cancer research in situ vaccination combined with androgen ...and reducing regulatory t-cell (treg)...

Published OnlineFirst April 20, 2010; DOI: 10.1158/0008-5472.CAN-09-2490

Microenvironment and Immunology
Cancer
Research

In situ Vaccination Combined with Androgen Ablation andRegulatory T-Cell Depletion Reduces Castration-ResistantTumor Burden in Prostate-Specific Pten Knockout Mice

Elizabeth J. Akins1, Miranda L. Moore2, Shuai Tang2,3, Mark C. Willingham2,Janet A. Tooze4, and Purnima Dubey1,2,5,6,7

Abstract

Authors' AGraduate3MolecularSciences, 5

and ImmuUniversity H

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E.J. Akins a

CorrespoHealth ScSalem, NCE-mail: pdu

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There is no effective treatment for prostate cancer arising after androgen ablation. Previous studies haveanalyzed the short-term effects of androgen ablation on the immune system and suggest an abatement ofimmune suppression by hormone removal. Because castration-resistant disease can arise years after treat-ment, it is crucial to determine the duration of immune potentiation by castration. Because immunothera-peutic efficacy is determined by the balance of immune cell subsets and their location within the tumor, weassessed the acute and chronic effect of androgen ablation on the localization of T-cell subsets withincastration-resistant murine prostate cancer. We observed a transient increase in CD4+ and CD8+ T-cellnumbers at the residual tumor after androgen ablation. More than 2 months later, regulatory T cells (Treg)were increasingly found within prostate epithelium, whereas CTLs, which were evenly distributed beforeandrogen ablation, became sequestered within stroma. Anti-CD25 antibody administration along withcastration enhanced CTL access to cancerous glands but did not increase effector function. Intraprostaticinjection of LIGHT-expressing tumor cells increased the proportion of CD8+ T cells with functional capacitywithin the cancerous gland. In addition, Treg depletion within the tumor was enhanced. Together, these ma-nipulations significantly reduced castration-resistant tumor burden. Thus, our results indicate that immunemodulations, which prevent Treg accumulation and augment effector cell infiltration of prostatic epithelium,may be effective in reducing tumor burden or preventing tumor recurrence after androgen ablation therapy. CancerRes; 70(9); 3473–82. ©2010 AACR.

Introduction

Androgen ablation therapy of prostate cancer causes apo-ptotic cell death, significantly reducing tumor burden (1).However, the benefit is often short-lived, and many patientsdevelop fatal androgen-independent disease (2).Systemic androgen removal modulates T-cell number and

function, increasing peripheral T-lymphocyte numbers (3, 4)and reducing regulatory T-cell (Treg) numbers (5). Androgenablation caused an early and transient increase in T-cell

ffiliations: 1Molecular Medicine and Translational ScienceProgram, 2Department of Pathology-Tumor Biology,Pathology Graduate Program, 4Department of BiostatisticalDepartment of Cancer Biology, 6Department of Microbiologynology, 7Comprehensive Cancer Center, Wake Forestealth Sciences, Winston-Salem, North Carolina

lementary data for this article are available at Cancer Research://cancerres.aacrjournals.org/).

nd M.L. Moore contributed equally to this work.

nding Author: Purnima Dubey, Wake Forest Universityiences, Medical Center Boulevard, 2104 Gray, Winston-27157-1092. Phone: 336-716-7078; Fax: 336-716-6757;[email protected].

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infiltration of human prostate tumors (6) and removed tole-rance to prostate cancer antigens in a transgenic mousemodel (7), suggesting that systemic removal of androgencan enhance prostate antitumor immunity.The presence of lymphocytes within prostate tumors (8, 9)

and circulating tumor-reactive T cells in the peripheral bloodof prostate cancer patients (10–14) have been reported. Effec-tor CD8+ T cells (6, 15), regulatory FoxP3+ cells (16), andTh17 cells (17) have been observed within prostate tumors.Whether the composition of lymphocytes favors effector orregulatory cells serves as a prognostic indicator of diseaseoutcome (18–20).In mouse models, dysfunctional T cells both systemically

and at the tumor site (21–25) indicate that CTL may fail tobecome fully functional or may receive one or more suppres-sive signals. The presence of Tregs at the tumor site can in-hibit effector cell trafficking and function. However, studiesdepleting Tregs have had mixed results in mouse models(26, 27). Human and animal studies suggest that androgenablation may overcome some suppressive signals, resultingin increased effector cell presence at the tumor site. Forthe postcastration influx of lymphocytes to be advantageous,the balance should favor effector cells and be maintainedover time. However, immune potentiation must be insuffi-cient because the tumor recurs.

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Malignant cells within prostate glands are surrounded by astroma that impedes lymphocyte infiltration of the tumorand presents a significant barrier (28), which functionalCTL must cross to access the tumor. Whereas a study in pa-tients with non–small cell lung cancer showed high levels ofepithelial infiltration by CD8+ cells and stromal infiltrationby CD4+ cells and suggested both as independent, positiveprognostic indicators of patient survival (18), such a studyhas not been reported for prostate cancer.We hypothesized that androgen ablation enhances antitu-

