A2A adenosine receptor protects tumors from antitumor T cells

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1 A2A denosine reeptor protets tumors from ntitumor T ells Akio Oht*, Elieser Gorelik, Simon J. Prsd, Frn Ronhese, Dmitriy Lukshev*, Mihel K. K. Wong, Xiojun Hung, Sheil Cldwell**, Kein Liu**, Ptrik Smith*, Jing-Fn Chen, Edwin K. Jkson, Sergey Apsov*, Sott Arms **, nd Mihil Sitkovsky* *Lortory of Immunology, Ntionl Institute of Allergy nd Infetious Diseses, Ntionl Institutes of Helth, Bethesd, MD 2892; New Englnd Inflmmtion nd Tissue Protetion Institute, Northestern University, Boston, MA 2115; Deprtment of Pthology nd University of Pittsurgh Cner Institute, University of Pittsurgh, Pittsurgh, PA 15213; Mlghn Institute of Medil Reserh, Wellington, New Zelnd; Deprtment of Mediine nd Deprtment of Phrmology nd Therpeutis, Roswell Prk Cner Institute, Bufflo, NY 14263; **Lortory of Tumor Immunology nd Biology, Center for Cner Reserh, Ntionl Cner Institute, Ntionl Institutes of Helth, Bethesd, MD 2892; Deprtment of Biohemistry nd Moleulr Biology, Medil College of Georgi, August, GA 3912; Deprtment of Neurology, Boston University Shool of Mediine, Boston, MA 2118; nd Center for Clinil Phrmology, Deprtments of Phrmology nd Mediine, University of Pittsurgh Cner Institute, University of Pittsurgh, Pittsurgh, PA Communited y Willim E. Pul, Ntionl Institutes of Helth, Bethesd, MD, June 22, 26 (reeived for review Mrh 29, 26) The A2A denosine reeptor (A2AR) hs een shown to e ritil nd nonredundnt negtive regultor of immune ells in proteting norml tissues from inflmmtory dmge. We hypothesized tht A2AR lso protets nerous tissues y inhiiting inoming ntitumor T lymphoytes. Here we onfirm this hypothesis y showing tht geneti deletion of A2AR in the host resulted in rejetion of estlished immunogeni tumors in 6% of A2ARdefiient mie with no rejetion oserved in ontrol WT mie. The use of ntgonists, inluding ffeine, or trgeting the A2 reeptors y sirna pretretment of T ells improved the inhiition of tumor growth, destrution of metstses, nd prevention of neovsulriztion y ntitumor T ells. The dt suggest tht effets of A2AR re T ell utonomous. The inhiition of ntitumor T ells vi their A2AR in the denosine-rih tumor miroenvironment my explin the prdoxil oexistene of tumors nd ntitumor immune ells in some ner ptients (the Hellstrom prdox ). We propose to trget the hypoxi3denosine3a2ar pthwy s ner immunotherpy strtegy to prevent the inhiition of ntitumor T ells in the tumor miroenvironment. The sme strtegy my prevent the premture termintion of immune response nd improve the vine-indued development of ntitumor nd ntivirl T ells. The oservtions of utoimmunity during melnom rejetion in A2AR-defiient mie suggest tht A2AR in T ells is lso importnt in preventing utoimmunity. Thus, lthough using the hypoxi3denosine3a2ar pthwy inhiitors my improve ntitumor immunity, the reruitment of this pthwy y seletive drugs is expeted to ttenute the utoimmune tissue dmge. utoimmunity ner therpy hypoxi inflmmtion The oexistene of tumors nd ntitumor immune ells is urrently explined y the inhiition of immune ells in poorly understood hostile tumor miroenvironment (1 3). This unidentified immunosuppressive mehnism limits promising ner therpies using ntitumor T ells (4 14). We hypothesized tht nerous tissues re proteted from ntitumor T ells euse of immunosuppressive signling vi T ell A2A denosine reeptor (A2AR) (15 17) tivted y extrellulr denosine produed from hypoxi tumor (Fig. 1). Indeed, hypoxi nerous tissues my e proteted y the sme hypoxi3denosine3a2ar pthwy tht ws reently shown to e ritil nd nonredundnt in preventing exessive dmge of norml tissues y overtive immune ells in vivo (18). It is well estlished tht some res of solid tumors often hve trnsient or hroni hypoxi (19, 2), whih is onduive to extrellulr denosine umultion (21). Hypoxi hs een implited in mehnisms of tumor protetion ginst ionizing rdition nd some hemotherpeuti gents (19) nd is ssoited with poor prognosis (2). T ells, inluding ntitumor T ells, do predominntly express -elevting Gs protein-oupled high-ffinity A2AR nd or low-ffinity A2B denosine reeptors (A2BR) (16, 17, 22 24); the numer of A2AR per T ell my determine the intensity of mximl T ell response to denosine (25, 26). Wheres we foused on A2AR, others hve disounted A2 reeptors nd suggested the A3 denosine reeptors s responsile for inhiition of ntitumor killer T ells (27, 28). Here we report tht geneti deletion of A2AR omplishes the omplete rejetion of immunogeni tumors y ntitumor CD8 T ells in the mjority ( 6%) of mie, wheres the ntgonists of A2 reeptors filitte CD8 T ell-medited retrdtion of tumor growth. Results The Grdient of T Cell-Inhiiting Extrellulr in Tumors. It ws importnt to onfirm the presene of elevted extrellulr denosine levels in nerous tissues using relile method (29). The HPLC nlysis nd the use of equilirium dilysis proes demonstrted higher levels of extrellulr denosine (Fig. 1), inresed denosine metolism, nd the onomitnt inrese in (29) in solid tumor miroenvironment (Fig. 7, whih is pulished s supporting informtion on the PNAS we site). We lso onfirmed tht ntitumor CD8 T ells used in this study do express the -elevting funtionl A2AR nd A2BR (Fig. 1). To diretly test whether A2AR inhiit ntitumor T ells in vivo,we studied the effets of A2AR gene deletion or ompetitive ntgonists on tumor growth in mie using different CD8 T elldependent ner immunosurveillne nd doptive immunotherpy models. A2AR Defiieny in the Host Leds to Complete Tumor Rejetion. To provide geneti evidene of the role of A2AR in proteting tumors from ntitumor T ells, we ompred the growth of immunogeni CL8-1 melnom (Fig. 2) nd RMA T lymphom (Fig. 3) in C57BL 6-kground A2AR gene-defiient (A2AR / ) nd in WT ontrol C57BL 6 mie. We seleted these well estlished tumor models euse the growth of CL8-1 melnom (3, 31) nd RMA T lymphom (32) is ontrolled y endogenous ntitumor CD8 T ells up to ertin size, nd then the tumor kills 1% of tumor-ering mie (see Supporting Text, whih is pulished s supporting informtion on the PNAS we site). The key role of Conflit of interest sttement: No onflits delred. Arevitions: A2AR, A2A denosine reeptor; A2BR, A2B denosine reeptor. A.O., E.G., F.R., S. Apsov, nd S. Arms ontriuted eqully to this work. To whom orrespondene should e ddressed t: New Englnd Inflmmtion nd Tissue Protetion Institute, Northestern University, 36 Huntington Avenue, 113 Mugr Life Sienes Building, Boston, MA E-mil: m.sitkovsky@neu.edu. 26 y The Ntionl Ademy of Sienes of the USA PNAS August 29, 26 vol. 13 no gi doi pns

2 TCR A2R Inhiition Inhiits Anti-Tumor Effets of T ells Fluoresene Signl (Aritrry Units) (pmol/1 6 ells) Geneti trgeting TCR Norml Tissue Edge of Tumor Center of Tumor No Inhiition No Inhiition Resue of Anti-Tumor Effetor Funtions of T ells CD8 T ells in response to CL8-1 is onfirmed in ontrol experiments (Fig. 8, whih is pulished s supporting informtion on the PNAS we site), where similr elertion of the onset of CL8-1 growth ws oserved in RAG-1 / nd CD8 / mie, ut not in A2R Internl Stndrd Internl Stndrd Internl Stndrd Antgonism ANTAGONIST TCR A2R Retention Time (Minutes) gp33-tcr-tg CTL - A2BR Ado Ado CGS CGS + ZM + ZM (pmol/1 6 ells) A2BR Control CMS4 CTL A2AR NECA CGS Agonist Agonist + ZM Fig. 1. Overview of experimentl strtegy to test the hypothetil mehnism of tumor protetion. () It is ssumed tht denosine nd A2AR, whih inhiit overtive immune ells to protet norml tissues (18), my protet mlignnt tissues from ntitumor T ells. The trnsient or hroni hypoxi in the tumor miroenvironment (19, 2) ould e onduive to umultion of denosine (15, 27), whih then my inhiit ntitumor CD8 T ells y inresing their immunosuppressive intrellulr levels (15 17). Geneti trgeting of A2AR my deinhiit CD8 T ells nd therey filitte their ntitumor effetor funtions, s ws shown in models of T ell-dependent virl nd utoimmune heptitis (18). A similr outome ould e omplished y using A2AR ntgonists, e.g., ZM241,385, whih were shown to prevent denosinetriggered elevtion (26), reverse the denosine-medited inhiition of tivted CD8 T ells (24) in vitro, nd deinhiit tivted immune ells in vivo (18). Evidene for suh tumor-proteting mehnism my lso e interpreted s proof of priniple for the fesiility of novel strtegy of tumor destrution, where