LETTERS. Adaptive immunity maintains occult cancer in an equilibrium state

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1 Vol 45 6 Decemer 27 doi:1.138/nture639 LETTERS Adptive immunity mintins occult cncer in n equilirium stte Ctherine M. Koeel 1, Willim Vermi 1,2, Jeremy B. Swnn 3,4, Ndeen Zerf 3, Scott J. Rodig 5, Lloyd J. Old 6, Mrk J. Smyth 3,4 * & Roert D. Schreier 1 * The cpcity of immunity to control nd shpe cncer, tht is, cncer immunoediting, is the result of three processes 1 8 tht function either independently or in sequence 9 : elimintion (cncer immunosurveillnce, in which immunity functions s n extrinsic tumour suppressor in nive hosts); equilirium (expnsion of trnsformed cells is held in check y immunity); nd escpe (tumour cell vrints with dmpened immunogenicity or the cpcity to ttenute immune responses grow into cliniclly pprent cncers). Extensive experimentl support now exists for the elimintion nd escpe processes ecuse immunodeficient mice develop more crcinogen-induced nd spontneous cncers thn wild-type mice, nd tumour cells from immunodeficient mice re more immunogenic thn those from immunocompetent mice. In contrst, the equilirium process ws inferred lrgely from clinicl oservtions, including reports of trnsplnttion of undetected (occult) cncer from orgn donor into immunosuppressed recipients 1. Herein we use mouse model of primry chemicl crcinogenesis nd demonstrte tht equilirium occurs, is mechnisticlly distinguishle from elimintion nd escpe, nd tht neoplstic cells in equilirium re trnsformed ut proliferte poorly in vivo. We lso show tht tumour cells in equilirium re unedited ut ecome edited when they spontneously escpe immune control nd grow into cliniclly pprent tumours. These results revel tht, in ddition to destroying tumour cells nd sculpting tumour immunogenicity, the immune system of nive mouse cn lso restrin cncer growth for extended time periods. To ssess whether n equilirium stte cn develop during primry tumourigenesis, groups of sex- nd ge-mtched wild-type C57BL/6 or 129/SvEv mice were injected with single low dose of the chemicl crcinogen 39-methylcholnthrene (MCA) (Fig. 1) nd monitored for the ppernce of progressively growing srcoms for the next 2 or 23 dys, fter which new tumour formtion decreses precipitously in C57BL/6 nd 129/SvEv mice, respectively (Supplementry Fig. 1). Mice developing progressively growing srcoms during the forementioned times were removed from the experiments. The remining mice, which often displyed smll stle msses t the site of MCA injection, were plced on weekly injections of either control monoclonl ntiody or monoclonl ntiodies tht deplete or lock specific immunologicl components, nd therefter monitored for the ppernce of progressively growing tumours. When two cohorts of wild-type C57BL/6 mice were treted with 25 mg MCA, 3/16 (19% cohort 1, Fig. 1) nd 4/19 (21%, cohort 2, Fig. 1c) developed progressively growing srcoms y dy 2 nd were removed from the experiment. When the remining mice in cohort 1 were treted with control immunogloulin, none developed dditionl tumours (Fig. 1). In contrst, when the remining mice in cohort 2 were treted with mixture of monoclonl ntiodies tht deplete CD4 1 nd CD8 1 cells nd neutrlize interferon-gmm (IFNc), 6% (9/15) developed progressively growing srcoms t the MCA injection site (Fig. 1c). Vritions of this experiment were repeted severl times in two lortories, using mice with either homogeneous C57BL/6 or 129/SvEv ckgrounds. The results were in complete greement nd reveled tht nti-cd4/cd8, nti-ifnc, or nti-il-12p4 (which is criticl for IFNc production) were eqully effective in inducing srcom outgrowth nd were not more effective when used in comintion (Fig. 1d, nd Supplementry Fig. 2). In totl, of 187 mice treted with low-dose MCA, 86 (46%) developed progressively growing srcoms following depletion of CD4/CD8 cells, IFNc nd/or IL-12 components tht prticipte in dptive immunity. In contrst, tumour outgrowth did not occur when monoclonl ntiodies were used tht deplete nturl killer cells (nti-nk1.1), lock nturl killer cell recognition (nti-nkg2d) or inhiit one nturl killer cell effector function (nti-tnf-relted poptosis inducing lignd (TRAIL)) (n 5 55) (Fig. 1d). These dt suggest tht