Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity

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1 2 Nture Pulishing Group Inhiiting Stt3 signling in the hemtopoieti system eliits multiomponent ntitumor immunity Mrin Kortylewski 1,4, Miej Kujwski 1,4, Tinhong Wng 2, Sheng Wei 1, Shumin Zhng 1, Shri Pilon-Thoms 1, Guilin Niu 1, Heidi Ky 3, Jmes Mulé 1, Willim G Kerr 1, Rihrd Jove 1,4, Drew Prdoll 2 &HuYu 1,4 The immune system n t s n extrinsi suppressor of tumors. Therefore, tumor progression depends in prt on mehnisms tht downmodulte intrinsi immune surveillne. Identifying these inhiitory pthwys my provide promising trgets to enhne ntitumor immunity. Here, we show tht Stt3 is onstitutively tivted in diverse tumor-infiltrting immune ells, nd lting Stt3 in hemtopoieti ells triggers n intrinsi immune-surveillne system tht inhiits tumor growth nd metstsis. We oserved mrkedly enhned funtion of dendriti ells, T ells, nturl killer (NK) ells nd neutrophils in tumor-ering mie with hemtopoieti ells, nd showed tht tumor regression requires immune ells. Trgeting Stt3 with smll-moleule drug indues T ell nd NK ell dependent growth inhiition of estlished tumors otherwise resistnt to diret killing y the inhiitor. Our findings show tht Stt3 signling restrins nturl tumor immune surveillne nd tht inhiiting hemtopoieti Stt3 in tumor-ering hosts eliits multiomponent therpeuti ntitumor immunity. Reent studies suggest ruil role of immune ells in suppressing tumor emergene nd sulpting immunogeni phenotypes of tumors (reviewed in ref. 1). It hs lso een shown tht elements of the tumor miroenvironment n promote immune tolerne, t lest in prt, y one mrrow derived dendriti ells (DCs) 2. Indeed, lthough the requirement for DCs in generting T-ell immunity is well estlished 6, DCs re immture nd dysfuntionl oth in individuls with ner nd in tumor-ering nimls 7,8. Evidene hs een umulting tht onogeni signling ontriutes to tumor immune evsion y inhiiting DC mturtion For exmple, we reently showed tht signl trnsduer nd tivtor of trnsription 3 (Stt3), whih is frequently tivted in diverse ners y ommon onogeni pthwys, medites immune suppression y inhiiting expression of proinflmmtory ytokines nd hemokines neessry for DC tivtion 11.Moreover, tumor Stt3 tivity promotes prodution of multiple ftors, mong them vsulr endothelil growth ftor (VEGF) nd interleukin (IL)- 1, tht tivte Stt3 signling nd inhiit funtionl DC mturtion in ulture 11. Inhiition of Stt3 tivity in DC progenitors hs lso een shown to redue umultion of immture DCs y tumorderived ftors in vitro 12. Furthermore, mrophges devoid of Stt3 signling do not effiiently indue T-ell nergy, supporting negtive regultory role of Stt3 in ntigen-presenting ells 13. These findings suggest tht Stt3 inhiition ould potentilly restore DC funtions in tumor-ering hosts. But reent study showed tht DC numer is mrkedly redued in mie with ongenitl hemtopoieti ells 14, implying tht trgeting Stt3 in hemtopoieti ells in vivo might negtively ffet DC funtion nd, therefore, ntitumor immune responses. We sought to diretly determine the role of hemtopoieti Stt3 signling on immune surveillne of tumors nd investigte how inhiiting Stt3 in host immune ells in vivo might ffet the funtion of vrious ell susets nd their ility to engge in ntitumor immune responses. To ypss the requirement of Stt3 during emryogenesis 1 nd to model therpeutilly relevnt setting for individuls with ner, we tested the effets of indued Stt3 ltion in hemtopoieti ells in dult mie efore nd fter tumor estlishment. Our dt show tht Stt3 is onstitutively tivted in tumor-infiltrting immune ells. Altion of Stt3 in hemtopoieti ells in dult nimls does not redue DC numers, ut rther enhnes DC