibody (anti-CD4 or -CD8) Dickinson), anti-CD8 PerCP-Cy5.5 (eBioscience), anti-CD25 was detected with Alexa Fluor 488 anti-rat then fixed in 1% PE (eBioscience) were used. Then cells were fixed, permeabi- paraformaldehyde for 10 minutes before using a biotinylated lized, and stained for intracellular antigens using the Foxp3 second rat primary antibody (anti-PNAd), which was detected Fix/Perm buffer set (eBioscience) and the following fluores- by streptavidin-Alexa Fluor 555. All sections were counter- cently labeled mAbs: anti-Foxp3 PE-Cy7 (eBioscience), anti- stained with TOTO-3 iodide (Invitrogen) before fixing with 1% Ki67 Alexa Fluor 647 (Becton Dickinson), anti-IFNg APC paraformaldehyde for 10 minutes and mounting with Vecta- (eBioscience), anti-IL2 PE (eBioscience), anti-granzyme B Alexa shield, containing 4', 6-diamidino-2-phenylindole (Vectorlabs). Fluor 647 (eBioscience), and anti-CD107a PE (eBioscience). Images were collected with a Zeiss LSM5 Pascal confocal For flow cytometric analysis, samples were acquired using a microscope. Images recorded in the far-red channel were FACS Canto II flow cytometer (Becton Dickinson) and were pseudocolored blue. Images were assembled in Adobe Photo- analyzed using FACSDiva software (Becton Dickinson). shop software.

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Gene expression analysis Samples 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 were corrected by background subtraction in GenomeStudio software and subsequently log-2 and baseline (median) trans- formed using Genespring software (Agilent) before analysis of genes.

