interleukin-15/interleukin-15ra complexes promote ... · in this process by directly killing...

Interleukin-15/Interleukin-15RA Complexes Promote Destruction of

Established Tumors by Reviving Tumor-Resident CD8+ T Cells

Mathieu Epardaud,1,5Kutlu G. Elpek,

1Mark P. Rubinstein,

6Ai-ris Yonekura,

1,2

Angelique Bellemare-Pelletier,1Roderick Bronson,

3Jessica A. Hamerman,

7

Ananda W. Goldrath,6and Shannon J. Turley

1,4

1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute; 2Harvard-Massachusetts Institute of Technology Division ofHealth Sciences and Technology; 3Harvard Medical School; 4Department of Pathology, Harvard Medical School, Boston, Massachusetts;5Department of Virologie et Immunologie Moleculaires UR892, INRA, Domaine de Vilvert, Jouy-en-Josas, France; 6Division of Biology,University of California at San Diego, La Jolla, California; and 7Immunology Program, Benaroya Research Institute, Seattle, Washington

Abstract

Tumors often escape immune-mediated destruction by sup-pressing lymphocyte infiltration or effector function. Newapproaches are needed that overcome this suppression andthereby augment the tumoricidal capacity of tumor-reactivelymphocytes. The cytokine interleukin-15 (IL-15) promotesproliferation and effector capacity of CD8+ T cells, naturalkiller (NK) cells, and NKT cells; however, it has a short half-lifeand high doses are needed to achieve functional responsesin vivo. The biological activity of IL-15 can be dramaticallyincreased by complexing this cytokine to its soluble receptor,IL-15RA. Here, we report that in vivo delivery of IL-15/IL-15RAcomplexes triggers rapid and significant regression of estab-lished solid tumors in two murine models. Despite a markedexpansion of IL-2/IL-15RB+ cells in lymphoid organs andperipheral blood following treatment with IL-15/IL-15RAcomplexes, the destruction of solid tumors was orchestratedby tumor-resident rather than newly infiltrating CD8+ T cells.Our data provide novel insights into the use of IL-15/IL-15RAcomplexes to relieve tumor-resident T cells from functionalsuppression by the tumor microenvironment and havesignificant implications for cancer immunotherapy andtreatment of chronic infections. [Cancer Res 2008;68(8):2972–83]

Introduction

Cancer immunosurveillance is the process whereby innate andadaptive immune mechanisms suppress the growth of tumors(1, 2). CD8+ T cells and natural killer (NK) cells play important rolesin this process by directly killing malignant cells (1, 3–5). Cancerimmunosurveillance is regulated not only by the immune systembut also by elements of the tumor microenvironment, includingmalignant cells, tumor stroma, and the vasculature (6). Indeed,tumors can escape immunosurveillance by disabling the functionof cytolytic lymphocytes and antigen-presenting cells, by prevent-ing blood-borne lymphocytes from infiltrating malignant tissue orby inducing tolerance (1, 7, 8).Various immunotherapeutic strategies have been developed for

the treatment of human cancers. Cancer vaccines strive to incite

robust antitumor immunity by immunizing the cancer patient withdifferent forms of tumor antigens; however, their effect on tumorburden has been modest (9–11). In adoptive cell therapy (ACT),patients are infused with autologous, tumor-specific T cells thatcan be derived from tumor-infiltrating lymphocytes (TIL) or fromperipheral blood lymphocytes engineered to express a tumor-specific T-cell receptor (12, 13). Although ACT has been successfulin inducing objective responses in select cancers such as metastaticmelanoma, most patients still fail to respond despite increasedfrequencies of circulating, tumor-specific lymphocytes (14, 15). It isbecoming increasingly clear that clinical efficacy in cancerimmunotherapy may be more dependent on the ability of immuneeffector cells to access the tumor and to exert their tumoricidalfunctions therein rather than on the numbers of circulating, tumor-specific lymphocytes (16–19). Unfortunately, fewer efforts havefocused on designing therapies that target tumor-resident T cellsand boost their effector function in situ . Thus, new approaches areneeded that either facilitate the infiltration of circulatingleukocytes into solid tumors or that effectuate the tumoricidalfunction of TILs that persist in a functionally suppressed state inthe malignant lesion.The administration of cytokines to augment immunosurveillance

has proven efficacious in the treatment of select cancers (20). Forexample, IL-2 is Food and Drug Administration approved for thetreatment of renal cell carcinoma and metastatic melanoma.However, IL-2 therapy is limited by systemic toxicity, poorbiological activity, and an inability to induce antitumor activityin most cancer patients (21) due to selective promotion ofT-cell activation–induced cell death (AICD) and expansion ofT regulatory cells (Treg; refs. 22–26). Highly related to IL-2 is thecytokine IL-15 (27, 28), which lacks these adverse effects. Inaddition to sharing the use of two receptor subunits (IL-2Rh/CD122 and IL-2Rg/CD132) and inducing similar intracellularsignaling events, both IL-15 and IL-2 induce the mild expansionof memory CD8+ T cells, NK cells, and NKT cells (22). IL-15 hasshown antitumor efficacy and enhances the effects of chemother-apy and ACT (29–32). However, like IL-2, IL-15 has a short half-lifeand high doses are needed to achieve biological responses in vivo(33, 34). Recently, it was shown that the biological activity of IL-15could be increased f50-fold by administering preformed com-plexes of IL-15 and its soluble receptor, IL-15Ra (35, 36). Thisincrease in activity is likely due to an increased half-life of thecomplex compared with IL-15 alone and that IL-15 is beingpresented by IL-15Ra to CD122+ cells similarly to how it is thoughtto be presented by dendritic cells in vivo . Compared with IL-15,IL-15/IL-15Ra complexes induce a dramatic expansion of CD122hi

cells, including antigen-experienced CD44hi CD8+ memory and

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

M. Epardaud and K.G. Elpek contributed equally to this work.Requests for reprints: Shannon J. Turley, Department of Cancer Immunology and

AIDS, Dana-Farber Cancer Institute, 44 Binney Street, D1440a, Boston, MA 02115.Phone: 617-632-4990; Fax: 617-582-7999; E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-08-0045

Cancer Res 2008; 68: (8). April 15, 2008 2972 www.aacrjournals.org

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memory phenotype (MP) T cells, NK cells, and NKT cells (35, 36).Given these promising data, we hypothesized that IL-15/IL-15Racomplex–driven expansion of such lymphocytes may promoteimmune-mediated destruction of established solid tumors thatwould otherwise escape immunosurveillance.Here, we report that systemic administration of IL-15/IL-15Ra

complexes relieves tumor-resident CD8+ T cells from control by thetumor microenvironment, allowing these cells to expand anddestroy advanced solid tumors without the need for vaccination orACT. Unexpectedly, we found that tumor destruction occurredindependently of circulating CD122+ lymphocytes or tumor-resident NK cells despite their marked expansion in lymphoidorgans and peripheral blood. Our results have significantimplications for immunotherapeutic intervention of humancancers and reveal a novel mechanism for reinvigorating thecytotoxic potential of TILs that persist within solid tumors.

