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Research Article

Whole-Body Irradiation Increases the Magnitude andPersistence of Adoptively Transferred T Cells Associatedwith Tumor Regression in aMouseModel of Prostate Cancer

Lindsay K. Ward-Kavanagh1, Junjia Zhu2,5, Timothy K. Cooper3,4, and Todd D. Schell1,5

AbstractAdoptive immunotherapy has demonstrated efficacy in a subset of clinical and preclinical studies, but the T

cells used for therapy often are rendered rapidly nonfunctional in tumor-bearing hosts. Recent evidence indicatesthat prostate cancer can be susceptible to immunotherapy, but most studies using autochthonous tumormodelsdemonstrate only short-lived T-cell responses in the tolerogenic prostate microenvironment. Here, we assessedthe efficacy of sublethal whole-body irradiation (WBI) to enhance the magnitude and duration of adoptivelytransferred CD8þ T cells in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Wedemonstrate that WBI promoted high-level accumulation of granzyme B (GzB, Gzmb)–expressing donor Tcells both in lymphoid organs and in the prostate of TRAMP mice. Donor T cells remained responsive tovaccination in irradiated recipients, but a single round ofWBI-enhanced adoptive immunotherapy failed to affectsignificantly the existing disease. Addition of a second round of immunotherapy promoted regression ofestablished disease in half of the treated mice, with no progression observed. Regression was associated withlong-term persistence of effector/memory phenotype CD8þ donor cells. Administration of the second round ofadoptive immunotherapy led to reacquisition of GzB expression by persistent T cells from the first transfer. Theseresults indicate that WBI conditioning amplifies tumor-specific T cells in the TRAMP prostate and lymphoidtissue, and suggest that the initial treatment alters the tolerogenic microenvironment to increase antitumoractivity by a second wave of donor cells. Cancer Immunol Res; 2(8); 777–88. �2014 AACR.

IntroductionProstate cancer is the most commonly diagnosed form of

cancer, and the second leading cause of cancer-related deathsamong American men (1). Although early-stage prostate can-cer tends to respondwell to radiation or surgical excision, thereare few options for men with advanced or recurrent prostatecancer. FDA approval of Sipuleucel-T, the first therapeuticcancer vaccine (2), has rekindled the pursuit of immunother-apy for the control of prostate cancer. Adoptive T-cell transferstrategies have demonstrated efficacy in delaying onset ofadenocarcinoma (3–7), or increasing survival through diseaseregression (8) in autochthonous murine prostate tumor mod-els. Together, these results suggest that prostate cancer is agood candidate disease for further development of immuno-

therapeutic treatment strategies, but a better understanding ofthis complex tumor microenvironment is needed to promotedurable antitumor immunity.

The transgenic adenocarcinoma of the mouse prostate(TRAMP) model recapitulates the disease progressionobserved in humans, providing a realistic but challengingmodel in which to develop prostate cancer therapies. TRAMPmice express the oncogenic SV40 large T antigen (T Ag) underthe control of the rat probasin promoter in the dorsolateral andventral lobes of the prostate (9). Tumor progression in TRAMPmice on the C57BL/6 background is characterized by theappearance of prostatic intraepithelial neoplasia (PIN) at week8, withwell-differentiated adenocarcinoma becoming predom-inant in the dorsal prostate as early as 16 weeks (9, 10).Concurrent with the onset of T Ag expression at 6 weeks ofage (11), TRAMP mice develop tolerance to T Ag within theCD8þ T-cell compartment (5, 6, 11, 12), with the residualpopulation of endogenous T Ag–specific CD8þ T cells exhibit-ing little impact on tumor progression (13–15). Tumors main-tain T Ag expression throughout disease progression (5),indicating the importance of oncogene expression in tumor-cell survival, and providing a tumor-associated immune target.

Previous studies in TRAMP mice have demonstrated thattransferred tumor-specific CD8þT cells are rapidly tolerized orconverted to a suppressor phenotype (3, 7, 16, 17). However, arecent study using adoptive transfer of genetically modified Tcells into irradiated TRAMPmice resulted in extended survival

Authors' Affiliations: Departments of 1Microbiology and Immunology,2Public Health Sciences, 3Comparative Medicine, and 4Pathology, PennState Hershey College of Medicine; and 5Penn State Hershey CancerInstitute, Hershey, Pennsylvania

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Todd D. Schell, Department of Microbiology &Immunology, Penn State Hershey College of Medicine, 500 UniversityDrive, H107, Hershey, PA 17033. Phone: 717-531-8169; Fax: 717-531-6522; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0164

�2014 American Association for Cancer Research.

CancerImmunology

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(8), suggesting that whole-body irradiation (WBI) conditioningcould maximize the antitumor effect. WBI has been shown toenhance antitumor immunity of CD8þT cells inmousemodels(18–21) and in human patients with cancer (22). Our grouppreviously demonstrated that WBI-enhanced adoptive T cell–mediated immunotherapy targeting T Ag produced durableregressions of established T Ag–induced choroid plexustumors, and persistence of donor T cells at the tumor site(23). We hypothesized that conditioning TRAMP mice withWBI would augment T-cell activity and persistence within thenegative regulatorymicroenvironment of the TRAMPprostate.Thus, we investigated the impact of a sublethal dose ofWBI onthe accumulation and maintenance of adoptively transferredCD8þT cells in TRAMPmice, and the associated impact on thedisease.