mor immunity by transiently shifting the balance of lympho-cyte subsets to favor effector CD8+ T cells and that, overtime, the lymphocyte composition changes to favor cells thatinhibit tumor rejection. We investigated this hypothesis in aprostate-specific Pten (phosphatase and tensin homologueon chromosome 10) knockout mouse model of prostate can-cer, a model that recapitulates human prostate cancer deve-lopment and progression (29). Androgen ablation in thismodel leads to apoptotic death in the primary tumor withpersistence of invasive disease. We characterized theT-lymphocyte infiltrate within these androgen-independentprostate tumors and found an overall increase in boththe number of T cells within the prostate and the ratio ofCD8+/FoxP3+ T cells.Using immunohistochemical analysis, we determined

that although Treg depletion did not increase the percen-tage of functional CD8+ T cells, it increased their access tocancerous glands. Vaccination with tumor cells expressingthe tumor necrosis factor (TNF) superfamily memberLIGHT (lymphotoxin-like, exhibits inducible expression,and competes with herpes simplex virus glycoproteinD for HVEM, a receptor expressed by T lymphocytes;ref. 30) along with castration and anti-CD25 administrationreduced tumor volume and sustained Treg depletion withinthe tumor. Thus, CTL infiltration of prostatic epithelium andin situ vaccination were crucial to an effective antitumorresponse.

Materials and Methods

Mice and cell lines. All animal work followed Wake ForestUniversity Health Sciences (WFUHS) Institutional AnimalCare and Use Committee regulations. PtenloxP/loxP mice wereobtained from Dr. Yong Chen (WFUHS) with permissionof Dr. Hong Wu (University of California at Los Angeles;ref. 29). A PB-Cre4 transgenic mouse breeding pair (31) wasobtained from the National Cancer Institute Mouse Modelsof Human Cancer Consortium, and the line was maintainedand intercrossed with PtenloxP/loxP mice to generate Pten−/−

male mice as described previously (29).Animals were killed at the indicated time points, and

wet weights of pooled prostate lobes were obtained.Tumors and spleens were embedded in OCT medium.H&E-stained sections were evaluated for the presenceof tumor by M.C.W. in a blinded fashion.UV-8101-RE sarcoma cells (32) were maintained in

DMEM + 10% fetal bovine serum (both from Lonza). PtenCaP8 cells were maintained in Pten medium, as described

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previously (33). TRAMP C-1 cells were maintained in TRAMPmedium (34).Castration. Mice were anesthetized with an i.p. injection

of 100 μL/25 g of a ketamine/xylazine mixture (23.75 mg/mLketamine + 1.25 mg/mL xylazine) and castrated by surgicalremoval of both testicles.Treg depletion. Tregs were depleted by a single i.p. injec-

tion of 0.5 mg anti-CD25 antibody (ref. 35; clone PC61,BioXCell).Antibody sources. The antibodies used are as follows: anti-

CD4 (clone GK1.5), anti-FoxP3 (clone FJK-16s), and IgG2b iso-type control (eBioscience); polyclonal rabbit anti-granzyme Band rabbit IgG (Abcam); anti-CD8 (clone 53-6.7), IgG2a iso-type control, and goat anti-rat IgG (BD Biosciences); biotiny-lated donkey anti-rabbit and goat anti-mouse Fab (JacksonImmunoResearch); and goat anti-rat-AF546 and donkeyanti-rat-AF488 (Invitrogen).Immunohistochemistry and immunofluorescence. Eight

micrometer cryosections were stained using standard im-munohistochemical techniques. For single staining, slideswere incubated with anti-CD4 (5 μg/mL), anti-CD8 (5 μg/mL),or anti-Foxp3 (10 μg/mL) antibody for 1 hour and biotiny-lated goat anti-rat immunoglobulin (1:300). Signal wasamplified using ABC kit (Vector Labs), developed with3,3′-diaminobenzidine (DAB), and mounted using Permount(Fisher).Granzyme B+/CD8+ cells were detected by immunohisto-

chemical analysis or immunofluorescence double staining.CD8 staining was developed with DAB + nickel, followed byanti-granzyme B (5 μg/mL, 30 minutes), donkey anti-rabbitbiotin (1:250), and avidin-biotin complex, developed with AEC(Vector Labs). For immunofluorescence, granzyme B was de-tected with red substrate 1 kit (Vector Labs), and CD8 wasdetected with Alexa Fluor-488 (1:100, Molecular Probes,Invitrogen Corp.).For double staining of CD4/FoxP3, slides were fixed in ice-

cold methanol, washed in PBS, and blocked with 1% bovineserum albumin/PBS. Endogenous mouse immunoglobulinwas blocked with goat anti-mouse Fab (100 μg/mL,30 minutes) washed, followed with rat anti-mouse FoxP3(40 μg/mL, overnight, room temperature). Slides were devel-oped with goat anti-rat AF546 (25 μg/mL), washed, andblocked, and anti-CD4 (25 μg/mL, 30 minutes) was followedby donkey anti-rat AF488 (25 μg/mL), counterstained with4′,6-diamidino-2-phenylindole,and mounted in 90% glycerol/PBS.Enumeration of T-cell subsets. Pictures of three to four

fields per prostate were quantified using the ImageJ CellCounter Plug-in (NIH). The total number of positive cellsper field and the number of positive cells in the glands weredetermined. Pictures were taken by E.J.A. or P.D. Cell countswere blinded or independently verified by P.D.Statistical analysis. One to four 8-μm sections of tissue

were measured on each mouse. The analysis used all of themeasurements and accounted for the correlation of havingrepeated measures in a mouse by using a mixed effects mo-del, with a random mouse effect. Differences in prostate tu-mor weight and the number of cells at each time point andtreatment were compared using pairwise comparisons from