Statistical analyses All statistical analyses were conducted using Prism 5 (GraphPad). Unless stated otherwise, data groups were compared using an unpaired, nonparametric Mann–Whitney test and displayed as median. Where stated, data groups were þ t fi Figure 1. Depletion of Foxp3 Tregs reduces tumor growth rate. Tumor compared by an unpaired test following con rmation that bearing Foxp3DTR mice received either DT (n ¼ 43) or control treatment the set was normally distributed using the D'Agostino and (n ¼ 24). Tumors were measured every other day during treatment and – Pearson omnibus normality test. In this instance the mean was tumor growth rates (k, days 1) were calculated. , P < 0.001; Mann– graphed. Correlation analyses were conducted using the Pear- Whitney test. Data from at least 3 independent experiments. son method and Pearson correlation coefficients (r2) are displayed. significant correlation between tumor growth rate and any of these markers (Fig. 2 and Supplementary Fig. S4), indi- þ Results cating that following Treg-depletion, the extent of CD8 or þ Activation of tumor-infiltrating lymphocytes after Treg CD4 T-cell activation is not a critical bottleneck to suc- depletion and impact on tumor progression cessful control of tumor growth. We also evaluated whether þ Previously we have found that Foxp3 Tregs are signifi- tumorsizeatthestartoftreatment correlated with treat- cantly enriched in all MCA-induced fibrosarcomas (2, 11). ment success. The data, shown in Fig. 3, clearly indicate that Tumors were induced using the chemical carcinogen MCA this is not the case, as the impact of Treg depletion on tumor þ in transgenic mice expressing the primate DTR on Foxp3 growth kinetics was not influenced by the size of the tumor cells (Foxp3DTR mice; ref. 5). Optimal Treg depletion in at the start of treatment. Foxp3DTR mice bearing MCA-induced tumors was determined by injecting increasing doses of DT (0.1 – 5 mg/kg) Tumor control is associated with accumulation of TILs þ every other day. Successful depletion of Foxp3 Tcellswas Next, we addressed whether the extent of T-cell infiltration þ obtained in all mice after injecting 5 mg/kg DT (<0.5% CD4 following Treg depletion was associated with control of tumor cells expressing Foxp3 remain; Supplementary Fig. S1 and growth. To enumerate tumor-infiltrating T cells, we conducted þ þ ref. 5); concomitant activation of CD4 FoxP3 and CD8 T extensive immunohistochemical analyses of 14 Treg– and 8 cells was confirmed by examining expression of Foxp3, control, Tregþ tumors with representative examples of CD8- CD25, and Ki67 (Supplementary Fig. S2 and ref. 5). A highly and CD4-specific staining shown in Fig. 4A and B. A significant þ significant reduction in tumor growth rate was observed in increase in the number of CD8 cells (P ¼ 0.0140) and an þ DT-treated (Treg–; n ¼ 43) compared with control-treated overall increase in the number of CD4 cells (P ¼ 0.0818) was (Tregþ; n ¼ 24) mice (P ¼ 0.0008; Fig. 1) and tumor observed in the tumors of Treg– versus control Tregþ mice regression was observed in 12% (n ¼ 5) of mice (tumor (Fig. 4C and D). However, the accumulation of tumor infiltrat- growth rate <0; Fig. 1). However, despite the overall reduc- ing T cells was not universal among treated mice, as many of tion in tumor growth rate, no difference in growth rate was these did not differ significantly from the control group (Fig. 4C observed in many of the treated mice. and D). Indeed, the Treg– mice could clearly be split into 2 As T cells have been shown to control MCA-induced tumors subgroups: those with less than 100 (TILlo) and those with þ þ following Treg depletion (7), we postulated that control of greater than 100 (TILhi) CD4 or CD8 cells per high-power þ tumor growth would correlate with the extent of T-cell acti- field (Fig. 4A–D). The correlation between the level of CD8 þ vation following depletion of Tregs. Indeed, analysis by flow and CD4 cell infiltration and tumor growth rate in Treg– mice cytometry revealed a highly significant increase in the per- was striking (P ¼ 0.0238 and P ¼ 0.0047, respectively; Fig. 4E þ þ centage of both CD8 and CD4 T cells in the spleen and and F) whereas no correlation was observed in control Tregþ tumors of Treg-depleted compared with control mice (Sup- mice (Fig. 4G and H). Although we do not rule out a role for T þ plementary Fig. S3). However, when we examined CD4 and cells in controlling tumors in any Tregþ tumor (particularly þ CD8 tumor-infiltrating lymphocytes (TIL) for expression of those with lower growth rates), these data indicate that the activation markers CD25 and Ki67 and also for functional following Treg-depletion, T-cell infiltration determines suc- markers, IFNg, IL-2, granzyme-B, and CD107a, we found no cessful control of tumor growth.

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þ Figure 2. Reduced tumor growth rate following depletion of Foxp3 Tregs does not correlate with the extent of T-cell activation. The proportion of þ tumor infiltrating CD8 T cells expressing CD25, Ki67, the cytokines IFNg and IL-2, and the functional markers granzyme B and CD107a were correlated with tumor growth rate (A–F). Statistical significance was evaluated using the Pearson correlation method. Data from at least 3 independent experiments. ns, not statistically significant.

HEV neogenesis in Treg-depleted, but not Treg-replete carbohydrate epitope expressed on a variety of L-selectin tumors ligands that is specifically expressed by HEV (12). No PNAd We hypothesized that those tumors that are controlled after staining was observed in any of the tumors recovered from depleting Tregs would differ from those that continue to Tregþ control animals (Fig. 5A, n ¼ 14). However, PNAd progress with respect to mechanisms governing T-cell traf- staining was identified in 50% of tumors recovered from ficking. Thus, tumors were examined by immunohistochem- animals depleted of Treg (Fig. 5B–E, n ¼ 14). The cuboidal istry for markers associated with HEVs, specialized postcapil- morphology of the cells (arrowhead) expressing PNAd in lary venules found in secondary lymphoid tissues that facilitate the tumors was consistent with HEV morphology (Fig. 5D). extravasation of naive and central memory T cells from blood Furthermore, PNAd expression often colocalized with MAd- to lymphoid organs (9). For this purpose, tumors were stained CAM-1, an HEV-associated molecule, normally expressed on using antibody clone MECA-79, which recognizes PNAd, a immature HEV, adult mucosal lymphoid tissue, and lamina