mturtion in tumor-ering mie. T ells, nturl killer (NK) ells nd neutrophils in tumor-ering mie with hemtopoieti system show enhned ntitumor tivity. Alting Stt3 in multiple hemtopoieti elements ultimtely results in immune ell medited ntitumor responses. Bloking Stt3 signling with smll-moleule drug in vivo indues immune-medited ntitumor effets. These findings suggest tht Stt3 is key negtive regultor of tumor immune surveillne nd tht it n e mnipulted to ugment innte nd dptive immune responsiveness to tumors. RESULTS Effets of Stt3 ltion in DCs in dult mie Using the Mx1-Cre-loxP system, ltion of Stt3 in hemtopoieti ells is induile in dult mie, overoming the requirement of Stt3 1 H. Lee Moffitt Cner Center nd Reserh Institute, 1292 Mgnoli Drive, Tmp, Florid 33612, USA. 2 Sidney Kimmel Cner Center, Johns Hopkins University Shool of Mediine, 16 Orlens Street, Bltimore, Mrylnd 21231, USA. 3 College of Puli Helth, University of South Florid, 1321 Brue B. Downs Boulevrd, Tmp, Florid 33612, USA. 4 Present ddress: City of Hope Ntionl Medil Center, 1 Durte Rod, Durte, Cliforni 911, USA. Correspondene should e ddressed to D.P. (dmprdol@jhmi.edu) or H.Y. (hyu@oh.org). Reeived 28 June; epted 13 Otoer; pulished online 2 Novemer 2; doi:1.138/nm VOLUME 11 [ NUMBER 12 [ DECEMBER 2 NATURE MEDICINE

2 2 Nture Pulishing Group Reltive ell numer PCR + IL MW mrker phospho-stt3 Stt3 loxp (full length) Stt3 (trunted) + IL EMSA (SIE) EMSA (MGF) GM-CSF IL-1 Stt3:3 Stt1:3 Stt1:1 Stt: in emryogenesis 16,17. Injetion of poly(i:c) into mie rrying the Mx1-Cre trnsgene (Cre) nd Stt3 lleles flnked y loxp sites (Stt3 loxp/loxp ) led to effetive Stt3 deletion in one mrrow ells, s determined y PCR-sed genotyping ssy 18 (Fig. 1), further onfirmed y lk of Stt3-DNA inding nd phosphorylted Stt3 even fter IL-1 stimultion (Fig. 1,). Results from flow ytometri nlysis of spleni DCs showed tht Stt3 ltion in hemtopoieti ells of dult mie did not redue the numer of CD11 + DCs over 4-week period (Fig. 2). Beuse poly(i:c) is Toll-like reeptor 3 lignd tht stimultes interferon (IFN)- prodution nd tivtes DCs, there exists the potentil tht inflmmtory responses ssoited with poly(i:c) tretment might osure the funtionl effets resulting from Stt3 deletion. The effets of poly(i:c) on DCs nd other immune ells re onfined to the 48 h of tretment (Supplementry Fig. 1 online). Thus, the immunologi nd tumor protetion nlyses in this study were performed Z d fter Figure 2 Effets of Stt3 ltion on DCs. () Totl numer of spleni DCs from mie is not redued s ompred to in tumor ering mie. Top, representtive nlysis of totl splenoytes y flow ytometry. Shown re the perentges of CD11 + nd CD11 + MHC II + low or high susets. Bottom, dt from FACS re presented s mens ± s.d.; n ¼. () IL-12 ELISA of CD11 + DCs purified from spleens of tumor-ering nd mie fter lipopolyshride stimultion (left pnel). Prolifertion of OTII CD4 + Tells stimulted y nd DCs ± OVA (right pnel). Dt re shown s mens ± s.d.; n ¼ 3. () Stt3 is onstitutively tivted in tumor-infiltrting DCs s determined y intrellulr stining with ntiody to phosphorylted Stt3 (phospho-stt3), followed y flow ytometry. Representtive results of three independent experiments with three to four mie per group re shown. (d) Perentges of MHCII hi CD8 hi or MHCII hi CD86 hi tumorinfiltrting CD11 + DCs re higher in mie. Shown re representtive results of FACS nlysis from one of three independent experiments with four to six mie per group. CD % ompletion of poly(i:c) tretment in order to fous speifilly on the effets of Stt3 ltion. We next evluted how Stt3 deletion might ffet DC tivtion. Although spleni DCs from tumor-ering nd hemtopoietilly Stt3-lted ( ) mie did not produe IL-12 spontneously (dt not