the interruption of hypoxi3denosine3a2ar signling in tumors my resue the ntitumor immune response from inhiition in the hostile tumor environment. () Demonstrtion of grdient of inresed levels of extrellulr denosine nd in solid tumor environment using n equilirium mirodilysis proe (see Supporting Text). () Expression of funtionl A2AR nd or A2BR on tumor-speifi CD8 T ells. CMS4 sromspeifi CD8 T ells nd nti-gp33 CD8 T ells were used in the experiments of Figs. 4 nd 11. The levels of A2AR-seletive gonist CGS2168-indued reflet the expression of A2AR, nd the denosine- or 5 -(Nethylroxmido) denosine (NECA)-indued represents the sum of signling y oth A2AR nd A2BR. The differene etween denosine NECA nd CGS2168 provides n indition s to the reltive ontriution of A2BR. Tumor Volume, mm mie e mie Dys of Tumors Growth Dy Dy 3? Dys fter CL8-1 inoultion CD4 / mie, s ompred with WT mie. This finding ws lso onfirmed in WT mie depleted of either CD4 or CD8 T ells y injeting nti-cd8 or nti-cd4 ntiodies (see Supporting Text). Remrkly, in 6% of A2AR / tumor-ering mie, oth CL8-1 (Fig. 2) nd RMA (Fig. 3) tumors hve een ompletely rejeted fter rehing reltively lrge size. The elimintion of tumor resulted in mouse survivl (Figs. 2 nd 3). In ontrst, no tumor rejetion nd no mie survivl ws oserved in prllel ontrols with tumor-inoulted WT mie (Figs. 2 nd 3), whih d % Mie survived % Mie survived Immunogeni CL8-1 P <.5 Wekly Immunogeni B16 (prent of CL8-1 ) Dys fter B16 inoultion CD8+ T ells CD8+ T ells Dy 5 Dy 7 Dy Dys fter tumor inoultion Dy 14 Dy 17 Dy 21 Dy 3 Fig. 2. Geneti defiieny of A2AR my led to omplete rejetion of estlished CL8-1 melnom nd to survivl of tumor-ering mie. () A2AR intivtion y geneti muttion my led to omplete tumor rejetion. The A2AR / mie or WT mie were inoulted s.. with CL8-1 melnom ells. The mouse fes on the grph indite omplete tumor rejetion nd mouse survivl, wheres the ross indites tht the mouse hd to e euthnized ording to n niml re protool when tumors rehed 2 m in dimeter. The question mrk in the lowest grph of tumor rejetion y A2AR / mie indites tht this mouse would likely hve ompletely rejeted tumor nd survived; even though the tumor itself ws eing destroyed y CD8 T ells, the wounded tissue ws 2 m in dimeter, nd mie hd to e euthnized ording to the niml re protool. Shown re representtive results of two experiments. () Geneti evidene tht intivtion of A2AR my led to survivl of mie with inoulted CL8-1 melnom (the sme experiment s in ). () A2AR intivtion y geneti muttion is not suffiient to ensure tumor rejetion nd survivl of mie with inoulted nonimmunogeni B16 melnom. Groups of A2AR / mie or WT mie were inoulted s.. with B16 melnom ells, nd tumor growth ws monitored. No rejetion of B16 hs een oserved in ny mie, nd no differenes in survivl were oserved etween WT C57BL 6 nd A2AR / mie. Shown re representtive results of two experiments. (d) Photogrphs of typil tumor s wound-heling proess during nd fter n immune ttk y nti-cl8-1 CD8 T ells in A2AR / mie. (e) Unusul pperne of mie with CL8-1 melnom tumor, whih grew to lrge size nd then ws rejeted. Two mie with suh phenotype were oserved mong five A2AR / survivors of CL8-1 melnom. IMMUNOLOGY Oht et l. PNAS August 29, 26 vol. 13 no

3 Tumor Volume, mm mie Tumor Volume, mm mie Survivl (%) mie (n = 11) mie (n = 9) P < Dys fter Tumor Inoultion Dys of tumors growth Dys of tumors growth Fig. 3. A2AR intivtion y geneti muttion my led to omplete rejetion of estlished RMA T lymphom y ntitumor CD8 T ells, ensuring survivl of tumor-ering mie. ( nd ) A2AR intivtion y geneti muttion my led to omplete RMA T lymphom tumor rejetion. The A2AR / mie (n 9) or WT mie (n 11) were inoulted s.. with RMA T lymphom ells. Representtive results of two experiments re shown. () A2AR intivtion y geneti muttion my led to survivl of mie with inoulted RMA lymphom. Shown re representtive results of two experiments. do express the tumor-proteting A2AR on their ntitumor CD8 T ells. In n importnt internl ontrol, no tumor rejetion or mouse survivl ws oserved when the prent of CL8-1, the nonimmunogeni B16 melnom, ws inoulted into either WT or A2AR / mie (Fig. 2). The nti-cl8-1 