Materials and Methods

Mice. The RIP1-Tag2 transgenic mouse line was previously described(37). RIP1-Tag2 mice were obtained from the Mouse Model of Human

Cancer Consortium (National Cancer Institute) and were maintained on a

C57BL/6 background. Perforin knockout mice, strain C57BL/6-Prf1tm1Sdz/J,

were obtained from The Jackson Laboratory, were maintained on a C57BL/6background, and were crossed to the RIP-Tag2 mice. C57BL/6 mice were

obtained from The Jackson Laboratory. All mice were bred and maintained

under barrier conditions in the Dana-Farber Cancer Institute Animal

Facility in accordance with NIH guidelines.Tumor cell lines. B16F10 melanoma cells were maintained in DMEM

with high glucose (Invitrogen Life Technologies) supplemented with 10%

fetal bovine serum (FBS), 1� penicillin/streptomycin. Cells were harvestedusing 0.25% trypsin/EDTA (Invitrogen Life Technologies) when 50% to 90%

confluent. MIN6 insulinoma cells were maintained in DMEM with high

glucose (Invitrogen Life Technologies) supplemented with 10% FBS,

1� penicillin/streptomycin, and 0.1% h-mercaptoethanol (38). Cells wereharvested using 0.25% trypsin/EDTA (Invitrogen Life Technologies) when

50% confluent and were labeled with 10 Amol/L of 5,6-carboxyfluorescein

diacetate succinimidyl diester (CFSE; Molecular Probes) for 10 min at 37jC.Cells were then cultured for 4 d with graded doses of IL-15/IL-15Racomplexes (per 1 mL): 0 = medium only, 0.1� = (0.1 Ag of IL-15 + 0.466 Ag ofIL-15Ra), 1� = (1 Ag of IL-15 + 4.66 Ag of IL-15Ra), 10� = (10 Ag of IL-15 +

46.6 Ag of IL-15Ra), and 100� = (100 Ag of IL-15 + 466 Ag of IL-15Ra).Cells were harvested and stained with 7-amino-actinomycin D (BD

PharMingen) to evaluate their viability. Cells were analyzed immediately,

without fixation, using a FACSCalibur (BD Biosciences). Data were analyzed

using FlowJo Software (Tree Star).Treatment with IL-15/IL15-RA complexes and assessment of tumor

burden. Recombinant murine IL-15 (eBioscience) and recombinant soluble

murine IL-15Ra-Fc (R&D Systems) were suspended in 0.1% bovine serum

albumin (BSA)/PBS, mixed, and incubated for 30 min at 37jC beforeinjection. Unless specifically noted, RIP1-Tag2 mice at 10 to 11 wk of age

received one injection of IL-15/IL-15Ra complexes, IL-15 alone, or PBS per

day on 2 consecutive days. For the IL-15/IL-15Ra complexes, 2 Ag of IL-15were precomplexed with 12 Ag of IL-15Ra-Fc in 300 AL PBS and injected i.v.

For IL-15 alone, 2 Ag of IL-15 were injected i.v. in 300 AL PBS. Mice were

sacrificed 4 d after the first injection of IL-15/IL-15Ra (schematized in

Fig. 1). For long-term treatments, RIP1-Tag2 mice at 10 to 11 wk of agereceived one injection of the IL-15/IL-15Ra complexes per day for 2

consecutive days i.v. followed by a total of 13 i.p. injections every 3 d. To

measure pancreatic tumor burden, pancreata were excised from euthanized

subjects and solid tumors were dissected away from surrounding exocrinetissue. Tumor diameters (d) were then measured and tumors were classified

in four categories: A, d < 1 mm; B , 1 V d V 2 mm; C, d = 2 mm; and D,

d > 2 mm. Pancreatic tumor burden was calculated as [(A � 1) + (B � 2) +

(C � 3) + (D � 4)]. For B16 transplantation, 5 � 105 cells were injected s.c.

in 200 AL of PBS into the abdomen of 6- to 12-wk-old C57BL/6 mice. Tendays later, or when the tumors reached 2 to 8 mm in diameter, mice were

treated with one injection of the IL-15/IL-15Ra complex per day on 2

consecutive days. Each injection was performed i.v. and consisted of 2 Ag ofIL-15 precomplexed with 12 Ag of IL-15Ra in 300 AL PBS. Mice weresacrificed 4 d after the first injection of IL-15/IL-15Ra. B16 tumor burden

was assessed by measuring the diameter (d) of each tumor using a caliper

and percent tumor growth was calculated as (dday4 � dday0) / dday0.

Phenotypic characterization of host leukocytes by cytofluorimetry.Spleens were processed into single-cell suspensions using glass slide

disruption followed by RBC lysis. Solid tumors were removed from the

pancreas and processed into single-cell suspension by gentle mechanical

disruption with forceps followed by enzymatic digestion using the followingdigestion medium: 0.2 mg/mL collagenase P (Roche), 0.8 mg/mL dispase

(Invitrogen), and 0.1 mg/mL DNase I (Sigma) in RPMI. Tumor suspensions

were incubated in digestion medium at 37jC for 25 min. Released cells werecollected and the remaining tumor tissue was subjected to further

processing by incubation in fresh digestion medium for an additional

25 min at 37jC. After digestion, single-cell suspensions were filtered with

80-Am nylon mesh; RBCs were lysed; and suspensions were filtered again.Blood was collected into tubes containing 10 mmol/L EDTA PBS to prevent

coagulation and RBCs were subsequently lysed. For staining, cells were

suspended in PBS/5% FBS/2 mmol/L EDTA (FACS buffer) and Fc receptors

were saturated using FcR-block antibody (2.4G2). Single-cell suspensionsfrom spleen, blood, and tumors were stained with fluorescently labeled

monoclonal antibodies specific for NK1.1 (PK136), CD3q (145-2C11), CD45

(30-F11), CD44 (1M7), CD69 (H.1.2F3; Biolegend), CD8a (53-6.7; BDPharMingen), and CD122 (IL-2/IL-15Rh; TM-B1; Caltag) at 4jC for

30 min; washed in FACS buffer; and analyzed immediately, without fixation,

using a FACSCalibur (BD Biosciences). Data were analyzed using FlowJo

Software (Tree Star).For intracellular IFNg analysis, total spleen or tumor cells were plated at

2 � 106/mL in complete medium [DMEM supplemented with L-glutamine

(2 mmol/L), 1� penicillin/streptomycin, and 10% FBS] and activated with

phorbol 12-myristate 13-acetate (PMA; 5 ng/mL) and ionomycin (500 ng/mL)for 5 h, and with monensin in the last 3 h. After cell surface staining with

anti–CD8-PerCP and anti–CD45-FITC antibodies, cells were fixed with

4% paraformaldehyde. Cells were then permeabilized with 0.1% saponinand stained with anti–IFNg-APC or isotype control antibody. Cells were

washed again and analyzed by flow cytometry. For granzyme B detection,

intracellular staining was done the same way using anti-granzyme

B–phycoerythrin antibody but without PMA and ionomycin stimulation.Fluorescence microscopy. Pancreas or pancreatic tumors were fixed in