shown), those from mie expressed higher levels of IL-12 upon ex vivo stimultion with lipopolyshride (Fig. 2). Moreover, spleni DCs isolted from mie ering lrger tumors showed enhned ility to present ntigen nd tivte ntigen-speifi CD4 + T ells ex vivo (Fig. 2). Reent work hs foused ttention on tumor-infiltrting immune ells s more diret mens of ssessing effets of tumor on its immunologi miroenvironment We therefore isolted tumor-infiltrting DCs from ontrol nd mie nd nlyzed expression of relevnt DC tivtion moleules. Flow ytometri nlysis fter stining with ntiody speifi for phosphorylted Stt3 showed tht Stt3 is % 3.9% 2.% MHC lss ll MHC lss ll Perent of totl splenoytes Reltive ell numer Figure 1 Induing Stt3 ltion in hemtopoieti ells of dult mie. () PCR nlysis of genomi DNA from one mrrow fter poly(i:c) tretment with primer sets tht distinguish full-length Stt3 loxp nd Stt3- deleted lleles. () EMSA onfirmed lk of Stt3-DNA inding tivity in the one mrrow ells. SIE is DNA proe for deteting Stt3 nd Stt1 DNA-inding tivity, wheres MGF detets Stt-DNA inding. Positions of Stt homo- nd heterodimers re indited. () Intrellulr flow ytometri nlysis using n ntiody to detet tyrosine-phosphorylted Stt3 (phospho-stt3) shows sene of tivted Stt3 in DCs. CD11 + DCs purified from spleens of nd mie were stimulted with IL-1, followed y intrellulr stining with phospho- Stt3 speifi ntiody. FL4-H: FL4 CD11-APC CD CD11 + MHCII + n = n = Spleen (no tumor) Spleen (with tumor) Tumor d MHCII MHCII % CD8 CD IL-12 (pg/ml) 1,4 12 1,2 1, % [ 3 H] inorportion ( 1 3 ounts) OVA + OVA % % phospho-stt CD86 CD86 NATURE MEDICINE VOLUME 11 [ NUMBER 12 [ DECEMBER 2 131

3 2 Nture Pulishing Group Figure 3 Role of Stt3 signling in neutrophils nd NK ells. () Numer of neutrophils from mie is higher, nd neutrophils hve stronger ytotoxiity thn those from mie. FACS nlysis of neutrophils (Gr1 + CD11 + )from nd mie hllenged with B16 tumor (left). Shown re the mens ± s.d.; n ¼. P o.1. 1 Cr-relese ssy with neutrophils: FgR + mouse mstoytom (P81) ells re used s trgets (right). Shown re representtive results from one of three independent experiments done in triplites; mens ± SD. Two mie were used for eh experiment. () NK ells from tumor-ering mie showed enhned ytotoxiity. Spleni NK ells from nd mie with nd without B16 tumor s in were nlyzed y FACS using CD49-speifi ntiody (left). Shown re the mens ± s.d.; n ¼. P o.1. NK ytotoxiity ws ssessed in 1 Cr-relese ssy using YAC-1 ells s trgets (right). Shown re the representtive results from one of three independent experiments done in triplite s in. () Tumorinfiltrting NK ells (CD49 + ) nd grnuloytes (Gr1 + ) from wild-type mie showed inresed phospho-stt3 ompred with their spleni ounterprts. Shown re FACS nlyses fter intrellulr stining with ntiody to phospho-stt3. The dt presented were onfirmed y two dditionl experiments. onstitutively tivted in tumor-infiltrting DCs (Fig. 2). Furthermore, exmintion of tumor-infiltrting DCs showed tht expression of mjor histoomptiility omplex (MHC) lss II, CD8 nd CD86 is onsiderly inresed in mie when ompred to those from mie (Fig. 2d). These results support the notion tht Stt3 tivtion is n importnt ontriutor to the impired tivtion stte of tumor-infiltrting DCs. We further nlyzed susets of tumor-infiltrting DCs in oth nd mie. Tumorinfiltrting DCs were essentilly ll CD8 in oth nd mie (Supplementry Fig. 2 online). The perentge of B22 + plsmytoid DCs ws higher in tumor thn in spleen, nd derese in tumor-infiltrting B22 + plsmytoid DCs in mie ws deteted (Supplementry Fig. 2 online). Notly, plsmytoid DCs re thought to e involved in tolerogeni funtions through genertion of regultory T ells 24. Stt3 signling nd ntitumor innte immunity Innte immunity