melnom response of CD8 T ells in the A2AR / host ws ompnied y different pperne of tumors nd of tumor-rejeting mie s ompred with WT mie (Fig. 2 d nd e). Wheres the solid, spheril, nd well defined tumors were ontinuously inresing in size in WT mie, the soft, flt, poorly defined tumors in A2AR / mie often showed entrl nerosis, nd, in some mie, their dispperne nd heling were ompnied y hir loss. In ddition, the signs of spontneously resolved utoimmunity were oserved in some A2AR / mie, whih rejeted tumors. As shown in Fig. 2e, these tumor-rejeting mie lost hir round the eyes round dy 3, nd then (dy 7) eme nude -like. On dy 9 the hir ws regrown. These oservtions resemle reports of utoimmunity in melnom-rejeting mie (13) nd melnom ptients who were undergoing immunotherpy with melnom ntigen-speifi T ells (14). Importntly, the defiieny in A2AR did not prevent the estlishment or the erly growth of inoulted tumors; rther, it hs improved the destrution of lrger, developed tumors (Figs. 2 nd 3). In some experiments the CL8-1 tumors strted growing similrly in oth WT nd A2AR / host, nd then the tumor seemed to dispper (round dy 14) in oth WT nd A2AR / mie only to repper in WT mie ut not in A2AR / mie (dt not shown). The erly time ourse of growth nd rejetion of RMA tumors in A2AR / mie, ut not in WT ontrols, is shown in Fig. 9, whih is pulished s supporting informtion on the PNAS we site. The outome desried here depended on the size of the tumor inoulum, whih determines the pility of ntitumor CD8 T ells to ompletely rejet tumors. A smller numer of injeted ells resulted in rejetion of tumors even in WT mie, lthough the rejetion ws sttistilly signifintly elerted in A2AR / mie (Fig. 9). A2AR nd A2BR Antgonists Filitte the Retrdtion of Tumor Growth Medited y Antitumor CD8 T Cells. To test whether the phrmologil inhiition of A2AR would render ntitumor CD8 T ells resistnt to inhiition y tumor-produed denosine (Fig. 1), we studied the effets of ZM241,385 [ ompetitive nd seletive ntgonist of oth A2AR nd A2BR (23, 33, 34)] or of ffeine (1,3,7-trimethylxnthine) [whih t physiologilly relevnt onentrtions preferentilly ntgonizes A2AR (35)]. The effets of ntgonists were tested in models of ner immunosurveillne y endogenous CD8 T ells nd in doptive immunotherpy models using in vivo indued nd ex vivo expnded ntitumor CD8 T ells of defined ntigen speifiity (3, 31, 36 39). Effet of A2 Reeptor Antgonists on Adoptively Trnsferred Antitumor CD8 T Cells. The A2 reeptors ntgonists ZM241,385 nd ffeine were found to enhne the ntitumor effets of CD8 T ells in studies of nti-cms4 srom CD8 T ells in lung metstsis model. Controls onfirmed tht CD8 T ells used re tumor peptide-speifi (dt not shown), nd they do express funtionl A2AR nd A2BR (Fig. 1) euse the denosine-indued inrese of ould e loked y either ZM241,385 or ffeine (Fig. 1, whih is pulished s supporting informtion on the PNAS we site). The omined tretment of mie with doptive trnsfer of CD8 T ells nd ntgonists resulted in sttistilly etter destrution of lung metstsis thn with CD8 T ells lone (Figs. 4 nd nd 1). A2 denosine reeptor ntgonists lso improved the ntitumor tivity of effetor CD8 T ells speifi for the poorly immunogeni tumor LL-LCMV s evidened y strongly enhned CD8 T ell-medited destrution of LL-LCMV tumor nd tumor growth retrdtion fter dministrtion of ffeine (Fig. 11, whih is pulished s supporting informtion on the PNAS we site). Effet of A2 Reeptor Antgonist on Endogenously Developed Antitumor CD8 T Cells. The ntgonists signifintly delyed the onset of rpid growth of CL8-1 melnom, even if injetions of ZM241,385 strted fter tumors rehed reltively lrge size (Fig. 4). No tumor rejetion or mouse survivl ws oserved during the ourse of tretments with ntgonists in ny tested model, lthough oth tested ntgonists did signifintly improve the CD8 T ell-medited dely of the onset of rpid tumor growth. Further improvement of the effets of ntgonists my require ompounds with signifintly extended hlf-lives in vivo, euse the ntgonists used here hve very short hlf-life in mie ( 3 5 min; see Fig. 12, whih is pulished s supporting informtion on the PNAS we gi doi pns Oht et l.