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CTLs Are Sequestered in Tumor Stroma after Androgen Removal


the mixed effects ANOVA model. Differences within mice inthe percentage of cells expressing granzyme B+, granzymeB+ percentage in stroma versus gland, and the density(cells/mm2) of CD4+, CD8+, and FoxP3+ in stroma versusgland were compared with 0 using a t test contrast in themixed effects ANOVA model. All analyses were performedin SAS (v 9.2), with a two-sided α level of 0.05. Data areshown as mean ± SEM and are compiled from at least fiveanimals per group.Flow cytometry. Splenocytes were dissociated into a single-

cell suspension and stained using the mouse Treg staining kit(eBioscience), following the manufacturer's instructions. Cellswere analyzed on a FACSCalibur using Cellquest PRO software(BD Biosciences).MLTC and 51Cr release assay.MLTC cultures were set up,

and lytic activity was assayed, as described previously (32),and assessed on days 6 to 7 of culture. Supernatant washarvested and measured after 4.5 hours.

Results

Deletion of exon 5 of the Pten tumor suppressor gene inthe prostate causes invasive prostate adenocarcinoma in100% of the mice (29) by 9 weeks of age. Although castrationat 16 weeks of age induced apoptosis within the primary tu-mor, invasive adenocarcinoma was maintained in the residualtissue. We analyzed animals between 2.5 and 10 weeks post-castration to span different stages of androgen-independentprostate growth. Prostate weights did not change significantlyover time (Fig. 1A), and carcinoma was detected at all timepoints by H&E staining (Supplementary Fig. S1).T-lymphocyte ratios favor CD8+ T cells early after

castration. Previous studies (6, 15) reported an increase inlymphocyte numbers at the prostate 1 to 4 weeks after hor-mone ablation. To determine the effect of castration on thenumber and proportion of T lymphocytes, we analyzed theprostates of Pten knockout mice by immunohistochemicalanalysis (Fig. 1B). Quantitative analysis showed that com-pared with intact 8- to 16-week-old mice, 2.5 weeks after cas-tration, there were ∼6-fold and 4-fold increases in thenumber of CD8+ and CD4+ T cells in the prostate, respectively(Fig. 1C). However, by 5 weeks postcastration, the numbers ofboth subsets were not significantly different from precastra-tion numbers (Fig. 1C), suggesting that the effects of hormoneremoval on the immune system are acute. The small numberof FoxP3+ T cells present did not change significantlythroughout tumor progression (Fig. 1C).The ratio of CD8+ to FoxP3+ T cells within the tumor is a

prognostic indicator of immune efficacy (20, 36, 37). Al-though not statistically significant, we observed an ∼7-foldincrease in the CD8+/FoxP3+ T-cell ratio at 2.5 weeks aftercastration (data not shown), showing that androgen ablationhad an immune potentiating effect that favored effector cells.Excluded from this analysis were distinct lymphoid aggre-

gates, which were found in approximately one-third of thesamples (Supplementary Fig. S2), because their functionalstatus may be different from tumor-infiltrating lymphocytes(38–40).

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Figure 1. Castration induces a transient increase in T-cell localization tothe prostate without altering prostate tumor weight. A, prostate wet
weights at indicated time points. B, CD8, CD4, and FoxP3 staining atpre-castration (pre-Cx) and 2.5, 5, and 10 wks after castration. Scale bar,100 μm (pre-Cx to 10 wks) and 10 μm (high magnification). Arrowsmark FoxP3+ cells. C, CD8+ and CD4+ T-cell numbers increased at2.5 wks postcastration. Quantification was performed as describedin Materials and Methods. , Pre-Cx;▪, 2.5 wks; , 5 wks;
□,10 wks; #, P < 0.05 versus precastration, 5 wks, and 10 wks;a, P < 0.05 versus precastration and 10 wks.

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For most tumors, rejection is mediated by effector CTL.Therefore, we evaluated the functional status of the CD8+T cells infiltrating the prostate gland after castration to as-sess whether there was an increase in functional cells. Theproportion of CD8+ T cells that expressed granzyme B, amarker of lytic capacity, was evaluated (Fig. 2A) and wasnot significantly different before castration and at 2.5 weeksfollowing castration (Fig. 2B). Because CD8+ T-cell numbersincreased, the number of granzyme B+ cells increased ∼10-fold (data not shown). However there was no effect on tumorvolume. By 10 weeks postcastration, the percentage of gran-zyme B+ cells in the tumor had declined significantly (Fig. 2B),indicating that enhancement of immune function was not sus-tained. Notably, about one-third of the CD8+ T cells at the tu-mor site before castration expressed granzyme B, suggestingthat the growing tumor may have elicited a CTL response.Effector T cells localized to the prostate do not efficiently

access tumor epithelium. Random distribution of lympho-cytes within a tissue may indicate that they are not activelyinvolved in immune responses (41). There was no preferen-tial localization of CD8+ and CD4+ cells within the prostateat any time point (Fig. 3A). In contrast to the CD4+ popula-tion, FoxP3+ cells were predominantly located in the tumorstroma early after castration (Fig. 3A). However, as the tumorprogressed, Tregs were increasingly detected in the epitheli-um, suggesting that glandular localization of Tregs may beone hallmark of prostate cancer progression.To assess whether functional CD8+ T cells were able to

overcome the stromal barrier, we calculated the percentageof CD8+ T cells within stroma and gland that expressed gran-zyme B. Whereas there was no difference in the location ofgranzyme B+CD8+ cells in noncastrated mice, at both 2.5 and5 weeks following castration, there was a significantly higher