hs n tive role in immune surveillne s well s indued ntitumor effets 1,2,26. Beuse grnuloytes n ontriute to tumor-ell killing nd tumor regression 27,28, we investigted how Stt3 defiieny might ffet their funtion. In greement with reent study 17, indution of Stt3 deletion onsiderly inresed the numer of spleni grnuloyti linege ells (Fig. 3). Anlysis of neutrophil ytolyti tivity in 1 Cr-relese ssy using FgR + mouse mstoytom (P81) ells s trgets indited tht redued Stt3 signling in neutrophils enhnes their ytolyti tivity ginst trget tumor ells, regrdless of whether they were isolted from tumor-free or tumor-ering mie (Fig. 3). As NK ells re importnt ytolyti effetors ginst tumors 29,we investigted whether loking Stt3 in hemtopoieti ells might ffet NK-ell numers nd their ility to kill trget ells. Immunostining splenoytes from ontrol or B16 tumor-ering mie using pn-nk mrker, CD49, indited no signifint hnge in the numer of NK ells etween nd hemtopoietilly Stt3-lted mie (Fig. 3). But, in the presene of tumor, NK-ell numers were deresed y out 2% in oth groups. We ssessed spleni NKell ytolyti tivity using 1 Cr-leled YAC-1 ells s trgets. The ytolyti tivity of NK ells isolted from tumor-free mie did not differ etween poly(i:c)-treted Stt3 loxp/loxp nd Mx1-Cre- Stt3 loxp/loxp mie. In ontrst, fter tumor hllenge, NK ells from mie showed enhned ytolyti tivity (Fig. 3). Anlysis of Perent of totl splenoytes Reltive ell numer Perent of totl splenoytes Tumor Tumor + + n = Stt3 6 +/+ + tumor + tumor n = Perent of ytotoxiity Perent of ytotoxiity CD phospho-stt3 12:1 2:1 :1 1:1 + tumor + tumor 12:1 2:1 :1 1:1 Gr1 + Spleen (no tumor) Spleen (with tumor) Tumor phosphorylted Stt3 y flow ytometry showed tht Stt3 ws lso onstitutively tivted in tumor-infiltrting NK ells nd neutrophils (Fig. 3). Mny tumor-ssoited ftors, inluding IL-1, VEGF nd IL-6, re tivtors of Stt3, nd IL-1 is undntly produed y mrophges in mie ering MB49 tumors 3. Flow ytometry nd eletrophoreti moility shift ssy (EMSA) indited tht IL-1 tivtes Stt3 in NK ells (CD49 + CD3 )nd grnuloytes (Gr1 + ; Supplementry Fig. 3 online). Furthermore, our dt showed tht FsL expression ws inresed in Gr1 + CD11 + neutrophils (Supplementry Fig. 4 online). Upregultion of FsL hs een ssoited with inresed ytotoxiity of neutrophils, nd n inhiitory role of Stt3 on FsL expression in tumor ells hs een previously reported 31. The findings of inresed funtion of purified NK ells nd neutrophils from tumor-ering mie together with n inrese in Stt3 tivity diretly within these popultions in tumor indites tht the role of Stt3 signling in downregulting funtion of these ell types is t lest, in prt, ell intrinsi. Given the omplex interellulr intertions tht our in vivo mong different immune-ell popultions, mplifying ell-extrinsi ffets re lso possiility. T-ell responses in hemtopoietilly mie Although ltion of Stt3 lleles in one mrrow ells led to redution of solute numers of T ells in tumor-free mie s previously reported 17, we oserved no differene in the numers of 1316 VOLUME 11 [ NUMBER 12 [ DECEMBER 2 NATURE MEDICINE

4 2 Nture Pulishing Group Perent of totl splenoytes 3 n = n = Foxp3 7.7% Tumor + IFN-γ + (dots/ 1 ells) T ells in the tumor-ering versus mie (Fig. 4). To ddress whether T ells isolted from mie ould mount tumor ntigen speifi immune responses, we treted Stt3 loxp/loxp nd Mx1-Cre-Stt3 loxp/loxp mie with poly(i:c), followed y B16 tumor hllenge. Two weeks fter tumor hllenge, we nlyzed splenoytes for T-ell response to p1e, n endogenous retrovirl gene produt of the B16 tumor. IFN-g ELISPOT ssy indited tht T ells from mie mounted stronger responses ginst tumor ntigen thn their ounterprts (Fig. 4). Beuse the ility of T ells to infiltrte tumors is