Materials and MethodsMice

TRAMP mice on the C57BL/6J background were purchasedfrom The Jackson Laboratory [C57BL/6-Tg(TRAMP)8247Ng/J,stock number 003135], bred, and maintained under barrierspecific pathogen-free (SPF) conditions in the animal vivariumat the Penn State Hershey Medical Center (Hershey, PA). Micewere screened by PCR for the presence of the T Ag transgene asdescribed previously, and transgene-positive males were used inexperiments at 18 weeks of age (9, 24). gBT-I.1 transgenic miceon the C57BL/6 background were generously provided by Dr.Francis Carbone (University of Melbourne, Australia; ref. 25).TCR-IV mice on the C57BL/6 background have been describedpreviously (23). In some cases, TCR-IV hemizygote males werebred with homozygous C57BL/6-Tg(UBC-GFP)30Scha/J (stocknumber 004353) or B6.PL-Thy1a/CyJ (stock number 000406)females purchased fromThe Jackson Laboratory to yieldmarkedTCR-IV cells. All animal studieswere approved by the Penn StateHershey Institutional Animal Care and Use Committee.

Cell lines and reagentsB6/WT-19 cells expressing full-length wild-type SV40 T Ag

were derived by transformation of primary mouse kidney cellswith wild-type SV40 (26), and were originally provided byDr. Satvir S. Tevethia (Penn State University College of Medicine,Emeritus, Hershey, PA). Cells were authenticated by Westernblotting and flow cytometry for the expression of full-lengthSV40 T Ag and mouse H-2Kb and H-2Db MHC class I molecules,respectively, and were grown as previously described (23). Pro-cessing and short-term culture of single-cell lymphocyte suspen-sions were performed using RPMI-1640 media supplementedwith 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mmol/LL-glutamine, 10 mmol/L HEPES, 50 mmol/L 2-ME, and 2% FBS.

Adoptive immunotherapyEighteen-week-old male TRAMP mice were exposed to 475

cGy g-ray WBI using a 60Co-source GammaCell 220 irradiator(Nordion International) 1 day before adoptive transfer. CD8þ

donor cells from the spleens and lymph nodes (LN) of na€�veTCR-IV or gBT-I.1 mice were enriched by positive magneticsorting using an autoMACS cell sorter (Miltenyi Biotec). Purity

was determined by flow cytometry, and ranged between 85%and 95%. Irradiated andage-matchedunirradiatedmale TRAMPmice received 1 � 106 CD8-enriched cells in sterile PBS byintravenous tail vein injection. For some experiments, CD90.1þ

donor cells were labeled with 5 mmol/L carboxyfluoresceinsuccinimidyl ester (CFSE) in 0.1% BSA in PBS before transfer.

Staining and flow cytometric analysis of lymphocytepopulations

Spleens and prostates were removed and processed indi-vidually, whereas medial iliac LNs, considered the tumor-draining LN (TLN), were pooled by treatment group. Single-cell suspensions of splenocytes and LN cells were generated bymechanical disruption and depletion of red blood cells asdescribed previously (23). Prostates were digested using a 1mg/mL collagenase (Life Technologies) and 50 U/mL DNase I(Roche) mixture prepared in RPMI-1640 complete media sup-plemented with 1% FBS for 1 hour at 37�C with rocking. Thecell suspension was passed through a wire mesh to removeparticulates, and the single-cell suspensionwas treated accord-ing to the protocol used for splenocytes.

Aliquots of 2 � 106 cells were stained with commerciallyavailable antibodies to cell-surface and intracellular proteins,as well as phycoerythrin (PE)-labeled H-2Kb/IV (TetIV) or H-2Kb/gB tetramers that recognize T Ag site IV- and HSV gB 498-505–specific T cells, respectively (23). Cells stained only forsurface markers were fixed with 2% paraformaldehyde, where-as the Cytofix/Cytoperm Kit (BD Pharmingen) was used topermeabilize and intracellularly stain cells for granzyme B(GzB). Samples were run on a FACSCanto II or BD LSRIIinstrument (BD Biosciences), and data were analyzed usingthe FlowJo software (TreeStar, Inc.). The following antibodycocktails were used: CD8-V450 and CD45.2-V500 (BD Phar-mingen), TetIV-PE, CD90.1-APC, CD62L-APCe780, CD44-FITC,CD4-PECy7, CD127-PerCP Cy5.5, or GzB-PerCP Cy5.5 (Invitro-gen). All antibodies were purchased from eBioscience unlessotherwise noted.

Intracellular cytokine stainingCells (2 � 106) from the spleen- or prostate-derived cell

suspensions were stimulated with 1 mmol/L site-IV C411L-substituted peptide (VVYDFLKL) or the unrelated H-2Kb/HSVglycoprotein B (gB) peptide (SSIEFARL) plus 1mg/mL brefeldinA for 4 hours at 37�C. Cells were stained for surface expressionof CD8 and CD90.1, and permeabilized with the Cytofix/Cytoperm Kit for intracellular staining with antibodies to IFNgand TNFa as per the manufacturer's instructions. The per-centage of cytokine-producing CD8þ T cells capable ofresponding to the site IV determinant was calculated bysubtracting the frequency of gB-reactive T cells from thefrequency of C411L-reactive T cells.

HistologyTRAMP mice were euthanized by CO2 asphyxiation and

perfused with 10 mL of sterile PBS followed by 10 mL of 10%neutral buffered formalin (NBF). The entire urogenital tractwas removed for immersion fixation in NBF overnight. Tissueswere then transferred into 70% ethanol for at least 24 hours

Ward-Kavanagh et al.