4% PFA/10% sucrose/PBS for 2 h and then incubated in 30% sucrose/PBS

for 1 h. Tissues were snap-frozen in optimum cutting temperature (OCT)

medium (Sakura Finetek). Sections (6 Am) were fixed in cold acetone for

10 min and blocked with 10% goat serum in PBS for 1 h. To visualize tumor-

associated leukocytes, tissue sections were incubated with biotinylated anti-

CD45 (Biolegend) or anti-CD8 (Caltag) for 30 min at room temperature,

washed, and then incubated with streptavidin-Cy3 (Invitrogen) for 30 min

at room temperature. All dilutions were made in 2% goat serum/PBS and

sections were washed thrice with 2% goat serum/PBS after incubation with

primary and secondary reagents. To visualize apoptotic cells within tumors,

we used the In Situ Cell Death Detection Kit, Fluorescein [terminal

deoxyribonucleotide transferase–mediated nick-end labeling (TUNEL);

Roche] to label DNA strand breaks. The permeabilization step was

performed at room temperature instead of 4jC with a modified

permeabilization solution of 0.25% Triton-X 100/0.1% sodium citrate.

Sections were then dried, mounted on glass coverslips with anti-fade

mounting medium containing 4¶,6-diamidino-2-phenylindole (DAPI; Molec-

ular Probes), and examined on a fluorescence microscope. Images were

captured using the Spot RT-Slider Digital Imaging System (Diagnostic

Instruments).

Leukocyte depletion. Cells expressing CD8+ and NK1.1+ were depleted

using monoclonal antibodies specific for CD8a (53-6.7) orNK1.1 (PK136), respectively. For depleting NK1.1+ cells, RIP1-Tag2 mice

received one i.v. injection of anti-NK1.1 (500 Ag) 24 h before treatment with

IL-15/IL-15Ra Complexes in Tumor Therapy

www.aacrjournals.org 2973 Cancer Res 2008; 68: (8). April 15, 2008



IL-15/IL-15Ra complexes and two subsequent antibody injections of 75 Ageach on days 0 and 1 of the regimen schematized in Fig. 1. For depletion of

CD8+ cells, RIP1-Tag2 mice received one i.v. injection of anti-CD8 (100 Ag)24 h before treatment with IL-15/IL-15Ra complexes and two subsequent

antibody injections of 50 Ag each on days 0 and 1 of the regimen. At the end

of the experiment, leukocyte depletion was verified by cytofluorimetry.

Adoptive transfer of RIP1-Tag2 leukocytes. Spleen and lymph nodes

were removed from 10.5-wk-old CD45.1+ or CD45.2+ RIP1-Tag2 mice and

Figure 1. Significant therapeutic effect of systemically administered IL-15/IL-15Ra complexes on solid tumors of multiple origins. A, top, schematic of theIL-15/IL-15Ra complexes regimen in mice bearing transplanted B16 melanoma tumors. B16 melanoma cells (5 � 105) were transferred s.c. into C57BL/6 recipients andtreatment with IL-15/IL-15Ra complexes commenced f10 d later upon detection of palpable tumors. Mice received one i.v. injection of IL-15/IL-15Ra complexes(2 Ag IL-15 + 12 Ag IL-15Ra in 300 AL PBS) per day for 2 d and tumor progression was evaluated 3 d later. Bottom, quantification of tumor growth in IL-15/IL-15Racomplex–treated (n = 8) and control (n = 9) C57BL/6 mice bearing B16 melanoma tumors. Columns, mean of individual mice (indicated above as n) per group;bars, SE. B, top, schematic of the IL-15/IL-15Ra complex regimen in RIP1-Tag2 mice bearing pancreatic tumors that arise spontaneously. Ten- to 11-wk-old RIP1-Tag2mice were i.v. injected with IL-15/IL-15Ra complexes (2 Ag IL-15 + 12 Ag IL-15Ra in 300 AL PBS), IL-15 alone (2 Ag in 300 AL PBS), or PBS (300 AL) onceper day for 2 d and analyzed 3 d later. Bottom, quantification of tumor burden in IL-15/IL-15Ra complex–treated (n = 39), IL-15–treated (n = 4), and control-treated(n = 47) RIP-Tag2 mice. Columns, mean of individual mice (indicated above as n) per group; bars, SE. C, effect of long-term treatment with IL-15/IL15-Ra complexeson the survival of RIP-Tag2 mice. RIP-Tag2 mice were treated with IL-15/IL15-Ra complexes as in B and received 13 i.p. injections for long-term treatment(., n = 5). Survival (%) of treated mice was compared with untreated control mice (o, n = 10). *, P < 0.05 in log-rank test for prolonged survival. D, fluorescencemicroscopic analysis of apoptotic cells in tumors from control and IL-15/IL-15Ra complex–treated RIP1-Tag2 mice. Cryosections of pancreatic tumors were stainedwith DAPI (blue ) and TUNEL (green ). Representative micrographs are shown for each condition (�20). Graph depicts the number of TUNEL+ cells per 100 mm2.Bottom right, cryosections of pancreatic tumors from IL-15/IL-15Ra complex–treated RIP1-Tag2 mice were stained with insulin (red) to identify h cells, DAPI (blue ),and TUNEL (green ). A representative micrograph is shown for this condition (�80).

Cancer Research




processed into single-cell suspension using glass slide disruption followedby RBC lysis. Cells were then incubated in RPMI containing 0.1% BSA for

10 min at 37jC with 10 Amol/L of CFSE. The labeling reaction was stopped

with RPMI/20% FBS on ice and the cells were washed twice in PBS/5%

FBS/2 mmol/L EDTA. Ten million cells were injected i.v. in 200 AL of PBSinto 10.5-wk-old CD45.2+ RIP1-Tag2 mice.

Assessment of host leukocyte proliferation. Cellular proliferation of

host leukocytes was measured using (+)-5-bromo-2¶-deoxyuridine (BrdUrd)

incorporation. Mice were maintained on BrdUrd (Sigma) in drinking water(0.8 mg/mL) with 1% glucose throughout the experiment starting 1 d before

IL-15/IL-15Ra complex treatment. Mice were injected on days 0 and 1 with

2 Ag of IL-15 precomplexed with 12 Ag of IL-15Ra in 300 AL PBS i.v. and

sacrificed between 1 and 3 d later. Spleen, lymph nodes, and tumors wereharvested and processed into single-cell suspension to evaluate BrdUrd

incorporation within total leukocytes (CD45+) and CD8+ populations

by cytofluorimetry using phycoerythrin-conjugated anti-BrdUrd antibody(BD PharMingen).