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lymph nodes, T and B cells were intermingled throughout the tumor mass and there was no FDC network (Fig. 6B and þ þ þ C).Furthermore,CD4 CD3 IL7R ,RORg lymphoid tissue inducer (LTi) cells, which are required for secondary lymph node development were not detected in HEVþ tumors (Supplementary Fig. S5; 18–20). Collectively, these data indicate that although HEV neognesis is accompanied by expression of the lymphoid chemokines CCL19, CCL21, and CXCL13, it takes place without the need for LTi cells or for lymphoid neo-organogenesis. It is however possible that HEV development and expression of lymphoid chemokines represent the early stages of this process, preceding development of TLO.

HEV neogenesis promotes T-cell infiltration and tumor þ Figure 3. Reduced tumor growth rate following depletion of Foxp3 Tregs control does not correlate with tumor size at start of treatment. Mice were HEV neogenesis has been reported in the context of monitored for tumor development weekly and injected with DT typcially chronic inflammation and autoimmune disease in both 80 to 150 days after MCA injection. Tumor size was evaluated at the start mouse models and human disease (9, 21, 22). Consistent of treatment and measured every 2 days thereafter as described in Materials and Methods. Statistical significance was evaluated using the with their ability to facilitate increased lymphocyte extrav- Pearson correlation method. Data from at least 3 independent asation, HEV are found in regions of extensive lymphocytic experiments. ns, not statistically significant. infiltrate. Thus, we compared the number of tumor-infiltrating T cells in HEVþ and HEV– tumors. A clear associ- propria (Fig. 5F). To determine whether PNAd was expressed ation was observed between presence of HEV within tumor þ on endothelial cells, we costained tumor sections with the tissue and a high number of CD8 T cells (Fig. 7A; P ¼ endothelial cell marker CD31 (PECAM-1), and PNAd. 0.0034). When we compared the presence of HEV with tumor þ Although many CD31 cells do not express either MAd- growth rate, HEVþ tumors showed significantly reduced CAM-1 or PNAd, our data clearly show coexpression of CD31 tumorgrowthratecomparedwithHEV– tumors (Fig. 7B; þ on all MAdCAM-1 or PNAd cells (Fig. 5G). Collectively, P ¼ 0.0082). Interestingly, no correlation was observed these data indicate the presence of HEV within tumor tissue. between the extent of CD31 staining and numbers of þ Under nonpathologic conditions, HEVs are confined to CD8 T cells, indicating that the development of HEV and secondary lymphoid tissues. HEV develop as an integral not blood vessel development per se, is central to the part of the stromal component of lymph nodes during increase in T-cell infiltration and control of tumor growth lymphoid neogenesis in embryonic life. Lymphotoxins a (Fig. 7C). In support of the premise that tumor HEV allow (LTa)andb (LTb) are required for the development of all the infiltration of T cells into the tissue, we frequently found HEV-containing lymph nodes in mice and the lymphoid T cells attached to or extravasating through the luminal wall chemokines, CCL21, CCL19, and CXCL13 segregate incom- of the vessel (Fig. 7D and E, arrowheads). Moreover, when ing lymphocytes into discrete T- and B-cell containing areas expression of CD3, CCR7, L-Selectin, and the transcription (9, 13–15). To determine whether the same mechanisms factor, KLF2 were assessed and compared between HEV– contribute to HEV neogenesis in tumors, expression of these and HEVþ tumors, there was a clear increase in the expres- molecules, as well as the PNAd scaffold protein GlyCAM-1, sion of these molecules in the HEVþ tumors (Fig. 7F). was compared in tumors containing HEVs (Treg–,HEVþ) Thus, these data suggest that development of HEV in þ andintumorswithnodetectableHEVs(Tregþ,HEV– and tumors, allows infiltration of L-selectinhiCCR7 T cells, Treg–,HEV–). Expression of each molecule was increased in which includes both central memory and naive T cells. In the HEVþ tumors (Fig. 6A) indicating that mechanisms support of this, we have observed clonal expansion within facilitating development of HEV-containing lymph nodes tumor-infiltrating T cells, suggesting that an antigen-driven also exist during HEV neogenesis in tumors (reviewed in response is generated intratumorally (Supplementary Fig. ref. 9). HEV neogenesis has also been reported under con- S6). Overall, these data strongly support the premise that ditions of chronic inflammation and in autoimmune lesions, following Treg depletion, the formation of HEV shapes the where their presence is often associated with the develop- size and composition of the T-cell infiltrate resulting in ment of tertiary lymphoid organs (TLO; refs. 9, 16, 17). The tumor control. events that govern HEV neogenesis and TLO development at thesesitesareundefined. However, TLOs, such as secondary lymph nodes are characterized by discrete T- and B-cell Discussion þ areas with a follicular dendritic cell (FDC) network forming Selective depletion of Foxp3 Tregs can result in immune- the center of the B-cell area. We therefore conducted exten- mediated control of tumors. We examined the distinguishing sive immunohistochemical staining for T and B cells and features of controlled versus progressing tumors following FDCs but found that in contrast to the organization found in therapeutic Treg depletion in mice bearing palpable