Cancer Immunol Res; 2(8) August 2014 Cancer Immunology Research778




before embedding in paraffin and sectioning. Prostate tissuesections were stained with hematoxylin and eosin (H&E),provided in an anonymous fashion to a board-certified veter-inary pathologist for tumor-stage scoring according to theBerman-Booty scoring system (27). Images were obtainedusing an Olympus BX51 microscope with a �4, �40, or�100 (oil-immersion) objective fitted with an Olympus DP71digital camera and CellSens Standard 1.6 imaging software(Olympus).

Statistical analysisThe two-sample Student t test or one-way ANOVAwere used

to compare the quantitative outcome variables between treat-ment groups at individual time points. A linear mixed-effectmodel was used to analyze the effect of treatment and time ontumor scores. Grades measured in different lobes of the samemouse were used as repeated measure inputs in the statisticalmodel. The interaction effect between time and treatment wasremoved from themodel as it was not significant. The Dunnetttest was used for multiple comparisons between a specifiedtreatment group (serving as control) and other groups. Thefamily-wise type I error rate was controlled. All statisticalanalyses were performed using the SAS software version 9.3(SAS Institute) or Prism software (GraphPad Software, Inc.).The significance level was set at 0.05 for all analyses.

ResultsThe initial donor T-cell response in the tumor-drainingLN is not altered by WBIWe initiated therapy of TRAMPmice at 18weeks of agewhen

the epithelium transitions from high-grade PIN to overt car-cinoma (9, 10). One million CD8þ donor cells enriched from TAg site IV–specific TCR-IV transgenic mice were transferredinto TRAMP mice 1 day after recipient mice received a lym-phodepleting dose of 475 cGyWBI. Transfer of na€�ve T cells wasused to allow visualization of early T-cell triggering in thelymphoid organs. WBI had no significant effect on totalaccumulation of TCR-IV cells in the tumor-draining LN(TLN; Fig. 1A). However, TCR-IV cells composed a largerproportion of cells in irradiated mice due to the depletion ofendogenous populations (Fig. 1B and C). We observed areproducible increase in the accumulation of endogenousCD8þ T cells within the TLN of unirradiated mice immediatelyfollowing transfer (Fig. 1C), suggestive of a brief period ofinflammation.CFSE-labeled TCR-IV cells showed minimal proliferation 2

days after transfer, but substantial dilution of CFSE by day 5(Fig. 1D and E) when the majority of cells in both treatmentgroups exhibited a differentiated CD44hiCD62Llo phenotype(Fig. 1F and G). In contrast, distant LNs contained few donorcells that had undergone multiple rounds of division (unpub-lished data), consistent with previous reports (13, 17). Prolif-eration was dependent upon antigen recognition, as TCR-IVcells transferred into irradiated or unirradiated C57BL/6 hostsremained undivided (Fig. 1H, left), whereas immunization withT Ag–expressing cells produced strong proliferation and effec-tor differentiation (Fig. 1H, right). These results demonstrate

that the kinetics of T-cell priming in the TLN of TRAMP miceare not altered by WBI despite host lymphodepletion.

WBI promotes increased donor T-cell accumulation inthe spleen of TRAMP mice

In the spleen, we observed an initial drop in the total numberof TCR-IV cells between days 2 and 3 after transfer (Fig. 2A).TCR-IV cells then expanded to high levels in irradiated micebetween days 5 and 7, and remained at increased levelsbetween 7 and 21 days, before contracting to baseline levelsby day 28 (Fig. 2A). Endogenous CD8þ T cells remained atreduced levels over this time period (Fig. 2B). In contrast,contraction of TCR-IV cells in unirradiated recipients occurredmuch earlier, and with faster kinetics (Fig. 2A).

To evaluate whether this discrepancy could be explained bydifferences in T-cell proliferation, CFSE dilution was moni-tored among TCR-IV cells that accumulated in the spleen.Unlike results in the TLN (Fig. 1C), accumulation of TCR-IVcells that had undergone multiple rounds of division wasaccelerated in irradiated mice at both days 5 and 7 aftertransfer (Fig. 2C and D). Nearly half of TCR-IV cells in thespleen at day 5 retained CD62L expression (Fig. 2E and F). ThisCD62Lhi population was predominantly CFSEþ (Fig. 2E), con-sistent with previous findings that T-cell differentiation cor-relates with the extent of cell proliferation (28). By day 7, themajority of TCR-IV cells in both groups downregulated CD62L(Fig. 2E), despite the lack of accumulation of CFSElo cells inunirradiated mice (Fig. 2D). No CD127þ cells were detectedamong TCR-IV cells that persisted at day 21 (Fig. 2G), suggest-ing the absence of classical memory T-cell development inTRAMP mice. Extensive proliferation of donor T cells wasantigen dependent, because CD8þ T cells specific for theherpes simplex virus gB underwent only limited proliferationin the spleens of irradiated TRAMP mice by day 7 (Supple-mentary Fig. S1A). Thus, WBI promotes the systemic accumu-lation of recently divided TCR-IV cells, while such cells areconfined mainly in the TLN of unirradiated TRAMP mice. Ourresults are consistent with previous studies demonstratingabortive proliferation of tumor-specific CD8þ T cells in unir-radiated TRAMP mice (7), and suggest that WBI conditioningenhances T-cell expansion or protects a population of antigen-experienced T cells from deletion.