4 % of CD31-mA stined field Numer of metstti nodules > ZM - +ZM No CTL 5 x 15 CTL Numer of metstti nodules P <.5 - +ZM Tumor Volume (mm 3) Cffeine * P <.5 ** P <.1 n = 9 26 % 56 % 9 % n = P <.5 * ** n = 1 - +ZM +Cffeine Control ZM Dys of Tumor Growth Tumor Volume, mm 3 d * P <.1 IFN-γ R -/- Dys fter CL8-1 inoultion Cffeine + - Dy Dy 28 Dy 5 Tumor inoultion (Tumors ~2 mm3) +Antgonist Tumor exmintion * WT P <.1 Control Ado Ado+ZM Ado+ Cffeine LT- β TNF-α IFN-γ TGF-β1 MIF L32 GAPDH Angiogenesis (CD31+) Apoptoti Tumor Cells Vitl Cell Stining No CTL 1 x 16 CTL Fig. 4. Tretment of mie with A2AR A2BR ntgonist enhned the destrution of estlished tumors y tumor ntigen-speifi CD8 T ells. () The ntgonist ZM241,385 improved destrution of CMS4 lung metstsis y ntitumor CD8 T ells. () Enhnement of destrution of CMS4 lung metstsis y doptively trnsferred CD8 T ells in mie tht onsumed ffeine in drinking wter. Bsed on the ggregtion of the dt points in the ontrol only tumor nd ffeine lone groups, n ritrry utoff vlue ( more thn 2% derese in the numer of metstti nodules) ws ssigned to illustrte potentil therpeuti effiy threshold (shown s the perentge of mie with deresed numer of metstses) for those groups of mie tht were treted with oth CD8 T ells nd A2 reeptor ntgonists. () ZM241,385, n ntgonist of A2AR nd A2BR, enhnes CD8 T ell-medited ntitumor immune response in mie with estlished s.. solid CL8-1 melnom. Dt represent men SEM. site). Of promise, ffeine hs muh longer hlf-life in vivo in humns (4). A2AR A2BR Antgonists My Deinhiit the Prodution of IFN- y Antitumor T Cells in the Tumor Miroenvironment. The neovsulriztion-inhiiting properties of IFN- were shown to e ruil for ntitumor tion of T ells in vivo (8), nd etter tumor destrution in mie with intivted A2AR A2BR (Figs. 2 4) ould e t lest prtilly explined y the relese of CD8 T ells from A2ARmedited inhiition of IFN- prodution in the denosine-rih tumor miroenvironment (Fig. 1). The elertion of CL8-1 melnom growth in IFN- reeptor gene-defiient mie (Fig. 5) nd oservtions of pity of A2AR A2BR-medited signling to inhiit the T ell reeptor-triggered up-regultion of IFN- mrn, lymphotoxin- mrna, nd TNF- mrna in ntitumor T ells (Fig. 5) re onsistent with this hypothesis. The inresed levels of IFN- ner or within tumors in mie with intivted A2AR A2BR were, in turn, expeted to inhiit neovsulriztion nd therey enhne tumor ell deth. This expettion ws onfirmed y oservtions of ntgonist (ffeine)- medited inhiition of tumor neovsulriztion nd y inresed poptosis of tumor ells (Fig. 5). Signifintly more lood vessels nd sprouting pillries were oserved in tumors from untreted mie thn in tumors from ffeine-treted mie (Fig. 5 nd d). The lrge estlished lood vessel visile in the enter of Fig. 5 Lower Right (red) reflets the ft tht tretment with ffeine strted t Fig. 5. Tretment with ffeine inhiits neovsulriztion nd inreses poptosis of CL8-1 melnom in mie. () Geneti evidene tht growth of inoulted CL8-1 melnom ells depends on funtionl IFN- reeptors (IFN- R). The IFN- R / or WT C57BL 6 mie (8 1 mie per group) were inoulted s.. with CL8-1 melnom ells. All dt represent men SEM. () Extrellulr denosine inhiits up-regulted IFN- gene trnsription in tivted ntitumor T ells, nd this inhiition is prevented y denosine reeptor ntgonists ZM241,385 (ZM) nd ffeine. mrna levels were nlyzed y n RNse protetion ssy. () Immunohistohemil demonstrtion of inresed tumor destrution nd inhiition of ngiogenesis in tumors in mie treted with ffeine. Mie were inoulted with CL8-1 nd treted with ffeine. CL8-1 melnoms growing in C57BL 6 mie were removed t dy 5 from ontrol (Lower Left) nd ffeine-treted (Lower Right) mie. (d) The degree of ngiogenesis ws determined s the perentge of CD31 stining to the totl field. The y xis (perentge of fields positive for CD31) reflets the perentge of re oupied y lood vessels in omprison to the totl tumor re. The summry of