dptive immunity plys prticulrly importnt prt in preventing lte MCA-induced srcom outgrowth. Two possiilities could explin the lte tumour outgrowth we oserved on lting dptive immunity: either the tretment permitted delyed de novo trnsformtion or it fcilitted expnsion of pre-formed occult cncer cells. To explore the former nd further sustntite the selective importnce of dptive immunity in preventing lte tumour outgrowth, experiments were conducted using mice lcking either recomintion-ctivting gene (Rg)-1 or Rg2, which possess n intct innte immune comprtment ut lck lymphocytes tht medite dptive immunity (tht is, T cells, nturl killer T cells nd B cells). In strk contrst to the results otined with wild-type mice, very few lte-forming tumours were oserved in Rg1 2/2 C57BL/6 mice exposed to 5 mg MCA ( dose tht induces tumour outgrowth comprle to tht in wild-type mice exposed to 25 mg MCA) nd susequently treted on dy 2 with control immunogloulin, nti-cd4/-cd8/-ifnc (Fig. 2, ), or nti- NK1.1 (Fig. 2c). Very few lte-forming tumors were lso oserved in Rg2 2/2 129/SvEv mice treted with 25 mg MCA (so s to provide crcinogenic insult comprle to tht used for wild-type mice) nd susequently treted on dy 23 with control immunogloulin or nti-ifnc (to ssess the effects of nturl-killer-cell-produced IFNc on cells of innte immunity) (Fig. 2d). These results show tht srcom formtion in immunodeficient Rg 2/2 mice ws essentilly 1 Deprtment of Pthology nd Immunology, Wshington University School of Medicine, St. Louis, Missouri 6311, USA. 2 Deprtment of Pthology, University of Bresci/Spedli Civili di Bresci, Bresci 25123, Itly. 3 Cncer Immunology Progrm, Sir Donld nd Ldy Trescowthick Lortories, Peter McCllum Cncer Centre, Est Melourne, Victori 32, Austrli. 4 Deprtment of Pthology, University of Melourne, Prkville, Victori 31, Austrli. 5 Deprtment of Pthology, Brighm nd Women s Hospitl Hrvrd Medicl School, Boston, Msschusetts 2115, USA. 6 Ludwig Institute for Cncer Reserch t Memoril Slon Kettering Cncer Center, New York 121, USA. *These uthors contriuted eqully to this work. 93

2 LETTERS NATURE Vol 45 6 Decemer 27 complete within 2 dys of MCA exposure. In ddition, the men time to tumour formtion in MCA-treted Rg1 2/2 C57BL/6 mice (15 6 5(6 s.e.m.) dys) differed strikingly from tht in MCAtreted wild-type mice rendered immunodeficient t dy 2 using the nti-cd4/-cd8/-ifnc mixture ( dys) (Fig. 2e). Similr findings were mde using 129/SvEv strin mice: dys for MCA-treted Rg2 2/2 mice versus dys for MCA-treted wild-type mice immunodepleted t dy 23 (Fig. 2f). Tken together, these dt rgue strongly ginst continuous de novo trnsformtion c d Are (cm 2 ) Tumour-positive mice (frction) MCA 1. Control monoclonl ntiody Anti-CD4/ Time post MCA (dys) /23 dys WT + cig WT + nti-cd4/ cig Anti-NK1.1 Anti-TRAIL Anti-NKG2D Anti-CD4/-CD8 Anti-IFNγ Anti-IL-12 Anti-CD4/-CD8/-IL-12 Anti-CD4/ Figure 1 The dptive immune system promotes n equilirium stte in primry MCA-induced srcoms., Protocol to test for the existence of n equilirium stte during MCA tumourigenesis. Detils re descried in the text nd Methods., Sixteen wild-type (WT) C57BL/6 mice were treted with 25 mg MCA. At 2 dys, the 13 tumour-free mice were treted weekly with control immunogloulin (cig) nd monitored for the ppernce of lte-forming tumours. c, Nineteen WT C57BL/6 mice were treted s in. At 2 dys, the 15 tumour-free mice were injected weekly with nti-cd4/ -CD8/-IFNc (P 5 8 Fisher s exct test etween nti-cd4/-cd8/-ifnc nd cig). d, Frction of WT C57BL/6 mice treted with 25 mg MCA tht develop lte-forming tumours fter tretment with the following monoclonl ntiodies strting t dy 2: cig (n 5 76), nti-nk1.1 (n 5 3), nti-trail (n 5 16), nti-nkg2d (n 5 15), nti-cd4/-cd8 (n 5 29), nti-ifnc (n 5 29), nti-il-12p4 (n 5 12), nti-cd4/-cd8/-il- 12 (n 5 12) nd nti-cd4/-cd8/-ifnc (n 5 13). 