WBI enhances accumulation of TCR-IV cells in theprostate

Accumulation of TCR-IV cells in the prostate was signifi-cantly enhanced in irradiatedmice as early as day 5 (Fig. 3A andinset), but dramatically increased by day 7 coincident withpeak accumulation of TCR-IV cells in the spleen (Fig. 2A). TCR-IV cells in the prostate of irradiated mice contracted over thenext 3 weeks, similar to the kinetics observed in the spleen(Fig. 3A). Prostate-infiltrating TCR-IV cells were restricted toCFSElo, differentiated cells independent of irradiation (Fig. 3Cand D).

The influx of TCR-IV cells into the prostate of irradiatedTRAMPmice was paralleled by a smaller-magnitude increase inendogenous CD8þ T cells (Fig. 3B), suggesting that TCR-IV cellinfiltration promotes inflammation within the prostate. T-cell

T-cell Persistence in Irradiated TRAMP Mice

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accumulationwas antigen dependent, however, as gB-specific Tcells failed to accumulate or drive recruitment of endogenousCD8þ T cells in the prostate of irradiated TRAMP mice (Sup-plementary Fig. S1C and S1D). These results demonstrate thatWBI promoted a significant but transient spike in antigen-specific T-cell accumulation in the prostate that was accompa-nied by an increase in endogenous CD8þ T-cell accumulation.

Irradiation facilitates the survival of tumor antigen–responsive GzBþ CD8þ T cells in TRAMP mice

Previous studies in TRAMPmice have demonstrated a blockin T-cell effector function despite the initial T-cell priming, andaccumulation in the prostate (3, 6, 7, 13, 17). Likewise, we found

that few CD8þ T cells recovered from either the spleen orprostate of TRAMP mice produced IFNg or TNFa in responseto tumor antigen (Fig. 4A and B). Furthermore, WBI failed toenhance the production of either cytokine at any time testeddespite enhanced T-cell accumulation (Fig. 4A and B andunpublished data).

Despite limited IFNg and TNFa production, high propor-tions of TCR-IV cells expressed GzB, particularly prostate-infiltrating TCR-IV cells (Fig. 4C). Although WBI failed tosignificantly enhance the total number of GzBþ TCR-IV cellsin the spleen (Fig. 4D), irradiation enhanced the accumulationof GzBþ TCR-IV cells in the prostate by greater than 100-foldat day 7 (Fig. 4E). Accumulation of GzBþ TCR-IV cells was

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Figure 1. WBI enhances the frequency of TCR-IV cells within the TLN. Eighteen-week-old male TRAMP mice received 475 cGy WBI or were left unirradiated,followed 1 day later by transfer of 1 � 106 CD8-enriched, CD90.1þ TCR-IV cells. All panels represent cells isolated from the TLN and evaluated by flowcytometry at the indicated times using TetIV staining. CD8þ TetIVþ TCR-IV cells plotted as total number (A) and the percentage of total cells (B). C,total endogenousCD8þTcells. Solid line, the averageCD8þT-cell count in the TLNof untreated TRAMPmice. D,CFSEdilution in TCR-IV cells recovered fromunirradiated (top) or irradiated (bottom) TRAMP mice. Dotted line, C57BL/6 control recipients. E, CFSElo TCR-IV cells were quantified on day 5 usingthe gate shown inD; n.s., no statistical difference. F andG,CD44andCD62LexpressionbyTCR-IV cells fromunirradiated (top) and irradiated (bottom) TRAMPmice 5 days after transfer. H, CFSE dilution in TCR-IV cells from C57BL/6 mice 5 days after transfer into irradiated (middle), or unirradiated mice without (left)or with B6/WT-19 immunization (right). The marker on the TCR-IVþWT-19 indicates the CFSElo threshold. CD44 and CD62L expression (far right) byTCR-IV cells in unirradiated, immunized C57BL/6 mice. Data are pooled from two independent experiments with 5 to 7 mice per group.






antigen dependent, as TCR-IV cells recovered from nontrans-genic littermates did not express GzB (Fig. 4D and E). Althougha population of TCR-IV cells persisted in the prostates ofirradiated mice at day 21 (Fig. 3A), these cells no longerexpressed GzB (Fig. 4E). This observation suggests thatGzBþ cells were either eliminated, became quiescent, or con-verted to an anergic/suppressor phenotype in the tumormicroenvironment (7, 16).To assess the functionality of the persisting cells, TRAMP

mice were immunized with B6/WT-19 cells 3 weeks afteradoptive transfer (Fig. 4F). TCR-IV cells in unirradiatedTRAMP mice failed to expand in either the spleen or prostatefollowing immunization (Fig. 4G), and exhibited no effectorfunctions (unpublished data), consistentwith the developmentof T-cell anergy. Although we cannot rule out that TCR-IV cellsare redistributed to unrelated tissues in these mice, such cellswere not recruited by immunization. Conversely, immuniza-tion significantly increased the number of TCR-IV cells inthe spleen of irradiated TRAMP mice (Fig. 4G). Immunizationalso significantly increased the proportion of donor cellsexpressing GzB in the spleen and prostate of irradiated mice(Fig. 4H), but promoted only a minimal increase in IFNg-

producing cells in the prostate (Fig. 4I). These data indicatethat irradiation facilitates the persistence of a subset of TCR-IVcells that retain responsiveness to antigenic challenge, and canacquire effector functions late in the antitumor response.