In vitro cytotoxicity assay. Cytolytic activity was determined by 5-h 51Cr

release assay, as previously described (39). Briefly, target cells, RMA, and

RMA pulsed with 1 Amol/L SIINFEKL (OVA peptide), were labeled for1 h with 51Cr. Effector cells were harvested on day 4 from spleens of mice

injected with IL-15/IL-15Ra complexes on days 0 and 1. For OT-I effector

cells, spleens from OT-Itg/RAG1�/� TCR transgenic mice (40) were

harvested and the percentage of OT-I cells was identified based oncytofluorimetric analysis of CD8 and Va2 expression. Target cells were

incubated with effector cells in 96-well round-bottomed plates for 5 h at

37jC with 5% CO2 at the indicated ratios. The percentage of specific 51Crrelease was calculated as follows: percentage of specific lysis = [(experi-

mental release � spontaneous release) / (maximum release � spontaneous

release)] � 100.

Histologic assessment. Tissue samples were collected, directly fixed inBouin’s solution, paraffin-embedded, and processed for H&E staining.

Histologic analysis was performed in a blinded fashion.

Statistical analysis. All data are presented as mean F SE. Statistical

analysis used the two-tailed unpaired Student’s t test for comparison of twoexperimental groups, and log-rank test for comparison of survival in

Kaplan-Meier survival plot. P values <0.05 were considered significant.

Results

In vivo delivery of IL-15/IL-15Ra complexes rapidly inducesa significant reduction in solid tumor burden. The biologicalactivity of IL-15 can be significantly increased when administeredas a complex with soluble IL-15Ra. The remarkable potency ofIL-15/IL-15Ra complexes could be beneficial in numerous clinicalsettings in which CD122+ cells (including NK cells, NKT cells, andCD8+ T cells) serve as critical effectors in immune surveillance ofsolid tumors and chronic infections; however, this has not beenformally tested to date. Stoklasek and colleagues recently reportedthat systemic administration of IL-15/IL-15Ra complexes couldprevent transplanted B16 melanoma cells from forming tumors,whereas IL-15 alone had no effect on tumor engraftment (36),providing evidence that prophylactic administration of IL-15/IL-15Ra complexes can prevent the generation of tumors. Whetherthis agent can affect immunosurveillance of established solidtumors had not been previously addressed. Thus, we tested theeffect of IL-15/IL-15Ra complexes on established solid tumors intwo different models: in one case, where tumors arise either fromtransplanted melanoma cells or in another where tumors arisespontaneously in the endocrine pancreas of transgenic animals. Tothis end, mice bearing palpable s.c. B16 tumors were treated withone i.v. injection of IL-15/IL-15Ra complexes per day for 2 days andtumor progression was evaluated 3 days later (Fig. 1A). Systemicadministration of IL-15/IL-15Ra complexes had a marked effect onthe overall tumor burden, impairing the growth of established B16

tumors in C57BL/6 mice by 75% compared with controls (30%versus 140%, P < 0.004; Fig. 1A).Having confirmed that in vivo delivery of IL-15/IL-15Ra

complexes could affect B16 tumors in a therapeutic setting, wenext sought to determine whether this regimen would have anyeffect on solid tumors that arise spontaneously in a vital organ. Tothis end, we used the RIP1-Tag2 transgenic mouse model in whichthe SV40 T antigen (Tag) is expressed under the control of the ratinsulin promoter (RIP), causing oncogenic transformation of themajority of pancreatic h cells (37). Tumor development in thesemice unfolds in several distinct stages (Fig. 1B). At 3 to 4 weeks ofage, h cells start proliferating in response to Tag expression(hyperplastic stage); at 7 to 8 weeks of age, new blood vessels formalong with alterations in the microvasculature (angiogenic switch);and by 10 weeks of age, solid tumors will have developed in 100% ofthe mice. Evidence for tumor-specific T-cell responses in pancreaticlymph nodes at early stages of tumorigenesis suggest thathyperplastic islets are subject to immunosurveillance; however,such mechanisms fail to prevent tumor growth (41) for reasonsthat remain unclear. To examine the therapeutic efficacy of IL-15/IL-15Ra complexes on established solid tumors of the pancreas, wetreated 10- to 11-week-old RIP1-Tag2 mice with the complexes orIL-15 alone once per day for 2 days and analyzed 3 days later(Fig. 1B). In vivo delivery of IL-15/IL-15Ra complexes caused arapid and significant reduction in the number and size of tumors(Fig. 1B, bottom left), diminishing pancreatic tumor burden by >50%(P < 0.0001) in RIP1-Tag2 mice (Fig. 1B, bottom right). In contrast,however, IL-15 at the same dose used in the complexes had nosignificant effect on tumor burden in RIP1-Tag2 mice (Fig. 1B,bottom right). Importantly, the body weight of treated subjects wasidentical to control littermates throughout the regimen (Supple-mentary Fig. S1A) and no observable toxicity or autoimmunity tonormal tissues was found in mice treated with IL-15 alone (datanot shown) or IL-15/IL-15Ra complexes (Supplementary Fig. S1Band data not shown). Together, these data indicate that systemicadministration of IL-15/IL-15Ra complexes inhibits the growth ofestablished B16 tumors and causes significant regression ofnaturally arising solid tumors of the pancreas.Having shown the rapid therapeutic effect of IL-15/IL-15Ra

complexes on tumor burden, we then tested the effect of this agenton the survival of RIP1-Tag2 mice. Subjecting RIP1-Tag2 miceharboring solid tumors to long-term treatment with IL-15/IL-15Racomplexes resulted in a significant prolongation of survival (P < 0.05)compared with control-treated animals (Fig. 1C). Taken together,these results show that treatment with IL-15/IL-15Ra complexesseems to be well tolerated and can have a significant clinicalbenefit for animals bearing advanced solid tumors.We then wanted to determine whether the reduction in

pancreatic tumor burden was due to specific destruction ofmalignant h cells or of other cells in the tumor stroma. To evaluatethis, tumors were harvested at various time points after injection ofIL-15/IL-15Ra complexes and processed for TUNEL analysis byimmunofluorescence microscopy. Within 48 hours of administeringIL-15/IL-15Ra complexes, the number of TUNEL+ cells increasedz7-fold (Fig. 1D), indicating that apoptosis was occurring in thetumors as a consequence of the treatment. To pinpoint the identityof the dying cells, we stained cryosections of the h-cell tumors withan insulin-specific antibody. The TUNEL+ cells were clearlycostained with an insulin-specific monoclonal antibody (Fig. 1D,bottom right), whereas insulin� cells were consistently TUNEL�,indicating that the apoptotic cells were malignant h cells rather