94 s the mechnism underlying the lte tumor outgrowth in wild-type mice fter immunodepletion. We therefore considered the possiility tht t lest some MCAtreted wild-type mice tht remined free of progressively growing tumours hroured fully trnsformed srcom cells, the outgrowth of which ws immunologiclly restrined. In the MCA model, Are (cm 2 ) c Tumour-positive mice (frction) Time to tumour formtion (dys) e Rg / + cig Rg1 / post MCA Rg / + nti-cd4/ 1 2 Time post MCA (dys) d Rg1 / C57BL/ Rg1 / 129/SvEv cig Anti-NK1.1 cig Anti-IFNγ C57BL/6 Post nti-cd4/ f Rg2 / post MCA 129/SvEv Post nti-cd4/ Figure 2 Antiody-induced equilirium disruption does not occur s result of prolonged de novo trnsformtion. Rg1 2/2 C57BL/6 mice were injected with 5 mg MCA nd, t dy 2, 21 tumour-free mice were treted weekly with cig (), wheres 2 other tumour-free mice were treted with nti- CD4/-CD8/-IFNc (). Mice were monitored for the ppernce of lteforming srcoms. The difference etween nti-cd4/-cd8/-ifnc nd cig groups is not significnt (P 5 16, Fisher s exct test). c, Thirty-two tumourfree Rg1 2/2 C57BL/6 mice, injected initilly with 5 mg MCA, were treted weekly with either cig (n 5 16) or nti-nk1.1 (n 5 16), strting t dy 2 nd monitored for srcom ppernce. The difference etween nti-nk1.1 nd cig groups is not significnt (P 5 84, Fisher s exct test). d, Twenty-seven tumour-free Rg2 2/2 129/SvEv mice, injected initilly with 25 mgmca,were treted weekly with either cig (n 5 15) or nti-ifnc (n 5 12) strting t dy 23 nd followed for srcom ppernce. The difference etween nti-ifnc nd cig groups is not significnt (P 5 5, Fisher s exct test). e,mentime to tumour formtion 6 s.e.m. for Rg1 2/2 C57BL/6 mice treted with 25 mg MCA (n 5 11) or tumour-free WT C57BL/6 mice treted initilly with 25 mg MCA nd susequently with nti-cd4/-cd8/-ifnc strting t dy 2 (n 5 8) (P 5 3, Mnn Whitney rnk sum test). f, Men time to tumour formtion 6 s.e.m. for Rg2 2/2 129/SvEv mice treted with 25 mgmca (n 5 3) or tumour-free WT 129/SvEv mice treted initilly with 25 mg MCA nd susequently with nti-cd4/-cd8/-ifnc strting t dy 23 (n 5 3) (P, 1, Mnn Whitney rnk sum test).

3 NATURE Vol 45 6 Decemer 27 LETTERS cellulr trnsformtion nd tumour development occur exclusively t the site of crcinogen injection. Exmintion of the injection site in 129/SvEv mice treted with 25 mg MCA reveled the presence of smll 2 8 mm msses tht ecme plple within 15 dys of MCA injection ut did not chnge in size during n dditionl 15 dys. When sectioned nd nlysed y hemtoxylin nd eosin stining, 47% (8/17) of the stle msses contined clusters of lrge cells showing vrile degrees of typi, with enlrged vesiculr nuclei, prominent nucleoli nd heterogeneous morphologies (Fig. 3, c), nd which lso expressed vimentin (Fig. 3c, inset). These fetures mirrored the immuno-cytologicl spectrum oserved in progressively growing primry MCA srcoms (Fig. 3, d). The remining msses from MCA-treted wild-type mice showed vrile degrees of inflmmtion ssocited with res of dense firosis nd lipid droplets (the oil diluent of the MCA) often surrounded y multinucleted gint cells (Supplementry Fig. 3, c). The MCA injection site in Rg2 2/2 mice lso contined firotic res nd lipid droplets ut lcked oth typicl cells nd multinucleted gint cells (Supplementry Fig. 3, d). Immunohistochemicl stining of stle msses contining typicl cells from MCA-treted wild-type mice nd growing primry MCA srcoms from wild-type mice reveled the presence of CD3 1 T cells, B22 1 cells nd F4/8 1 mononucler phgocytes infiltrting into regions contining typicl cells (Supplementry Fig. 4). When stle msses contining typicl cells were trnsiently cultured, popultion of typicl firolst-like cells grew out tht formed progressively growing tumours when trnsplnted into immunodeficient Rg2 2/2 mice (Fig. 3i). In contrst, norml skin firolsts from either MCA-treted mice (Fig. 3i) or untreted H&E H&E nd vimentin Ki-67 TUNEL c e g Stle mss d f h Srcom 125 i Rg2 / Tumour free (%) j Ki-67 + /typicl cells Srcoms Stle msses Norml firolsts Stle msses Time post trnsplnt (dys) Figure 3 Demonstrtion of occult cncer in immunocompetent mice in the equilirium phse of cncer immunoediting. Sections from representtive stle msses isolted from WT 129/SvEv mice treted with 25 mg MCA (, c, e, g) nd progressively growing srcoms from MCA-treted WT 129/SvEv mice (, d, f, h) stined for hemtoxylin nd eosin (H&E) (,, c, d), vimentin (inset in c nd d), the prolifertion mrker Ki-67 (e, f) nd the poptosis mrker TUNEL (g, h). In c nd e, rrowheds indicte typicl cells. Mgnifiction: 23 ; scle rs, 1 mm ( nd ), 63 ; scle rs, 33 mm (c f) nd 1,3 ; scle rs, 2 mm (g nd h). i, Frction of tumour-free Rg2 2/2 129/SvEv