Adoptive transfer with WBI fails to reduce the diseasescore at early times after treatment

We evaluated the impact of WBI-enhanced TCR-IV transferon the disease score in TRAMP mice. Therapy was initiated at18 weeks of age, and the disease score wasmeasured over time.Histologic scoring of the prostate lobes at 7 days after transferdemonstrated that mice in all groups remained in the high-grade PIN stage (Fig. 5F, day 7). At 21 days after transfer, micetreated with WBI or WBI-enhanced adoptive immunotherapyhad stable high-grade PIN, whereas a subset of mice in theother treatment groups had progressed to well-differentiatedinvasive adenocarcinoma (score 13) or poorly differentiatedneuroendocrine tumors (score 19 or 21; Fig. 5A–D and F, 21days). We noted a significant increase in inflammation withinthe prostate at day 21 for mice that received adoptive T-celltransfer either alone or in combination with WBI, but not inWBI-only–treated mice (Fig. 5E). However, disease scores did

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Figure 2. WBI enhances TCR-IV cellaccumulation in the spleen ofTRAMP mice. TRAMP mice weretreated as in Fig. 1, andsplenoctyes were analyzed by flowcytometry. Total number of TCR-IVT cells (A) and endogenous CD8þ Tcells (B). B, solid line, averageCD8þ

T-cell number in untreated TRAMPmice; �, statistical significance bythe Student t test. C and D, CFSEdilution in TCR-IV cells recoveredfrom unirradiated (top) or irradiated(bottom) TRAMP mice. C, dottedline, C57BL/6 control. E,representative CFSE dilution ofCD44hiCD62Lhi (solid line) andCD44hiCD62Llo (dashed line) TCR-IV populations in nonirradiated (top)and irradiated (bottom) TRAMPmice 5 days after transfer. F,quantification of CD44hiCD62Llo

TCR-IV cells. G, CD127 expressionby TCR-IV cells in nonirradiated(top) and irradiated (bottom)TRAMPmice 21 days after transfer.Quadrants were set usingendogenous CD8þ T cells. Data arepooled from at least twoindependent experiments with aminimum of 6 mice per group.






not significantly differ among the groups at either 7 or 21 daysafter treatment, and tumor regressions were not observedwithin this 21-day window.

A second round of adoptive transfer with WBI promoteshigh-level T-cell accumulation, T-cell persistence, anddisease regression

Because TCR-IV cell numbers in the prostate contractdramatically by 21 days after treatment (Fig. 3A), we admin-istered a second round of WBI-enhanced adoptive immuno-therapy to prolong the period of T-cell activity (Fig. 6A). T cellsfrom the first (CD90.1þ) and second (GFPþ) transfers wereevaluated 7 days after the second transfer. Analysis of GFPþ

TCR-IV cells revealed a significantly higher accumulation inboth the spleen and prostate of mice that received the initialtreatment at 18 weeks relative to mice that only receivedtreatment at 21 weeks (Fig. 6B and C). This result suggeststhat the first treatment alters the immune environment toallow amore robust T-cell responsewith the second treatment.Conversely, administration of the second treatment did notsignificantly alter the number of CD90.1þ cells detected in thespleen or prostate, indicating that T cells from the first transferdid not expand in response to the second treatment. Analysis ofeffector functions demonstrated that neither adoptive transferproduced a significant population of IFNg-producing TCR-IVcells (Fig. 6D), and the proportion of GFPþ T cells in theprostate that acquired GzB expression by 7 days after transferwas independent of treatment at 18 weeks (Fig. 6E, right).However, we observed a dramatic increase in the frequency ofCD90.1þGzBþ TCR-IV cells in the prostate following thesecond treatment (Fig. 6E, left; P < 0.005). Together, theseresults demonstrate that a second round of immunotherapypromotes high-level accumulation of donor T cells in thelymphoid organs and the prostate, and facilitates reactivationof persisting tumor-specific CD8þ T cells.

We also performed histologic analysis of the prostate lobesat 70 days after treatment for mice that received two rounds ofWBI and adoptive transfer or the individual components. Dataare reported as the histologic score (Fig. 5F, day 70) and diseaseoutcome (Fig. 5G), including mice that succumbed to tumorprogression before 28weeks of age.Histologic analysis revealedthat half of themice that received two rounds ofWBIwith TCR-IV cell transfer had Berman-Booty scores reduced to low- ormoderate-grade PIN (Fig. 5F, day 70), indicated by reducedherniated glands, and papillary and cribriform structures (Fig.6F). Given that all mice exhibited high-grade PIN at the time ofthe second treatment (Fig. 5F, day 21), the decrease in lesionscoring is consistent with disease regression (Fig. 5G), and wasdemonstrated to be widespread throughout the tissue (Sup-plementary Fig. S2). The remaining mice in the 2�WBIþTCR-IV transfer group showed stable high-grade PIN, with noneexhibiting disease progression (Fig. 5F and G). The 2�WBIþTCR-IV groupdiffered significantly fromall other groupsexcept for mice that received two rounds of TCR-IV cellswithout WBI, which was marginally insignificant (Fig. 5F; P¼ 0.0522). However, neither the 2� TCR-IV group (P¼ 0.8666)nor any other treatment group, excluding the 2�WBIþTCR-IVgroup, differed significantly from untreated mice (Fig. 5F).Instead, a proportion of the TRAMP mice in all other groupsexhibited progression to higher-grade prostate cancer (Fig. 5F,day 70 and 5G), including well-differentiated focally invasiveadenocarcinoma (Fig. 6G and I, grade 13), and poorly