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Figure 4. Tumor growth rate is þ associated with the extent of CD8 and CD4þ T-cell inﬁltration. Tumor sections were stained with anti- CD8 or -CD4 antibodies (green) and counterstained with TOTO-3 (blue). Representative images from control and Treg-depleted mice with low or high T-cell counts are shown (A and B). CD8þ and CD4þ T cells from Treg-depleted (n ¼ 14) and control mice (n ¼ 8) were compared (C and D) and correlated with individual tumor growth rates (E and F, DT-treated mice; G and H control untreated mice). ns, not statistically signiﬁcant.

carcinogen-induced tumors. Previously, using the tumor from blood. The significance of this effect is striking: HEV cell line, B16, it was reported that although Treg-depletion are not observed unless Tregs are depleted and there is an promoted systemic T-cell activation (as also shown absolute concordance between presence of HEV, high num- above), control of tumor growth was limited by failure of bers of tumor-infiltrating T lymphocytes, and control of the T cells to infiltrate the tumor (6). In this and previous tumor growth. studies, it was shown that sensitizing the tumor stroma As yet, we have not identified the mechanisms underpinning through irradiation or inflammation, induced vascular HEV neogenesis in tumors. Under nonpathologic conditions, remodeling and upregulation of adhesion molecules facili- HEV development is confined to secondary lymphoid tissue. tating T-cell infiltration into tumors (6, 23). The data pre- Ligation of the LTb receptor (LTbR) on stromal-organiser cells sented herein identify, for the first time, a previously unsus- by LTab expressing LTi cells represents a key event in the pected effect of Treg-depletion in facilitating neogenesis development of lymph nodes (18, 19). Although LTbR signaling of HEV, which enable T-cell recruitment into the tumor is required to maintain fully differentiated HEV (24, 25),