mice following sucutneous injection with 1 6 norml skin firolsts (n 5 1, dimonds) or 1 6 cells isolted from long-term stle msses from MCA-treted WT 129/SvEv mice (n 5 5, tringles). Ech cell line ws injected into four Rg2 2/2 recipients. j, Proportion of typicl cells stining positive for Ki-67 in formlin-fixed tissue from growing primry MCA-srcoms from WT mice (n 5 1, squres) or stle msses from MCA-treted WT 129/SvEv mice (n 5 9, tringles) (P 5 3 Mnn Whitney rnked sum test, difference etween prolifertion indices of tumour cells in stle msses versus growing primry MCA srcoms). wild-type mice (dt not shown) did not form tumours in Rg2 2/2 mice. Thus, typicl cells from stle msses tht formed in MCA-treted, wild-type mice were fully trnsformed. These results contrdict n erlier report stting tht MCA tretment of wild-type mice leds to encpsultion of MCA crystls without pprent cellulr trnsformtion 11. However, the former study filed to chrcterize cells with ltered morphology tht were noted in the MCA grnuloms nd tht my hve een identicl to the trnsformed typicl cells tht we descrie herein. Moreover, MCA crystls re not oserved in our mice, indicting tht the MCA might not hve een fully dissolved in the erlier study nd therefore my hve chieved extremely high in vivo concentrtions resulting in rpid cellulr trnsformtion nd tumour outgrowth, therey oscuring detection of trnsformed cells, the growth of which ws under immunologic restrint. The prdox tht stle msses from our MCA-treted immunocompetent mice often contined trnsformed cells ut did not increse in size in vivo suggested tht net tumour cell expnsion ws eing immunologiclly restrined. This possiility ws explored y compring sections of stle msses contining typicl cells or progressively growing primry srcoms for expression of mrkers of prolifertion (Ki-67) 12,13 (Fig. 3e, f) or poptosis (TUNEL) (Fig. 3g, h). Anlysis of 1 progressively growing MCA srcoms reveled strong nucler rectivity for Ki-67 in high proportion of tumour cells (men prolifertion index 5 4%) (Fig. 3f, j), nd detectle levels of cellulr poptosis (Fig. 3h). This result is consistent with the high histologicl grde of these lesions shown y cellulr pleiomorphism, numerous visile mitoses nd undnt necrosis. In contrst, only limited percentge of typicl cells in nine stle msses stined positively for Ki-67 (men prolifertion index 5 18%) (Fig. 3e, j) nd these smples lso showed 1.7-fold more TUNEL stining thn growing srcoms (Fig. 3g). Of note, oth smple sets contined popultions of proliferting, Ki-67 1 leukocytes. Thus, stle msses re chrcterized y comintion of incresed poptosis nd decresed tumour cell prolifertion. The concept tht the immune system could mintin tumours in dormnt stte originlly stemmed from experiments involving trnsplnttion of certin tumour cell lines into pre-immunized mice 14,15. In the current study, we show tht immunity cn restrin the outgrowth of occult tumour cells during primry tumour induction in nive mice nd dditionlly demonstrte tht the lck of net expnsion of dormnt tumour cells in MCA-treted immunocompetent mice is most proly the result of continuously ongoing comintion of cytosttic nd cytolytic immune effects. Therefore the term equilirium est descries the persistence of cncer tht we oserve in wild-type mice exposed to low-dose MCA. Throughout these experiments we noticed tht smll percentge of low-dose MCA-chllenged wild-type mice, either left untreted, or treted with control monoclonl ntiody, went on to develop lteppering srcoms (Fig. 1d, Supplementry Fig. 2). We therefore sked whether srcom cells tht spontneously escpe equilirium could e distinguished on the sis of immunogenicity from those remining in equilirium. As controls, we lso confirmed the immunogenicities of MCA srcom cell lines generted in the current study tht grew out with norml kinetics from MCA-treted wildtype mice (edited tumour cells) nd Rg2 2/2 mice (unedited tumour cells). In greement with previous reports 1, edited MCA-srcom cell lines from wild-type mice displyed dmpened immunogenicity ecuse they formed progressively growing tumours in either Rg2 2/2 or nive wild-type recipients (Fig. 4,, respectively), nd unedited MCA srcom cell lines from Rg2 2/2 mice were highly immunogenic ecuse, lthough they grew in Rg2 2/2 recipients (Fig. 4), 4% were rejected in nive wild-type mice (Fig. 4). Notly, tumour cells tht hd een mintined in equilirium (tht is, derived either directly from stle msses or from MCA-treted, wild-type mice tht hd undergone nti-cd4/-cd8/-ifnc-medited equilirium disruption) formed progressively growing tumours in 95