than tumor stromal cells. Given how rapidly this novel regimencauses tumor cell apoptosis, we tested the direct effect of IL-15/IL-15Ra complexes on the viability and proliferation of malignanth cells in vitro , and neither of these were directly affected by IL-15/IL-15Ra complexes, even at high concentrations (SupplementaryFig. S2A). In support of these functional data, CD122 surface expres-sion was undetectable on all tumor cells tested, whereas tumor-associated CD8+ T cells were CD122+ (Supplementary Fig. S2B).Thus, we conclude that tumor destruction following systemicadministration of IL-15/IL-15Ra complexes does not result from adirect effect on tumor cells.IL-15/IL-15RA complexes expand CD8+ T cells and NK1.1+

cells in lymphoid organs and tumors of RIP1-Tag2 mice.Treatment of tumor-free C57BL/6 mice with IL-15/IL-15Racomplexes is known to expand CD122+ cells in the spleen,including NK cells, NKT cells, and MP CD8+ T cells (35, 36). Toascertain whether CD122+ cells in mice harboring solid pancreatictumors respond in a similar manner, we treated 10- to 11-week-oldRIP1-Tag2 mice with IL-15/IL-15Ra complexes and then analyzedthe abundance and activation state of CD8+ T cells and NK1.1+ cellsin various tissues. In agreement with previous reports (35), wefound that the size and weight of the spleen increased significantlyas a result of the treatment (Supplementary Fig. S3). Cytofluori-metric analysis indicated that the relative abundance of NK1.1+

splenocytes increased from 3% in PBS-treated controls to 11% inIL-15/IL-15Ra complex–treated mice (Fig. 2A, left) with equalproportions of NK (CD3�) and NKT (CD3+) cells (SupplementaryFig. S4A). An expansion of the CD8+ T-cell compartment was alsoobserved, increasing from 11% in controls to 18% in IL-15/IL-15Racomplex–treated mice (Fig. 2A, left). A comparable expansion ofNK1.1+ cells and CD8+ T cells was also observed in peripheral blood(data not shown) and tumor-draining lymph nodes (data notshown), whereas surface expression of prototypical activationmarkers, such as CD44 and CD69, by circulating NK1.1+ cells andCD8+ T cells was only minimally affected (data not shown).Next, we evaluated the effect of systemically administered IL-15/

IL-15Ra complexes on tumor-associated leukocytes. For thesestudies, we established a sequential digestion method that allowsfor maximal release of single cells from large solid tumors that aresurgically excised from the surrounding pancreatic tissue. Applyingthis method in combination with cytofluorimetry, we detected apopulation of CD45+ cells that comprise f20% of the total tumorcell suspension in untreated mice (Fig. 2B, left). This observationwas somewhat surprising because it had been previously reportedthat the advanced-stage tumors in RIP1-Tag2 mice were largelydevoid of leukocytes (41). To confirm that the leukocytes detectedby cytofluorimetry were bona fide intratumoral cells rather thancontaminating peritumoral or extratumoral leukocytes that werephysically associated with the excised tumor, we examined thelocalization of CD45+ cells in pancreata of RIP-Tag2 mice usingimmunofluorescence microscopy. This analysis showed that theCD45+ cells detected by flow cytometry were indeed derived froman intratumoral leukocyte population that was evenly distributedthroughout the tumor parenchyma (Fig. 2B, right). Interestingly,systemic administration of IL-15/IL-15Ra complexes caused theintratumoral leukocytes to expand to 30% (Fig. 2B, left) of thetumor, and CD45+ cells were localized within the tumor bed oftreated mice similar to control tumors (Fig. 2B, right).Detailed analysis of the intratumoral leukocyte composition

indicated that a variety of leukocyte subsets are represented in thetumor under steady-state conditions, including CD8+ T cells, NK

cells, and NKT cells expressing CD122 (data not shown), as well asCD4+ T cells and Tregs. Treatment with IL-15/IL-15Ra complexescaused a 7- to 8-fold increase in intratumoral NK1.1+ cells (Fig. 2A,right), which are largely NK cells (88% NK1.1+CD3�; SupplementaryFig. S4B). Similarly, intratumoral CD8+ T cells increased 4- to 5-foldupon treatment with IL-15/IL-15Ra complexes (Fig. 2A, right).Surface expression of CD44 and CD69 by tumor-associatedleukocytes was only minimally affected (data not shown) by theadministration of IL-15/IL-15Ra complexes. The percentage oftumor-associated CD4+ T cells and Tregs was similar between PBSand complex-treated RIP1-Tag2 mice (data not shown). In sum,these results show that systemic delivery of IL-15/IL-15Racomplexes increases the frequency of CD8+ T cells and NK1.1+

cells in lymphoid tissues and solid tumors of RIP1-Tag2 mice, butdoes not markedly alter their activation state.Tumor destruction upon exposure to IL-15/IL-15RA com-

plexes is mediated by CD8+ T cells, not by NK or NKT cells.Having shown that NK1.1+ cells and CD8+ T cells were bothresponsive to IL-15/IL-15Ra complexes in RIP1-Tag2 mice bearingadvanced-stage solid tumors, we asked whether one or both ofthese cell populations was responsible for the tumor celldestruction. To address this, RIP-Tag2 mice were injected withmonoclonal antibodies that specifically deplete NK1.1+ cells orCD8+ cells or with isotype controls before the injection of IL-15/IL-15Ra complexes. Systemic depletion of NK1.1+ cells had no effecton the efficacy of IL-15/IL-15Ra complex therapy (Fig. 2C), whereasdepletion of CD8+ T cells completely abrogated the therapeuticefficacy of IL-15/IL-15Ra complexes (Fig. 2D). We concludethat CD8+ T cells, but not NK or NKT cells, are responsible forthe diminished tumor burden in IL-15/IL-15Ra–treated RIP1-Tag2 mice.Tumor-resident, not circulating, CD8+ T cells mediate tumor

destruction. Next, we sought to define the steps leading to tumorcell destruction by CD8+ T cells following in vivo delivery of IL-15/IL-15Ra complexes. We reasoned that these lymphocytes couldpromote tumor regression by two different mechanisms that werenot mutually exclusive. One possibility was that CD8+ T cells withinsecondary lymphoid tissues or blood would undergo expansion andactivation upon exposure to IL-15/IL-15Ra complexes, traffic viablood to tumors, infiltrate the tumor parenchyma, and finally killmalignant cells. A second possible mechanism was that tumor-resident CD8+ T cells would expand in the tumor itself uponsignaling by IL-15/IL-15Ra complexes and then destroy neighbor-ing tumor cells. To explore these possibilities, we first determinedwhether CD8+ T cells within solid tumors of RIP1-Tag2 mice divideupon exposure to IL-15/IL-15Ra complexes using BrdUrd incor-poration. Intratumoral CD8+ T cells incorporated BrdUrd within 24hours of treatment, whereas CD8+ T cells in lymphoid tissues didnot respond until day 2 (Fig. 3A). Furthermore, the amount ofBrdUrd incorporated by the intratumoral CD8+ T cells was higherthan CD8+ T cells within lymphoid organs (Fig. 3B). Theseobservations are consistent with the fact that 80% of the tumor-resident CD8+ T cells express CD122+, whereas only 24% of spleenCD8+ T cells are CD122+ in 10-week-old RIP1-Tag2 mice (Fig. 3C).Thus, tumor-resident CD8+ T cells rapidly proliferate in response tosystemically delivered IL-15/IL-15Ra complexes.Previous reports established that solid pancreatic tumors in