Figure 3. WBI enhances accumulation of TCR-IV cells in the prostate ofTRAMP mice. TRAMP mice were treated as in Fig. 1, and prostate cellswere analyzed by flow cytometry. A, total number of CD90.1þ TCR-IVcells and endogenous CD8þ T cells (B). Solid line indicates the averagenumber of CD8þ T cells in an untreated TRAMP. Bar graph, data fromday5 on a reduced scale; �, statistical significance by the Student t test. C,CD44hiCD62Llo and (D)CFSElo cellswithinCD90.1þTCR-IVpopulationat5 days after transfer. Data are pooled from at least two independentexperiments with a minimum of 6 mice per group.






differentiated neuroendocrine carcinomas (Fig. 5F, grade 21).Some mice in untreated, 2� WBI and 2� TCR-IV treatmentgroups succumbed to tumor-associated death before day 70(Fig. 5G), preventing their inclusion in the histologic scoring(Fig. 5F, x). In the case of untreatedmice, deaths before the endof the experiment explain the apparent drop in the Berman-Booty score between days 21 and 70. Taken together, these dataindicate that two rounds ofWBIwithTCR-IV transfer, but not asingle round, can promote disease regression in a subset ofTRAMP mice, and block disease progression for an extendedperiod of time.We determined the level of TCR-IV cell persistence in the

lymphoid organs of the same mice at 28 weeks, and found thatTCR-IV cells were present in mice that received two rounds ofWBI with adoptive transfer, but not in those that received only

two rounds of TCR-IV cells without WBI (Fig. 6J). Given that Tcells from a single treatment dropped to undetectable levels by4 weeks after transfer (Fig. 2A), these results indicate that twotreatments increased the persistence to at least 7 weeks.Recovered TCR-IV cells were predominantly CD44hiCD62Llo,and a subset of these cells expressed CD127, consistent withmemory T-cell development within the lymphoid organs (Fig.6J). We noted that total splenocyte numbers seemed to beslightly reduced 7 weeks after the second round of therapy, butwere not significantly below normal at this time after treat-ment (Supplementary Fig. S3B). Taken together, our resultsdemonstrate that autochthonous prostate lesions are suscep-tible to T-cell–based therapy, but disease regression requiresmultiple rounds of adoptive T-cell transfer with low-dose WBIconditioning. These data also suggest that disease regression is

Figure 4. WBI facilitates persistence of antigen-responsive TCR-IV cells. A and B, total number of CD8þ T cells in the spleen (A) or prostate (B) ofTRAMPmice that are TetIVþ, or produce IFNg or TNFa in response to ex vivo stimulationwith Site IV peptide (C411L) 7 days after transfer. C, the percentage ofCD90.1þ TCR-IV cells expressing GzB 7 days after transfer. Total numbers of GzBþ TCR-IV cells in the spleen (D) and prostate (E) of irradiated orunirradiated mice. F, schematic of challenge experiment in TRAMP mice. G to I, TRAMP mice treated as in (F) were harvested 28 days after transfer andevaluated for total number of TCR-IV cells (G), and expression of GzB (H) or production of IFNg (I). Statistical significance was evaluated by the Student t test.Data are pooled from two independent experiments with a minimum of 6 mice per group.






associated with a change in the immune microenvironmentthat allows the persistence of tumor-specific CD8þ T cells.

DiscussionOur results show that irradiation-enhanced adoptive T-cell

transfer can result in durable T-cell responses in TRAMPmice.

WBI increased the accumulation and persistence of GzBþ

effector phenotype TCR-IV cells in recipient TRAMP mice,particularly in the neoplastic prostate, and protected a subsetof donor cells from deletion or anergy. Furthermore, twosuccessive rounds of WBI-enhanced adoptive immunotherapyled to reactivation of persisting T cells, high-level accumulationof donor cells, and long-term survival of memory phenotype T

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Figure 5. Two rounds of WBI with adoptive transfer are associated with reduced disease score at late times after treatment. A–D, representative H&E sectionsof the most advanced lesion in the dorsal prostate of TRAMP mice at 21 days after transfer for mice shown in F. A, high-grade PIN with inflammation inWBIþTCR-IV (A) and TCR-IV (C) groups, high-grade PIN in the WBI-only group (B), and well-differentiated adenocarcinoma resembling high-grade PIN butwith focal invasion and stromal reaction (inset) in the untreated group (D). E, average inflammation score for each treatment group 21 days after transfer.Statistical significance was verified by one-way ANOVA. �,P < 0.05. F, Berman-Booty scoring of TRAMPmice at 19 (red), 21 (green), and 28 (blue) weeks of age.Each point indicates an individual mouse, and the most advanced lesion in the prostate; x, mice that succumbed to tumor burden before 28 weeks of age.Statistical significance was evaluated using the Dunnett test. G, categorization of disease outcome in TRAMP mice at 28 weeks of age using the followingscale: death, lethal tumor burden; progression, score > 9; stable disease, score 7–9; and regression, score < 7; n, number of mice per group.






cells. These immunologic findingswere associatedwith regres-sion of established autochthonous lesions or the maintenanceof stable disease through 28 weeks of age. We suggest that thecombination of WBI and T-cell transfer moderates theimmune-suppressive environment in the prostate, promotinglong-term immunity.A dominant impact of WBI conditioning in the TRAMP

model was increased T-cell accumulation in both the spleenand the prostate. Because TCR-IV T cells initially proliferated

in the TLN of both irradiated and unirradiated TRAMP mice,increased accumulation may be explained by better T-cellsurvival or prolonged T-cell proliferation in irradiated mice.This interpretation is supported by the accumulation of CFSElo