4 LETTERS NATURE Vol 45 6 Decemer 27 Rg2 2/2 mice (Fig. 3i nd 4, respectively) ut were highly immunogenic in wild-type mice ecuse 51% nd 31%, respectively, were rejected (Fig. 4). In contrst, cell lines produced from lte-forming srcoms, which grew out spontneously from mice in equilirium, formed tumours when trnsplnted into either Rg2 2/2 mice or nive wild-type recipients (Fig. 4,, respectively). Thus, tumour cells held in equilirium hve n unedited phenotype, wheres those tht spontneously progress to the escpe phse re edited. We therefore conclude tht t lest some tumour cell editing occurs t the temporl interfce etween equilirium nd escpe. In summry, in ddition to the immune system s cpcity to destroy nd shpe cncer, this study shows tht immunity cn lso control cncer for long time periods y process clled equilirium. We show tht equilirium is indeed component of cncer immunoediting ecuse tumour cells in equilirium re highly immunogenic (unedited), wheres those spontneously exiting equilirium tht ecome growing tumours hve ttenuted immunogenicity (edited) results tht plce this process temporlly etween elimintion nd escpe. We demonstrte tht elimintion nd equilirium cn e mechnisticlly distinguished ecuse, lthough the former requires the ctions of oth innte nd dptive immunity 8, equilirium is mintined solely y dptive immunity. Similrly, we show Tumour free (%) Tumour free (%) Rg2 / recipients Time post trnsplnt (dys) Wild-type recipients Time post trnsplnt (dys) WT + nti-cd4/ Rg2 / tumours Spont. lte WT tumours WT tumours Stle msses WT + nti-cd4/ Rg2 / tumours Spont. lte WT tumours WT tumours Figure 4 Srcom cells in equilirium show high immunogenicity, wheres those spontneously exiting equilirium hve ttenuted immunogenicity., Kpln Meier survivl nlysis of Rg2 2/2 129/SvEv mice injected with srcom cell lines from clssicl progressively growing MCA-srcoms isolted from WT (n 5 5, circles) or Rg2 2/2 129/SvEv mice (n 5 13, dimonds) or isolted from tumours forming in MCA-treted WT 129/SvEv mice fter nti-cd4/-cd8/-ifnc-medited equilirium disruption (n 5 16, tringles) or isolted from lte-ppering tumours from MCA-treted WT 129/SvEv mice tht spontneously progress from equilirium to escpe (n 5 7, squres). Ech cell line ws injected t 1 6 cells per mouse into two mice ech., Kpln Meier survivl nlysis s in except tht 5-memer groups of nive WT 129/SvEv mice were used s the recipients for ech cell line. Also tested were srcom cell lines grown directly out of stle msses from MCA-treted WT mice in equilirium (n 5 5, inverted tringles). 96 tht equilirium nd escpe re distinct ecuse, wheres equilirium represents time of tumour cell persistence without expnsion, escpe is chrcterized y progressive tumour growth. We do not envisge tht every tumour cell must pss through n elimintion process efore it enters equilirium, nor do we hold tht every progressively growing tumour must trnsit through n equilirium process. However, we predict tht mny cliniclly pprent tumours my progress through liner elimintionrequiliriumrescpe continuum, nd ongoing work is focused on otining moleculr signtures of tumour cells in ech step of this process. Nevertheless, our results confirm n importnt, ut heretofore untested, prediction of the cncer immunoediting hypothesis the existence of n equilirium stte therey providing dditionl support to the centrl premise of cncer immunoediting: tht immunity cn influence cncer development oth quntittively nd qulittively. Our findings in this mouse model lso hve potentil relevnce to humn cncers. First, they indicte tht mintining cncer in n equilirium stte my represent relevnt gol of cncer immunotherpy in which ugmenttion of dptive tumour immunity could result in improved tumour control. Second, they provide mechnistic underpinnings for the recent findings tht the qulity nd quntity of the immune rection within certin tumour types (for exmple, colorectl nd ovrin cncers) re