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chronically inflamed tissue was first reported around 15 years ago and more recently in tumors (26–28). As in lymph nodes, the presence of HEV is almost always associated with the development of a stromal cell network that organizes infiltrating lymphocytes into discrete T- and B-cell areas in so- called TLO (29–31). However, the mechanisms underlying HEV neogenesis and how it relates to the development of the stromal cell network of TLO are poorly defined. In the case of the HEVþ tumors described in this study, although we have found no evidence of LTi cells or TLOs, expression of inflammatory cytokines and lymphoid chemokines is increased. Collectively these data imply that HEV neogenesis in tumors occurs through mechanisms that promote inflammation (in this case, Treg depletion) and deviate from those operating in secondary lymph nodes, possibly in terms of the cellular source of LTab. An unexplained aspect of our findings is that HEV development, although tightly linked to Treg depletion, is only observed in around half of Treg-depleted mice. Several explanations may account for this finding, an exploration of which may offer vital clues to defining the mechanisms underpinning HEV neogenesis in tumors. First, the nature of the immune response stimulated following Treg depletion may affect whether HEV develop or not. Perhaps, following Treg depletion, T cells and/or B cells in some, but not all mice, produce important HEV inducing factors (e.g., LTab). Furthermore, the tumor may specifically impinge on the type of immune response induced following Treg depletion. Tumors that retain immunogenicity, perhaps as a result of Treg-induced im- munosuppression, may activate tumor-specificTandB cells, which, as described above, drive HEV development. On the other hand, tumors that have escaped the attention of the immune system through the loss of strong antigens (immunoediting), may fail, even after Treg depletion, to induce a sufficiently robust, HEV-promoting immune response. Indeed, Matsushita and colleagues recently showed that the outgrowth of MCA-induced tumors could result from T-cell dependent selection of tumors lacking strong antigens (32). Alternatively, tumor-intrinsic features may influence HEV development in a stochastic fashion. For instance, tumors may produce factors that either Figure 5. Staining of HEV associated molecules PNAd and MAdCAM-1 in actively inhibit or facilitate HEV development. Gaining an tumors from Treg depleted mice. Paraffin-embedded sections, from understanding of these complex issues will uncover the Treg-replete (Tregþ) and Treg-depleted (Treg–) tumors, were stained with mechanisms underpinning HEV neogenesis in tumors and anti-PNAd antibodies (brown) and counterstained with haematoxylin (blue; A–D). Representative low-power (A and B) and high-power (C and facilitate a better understanding of whether a given D) fields of view are shown. Cells stained with anti-PNAd antibodies immune response can promote tumor immunity through display a cuboidal morphology typical of HEV (arrowhead). Frozen blood vessel differentiation. sections from Tregþ (n ¼ 6) and Treg– (n ¼ 14) tumors were stained with The importance of understanding how HEV can develop in anti-PNAd antibodies and levels of PNAd expression were determined by peripheral sites has clear implications for cancer immunother- quantitating the percentage of PNAd-positive pixels per area of tumor (E). Frozen sections, from controlled tumors, were stained with anti-PNAd apy. Recently, in a study of more than 300 human tumors, (red) and anti-MAdCAM-1 (green) antibodies (F) and anti-PNAd (red) and Martinet and colleagues discovered a highly significant asso- anti-CD31(green) antibodies (G). Cells were counterstained with TOTO-3 ciation between the presence of HEV and the frequency of (blue). Data from at least 2 independent experiments. tumor-infiltrating T cells (26). Moreover, when a cohort of around 150 breast cancer patients were investigated, the same mechanisms underpinning HEV formation in lymph nodes and study reported a significant correlation between HEV and a whether they are related to those driving stromal cell differ- favorable prognosis. These data support a crucial role for HEV entiation remain incompletely defined. HEV neogenesis within neogenesis in tumor rejection. Our study highlights one avenue

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Figure 6. HEV neogenesis is associated with expression of lymphoid chemokines but not development of TLO. The relative expression of various HEV- associated genes were determined and 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 antibodies speciﬁc for B and T cells (red and green; anti-B220 and anti-CD3), FDCs (green; anti-CD35) or T cells and PNAd (green and red; anti-CD3 and anti-PNAd). Cells were counterstained with TOTO-3 (blue). Scale bar, 20 mm. Data from at least 2 independent experiments.