mesurements of two tumors per group is presented. Methods for the RNse protetion ssy nd immunohistohemistry re provided in Supporting Text. dy 28, when the tumor ws lredy lrge ( 2 mm 3 ). In ddition, fewer surrounding new smll vessels or endothelil ells were oserved in representtive field. Tumor ells (lue) round this vessel re re norml nd vile; however, frther wy from this vessel re there re lmost no new sprouting pillries, nd there re numerous poptoti tumor ells (Fig. 5 Lower Right, green). Antitumor Effets of A2AR Trgeting Are CD8 T Cell-Dependent. The geneti deletion or phrmologil ntgonism of A2AR resulted in omplete rejetion or growth retrdtion of tumors only if they were immunogeni nd generted ntitumor CD8 T ells in n immunoompetent host. The omplete tumor rejetion in n A2AR / host (Figs. 2 nd 3) or the dely of rpid tumor growth in WT mie y ntgonists (Fig. 4) ws due to ugmenttion of effets of CD8 T ells rther thn to effets on some other ells IMMUNOLOGY Oht et l. PNAS August 29, 26 vol. 13 no

5 Numer of metstti nodules CTL + ontrol sirna p =.116 * CTL + A2AR/A2BR sirna 2 n = Dys fter Tumor Inoultion tht re unrelted to CD8 T ell funtions. This finding is supported y severl lines of evidene. In one of the ontrols we ompred the genetilly engineered immunogeni CL8-1 melnom with the prent ell line, B16 melnom. The CL8-1 melnom ws estlished from nonimmunogeni B16 melnom y inresing expression of H-2K MHC lss I moleules to inrese CL8-1 immunogeniity (3, 41). The growth of CL8-1 in WT mie ws delyed ompred with growth of B16 ells euse of nti- CL8-1 CD8 T ells (Fig. 2 nd ). In ddition, mny CD8 T ells hve een oserved mong infiltrting lymphoytes in CL8-1 tumors, wheres no infiltrtion of ntimelnom T ells ws oserved in tumors developed fter inoultion of B16 melnom (3, 41). If the rejetion of CL8-1 tumor in A2AR / mie (Fig. 2) ws due to A2AR defiieny in ells other thn CD8 T ells or in ells tht re unrelted to CD8 T ell funtions, then omplete B16 melnom rejetion would e lso expeted in A2AR / mie, euse B16 differ from CL8-1 only in the ility to generte ntitumor CD8 T ells. The opposite ws found. B16 melnom ws growing eqully fst in oth WT nd A2AR / mie, nd none of A2AR / mie survived B16 inoultion (Fig. 2). It is shown tht ZM241,385 (or ffeine; dt not shown) signifintly delyed CL8-1 growth in WT mie, whih developed nti-cl8-1 CD8 T ells (Fig. 4), ut did not ffet tumor growth in ontrol group of nude mie with no nti-cl8-1 CD8 T ells (dt not shown). These ontrol experiments with immunodefiient mie support the view tht ntitumor effets of ntgonist require CD8 T ells. In ddition, the redution in tumor growth y ntgonists in doptive immunotherpy models hs een oserved when ntgonists were given in omintion with nti-cd8 T ells, ut not lone (Fig. 4). Finlly, we found improvement of doptive immunotherpy when A2AR nd A2BR expression in ntitumor CD8 T ells ws speifilly loked y sirna pretretment efore their doptive trnsfer (Fig. 6). The pretretment of ntitumor T ells with A2AR nd A2BR sirna redued CMS4 lung metstsis from 83 to 49 (P.116) nd lso improved survivl of RMA-inoulted mie (ontrol, 2 12; A2AR A2BR, 7 12; P.5), suggesting tht, indeed, the A2AR expressed on ntitumor CD8 T ells ply n inhiitory role during ttk on tumor. Tken together, these ontrols lso provide geneti evidene for the ritil role of A2AR in regultion of effetor funtions of ntigen-speifi CD8 T ells in vivo nd, possily, in ontrol of T ell-medited utoimmunity. Survivl (%) Srmle A2A/A2B p <.5 Fig. 6. Suppression of A2AR A2BR expression in T ells improved doptive immunotherpy. () sirna ginst A2AR nd A2BR ws trnsfeted into nti-cms4 CD8 T ells efore doptive trnsfer (see Supporting Text). Lung metstsis ws exmined 11 dys fter injetion of T ells (1 1 6 ells per mouse) s desried in Methods. T ell-speifi knokdown of A2AR A2BR filitted inhiition of lung metstsis y ntitumor T ells (*, P.116 y ANOVA). () Survivl