relile prognostic indictors of cncer ptient survivl Third, they explin how occult cncer cn e trnsplnted from orgn donor to recipient 1, ecuse tumour cells held in equilirium in the donor my grow in recipient who is nive to the ntigens of the trnsplnted tumour cells nd is immunosuppressed. Fourth, they provide mechnism tht cn explin the presence of occult tumour cells in orgns for exmple, the prostte of individuls lcking clinicl symptoms of disese Finlly, our study rises the possiility tht t lest some of the proposed tumour-promoting ctions of chronic inflmmtion my e result of interfering with dptive immunity s cpcity to hold occult cncers in equilirium 23,24. Our results thus provide foundtion for future work to define the moleculr mechnisms y which dptive immunity mintins cncer in n equilirium/dormnt/ persistent stte, perhps pving the wy for development of new therpeutic modlities to convert cncer into controllle chronic disese. METHODS SUMMARY Wild-type or Rg-null mice were injected sucutneously with low doses of MCA (Sigm Fine Chemicls), s descried 1,25, nd monitored for tumour development. After 2 dys (C57BL/6) or 23 dys (129/SvEv), MCA-treted mice ering progressively growing tumours were removed from the experiment nd the remining mice were treted weekly with either control immunogloulin or monoclonl ntiodies tht deplete or lock specific immune components nd were monitored for tumour development for the next 1 dys. Tumour trnsplnttion experiments were performed, s descried previously 1,25. For morphologic nd immunohistologic evlution of primry growing srcoms nd stle msses, sections of formlin-fixed, prffin-emedded tissue smples were evluted fter stining with hemotoxylin nd eosin or monoclonl ntiodies specific for either cell surfce mrkers or the Ki-67 protein. Apoptosis ws determined y TUNEL stining. The prolifertion index is defined s the percentge of lrge, typicl, cells showing enlrged nuclei with prominent nucleoli tht stined positively for Ki-67 in multiple high power (63) fields. Full Methods nd ny ssocited references re ville in the online version of the pper t Received 26 June; ccepted 24 Septemer 27. Pulished online 18 Novemer Shnkrn, V. et l. IFNc nd lymphocytes prevent primry tumour development nd shpe tumour immunogenicity. Nture 41, (21). 2. Dunn, G. P., Bruce, A. T., Iked, H., Old, L. J. & Schreier, R. D. Cncer immunoediting: from immunosurveillnce to tumor escpe. Nture Immunol. 3, (22). 3. Street, S. E., Trpni, J. A., McGregor, D. & Smyth, M. J. Suppression of lymphom nd epithelil mlignncies effected y interferon c. J. Exp. Med. 196, (22).

5 NATURE Vol 45 6 Decemer 27 LETTERS 4. Smyth, M. J. et l. NKG2D recognition nd perforin effector function medite effective cytokine immunotherpy of cncer. J. Exp. Med. 2, (24). 5. Dunn, G. P., Old, L. J. & Schreier, R. D. The immunoiology of cncer immunosurveillnce nd immunoediting. Immunity 21, (24). 6. Dunn, G. P. et l. A criticl function for type I interferons in cncer immunoediting. Nture Immunol. 6, (25). 7. Dunn, G. P., Old, L. J. & Schreier, R. D. The three Es of cncer immunoediting. Annu. Rev. Immunol. 22, (24). 8. Smyth, M. J., Dunn, G. P. & Schreier, R. D. Cncer immunosurveillnce nd immunoediting: the roles of immunity in suppressing tumor development nd shping tumor immunogenicity. Adv. Immunol. 9, 1 5 (26). 9. Dunn, G. P., Koeel, C. M. & Schreier, R. D. Interferons, immunity nd cncer immunoediting. Nture Rev. Immunol. 6, (26). 1. McKie, R. M., Reid, R. & Junor, B. Ftl melnom trnsferred in donted kidney 16 yers fter melnom surgery. N. Engl. J. Med. 348, (23). 11. Qin, Z., Kim, H. J., Hemme, J. & Blnkenstein, T. Inhiition of methylcholnthreneinduced crcinogenesis y n interferon c receptor-dependent foreign ody rection. J. Exp. Med. 195, (22). 12. Brown, D. C. & Gtter, K. C. Ki67 protein: the immculte deception? Histopthology 4, 2 11 (22). 13. Bullwinkel, J. et l. Ki-67 protein is ssocited with riosoml RNA trnscription in quiescent nd proliferting cells. J. Cell. Physiol. 26, (26). 14. Weinhold, K. J., Goldstein, L. T. & Wheelock, E. F. Tumour-dormnt sttes estlished with L5178Y lymphom cells in immunised syngeneic murine hosts. Nture 27, (1977). 