proportion of functional cells in the tumor-associated stromathan in the glands (Fig. 3B). In addition, the majority of func-tional cells were excluded from the prostate glands at alltime points examined (Fig. 3C). Thus, whereas androgen ab-lation induced a substantial increase in lymphocyte numbersin the prostate, their location within the organ was not con-ducive for targeting of malignant epithelial cells.Systemic PC61 administration does not deplete Tregs

within the prostate tumor. Persistence of Tregs may be animmune suppression mechanism critical to prostate cancerprogression. Therefore, we hypothesized that depletion ofTregs at the time of castration would enhance T-cell locali-zation to function within the prostate epithelium.In a melanoma model, prophylactic anti-CD25 treatment

promoted tumor rejection, whereas therapeutic anti-CD25treatment did not increase CD8+ T cell/Treg ratio or en-hance tumor infiltration by effector T cells (42). Because an-drogen ablation results in death of the majority of existingtumor cells, we reasoned that concurrent PC61 treatmentwould mimic the prophylactic phenomenon; therefore, wegave the anti-CD25 antibody, PC61, i.p. 2 days before castra-tion (35). Flow cytometry analysis of splenocytes showed thatTregs were depleted systemically 48 hours later (Fig. 4A).Because changes in lymphocyte function and number were

most evident at 2.5 weeks postcastration, we evaluated theeffect of a single anti-CD25 treatment at that time point.The number of CD4+ and CD8+ T cells that infiltrated theprostate was not different from untreated animals (Fig. 4B).Surprisingly, FoxP3+ T cell numbers were significantly higherthan castrated animals that were not treated with antibody(Fig. 4B), suggesting that PC61 administration did not de-plete intratumoral Tregs or that compensatory mechanismswithin the tumor maintained Tregs at the tumor site. We

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Figure 2. Androgen ablation doesnot alter granzyme B+ CD8+ T-cellproportions. A, representativesections of granzyme B+ cells (red)and CD8+ cells (black) at eachtime point. B, the percentage ofgranzyme B+ CD8+ cells wascalculated (#, P < 0.05 versus10 wks). , Pre-Cx;▪, 2.5 wks;

□, 5 wks; , 10 wks. Scale bar,100 μm.

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analyzed prostate tumors 3 days after antibody administra-tion and found that Treg numbers within the prostate tumorwere similar to those of untreated animals (Fig. 4B), sugges-ting that systemic antibody administration did not depleteintratumoral Tregs.Fluorescent double staining (Fig. 4C) showed that the

FoxP3+ cells within the tumor were also CD4+, indicatingthe accumulation and maintenance of classic Tregs at thetumor site (43).Treg depletion augments effector cell infiltration of tumor

epithelium without increasing effector cell function.We alsoexamined the effect of PC61 treatment on the localization ofT cells within the prostate microenvironment. Whereas theCD8+ cells continued to be randomly distributed throughoutthe organ, CD4+ cells were preferentially localized withintumor stroma (Fig. 4D).



Interestingly, the FoxP3+ cells were enriched in the glands,in contrast to prior significant enrichment in the stroma(Fig. 4D). Thus, PC61 treatment accelerated localizationof Tregs to the epithelium, which occurred by 10 weekspostcastration in the absence of antibody treatment.The percentage of granzyme B+ CD8+ T cells was signifi-

cantly lower in antibody-treated mice (Fig. 5A). BecauseCD8+ T-cell numbers did not decrease after antibodytreatment, systemic anti-CD25 administration may haveprevented acquisition of full effector function.CD8+ cells expressing granzyme B were evenly distributed

between stroma and gland after PC61 administration, sug-gesting that removal of Tregs facilitated tumor infiltrationby effector cells (Fig. 5B). However, compensatory move-ment of regulatory cells to the same compartment may keepeffector function in check and prevent tumor rejection.

Figure 3. Both effector CD8+ T cells andFoxP3+ T cells are localized to prostatetumor stroma after androgen ablation.A, FoxP3+ cells are preferentially localizedto tumor stroma after androgen ablationand shift to the cancerous glands over time(*, P < 0.05 compared with gland or stromaat the same time point). B, the percentageof CD8+ cells in stroma or gland expressinggranzyme B was determined (*, P < 0.05versus gland at the same time point). ,Pre-Cx;▪, 2.5 wks; , 5 wks; □,10 wks. C, the distribution of granzymeB+/CD8+ T cells in stroma or gland wasdetermined (*, P ≤ 0.05 versus gland at thesame time point).▪, stroma; □, gland.

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In situ vaccination with LIGHT-expressing tumor cellsdecreases tumor burden. Experiments in transplantedtumor models have shown that introduction of the TNFreceptor superfamily member LIGHT at the tumor sitecan recruit and activate naive T cells, causing rejection ofantigenically unrelated tumors. LIGHT is a ligand for herpesviral entry mediator expressed on T cells and lymphotoxin-β receptor expressed on stromal cells (28). Ligation of thesetwo receptors increased localization of effector T cellswithin the tumor (30) and may enhance extravasation oftumor stroma. We tested the hypothesis that intraprostaticinjection of LIGHT-expressing tumor cells would increaseinfiltration of the cancerous glands by functional CD8+T cells. The highly immunogenic C57BL/6-derived sarcomatumor cell line UV-8101-RE (32) was transduced with a rep-lication-incompetent retrovirus expressing mutant LIGHT(mLIGHT; Supplementary Fig. S3). Two days after injectionof PC61 antibody, Pten knockout mice were castrated andsimultaneously injected with 1 × 106 UV-8101-RE-mLIGHTor UV-8101-RE cells alone into both anterior and dorso-lateral prostate lobes. Prostates and spleens were harvestedand analyzed 2.5 weeks later. On the day of the surgery, pros-tate lobes and seminal vesicles were enlarged and clearly