RIP1-Tag5 mice become impenetrable by circulating leukocytesafter the angiogenic switch because of alterations in the localvasculature (6, 42–44). However, it has remained unclear whetheraltered tumor vessels (45) prevent circulating leukocytes from

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Figure 2. IL-15/IL-15Ra complexes expand CD8+ Tcells and NK1.1+ cells in spleen and solid tumors, but tumor destruction requires CD8+ Tcells but not NK1.1+ cells.A, cytofluorimetric analysis of NK1.1+ and CD8+ splenocytes (left) and intratumoral leukocytes (right ) from RIP1-Tag2 controls or RIP1-Tag2 littermates treatedwith IL-15/IL-15Ra complexes. Percentage of NK1.1+ cells or CD8+ cells among live-gated cells was assessed on day 4 of regimen. Columns, mean from threeindependent experiments (treated mice, n = 10; controls, n = 8); bars, SE. B, cytofluorimetric (left) and immunofluorescence microscopic (right ) analyses ofCD45+ cells in tumors of IL-15/IL-15Ra complex–treated RIP1-Tag2 mice compared with control mice. NK1.1+ (C ) or CD8+ T cells (D ) were depleted from RIP1-Tag2mice using monoclonal antibodies before injection of IL-15/IL-15Ra complexes. Depletion of cells was evaluated in spleen and tumors by cytofluorimetry. Left, dotplots depict anti-CD8 (X axis ) and anti-NK1.1 (Y axis ) staining, and values in plots indicate the percentage of the population among live-gated splenocytes or live-gatedtumor cells. Right, the effect of depleting NK1.1+ cells (C ) or CD8+ cells (D) on treatment with IL-15/IL-15Ra complexes was assessed as a measure of tumorburden. Columns, mean (n = 3 individual mice for each condition); bars, SE.





infiltrating advanced solid tumors in the RIP1-Tag2 line. If suchbarriers indeed develop during tumorigenesis in the RIP1-Tag2mouse model, then it would be imperative to evaluate whethersuch barriers persist after treatment with IL-15/IL-15Ra complexesso as to determine whether newly infiltrating or tumor-residentlymphocytes mediate the tumor destruction. In agreement with ourcytofluorimetric enumeration of leukocyte expansion in IL-15/IL-15Ra complex–treated RIP1-Tag2 mice, histologic analysis ofliver, kidney, and exocrine pancreas clearly showed an increase inleukocyte frequency in blood vessels of these tissues (Fig. 4A). Instriking contrast, tumor vessels seemed to lack leukocytes underboth conditions (Fig. 4B), suggesting that circulating leukocytes areexcluded from solid tumors in RIP1-Tag2 mice, even aftertreatment with IL-15/IL-15Ra complexes. These data are furthercorroborated by our observation that CD8+ T cells are present inthe tumor parenchyma (Fig. 4C) of control and IL-15/IL-15Racomplex–treated animals rather than in tumor blood vessels aspreviously reported for adoptively transferred cells (46).To test this notion more stringently, we directly monitored the

trafficking of circulating leukocytes in control and IL-15/IL-15Racomplex–treated RIP1-Tag2 mice. We transferred CFSE-labeledsplenocytes from CD45.1+ congenic RIP1-Tag2 donors into CD45.2+

RIP1-Tag2 mice. At various times after transfer, lymphoid organs,liver, and tumors were harvested from control and IL-15/IL-15Racomplex–treated recipients and processed for cytofluorimetricenumeration of the donor cells. Donor cells were readily detectedin spleen, lymph nodes, and liver of both IL-15/IL-15Ra complex–treated RIP1-Tag2 mice and PBS-treated control littermates(Fig. 5A). In contrast, the tumors in both conditions containedfew if any CD45.1+ donor cells (Fig. 5A) within the first 72 hoursafter transfer. Consistent with our BrdUrd studies, we found thatthe donor cells started dividing, as measured by CFSE dilution, 2 to

3 days after transfer in peripheral lymphoid organs and liver ofIL-15/IL-15Ra complex–treated mice (Fig. 5B). Tumor-infiltratingleukocytes were not detected until 3 days posttransfer and seemedto be progeny of donor cells that had divided elsewhere.Interestingly, these cells arrive after tumor burden is alreadysignificantly reduced in IL-15/IL-15Ra complexes (Fig. 5C), thusimplicating tumor-resident rather than circulating lymphocytes inthis process. These results establish two new points. First,advanced solid tumors in RIP1-Tag2 mice are not readily accessibleto any circulating leukocytes. Second, systemic treatment of 10- to11-week-old RIP1-Tag2 mice with IL-15/IL-15Ra complexes doesnot markedly increase leukocyte infiltration of solid tumors. Theseresults suggest that tumor-resident CD8+ T cells play a major rolein the tumor destruction that ensues after in vivo delivery of IL-15/IL-15Ra complexes.IL-15/IL-15RA complexes endow tumor-resident CD8+

T cells with tumoricidal potential. Despite the fact thatendogenous CD8+ T cells with a memory phenotype are presentwithin advanced RIP1-Tag2 tumors and that tumor-specificmemory CD8+ T cells have been observed in these animals, suchlymphocytes could be functionally impaired, unable to recognizeantigen or ignorant under steady-state conditions becausetumorigenesis is unchanged in T cell–deficient RIP1-Tag2 mice(47). However, systemic administration of IL-15/IL-15Ra complexesleads to the rapid expansion of tumor-resident CD8+ T cells that inturn destroy the surrounding tumor. To gain a better understand-ing of how this treatment leads to tumor cell destruction by CD8+

T cells, we initially examined the localization of CD8+ T cells withinthe tumors at early time points. Within 48 hours of treatment, wefound that the apoptotic tumor cells were often in close proximityto or even in direct contact with tumor-resident CD8+ T cells,suggesting that the lymphocytes were directly lysing the malignant

Figure 3. Tumor-resident CD8+ T cells rapidly expand after IL-15/IL-15Ra complex treatment. A, kinetics of BrdUrd incorporation in spleen, lymph node, and tumorsof control and IL-15/IL-15Ra complex–treated RIP1-Tag2 mice. BrdUrd incorporation among CD8+ cells was assessed at 24-h intervals throughout the treatment.*, P < 0.05, Student’s t test. Data are representative of two independent experiments (n = 6 individual mice per time point). B, histogram depicts BrdUrd+ cellsamong CD8+ cells in spleen, lymph nodes, and tumors at day 3. C, expression of surface CD122 and CD44 by CD8+ T cells in spleens and tumors of control andIL-15/IL-15Ra complex–treated RIP1-Tag2 mice. Values in dot plots indicate percentage of gated cells among CD8+ T cells.