15. Siu, H., Vitett, E. S., My, R. D. & Uhr, J. W. Tumor dormncy. I. Regression of BCL1 tumor nd induction of dormnt tumor stte in mice chimeric t the mjor histocomptiility complex. J. Immunol. 137, (1986). 16. Glon, J. et l. Type, density, nd loction of immune cells within humn colorectl tumors predict clinicl outcome. Science 313, (26). 17. Ohtni, H. Focus on TILs: Prognostic significnce of tumor infiltrting lymphocytes in humn colorectl cncer. Cncer Immun. 7, 4 12 (27). 18. Sto, E. et l. Intrepithelil CD8 1 tumor-infiltrting lymphocytes nd high CD8 1 /regultory T cell rtio re ssocited with fvorle prognosis in ovrin cncer. Proc. Ntl Acd. Sci. USA 12, (25). 19. Skr, W. A. & Prtin, A. W. Histologicl mrkers of risk nd the role of high-grde prosttic intrepithelil neoplsi. Urology 57, (21). 2. Welch, H. G. & Blck, W. C. Using utopsy series to estimte the disese reservoir for ductl crcinom in situ of the rest: how much more rest cncer cn we find? Ann. Intern. Med. 127, (1997). 21. Mutter, G. L. et l. Moleculr identifiction of ltent precncers in histologiclly norml endometrium. Cncer Res. 61, (21). 22. Bch, P. B. et l. Computed tomogrphy screening nd lung cncer outcomes. J. Am. Med. Assoc. 297, (27). 23. Blkwill, F. & Coussens, L. M. Cncer: n inflmmtory link. Nture 431, (24). 24. Lin, W. W. & Krin, M. A cytokine-medited link etween innte immunity, inflmmtion, nd cncer. J. Clin. Invest. 117, (27). 25. Smyth, M. J. et l. Differentil tumor surveillnce y nturl killer (NK) nd NKT cells. J. Exp. Med. 191, (2). Supplementry Informtion is linked to the online version of the pper t Acknowledgements The uthors re grteful for the dvice of E. Unnue, G. Dunn, H. Virgin, P. Allen, M. Colonn, J. Trpni, R. Uppluri, J. Bui nd ll memers of the Schreier lortory during the preprtion of this mnuscript. We lso gretly pprecite the technicl ssistnce of C. Arthur, M. White, J. Archmult nd J. Shrkey. This work ws supported y grnts to R.D.S. from the Ntionl Cncer Institute, the Ludwig Institute for Cncer Reserch, the Cncer Reserch Institute nd Atlntic Philnthropies, nd to M.J.S. from the Ntionl Helth nd Medicl Reserch Council of Austrli for Fellowship nd Progrm Grnt Support. C.M.K. ws supported y pre-doctorl trining grnt from the Cncer Reserch Institute. J.B.S. ws supported y n Austrlin Postgrdute Reserch Awrd. Author Contriutions The work in this pper reflects n equl contriution from the M.J.S nd R.D.S. lortories. C.M.K., M.J.S. nd R.D.S. were involved in ll spects of experimentl work, project plnning nd dt nlysis. W.V. nd S.J.R. were responsile for performing nd interpreting the histologicl nlyses. L.J.O. nd J.B.S. prticipted in project plnning nd N.Z. ws involved in the experimentl work. Author Informtion Reprints nd permissions informtion is ville t Correspondence nd requests for mterils should e ddressed to R.D.S. (schreier@immunology.wustl.edu) or M.J.S. (mrk.smyth@petermc.org). 97

6 doi:1.138/nture639 METHODS Mice. Wild-type C57BL/6 mice were purchsed from the Wlter nd Eliz Hll Institute of Medicl Reserch nd Rg1 2/2 C57BL/6 mice were red t the Peter McCllum Cncer Centre. Wild-type nd Rg2 2/2 129/SvEv mice were purchsed from Tconic Frms nd entered into the experiments t 8 to 12 weeks old. Mice were housed ccording to the Americn Assocition for Lortory Animl Science conditions in specific pthogen-free fcilities t the Wshington University School of Medicine, St. Louis, Missouri nd the Peter McCllum Cncer Centre, Est Melourne, Victori, Austrli. Regents. Anti-CD4 (GK1.5) 26, nti-cd8 (YTS-169.4) 27 nti-ifnc (H22) 28, nti-nkg2d (C7) 29, nti-nk1.1 (PK136) 3, nti-trail (N2B2) 31, nti-il- 12p4 (C17.8) 32 nd control immunogloulin (the PIP monoclonl ntiody specific for glutthione S-trnsferse 33 ) were generted from spent culture superntnt or hyridom scites nd purified in ggregte-, endotoxin-, nd protein A-free form. Antiodies were injected t n initil dose of mg ech with weekly (CD4, CD8, NK1.1, IFNc) or iweekly (NKG2D, TRAIL, IL-12p4) mintennce dose of 25 mg of ech. MCA tumour induction. Groups of wild-type nd Rg1 2/2 C57BL/6 mice were injected sucutneously in the hind flnk with 5 or 25 mg of MCA in.1 ml of corn oil, s descried 34. Mice were monitored every 7 dys for tumour development from 7 dys fter MCA tretment. Tumours..5 cm 2 in re nd demonstrting progressive growth were recorded s tumour positive. Wild-type nd Rg2 2/2 129/SvEv mice were injected sucutneously in the flnk with 25 mg MCA (Sigm Fine Chemicls) dissolved in.15 ml of penut oil (with gentle heting), s descried 35. Mice were monitored every 7 dys for tumour development from 9 dys fter MCA tretment. 