visible. At the time of harvest, seminal vesicles were shrunkenand prostate lobes were reduced in size (data not shown).Mixed lymphocyte tumor cell cultures of spleens from

immunized mice showed that both UV-8101-RE–vaccinatedand UV-8101-RE-mLIGHT–vaccinated mice elicited astrong and specific lytic response by 2.5 weeks postvacci-nation, which was indistinguishable from that of wild-typenon–tumor-bearing mice (Fig. 6A). Tumor wet weightsfrom animals injected with UV-8101-mLIGHT were signifi-cantly smaller compared with weights of tumors from ani-mals that were castrated and treated with PC61 antibody(Fig. 6B) and approaching significance compared with ani-mals 2.5 weeks after castration alone. In contrast, althoughPten knockout mice injected with UV-8101-RE cells alsomounted a specific CTL response to the vaccine, therewas no significant reduction in prostate tumor weight.Although CD8+ T-cell numbers in the tumors of vaccinated

mice were unchanged (Fig. 6C), the number of FoxP3+ T cellssignificantly decreased within tumors vaccinated withUV-8101-RE-mLIGHT or UV-8101-RE alone (Fig. 6C). Thus,generation of a strong and productive CTL response withinthe tumor may augment Treg depletion initiated by PC61treatment.The proportion of CD8+ cells expressing granzyme B was

significantly increased in the prostate glands of mice vacci-nated with UV-8101-RE cells, with or without the addition ofLIGHT (Fig. 6D), likely due to the generation of a strong CTLresponse against the UV-8101 tumor-specific antigen. Never-theless, only vaccination with LIGHT-expressing tumor cellsreduced tumor burden, suggesting that introduction ofLIGHT in an immunogenic context may enhance bystanderCTL responses to prostate tumor antigens. Two of five micevaccinated with UV-8101-RE-mLIGHT also lysed Pten CaP8cells, a tumor cell line derived from a Pten knockout mouseprostate tumor (one example shown in Fig. 6A, top left),whereas mice vaccinated with UV-8101-RE cells did not lysePten CaP8 cells (Fig. 6A, top right).

Discussion

We evaluated the short-term and long-term effect ofandrogen ablation on the localization of T-cell subsets toprostate tumors in a prostate-specific Pten knockout mousemodel. Pten deletion is frequently observed in human pros-tate cancers (44). Therefore, evaluation of the effects ofimmune modulations in a model that has parallels to humandisease is a particular strength of our studies.Previous studies in human and mouse models have re-

ported that androgen ablation, the preferred first-line therapy

Figure 5. Treg depletion alleviates sequestration of granzyme B+ cellsin the tumor stroma. A, percentage of granzyme B+ CD8+ cells in theprostate is reduced after PC61 administration. *, P < 0.05 comparedwith 2.5 wks. □, pre-Cx;▪, 2.5 wks; , 2.5 wks postcastration + PC61administration. B, granzyme B+ cells are equally distributedbetween tumor stroma and gland after PC61 treatment.▪, stroma;, gland. *, P < 0.05 compared with gland at the same time point.

Figure 4. Treg depletion changes T-cell distribution within the prostate tumor microenvironment. A, Tregs were detected by flow cytometry as described inMaterials and Methods. Top right quadrant, CD25+ FoxP3+ T cells (%). B, FoxP3+ cells are not depleted in the prostate after systemic PC61 administration,whereas CD8+ and CD4+ T cell numbers are unchanged.▪, 2.5 wks; , 2.5 wks + PC61 administration; □, 3 d postadministration of PC61.*, P < 0.05 compared with 2.5 wks and 3 d post-PC61. C, fluorescent double staining for CD4 and FoxP3. One representative sample from the2.5 wks + PC61 treatment group. Arrowheads, CD4/FoxP3 double-positive cells; arrow, single CD4+ cell. D, CD4+ T cells are preferentially localizedto prostate stroma, whereas FoxP3+ cells are within cancerous glands.▪, stroma; , gland. *, P < 0.05 compared with gland at the same time point;a, P = 0.05 compared with stroma at the same time point. Data for 2.5 wks time points in B and D are the same as in Figs. 1C and 3A, respectively.

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Figure 6. Vaccination with LIGHT-expressing tumor cells together with castration and PC61 administration reduced prostate tumor weight. A, splenocytesfrom vaccinated mice were stimulated in vitro with UV-8101-LIGHT or UV-8101-RE.▪, UV-8101-RE-LIGHT; ♦, UV-8101-RE; •, Pten CaP8;▴,TRAMP C-1. B, prostate wet weight of mice was determined. P = 0.057 between 2.5 wks and 2.5 wks + PC61 + UV-8101-LIGHT. P = 0.03between 2.5 wks + PC61 and 2.5 wks + PC61 + UV-8101-RE-LIGHT. C, FoxP3+ cells are significantly decreased in the tumor after vaccination withimmunogenic sarcoma cells.▪, 2.5 wks; □, 2.5 wks + PC61 treatment; , 2.5 wks + PC61 + UV-8101-RE-LIGHT; , 2.5 wks + PC61 + UV-8101-RE.*, P < 0.05 compared with 2.5 wks and 2.5 wks + PC61; a, P < 0.05 compared with castration and PC61 treatment alone. D, proportion of granzyme B+cells within the prostate glands is significantly increased after intraprostatic vaccination.▪, stroma; , gland. *, P < 0.05 compared with gland at thesame time point; #, P < 0.05 compared with gland at 2.5 wks and 2.5 wks + PC61 treatment. Data for 2.5 wks and 2.5 wks + PC61 are the same as inFigs. 1 and 4.