T cells in the spleen of irradiated, but not unirradiated, micebeginning at day 5 after transfer. Multiple mechanisms havebeen proposed to explain the immune-enhancing effects ofirradiation, including upregulation of costimulatory functionby dendritic cells (DC; ref. 29). Sublethal WBI has also been

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Figure 6. A second round of WBIwith TCR-IV transfer promotesenhanced T-cell accumulation,effector function, and persistence.A, schematic of consecutivetreatment regimen. Organs wereharvested at 22 weeks of age toassess the accumulation ofCD90.1þ or GFPþ TCR-IV cells inthe spleen (B) and prostate (C).Some mice received treatmentonly at week 18 or 21 forcomparison. D, IFNg productionand GzB expression by CD90.1þ

or GFPþ TCR-IV T cells (E). F–I,representative H&E sections of thelesions detected in the dorsalprostate at 28 weeks of age formice shown in Fig. 5F. F, healthyprostate lobe indicative of diseaseregression but showing a smallarea of low-grade PIN (asterisk) inmice that received 2� WBIþTCR-IV. Well-differentiatedadenocarcinoma with focalinvasion (inset) in mice thatreceived 2� WBI (G) or notreatment (I). Arrows, invasive cellpopulations. H, high-grade PIN in2� TCR-IV–only mice. J, analysisof splenocytes at 28 weeksshowing TetIVþ CD8þ T cells (left),and CD44 and CD62L (center) orCD127 (right) on CD8þTetIVþ cellsfrom the indicated treatmentgroups. The plots show resultsobtained from two independentexperiments with a minimum of5 mice per group.






shown to induce microbial translocation, critical for adoptiveimmunotherapy in a melanoma model, which promotes thesystemic circulation of bacterial lipopolysacharide and matu-ration of DCs (30). Such a mechanism may be particularlyimportant for achieving productive, rather than abortive,activation of na€�ve donor T cells. Administration of higherdoses of radiation (10–15 Gy) can induce immunogenic tumor-cell death, which may facilitate increased acquisition of tumorantigen by DCs to promote more efficient cross-presentationto T cells (31, 32). However, we have no direct evidence that thelower dose of WBI used in this study can induce immunogeniccell death in the TRAMP prostate.

A second impact of irradiation was increased persistence ofresponsive T cells. A subset of tumor-specific T cells remainedresponsive to immunization in irradiated mice 3 weeks aftertransfer. This finding contrasts with results from studiesshowing lack of response to vaccination in unirradiatedTRAMP mice following T-cell contraction (17). Increasedpersistence could be a product of the initially higher T-cellaccumulation in irradiated TRAMP mice. Indeed, TCR-IVT-cell numbers in unconditioned mice were highest immedi-ately following transfer, and did not increase following initi-ation of proliferation. Development of persisting T cells couldalternatively be explained by unique differentiation of TCR-IVT cells in irradiated versus unirradiated TRAMP mice. Lym-phopenia induced by WBI can enhance access to survivalcytokines, resulting in increased functional differentiationof T cells in tumor-bearing mice (33), and the promotion ofa memory-like phenotype (34–36). Thus, lymphopenia may beessential to develop long-term immunity in this model.

Despite strong GzB expression by donor TCR-IV cells recov-ered from irradiatedTRAMPmice, these cells produced limitedIFNg and TNFa, consistent with results in human and mouseprostate cancer (37, 38). In fact, the majority of studies inTRAMP mice show that tumor antigen-specific CD8þ T cellsare rapidly tolerized or converted to a suppressive phenotype(3, 7, 13, 16, 17, 39). Immunosuppression was delayed by eitherDC-based vaccination or CD4þ T-cell cotransfer, but wasrapidly reexerted once these supportive regimens were dis-continued (3, 7, 40). TGFb plays a major suppressive role in theTRAMP model (4, 8, 14, 17, 39). However, the effects of TGFbsignaling may not explain our observations of split anergy(GzBþ without cytokine production) because TGFb signalinghas been shown to activate the Smad complex and Atf1 toreduce the expression of both IFNg and GzB (41). Because onlya minor fraction of TCR-IV cells ever developed into IFNgþ

effectors in irradiated TRAMP mice, even as early as 3 daysafter transfer (unpublished data), this mechanism of T-cellanergymay be distinct from the sequential loss of cytokine andcytotoxic molecule expression described for T-cell exhaustion(42). Rather, our results suggest a block in T-cell differentiationfollowing initial antigen encounter. Alternatively, the impactof TGFb on the T cells may be temporarily reduced by WBI,allowing partial differentiation to GzB-expressing cells. Fur-ther studies are required to identify the upstream signals thatregulate this phenotype.

Na€�ve donor T cells were used to allow visualization of earlyevents in the lymphoid organs and to allow direct comparison

of our results with those obtained in previous studies withother T Ag–induced cancers (23). TCR-transduced T cells,which require in vitro activation, were recently shown to inducelocalized elimination of TAg–expressing cells in the prostate ofirradiated TRAMP mice (8), suggesting that irradiation alsocan enhance the effectiveness of in vitro expanded and redir-ected T cells in this system. From a translational perspective,the current requirement for large numbers of donor T cellsprecludes the use of antigen-specific na€�ve donor T cells forclinical use (43). However, na€�ve T cells are readily abundant inthe peripheral blood and, along with themore recently definedT-memory stem cells, have been shown to out-perform centraland effector memory T cells in experimental models of adop-tive T-cell therapy (44, 45) and as the starting population forgenetic engineering of donor T cells (46). This is likely due totheir enhanced ability to undergo differentiation into effectorT cells needed for tumor elimination while retaining theirrenewable potential (43). Current efforts in thefield are focusedon expanding T cells that retain a renewable phenotype,including genetic reprogramming of cells to retain character-istics of na€�ve T cells and T-memory stem cells to provide asource of donor cells (45, 47).