of RMA tumor-ering WT mie ws improved y the trnsfer of A2AR A2BR sirna-pretreted T ells (P.5 y log-rnk test). Anti-RMA T ells were otined from WT mie immunized with RMA tumor ells. Unseprted spleen nd lymph node ells (5 1 7 ) were trnsfeted with either ontrol or A2AR nd A2BR sirna nd injeted into tumor-ering mie 1 dys fter inoultion of RMA ells (2 1 5 ). Disussion The dt presented suggest tht ntitumor T ells re inhiited y tumor-produed extrellulr denosine euse of the A2ARtriggered elevtion of intrellulr levels of. Inrese of intrellulr indues protein kinse A-medited phosphoryltion nd tivtion of COOH-terminl Sr kinse (Csk). Csk then my phosphorylte nd inhiit Lk, whih, in turn, diminishes TCR signling nd IFN- prodution (22). Reent studies of CD26 (denosine deminse) (42) support our hypothesis tht derese in denosine signling is immunoenhning. Among the CD8 T ell responses tht ould e inhiited y A2AR A2BR re prolifertion (16), lethl hit delivery (12, 43), Fs lignd up-regultion (24), nd prodution of ytokines suh s IFN- (17, 44). IFN- hs een demonstrted to ply n importnt role in ntitumor effets of CD8 T ells euse of inhiition of tumor ngiogenesis (8). The dt presented suggest omined therpy in whih the ntineovsulriztion pproh (45) ould e omplemented y the A2AR A2BR-trgeting strtegy desried here to deinhiit nd therefore enhne IFN- prodution y ntitumor T ells. We interpret our dt s (i) evidene for the mjor role of A2AR in nerous tissue protetion from ntitumor T ells nd (ii) demonstrtion of the fesiility of the strtegy to enhne the immune-medited tumor destrution y geneti deletion or phrmologil ntgonism of A2AR nd possily A2BR. These oservtions lso suggest future studies to estlish whether A2AR my e involved s primry trigger (17, 44) of expression of other tumor-proteting immunosuppressive moleules (37). The proposed strtegy to ountert immunosuppressive signling y denosine ner solid tumors is omplementry to other pprohes direted to improve the development nd funtion of ntitumor T ells (4, 1, 11, 14). The limittion of this pproh is tht it is pplile only to immunogeni tumors (Fig. 2). In ddition, this strtegy so fr resulted in omplete tumor rejetion in only 6% of mie with genetilly trgeted A2AR; ntgonists used signifint, ut not omplete, tumor growth retrdtion. The tumors espe from CD8 T ells oserved in 4% of A2AR / mie (Figs. 2 nd 3) ws not due to the loss of ntigen-presenting moleules (dt not shown) ut ould e explined y the tumor-proteting A2BR, whih ould e expressed in the sene of A2AR on A2AR / CD8 T ells (Fig. 1 nd Supporting Text). Ativted A2BR my inhiit CD8 T ells in the denosine-rih tumor miroenvironment, nd oth A2AR nd A2BR re expressed on some ntitumor CD8 T ells (Fig. 1). It remins to e determined whether A2BR my ount for CD8 T ell filure to destroy tumors in the 4% A2AR / mie tht filed to rejet tumors (Figs. 2 nd 3) nd whether even etter tumor rejetion y CD8 T ells ould e omplished y intivtion of oth A2AR nd A2BR in ntitumor T ells. It is lso importnt to refully onsider the known rdiovsulr nd neurologil (35) [s well s possile proinflmmtory (17, 18, 44)] effets of A2AR nd A2BR ntgonists, inluding ffeine, s well s the effet inversion oserved when ompring ute versus hroni dministrtion of ffeine (17, 35, 46). Further studies of spontneous immunogeni tumors my determine whether A2 denosine reeptors lso ount for the filure of T ells to destroy spontneously rising tumors t erly stges. We propose to trget the hypoxi3extrellulr denosine3a2ar A2BR signling pthwy s ner immunotherpy strtegy to prevent the inhiition of ntitumor T ells in the tumor miroenvironment. The sme strtegy my prevent the premture termintion of immune response nd improve the vine-indued development of ntitumor nd ntivirl T ells. Methods Indution of Tumor-Speifi Cytotoxi T Cells. Anti-CMS4 srom CD8 T ells were prepred s desried previously (38). CMS gi doi pns Oht et l.