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for localized prostate cancer, increases the number and func-tional status of T cells at the tumor site. However, this effectmust be transient, because the tumor continues to grow. Theincrease in functional cell density we observed was not a resultof smaller prostate volume, because tumor weights early andlate after castration were similar. Androgen ablation alone wasinsufficient to prevent tumor recurrence, perhaps because theeffector cells remained sequestered in the stroma.The timing and dosage of anti-CD25 treatment is critical,

because CD25+ effector cells may also be depleted. In aprevious study, only prophylactic anti-CD25 administrationaugmented effector cell function (42). We reasoned thatanti-CD25 administration, along with castration, would be aprophylactic treatment for residual castration-resistantdisease. Because Tregs in the prostate tumor were not signifi-cantly depleted despite near complete systemic Treg deple-tion, intratumoral injection of PC61 may be necessary todeplete Tregs within the tumor.PC61 administration increased granzyme B+ CD8+ T-cell

access to the cancerous gland. However, CD8+ T-cell num-bers were slightly (but not significantly) reduced at the tu-mor site, indicating that CTL proliferation did not increasewithin the tumor. In addition, the percentage of granzymeB+ cells within the tumor was significantly decreased, eitherdue to depletion of effector cells or incomplete acquisition ofeffector function by CD8+ T cells. Thus, systemic administra-tion of anti-CD25 antibody may be detrimental to the antitu-mor response even when delivered in the setting of minimalresidual disease. A study using a transplantable model ofglioblastoma showed that Treg depletion by systemic PC61treatment reduced tumor burden when the tumors weresmall. Later administration of PC61 did not affect tumor bur-den and also depleted effector cells (45).One potential explanation for the inability of CD8+ T cells at

the tumor site tomount a rejection response is the lack of cost-imulation that is necessary for acquisition of full effector func-tion. Transduction of tumor cells with molecules, such as B7-1(46) and 4-1-BB ligand (47), provides costimulation in situ andaugments effector function. Subcutaneous challenge of micewith tumor cells expressing the costimulatorymolecule LIGHTincreased the immunogenicity of the transduced tumor andactivated T cells specific for an antigenically unrelated tumorpresent at a distant site within the same animal (30). Further-more, introduction of LIGHT in the tumor microenvironmentincreased effector T-cell infiltration of the tumor.We chose a highly immunogenic cell line as the vaccine to

deliver LIGHT to elicit a strong immune response within theprostate tumor. Reduction in prostate tumor weight was onlyobserved when LIGHT-expressing tumor cells were used as a



vaccine, indicating that introduction of a potent immunogenalone is insufficient to elicit a rejection response against unde-fined antigens expressed by the endogenous prostate tumor.As reported previously (30), we also observed that LIGHTcan enhance function of bystander T cells within antigenicallyand histologically unrelated tumor types. Approximately halfof the injected cells expressed LIGHT, and a further increase inretroviral transduction of the vaccine may increase the by-stander effect. To our knowledge, our study is the first toshow that LIGHT immunization has potential as a therapeuticmanipulation for endogenous tumors.Elicitation of a strong CTL response against the vaccine

may have resulted in movement of Tregs out of the organor alternately may have enhanced Treg depletion initiatedby anti-CD25 antibody treatment. We did not test whetherLIGHT vaccination alone may be sufficient to deplete Tregs.Additional experiments in this tumor model will address

whether repeated vaccinations with LIGHT-expressing tumorcells can sustain Treg depletion and increased effector func-tion within the tumor, further reducing tumor burden.In conclusion, our results imply that combination immu-

notherapy, which leads to an increased proportion of func-tional T cells that can efficiently access the cancerousglands, may be most effective at eliminating or preventingthe development of castration-resistant prostate cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Yong Chen (WFUHS) for the PtenloxP/loxP breeding pair;Dr. Yang-Xin Fu (University of Chicago) for the pcDNA3.1-mLIGHT plasmid;Karin Schreiber and Dr. Hans Schreiber (University of Chicago) for UV-8101-RE cells; Dr. Hong Wu for Pten CaP8 cells; Dr. Owen Witte (University ofCalifornia at Los Angeles) for TRAMP C-1 cells; Wei Du, Marie Plyler, andthe staff of Molecular Diagnostics laboratory of WFUSM Department ofPathology for their assistance; Dr. Weiwei Huang (WFUHS) for teaching usintraprostatic injections; Dr. Hong Wu for helpful suggestions on themanuscript; and Drs. Hans Schreiber and Yang-Xin Fu for helpfulsuggestions on the LIGHT experiments.

Grant Support

American Cancer Society Research Scholar Award grant RSG-07-196-01 andNIH grant R21CA124457 (P. Dubey) and NIH grant T32GM063485 (E.J. Akins).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 07/06/2009; revised 02/09/2010; accepted 02/23/2010; publishedOnlineFirst 04/20/2010.