Our finding that the disease score was reduced in TRAMPmice that received two rounds of WBI with adoptive transfer,but not by a single round of therapy, could be explained byseveral mechanisms. This result was associated with durableTCR-IV accumulation for at least 7 weeks, whereas T cellscontracted to baseline levels within 4 weeks after a singletreatment. This observation suggests that regression ofadvanced prostatic lesions requires an extended attack by theimmune system. Indeed, persistence of adoptively transferredT cells positively correlates with objective clinical responses(48). After the second treatment, but not the first, the TCR-IVpopulation included CD127þ cells (Figs. 2G and 6J), suggestingthat the second round of treatment uniquely produced mem-ory-like cells. This change in TCR-IV persistence with tworounds of WBI-enhanced adoptive immunotherapy may beexplained by reduced immunosuppression or increasedinflammation at the tumor site produced by the first roundof therapy (Fig. 5E), providing an environment that allows thedevelopment of a persistent T-cell population with the secondtransfer. Such a change in the tumor microenvironment is alsosuggested from our finding that some prostate-resident TCR-IV cells from the initial transfer regained GzB expressionfollowing the second round of therapy (Fig. 6C). Reactivationof tolerant prostate-resident T cells was previously observedfollowing intraprostatic injection of antigen-pulsed DCs (40),indicating that these T cells are not irreversibly tolerized. Theseencouraging results raise the possibility that endogenousprostate tumor-specific T cells might be rescued from toler-ance following immune intervention.

It remains to be determined whether two complete cyclesof WBI with TCR-IV cells are required to generate long-livedtumor-specific T-cell responses in TRAMP mice. For example,whether a second dose of irradiation before the second T-celltransfer is required to recapitulate both the long-lived T-cellresponse and disease regression is unknown. Clearly, theprovision of multiple doses of WBI is not desirable for a






translational setting due to increased potential for radiation-induced toxicities. Because WBI-induced lymphopenia is onlypartially recovered before the second treatment (Fig. 2B andSupplementary Fig. S3A), WBI may not be necessary toenhance T-cell activation and accumulation during the secondtransfer. Lymphodepletion or innate triggering might alterna-tively be induced by approaches such as chemotherapy toreduce potential side effects from radiation. In addition,hematopoietic stem cells could be provided at the time of thesecond transfer (48). Further studies are required to identifythe particular changes to the immune and prostate microen-vironment induced after each round of therapy.Previously, we used WBI with TCR-IV–adoptive transfer to

evaluate the impact of immunotherapy in the T Ag–drivenSV11 choroid plexus tumor model (23). In both the SV11 andTRAMPmodels, WBI enhanced the persistence of donor TCR-IV cells, and increased the therapeutic effect. However, in SV11mice, a single round of WBI-enhanced adoptive immunother-apy was sufficient to produce complete regression of estab-lished tumors, and conferred long-term protection from recur-rence (23). TCR-IV cells persisted at high frequencies in thebrains of irradiated SV11 mice for over 2 months, whereasthose localized to the TRAMP prostate after a single round oftherapy contracted to baseline detection after 21 days. TCR-IVcells in TRAMP mice also lacked IFNg production, whereasIFNg production by TCR-IV cells in SV11micewas essential fortumor elimination (23). This finding could explain the lessefficient elimination of tumors in TRAMP mice. This compar-ison indicates that a similar adoptive immunotherapyapproach may be applicable for tumors arising in distincttissues that share a common antigen. However, unique con-

tributions of the tissue microenvironments regulate the dura-tion of the response and impact of the therapeutic approach,thus requiring a more complete understanding of the uniquecontribution of each environment.

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

Authors' ContributionsConception and design: L.K. Ward-Kavanagh, T.D. SchellDevelopment of methodology: L.K. Ward-Kavanagh, T.K. Cooper, T.D. SchellAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.K. Ward-Kavanagh, T.D. SchellAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.K. Ward-Kavanagh, J. Zhu, T.K. Cooper, T.D. SchellWriting, review, and/or revision of the manuscript: L.K. Ward-Kavanagh,J. Zhu, T.K. Cooper, T.D. SchellAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Zhu, T.D. SchellStudy supervision: T.D. Schell

AcknowledgmentsThe authors thank Jeremy Haley and Ellen Mullady for outstanding technical

support, and the staff of the Penn State Hershey Flow Cytometry Core Facility forexpert assistance.

Grant SupportThis work was supported by research grant RO1 CA025000 from the National

Cancer Institute, NIH (to T.D. Schell). L.K. Ward-Kavanagh was supported bypredoctoral training grant T32 CA060395 from the National Cancer Institute,NIH.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received September 25, 2013; revised April 8, 2014; accepted April 26, 2014;published OnlineFirst May 6, 2014.

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2014;2:777-788. Published OnlineFirst May 6, 2014.Cancer Immunol Res Lindsay K. Ward-Kavanagh, Junjia Zhu, Timothy K. Cooper, et al. Regression in a Mouse Model of Prostate Cancerof Adoptively Transferred T Cells Associated with Tumor Whole-Body Irradiation Increases the Magnitude and Persistence

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