6 speifi CD8 T ells (1 1 6 ) were expnded y stimulting with irrdited CMS4 ells in the presene of BALB spleen ells nd IL-2. Anti-LCMV gp33 CD8 T ells were prepred from LCMV peptide-speifi 318 T ell reeptor trnsgeni mie s desried in Fig. 11. umultion in these ells ws mesured s desried in Supporting Text (26). Studies of Rejetion of Tumors y Endogenous CD8 T Cells in A2AR / Mie. CL8-1 melnom lone ws isolted fter trnsfetion of B16BL6 melnom lone BL6-8 with the H-2K gene to inrese immunogeniity (3, 41). No progressively growing tumors were deteted fter s.. inoultion of to CL8-1 ells in immunoompetent C57BL 6 mie. The ntitumor resistne in C57BL 6 mie n e overome y inoulting higher dose of CL8-1 (e.g., ) ells. With the sme reson, RMA T lymphom ells tht express the H-2K moleule were inoulted t ells to overome ntitumor resistne in C57BL 6 mie. Tumor ells were wshed nd suspended in PBS nd injeted s.. (1 l per mouse). Perpendiulr tumor dimeters were mesured nd tumor volumes were lulted ording to the formul 2.52, where is the smller nd is the lrger tumor dimeter (31). The experiment ws terminted when tumors rehed 2. m in dimeter or eme ulerted. Animl experiments were performed ording to the protool pproved y the institutionl niml re nd use ommittees of the University of Pittsurgh nd the Ntionl Institute of Allergy nd Infetious Diseses. Effets of Reeptor Antgonists on s.. Tumor Growth. CL8-1 ells ( ) were injeted s.. into C57BL 6 mie. When tumors rehed 8 mm in dimeter mie were divided rndomly into groups of 1 mie with similr tumor size ( 2 mm 3 ; CL8-1 tumors rehed this size round dy 28 in C57BL 6 mie). ZM241,385 (Toris, Ellisville, MO) tretment ws done y dily i.p. injetions of.2 mg per mouse per dy. Cffeine (Sigm, St. Louis, MO) ws given s drinking wter (.1% wt vol). The effets of these ntgonists in vivo serve only s n indition tht either individul A2AR or A2BR (23, 4) or oth re involved in down-regultion of ntitumor CD8 T ells in vivo. In ontrol ssys of ex vivo serum from ntgonist-treted mie (Fig. 12 nd dt not shown) we onfirmed tht, during tretment, the initil in vivo levels of ZM241,385 nd ffeine in serum re suffiiently high to prevent (ntgonize) the denosine3a2ar-indued umultion in ells. Effets of Reeptor Antgonists on Lung Metstsis. CMS4 srom ells were injeted i.v. into the lterl til vein of BALB mie ( ells in 1 l totl volume). Ten dys lter (4 dys fter in vitro stimultion), therpeuti CD8 T ells were injeted i.v (38). Tretment with ZM241,385 or ffeine strted on the dy of doptive trnsfer. One to 2 weeks fter doptive trnsfer, the lungs were removed nd proessed for enumertion of metstsis. Lungs were inflted with 15% solution of indi ink, reseted, nd fixed in Fekete s solution. The numer of pulmonry nodules ws enumerted under disseting mirosope in lind tests. Sttistis. The sttistil differenes etween survivl of mie in WT versus A2AR / mie were lulted ording to the log-rnk test. The sttistil differenes in the size of tumors were lulted y using the Student t test. For evlution of the lung metstsis experiment, the more relevnt Mnn Whitney test or ANOVA ws used, euse the lung metstsis my not e normlly distriuted. We thnk Drs. Willim Pul, Ronld Germin, Mihel Lenrdo, nd Ronld Shwrtz (Ntionl Institute of Allergy nd Infetious Diseses) nd Steven Rosenerg nd Alfred Singer (Ntionl Cner Institute, Ntionl Institutes of Helth) for support, disussions, nd help nd Dr. Jne Kinsel nd Brend Mrshll for help in preprtion of the mnusript. M.S. ws supported y n Intrmurl Ntionl Institutes of Helth Progrm (Ntionl Institute of Allergy nd Infetious Diseses) nd then y Ntionl Institutes of Helth Extrmurl Grnt 1 R1 CA NIH. 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