References
1. Huggins C. Endocrine-induced regression of cancers. Science 1967;
156:1050–4.2. Crawford ED. Challenges in the management of prostate cancer. Br J

Urol 1992;70Suppl 1:33–8.3. Aragon-Ching JB, Williams KM, Gulley JL. Impact of androgen-

deprivation therapy on the immune system: implications for combi-nation therapy of prostate cancer. Front Biosci 2007;12:4957–71.

4. Hess PR, Boczkowski D, Nair SK, Snyder D, Gilboa E. Vaccinationwith mRNAs encoding tumor-associated antigens and granulo-cyte-macrophage colony-stimulating factor efficiently primes CTLresponses, but is insufficient to overcome tolerance to a modeltumor/self antigen. Cancer Immunol Immunother 2006;55:672–83.

5. Page ST, Plymate SR, Bremner WJ, et al. Effect of medical castrationon CD4+ CD25+ T cells, CD8+ T cell IFN-γ expression, and NK cells:

Cancer Res; 70(9) May 1, 2010 3481



Akins et al.

3482


a physiological role for testosterone and/or its metabolites. Am JPhysiol Endocrinol Metab 2006;290:E856–63.

6. Roden AC, Moser MT, Tri SD, et al. Augmentation of T cell levels andresponses induced by androgen deprivation. J Immunol 2004;173:6098–108.

7. Drake CG, Doody AD, Mihalyo MA, et al. Androgen ablation mitigatestolerance to a prostate/prostate cancer-restricted antigen. CancerCell 2005;7:239–49.

8. McArdle PA, Canna K, McMillan DC, McNicol AM, Campbell R,Underwood MA. The relationship between T-lymphocyte subset in-filtration and survival in patients with prostate cancer. Br J Cancer2004;91:541–3.

9. Vesalainen S, Lipponen P, Talja M, Syrjanen K. Histological grade,perineural infiltration, tumour-infiltrating lymphocytes and apoptosisas determinants of long-term prognosis in prostatic adenocarcino-ma. Eur J Cancer 1994;30A:1797–803.

10. Chakraborty NG, Stevens RL, Mehrotra S, et al. Recognition of PSA-derived peptide antigens by T cells from prostate cancer patientswithout any prior stimulation. Cancer Immunol Immunother 2003;52:497–505.

11. Elkord E, Rowbottom AW, Kynaston H, Williams PE. Correlationbetween CD8+ T cells specific for prostate-specific antigen and le-vel of disease in patients with prostate cancer. Clin Immunol 2006;120:91–8.

12. Elkord E, Williams PE, Kynaston H, Rowbottom AW. Differential CTLsspecific for prostate-specific antigen in healthy donors and patientswith prostate cancer. Int Immunol 2005;17:1315–25.

13. McNeel DG, Nguyen LD, Ellis WJ, Higano CS, Lange PH, Disis ML.Naturally occurring prostate cancer antigen-specific T cell responsesof a Th1 phenotype can be detected in patients with prostate cancer.Prostate 2001;47:222–9.

14. Perambakam S, Xue BH, Sosman JA, Peace DJ. Induction of Tc2cells with specificity for prostate-specific antigen from patients withhormone-refractory prostate cancer. Cancer Immunol Immunother2002;51:263–70.

15. Mercader M, Bodner BK, Moser MT, et al. T cell infiltration of theprostate induced by androgen withdrawal in patients with prostatecancer. Proc Natl Acad Sci U S A 2001;98:14565–70.

16. Miller AM, Lundberg K, Ozenci V, et al. CD4+CD25high T cells areenriched in the tumor and peripheral blood of prostate cancer pa-tients. J Immunol 2006;177:7398–405.

17. Sfanos KS, Bruno TC, Maris CH, et al. Phenotypic analysis ofprostate-infiltrating lymphocytes reveals TH17 and Treg skewing.Clin Cancer Res 2008;14:3254–61.

18. Al-Shibli KI, Donnem T, Al-Saad S, PerssonM, Bremnes RM, Busund LT.Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer. Clin Cancer Res 2008;14:5220–7.

19. Monnier-Benoit S, Mauny F, Riethmuller D, et al. Immunohistoche-mical analysis of CD4+ and CD8+ T-cell subsets in high risk humanpapillomavirus-associated pre-malignant and malignant lesions ofthe uterine cervix. Gynecol Oncol 2006;102:22–31.

20. Sato E, Olson SH, Ahn J, et al. Intraepithelial CD8+ tumor-infiltratinglymphocytes and a high CD8+/regulatory T cell ratio are associatedwith favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A2005;102:18538–43.

21. Ciavarra RP, Holterman DA, Brown RR, et al. Prostate tumor micro-environment alters immune cells and prevents long-term survival inan orthotopic mouse model following flt3-ligand/CD40-ligand immu-notherapy. J Immunother 2004;27:13–26.

22. Degl'Innocenti E, Grioni M, Boni A, et al. Peripheral T cell toleranceoccurs early during spontaneous prostate cancer development andcan be rescued by dendritic cell immunization. Eur J Immunol 2005;35:66–75.

23. Mihalyo MA, Hagymasi AT, Slaiby AM, Nevius EE, Adler AJ. Dendriticcells program non-immunogenic prostate-specific T cell responsesbeginning at early stages of prostate tumorigenesis. Prostate 2007;67:536–46.