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Figure 4. Circulating leukocytes are undetectable in blood vessels of solid tumors in RIP1-Tag2 mice even after IL-15/IL-15Ra complex treatment. Histologicanalysis of blood vessels in parenchymal tissues and tumors of IL-15/IL-15Ra complex treatment and control RIP1-Tag2 mice. A and B, left, organs were fixed andprocessed for H&E staining. Images show representative histology of liver, kidney, exocrine pancreas, and pancreatic tumors in control and IL-15/IL-15Ra complex–treated RIP1-Tag2 mice. Right, quantification of leukocytes in blood vessels of liver, kidney, exocrine pancreas, and pancreatic tumors in control andIL-15/IL-15Ra complex–treated RIP1-Tag2 mice from two independent experiments (n = 7 individual mice per condition). C, immunofluorescence microscopicanalysis of CD8+ T cells in tumors of IL-15/IL-15Ra complex–treated and control RIP1-Tag2 mice. Tumors were excised 48 h after treatment with IL-15/IL-15Racomplexes and frozen in OCT. Cryosections from pancreata of treated and control mice were stained with DAPI (blue ) and monoclonal antibodies to CD8(red) and the pan-blood vessel marker, CD31 (green ). Left, images (�20) are representative of more than four independent experiments. Right, areas in whiteboxes are magnified.





Figure 5. Circulating leukocytes do not efficiently infiltrate advanced solid tumors in IL-15/IL-15Ra complex–treated RIP1-Tag2 mice. Cytofluorimetric tracking ofcirculating leukocytes in IL-15/IL-15Ra complex–treated RIP1-Tag2 mice. Spleen and lymph nodes cells from 10.5-wk-old CD45.1+ congeneic RIP1-Tag2 micewere stained with CFSE and transferred into age-matched CD45.2+ RIP1-Tag2 mice. Recipients were treated with IL-15/IL-15Ra complexes at the time of donorleukocyte infusion and the distribution of CD45.1+ CFSE-labeled donor leukocytes was subsequently evaluated 24, 48, and 72 h later by cytofluorimetry (n = 6 miceper condition). A, dot plots depict CD45.1+ donor cells (X axis ) versus forward size scatter (FSC ; Y axis ). B, histograms depict proliferation of CFSE+ donor cells(X axis). C, quantification of tumor burden in IL-15/IL-15Ra complex–treated (n = 16) and control (n = 21) RIP1-Tag2 mice at 48 h of treatment. Columns, meanfor individual mice (represents four independent experiments); bars, SE.

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h cells (Fig. 6A). Consistent with this notion, we found that thecytolytic activity of splenic CD8+ OT-I T cells toward OVA peptide-pulsed targets was potentiated by IL-15/IL-15Ra complexeswhereas control-treated CD8+ T cells exhibited no cytolyticpotential (Fig. 6B). Thus, signaling by the IL-15/IL-15Ra complexesseemed to activate the killing potential of CD8+ T cells.Next, we examined tumor-resident CD8+ T cells for their

expression of molecules that have been implicated in tumor cellkilling, including IFNg, granzyme B, and perforin. We found thattreatment with IL-15/IL-15Ra complexes lead to an increase in IFNgand granzyme B expression by tumor-resident and splenic CD8+

T cells (Fig. 6C); however, tumor-resident CD8+ T cells expressedhigher levels of both effector molecules under steady-stateconditions and in treated animals (Fig. 6C). Because our attemptsto examine perforin expression by intracellular flow cytometry weremetwith technical difficulties, wewere unable to quantify changes inperforin protein after treatment. Given the effect of the IL-15/IL-

15Ra complexes on expression of granzyme B, we generatedperforin-deficient RIP1-Tag2 mice to evaluate the contribution ofthe granzyme B/perforin pathway in the IL-15/IL-15Ra complex–stimulated tumor destruction. As shown in Fig. 6D , the reduction insolid tumor burden in IL-15/IL-15Ra complex–treated mice wasabrogated in the knockout animals, indicating that tumor destruc-tion is dependent on the granzyme B/perforin pathway. In sum,we conclude that in vivo delivery of IL-15/IL-15Ra complexesendows tumor-resident CD8+ T cells with the capacity to rapidlyexpand and directly kill their tumor cell neighbors.

Discussion

The potential use of IL-15 as a cancer immunotherapeutic agenthas been investigated in several mouse models of cancer (29–31).Transfer of B16 cells into IL-15 transgenic mice showed thatubiquitous cytokine expression can act prophylactically to

Figure 6. In vivo delivery of IL-15/IL15Ra complexes endows tumor-resident CD8+ T cells with cytolytic potential. A, cryosections of tumors from IL-15/IL-15Racomplex–treated mice were stained with DAPI (blue ), anti-CD8 (red ), and TUNEL (green ). Arrows, CD8+ Tcells in close proximity with apoptotic tumor cells. B, ex vivoassessment of cytolytic function among splenic CD8+ T cells in mice treated with IL-15/IL-15Ra complexes. OT-I/Rag1�/� TCR transgenic mice were injected withIL-15/IL-15Ra complexes daily with a 24-h interval (days 0 and 2). On day 3, OT-I CD8+ T cells were isolated from spleens of treated and control mice. Specificcytotoxicity was determined after 5 h of coculture with control (RMA ) or OVA peptide-pulsed (RMAOVAp ) target cells. Results are representative of two independentexperiments. C, cytofluorimetric analysis of IFNg (top ) and granzyme B (bottom ) expression by CD8+ T cells from tumors and spleens of control and IL-15/IL-15Racomplex–treated RIP1-Tag2 mice. Values in representative histograms indicate percentage of IFNg+ cells and mean fluorescence intensity (MFI ) of granzyme Bexpression among total CD8+ T cells within the tumors. Columns, mean from two independent experiments (treated mice, n = 5 ; controls, n = 6); bars, SE.D, quantification of tumor burden in control wild-type (WT ) RIP-Tag2 mice (n = 4), IL-15/IL-15Ra complex–treated WT RIP-Tag2 mice (n = 4), and IL-15/IL-15Racomplex–treated perforin-deficient (Prf�/�) RIP-Tag2 mice (n = 4). Columns, mean for each experimental condition; bars, SE.