129/SvEv mice were considered tumour positive when msses reched n verge dimeter of 9 mm nd continued to grow progressively. Cell lines. Cell lines were creted y mechnicl disruption of tissue, followed y 1 h tretment with collgense Type IA (.5 mg ml 21 ) nd culture in endotoxin-low RPMI medium (,1 EU ml 21 ) supplemented with 1% fetl clf serum (5 EU ml 21 ). Tumour trnsplnttion. Before use, vil of frozen srcom cells ws thwed nd cultured in vitro in RPMI medium supplemented with 1% fetl clf serum for two pssges. Tumour cells were collected y incution in 5% trypsin, wshed two times in RPMI medium nd one time in endotoxin-free PBS nd then injected sucutneously in volume of.15 ml PBS into the shved flnks of mice. All injected cell lines were more thn 9% vile, s determined y trypn lue exclusion. Tumour growth ws monitored y mesuring two perpendiculr dimeters. Histology, prolifertion index nd immunohistochemistry. Sections were otined from formlin-fixed, prffin-emedded tissue smples. For morphologicl evlution, sections were stined in hemotoxylin nd eosin. The prolifertion index ws clculted from tissue sections stined using n indirect immunoperoxidse technique tht employed s primry ntiody the clone Tec-3 (DkoCytomtion, 1:25 dilution), which is specific for the Ki-67 protein, mrker of proliferting cells, nd iotinylted rit nti-rt IgG secondry ntiody (Vector Lortories, 1:2 dilution). At lest 2 typicl/neoplstic cells were counted nd scored per smple. All other immunohistochemicl stins were performed on formlin-fixed, prffin-emedded 4 mm tissue sections using stndrd techniques. The following primry ntiodies were used: ntivimentin (Acm, 2828, 1:1 dilution following proteinse K digestion for 15 min.), nti-cd3 (Cell Mrque, CMC363, 1:1,5 dilution), nti-cd45r/ B22 (BD Phrmingen, no , 1:2 dilution), nd nti-f4/8 ntigen (Serotec, MCA497, 1:5, dilution following proteinse K digestion for 1 min.). Chromogenic terminl-deoxynucleotidyl-trnsferse-medited nickend lelling (TUNEL) stining with methyl green counterstining ws done s per the mnufcturer s instructions (ApopTg Peroxidse In situ Kit, no. S71, Chemicon Interntionl). Sttisticl nlysis. Fisher s exct test ws used to determine the significnce of the ssocition etween two vriles (tumour development nd ntiody tretment) in contingency tle. The Mnn Whitney rnk sum test ws used to ssess whether two smples of oservtions (time to tumour formtion nd prolifertion index) come from the sme distriution, without ssuming equl vrinces etween the two popultions. 26. Dilyns, D. P. et l. Chrcteriztion of the murine ntigenic determinnt, designted L3T4, recognized y monoclonl ntiody GK1.5: expression of L3T4 y functionl T cell clones ppers to correlte primrily with clss II MHC ntigen-rectivity. Immunol. Rev. 74, (1983). 27. Coold, S. P., Jysuriy, A., Nsh, A., Prospero, T. D. & Wldmnn, H. Therpy with monoclonl ntiodies y elimintion of T-cell susets in vivo. Nture 312, (1984). 28. Schreier, R. D., Hicks, L. J., Celd, A., Buchmeier, N. A. & Gry, P. W. Monoclonl ntiodies to murine c-interferon which differentilly modulte mcrophge ctivtion nd ntivirl ctivity. J. Immunol. 134, (1985). 29. Ho, E. L. et l. Costimultion of multiple NK cell ctivtion receptors y NKG2D. J. Immunol. 169, (22). 3. Koo, G. C. & Pepprd, J. R. Estlishment of monoclonl nti-nk-1.1 ntiody. Hyridom 3, (1984). 31. Kygki, N. et l. Expression nd function of TNF-relted poptosis-inducing lignd on murine ctivted NK cells. J. Immunol. 163, (1999). 32. Wysock, M. et l. Interleukin-12 is required for interferon-c production nd lethlity in lipopolyscchride-induced shock in mice. Eur. J. Immunol. 25, (1995). 33. Dunn, G. P. et l. A criticl function for type I interferons in cncer immunoediting. Nture Immunol. 6, (25). 34. Smyth, M. J. et l. Differentil tumor surveillnce y nturl killer (NK) nd NKT cells. J. Exp. Med. 4, (2). 35. Shnkrn, V. et l. IFNc nd lymphocytes prevent primry tumour development nd shpe tumour immunogenicity. Nture 41, (21).