24. Tseng-Rogenski SS, Arredouani MS, Neeley YC, Lu B, ChinnaiyanAM, Sanda MG. Fas-mediated T cell deletion potentiates tumorantigen-specific tolerance in a mouse model of prostate cancer.Cancer Immunol Immunother 2008;57:1357–65.



25. Zheng X, Gao JX, Zhang H, Geiger TL, Liu Y, Zheng P. Clonaldeletion of simian virus 40 large T antigen-specific T cells in thetransgenic adenocarcinoma ofmouse prostatemice: an important rolefor clonal deletion in shaping the repertoire of T cells specific for anti-gens overexpressed in solid tumors. J Immunol 2002;169:4761–9.

26. Degl'Innocenti E, Grioni M, Capuano G, et al. Peripheral T-cell tole-rance associated with prostate cancer is independent from CD4+CD25+ regulatory T cells. Cancer Res 2008;68:292–300.

27. Tien AH, Xu L, Helgason CD. Altered immunity accompanies diseaseprogression in a mouse model of prostate dysplasia. Cancer Res2005;65:2947–55.

28. Yu P, Fu YX. Tumor-infiltrating T lymphocytes: friends or foes? LabInvest 2006;86:231–45.

29. Wang S, Gao J, Lei Q, et al. Prostate-specific deletion of the murinePten tumor suppressor gene leads to metastatic prostate cancer.Cancer Cell 2003;4:209–21.

30. Yu P, Lee Y, Liu W, et al. Priming of naive T cells inside tumors leadsto eradication of established tumors. Nat Immunol 2004;5:141–9.

31. Wu X, Wu J, Huang J, et al. Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene abla-tion. Mech Dev 2001;101:61–9.

32. Dubey P, Hendrickson RC, Meredith SC, et al. The immunodominantantigen of an ultraviolet-induced regressor tumor is generated by asomatic point mutation in the DEAD box helicase p68. J Exp Med1997;185:695–705.

33. Jiao J, Wang S, Qiao R, et al. Murine cell lines derived from Pten nullprostate cancer show the critical role of PTEN in hormone refractoryprostate cancer development. Cancer Res 2007;67:6083–91.

34. Foster BA, Gingrich JR, Kwon ED, Madias C, Greenberg NM. Cha-racterization of prostatic epithelial cell lines derived from transgenicadenocarcinoma of the mouse prostate (TRAMP) model. Cancer Res1997;57:3325–30.

35. Cao X, Cai SF, Fehniger TA, et al. Granzyme B and perforin are im-portant for regulatory T cell-mediated suppression of tumor clea-rance. Immunity 2007;27:635–46.

36. Ladoire S, Arnould L, Apetoh L, et al. Pathologic complete responseto neoadjuvant chemotherapy of breast carcinoma is associated withthe disappearance of tumor-infiltrating foxp3+ regulatory T cells. ClinCancer Res 2008;14:2413–20.

37. Quezada SA, Peggs KS, Curran MA, Allison JP. CTLA4 blockade andGM-CSF combination immunotherapy alters the intratumor balanceof effector and regulatory T cells. J Clin Invest 2006;116:1935–45.

38. Dieu-Nosjean MC, Antoine M, Danel C, et al. Long-term survival forpatients with non-small-cell lung cancer with intratumoral lymphoidstructures. J Clin Oncol 2008;26:4410–7.

39. Gobert M, Treilleux I, Bendriss-Vermare N, et al. Regulatory T cellsrecruited through CCL22/CCR4 are selectively activated in lymphoidinfiltrates surrounding primary breast tumors and lead to an adverseclinical outcome. Cancer Res 2009;69:2000–9.

40. Harrison JC, Dean PJ, el-Zeky F, Vander Zwaag R. Impact of theCrohn's-like lymphoid reaction on staging of right-sided colon can-cer: results of multivariate analysis. Hum Pathol 1995;26:31–8.

41. Mrass P, Takano H, Ng LG, et al. Random migration precedes stabletarget cell interactions of tumor-infiltrating T cells. J Exp Med 2006;203:2749–61.

42. Quezada SA, Peggs KS, Simpson TR, Shen Y, Littman DR, Allison JP.Limited tumor infiltration by activated T effector cells restricts thetherapeutic activity of regulatory T cell depletion against establishedmelanoma. J Exp Med 2008;205:2125–38.

43. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the deve-lopment and function of CD4+CD25+ regulatory T cells. Nat Immunol2003;4:330–6.

44. Deocampo ND, Huang H, Tindall DJ. The role of PTEN in the progressionand survival of prostate cancer. Minerva Endocrinol 2003;28:145–53.

45. Curtin JF, Candolfi M, Fakhouri TM, et al. Treg depletion inhibits ef-ficacy of cancer immunotherapy: implications for clinical trials. PLoSOne 2008;3:e1983.

46. Chen L. Co-inhibitory molecules of the B7-28 family in the control ofT-cell immunity. Nat Rev Immunol 2004;4:336–47.

47. Driessens G, Kline J, Gajewski TF. Costimulatory and coinhibitoryreceptors in anti-tumor immunity. Immunol Rev 2009;229:126–44.

Cancer Research



2010;70:3473-3482. Published OnlineFirst April 20, 2010.Cancer Res Elizabeth J. Akins, Miranda L. Moore, Shuai Tang, et al.

Knockout MicePtenTumor Burden in Prostate-Specific Regulatory T-Cell Depletion Reduces Castration-Resistant

Vaccination Combined with Androgen Ablation andIn situ

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