stimulate potent antitumor immunity and prevent tumor engraft-ment (36). Other studies investigating the curative potential of thisagent showed that IL-15 could enhance the effect of chemother-apeutic agents and ACT on transplanted tumors, but had little orno effect when administered alone (29–32). IL-15 complexed withIL-15Ra exhibits remarkable potency compared with free IL-15(35, 36) and has been shown to prevent engraftment of B16 tumors(36). We report here that two injections of IL-15/IL-15Racomplexes leads to CD8+ T cell–mediated regression of autoch-thonous solid tumors of the pancreas and also severely retardsgrowth of established B16 tumors without significant toxicities.Remarkably, in vivo delivery of IL-15/IL-15Ra complexes promotesimmune-mediated destruction of established tumors by endoge-nous, tumor-resident CD8+ T cells. These results are particularlystriking as the therapeutic effect of IL-15/IL-15Ra occurs at arelatively low dose without the addition of chemotherapeuticagents, vaccination, ACT, or other cytokines. Although IL-15 lacksthe adverse effects of IL-2 such as Treg expansion and AICD, it is ofgreat interest to compare the effect of IL-15/IL-15Ra complexes tothat of IL-2/anti–IL-2 complexes in vivo (48).The superagonist activity of IL-15/IL-15Ra complexes is most

pronounced for CD8+ memory-phenotype cells (35). Our resultsshow that IL-15/IL-15Ra complexes stimulate the expansion ofCD122+ cells and enhanced the effector functions of NK and CD8+

T cells. Of particular interest was the ability of these complexes toarm a small population of naturally occurring intratumoral MPCD8+ T cells with the capacity to proliferate and kill neighboringcancer cells in an environment that otherwise seems to beimmunosuppressive. The existence of such cells in solid tumors ofthe pancreas in RIP1-Tag2 transgenic mice was unexpected asinsulinomas are often described as lacking leukocytic infiltrates(6, 41, 42, 45, 49). In retrospect, it is understandable how a popu-lation of long-lived CD8+ T cells might have been overlookedbecause these cells are sparsely distributed throughout the tumor.Furthermore, processing of tumors by multiple rounds of enzymaticdigestion allowed us to systematically analyze the cellularcomposition of these malignant lesions and the response of apopulation of MP CD8+ T cells to IL-15/IL-15Ra complexes withinthe tumor. Speiser et al. (41) also provided functional evidence for asmall pool of tumor-specific, memory CD8+ T cells in RIP-Tag2 micethat rapidly expanded upon vaccination with tumor antigen andacquired sufficient cytolytic potential to kill tumor cells. Thus, treat-ment with IL-15/IL-15Ra complexes enables MP CD8+ T cells thatpersist in tumors to rapidly divide and kill neighboring tumor cells.We and others have shown that circulating leukocytes cannot

access solid tumors of the endocrine pancreas because of anatypical vascular network (45, 49). Like RIP1-Tag2 tumors, solidtumors in RIP1-Tag5 mice develop vascular alterations thatmitigate leukocyte adhesion (6, 42–45). These observations suggestthat immunotherapies that aim to target established tumors withcirculating leukocytes, including vaccination and ACT, will not befully effective in the face of such vascular barriers (50). Identifyingagents that promote extravasation of blood leukocytes into tumorsis of considerable interest. For example, treatment with either CpG-ODN or irradiation were recently found to promote infiltrationof RIP1-Tag5 tumors by adoptively transferred tumor-specificT lymphocytes (6, 42). These vascular barriers may also be thecause of incomplete tumor destruction in both short- and long-term treatments. At any given time, RIP1-Tag2 mice containtumors of various sizes and composition. Although IL-15/IL-15Racomplexes will lead to expansion and activation of CD122+ CD8+ T

cells in large tumors, smaller tumors that contain fewer or lackCD122+ CD8+ T cells may be refractory to this treatment. On theother hand, tumor-resident CD8+ T cells may become functionallyexhausted after treatment because they cannot be reinforced bycirculating CD8+ T cells due to vascular barriers. Although IL-15/IL-15Ra complex treatment prolonged the survival of both RIP1-Tag2and B16 tumors (51) in a CD8+ T cell– and NK cell–dependentmanner, respectively, reversal of vascular barriers by other biologicsmay be required for efficient CD8+ T-cell infiltration and completecure. Alternatively, as we show here, treatment with agents thatsidestep vascular barriers by endowing intratumoral lymphocyteswith tumoricidal function may prove to be highly efficacious inreducing tumor burden while minimizing the risk of systemicinflammation or toxicity. According to a recent study addressingthe mechanism by which adoptively transferred CD8+ T cellspenetrate and destroy transplanted EL4 tumors, circulating CD8+ Tcells entered tumors from peritumoral vessels and began killingmalignant cells shortly after extravasation (46). Migration ofcytotoxic lymphocytes from the tumor margin to the tumor coreoccurred progressively as a result of tumor cell elimination (46). Incontrast, we found that MP CD8+ T cells of host origin aredistributed evenly throughout solid tumors of the pancreas andafter exposure to IL-15/IL-15Ra complexes begin killing malignantcells from their long-term posts within the tumor core. Thus, thestrategy by which adoptively transferred T cells infiltrate anddestroy tumors seems to differ rather significantly from that usedby naturally occurring effector cells that persist within solid tumorsto kill malignant cells following IL-15/IL-15Ra treatment. Despitethe marked increase in NK cell abundance in IL-15/IL-15Ra–treated RIP1-Tag2 mice, NK1.1+ cells do not contribute to the tumordestruction observed in our study. The cellular mechanism ofimmune-mediated tumor destruction is dictated in part by thestatus of MHC class I expression on the tumor cell targets (38).RIP1-Tag2 tumors are known to maintain expression of MHC class Imolecules (41); therefore, they are likely to inactivate local NK cellsby triggering inhibitory receptors and be targeted by CD8+ T cells.Complexing free IL-15 with its soluble receptor IL-15Ra allows a

novel mechanism for boosting the biological activity of thiscytokine in various clinical settings. Here, we report that systemicadministration of IL-15/IL-15Ra complexes can circumvent tumorimmune evasion by endowing tumor-resident CD8+ T cells with thecapacity to act as a ‘‘trojan horse’’ and destroy tumors from within.This is a particularly important mechanism as many tumors likethe RIP1-Tag2 tumors develop barriers for new immune cellinvasion. Given that immune effector cells have been detected invarious types of solid tumors (16), it will be of considerable interestto test the efficacy of IL-15/IL-15Ra complexes in other tumormodels and in human cancers.

Acknowledgments

Received 1/4/2008; revised 2/15/2008; accepted 2/19/2008.Grant support: American Cancer Society (S.J. Turley), Richard and Susan Smith

Family Foundation (S.J. Turley), V Foundation (A.W. Goldrath), Cancer ResearchInstitute (A.W. Goldrath), Arthritis Foundation (J.A. Hamerman), Institut Nationalde la Recherche Agronomique (M. Epardaud), and Prevent Cancer Foundation(M.P. Rubinstein).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Competing interests statement: The authors declare that they have no competingfinancial interests.

We thank K. Wucherpfennig, G. Dranoff, R. Johnson, and A. Doedens for helpfuldiscussions of this work and L. Lanier (University of California San Francisco) andM. Kronenberg (La Jolla Institute of Allergy and Immunology) for the gifts of PK136.

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2008;68:2972-2983. Cancer Res Mathieu Epardaud, Kutlu G. Elpek, Mark P. Rubinstein, et al.

T Cells+Tumor-Resident CD8Destruction of Established Tumors by Reviving

Complexes PromoteαInterleukin-15/Interleukin-15R

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