toward achieving this goal, namely the depletion of Tregs. It into the tumor (33). In both cases, this resulted in efficient would be extremely interesting therefore to examine whether priming of tumor-specific T cells, presumably because of in large cohorts of human tumors such as those used in the the high probability of these T cells meeting their cognate above studies, there is an inverse association between Tregs antigenwithinthetumormass.Wesuggestthatdepleting and the presence of HEV. Tregs can lead to HEV neogenesis; a process that is critical Although the precise mechanism of HEV induction in in determining the composition and size of the T-cell our study remains to be elucidated, the significant increase infiltrate thus permitting tumor destruction. Although the in expression of LTb in HEVþ tumors and the previously mechanisms of HEV induction and the influence of Tregs described role for LTab in maintaining fully functional on this process are poorly understood, it is clear that a HEV, support an important role for this cytokine. A study better understanding of these mechanisms will present by Schrama and colleagues showed that administration of new opportunities for more effective antitumor therapy. an LTa fusion protein into B16 melanoma-bearing mice Furthermore, HEV neogenesis may play deleterious role resulted in HEV neogenesis, which subsequently promoted in autoimmunity and chronic inflammation (9). Our find- entry of naive T cells that were primed intratumorally and ings may therefore point to a hitherto unidentified mech- which contributed to tumor destruction (10). Similarly, Yu anism through which a defect in Treg activity drives and colleagues reported the same effect in mice inoculated autoimmune disease and chronic inflammation by allowing with LIGHT-expressing tumors; this effect was attributed HEV induction, hence sustained immune cell infiltration to the ability of LIGHT, such as LTa to facilitate generation and destruction of the affected tissue. Harnessing this of lymphoid structures, which enabled entry of naive T cells activity of Tregs therefore has implications for both cancer

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Figure 7. Increased T-cell infiltration and reduced tumor growth rate in HEVþ Treg-depleted tumors. The þ number of tumor infiltrating CD8 cells (A) and the tumor growth rates (B) of Tregþ HEV–, n ¼ 8, Treg– HEV–, n ¼ 6, and Treg– HEVþ, n ¼ 7, tumors were compared. , P < 0.01; Mann–Whitney test. Frozen sections were stained with CD31- and CD8- specific antibodies. Levels of CD31 expression were determined by quantitating the percentage of CD31-positive pixels per area of tumor and compared with numbers of CD8þ T cells (C). Frozen sections, from HEVþ tumors, were stained with anti-PNAd (red) and anti-CD8 (green) antibodies (D) or anti-PNAd (red) and anti-CD4 (green) antibodies (E). Cells were counterstained with TOTO-3 (blue). CD8þ and CD4þ cells are seen attached to and extravasating through the vessel wall (arrowheads; D and E). Gene expression analysis of T-cell associated genes was compared between tumor samples (F). Data from at least 2 independent experiments. ns, not statistically significant.

immunotherapy and the treatment of chronic inflamma- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.P. Hindley, E. Jones, S. Cutting, K. Ladell, D. Price, A. tory diseases. J. Godkin, A. Gallimore Writing, review, and/or revision of the manuscript: J.P. Hindley, E. Jones, K. Disclosure of Potential Conflicts of Interest Ladell, D. Price, A. Ager, A.J. Godkin, A. Gallimore No potential conflicts of interest were disclosed. Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): J.P. Hindley, E. Jones, K. Smart, H. Bridgeman, A. Gallimore Authors' Contributions Study supervision: J.P Hindley, E. Jones, A. Gallimore Conception and design: J.P. Hindley, E. Jones, B. Ondondo, A. Gallimore Development of methodology: J.P. Hindley, E. Jones, B. Ondondo, K. Ladell, D. Price, A. Ager Acquisition of data (provided animals, acquired and managed patients, Acknowledgments provided facilities, etc.): J.P. Hindley, E. Jones, S.N. Lauder, S. Cutting, K. Ladell, The authors are grateful to the staff at Cardiff University JBIOS for their K. Wynn, D. Withers, D. Price, A. Gallimore continued support.

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Hindley et al.

Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate This work was supported by a project grant from the Medical Research this fact. Council (G0801190) and a University Award (A. Gallimore) from the Wellcome Trust (086983/Z/08/Z). The costs of publication of this article were defrayed in part by the Received May 16, 2012; revised August 22, 2012; accepted August 24, 2012; payment of page charges. This article must therefore be hereby marked published OnlineFirst September 7, 2012.

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5482 Cancer Res; 72(21) November 1, 2012 Cancer Research

T-Cell Trafficking Facilitated by High Endothelial Venules Is Required for Tumor Control after Regulatory T-Cell Depletion

James P. Hindley, Emma Jones, Kathryn Smart, et al.

Cancer Res 2012;72:5473-5482. Published OnlineFirst September 7, 2012.

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