onsidered ruil for indution of tumor regression 32,33, we determined whether T ells from mie ould effiiently infiltrte tumors. Immunohistohemil stining of B16 nd MB49 mouse tumors showed onsiderly higher infiltrtion y T lymphoytes in tumor tissues from mie (Fig. 4). As erdition of tumors, inluding B16 tumors, is thought to e inhiited y T regultory (T reg ) ells 23,34,we exmined whether Stt3 defiieny in hemtopoieti ells might ffet tumor-infiltrting T reg ells. Indeed, the proportions of tumorinfiltrting CD4 + T ells expressing T reg mrkers (CD2, Foxp3 nd Lg-3) in mie were onsiderly redued (Fig. 4d). Chnges in tumor-infiltrting T reg ells might e the result of Stt3 ltion in T ells or indiret effets influened y Stt3 signling in other immune ells. Although PCR nlyses indite tht Stt3 is deleted in one mrrow ells from poly(i:c)-treted Mx1-Cre-Stt3 loxp/loxp mie, Stt3 is not effiiently lted in their thymus (dt not shown). Stt3 in hemtopoieti ells nd ntitumor immunity The ility to indue hemtopoieti Stt3 ltion in our system llows us to determine how Stt3 signling in hemtopoieti ells ultimtely ffets tumor growth independent of Stt3 tivity within tumor ells. We first exmined whether lting Stt3 in hemtopoieti ells might ffet tumor estlishment. Alting Stt3 lleles in hemtopoieti ells efore suutneous tumor hllenge signifintly inhiited B16(F1) tumor growth (Fig. ). Some of the MB49 tumors initilly grew in the Stt3-lted mie, ut they eventully d CD2 Lg % 1.4% 26.2% % % 1.1 CD Figure 4 Chrteriztion of T ells from tumor ering mie with hemtopoieti system. () Perentge of T ells (CD3 + ) in spleens of tumor-free nd B16 tumor-ering mie with nd without Stt3 in their hemtopoieti system s shown y flow ytometry. Shown re the mens ± s.d.; n ¼, P o.1. () T ells from B16 tumor-ering mie with hemtopoieti ells were le to mount stronger responses ginst n endogenous B16 tumor ntigen thn their ounterprts, s ssessed y IFN-g ELISPOT. Dt shown re men numers of p1e-speifi IFN-g produing spots from seven to nine seprte mie per group nlyzed individully. P ¼.92. () T ells in mie with hemtopoieti ells infiltrte tumors more effiiently. Immunohistohemil nlysis of B16 (top) nd MB49 (ottom) tumor tissue setions prepred from nd mie. Red indites CD8 stining; lue indites CD4 stining. (d) The perentges of tumorinfiltrting T reg ells from mie re redued, s shown in B16 tumors. The surfe expression of CD2 nd Lg-3 or the intrellulr levels of Foxp3 ws evluted in tumor-infiltrting CD4 + T ells y flow ytometry. Shown re perentges of doule-positive ells, representtive of three independent experiments. ll regressed (Fig. ). Beuse essentilly ll of the immune effets of poly(i:c) tretment hve ompletely susided y 48 h fter tretment (Supplementry Fig. 1 online), we performed tumor hllenge experiments d fter the lst poly(i:c) tretment, nd ll ontrol groups inluded poly(i:c) injetion to minimize potentil ntitumor effets resulting from poly(i:c) itself. Deletion of Stt3 in the Mx1-Cre-Stt3 loxp/loxp lso ours, leit to lesser degree, in some tissues other thn those of hemtopoieti linege 16,3. To prove tht the oserved ntitumor effets were ontriuted y Stt3 ltion in the hemtopoieti omprtment, we reonstituted lethlly irrdited wild-type reipients with or one mrrow. We hllenged mie with engrfted or one mrrow with MB49 tumors 2 months fter one mrrow trnsplnt (Fig. ). We oserved growth inhiition followed y omplete rejetion of tumors in mie with reonstituted one mrrow, wheres tumors in mie grew progressively. These results onfirmed tht loss of Stt3 in the hemtopoieti system medites the oserved ntitumor effets. We next determined whether loking Stt3 signling in immune ells would hve therpeuti ntitumor effets. We first hllenged Stt3 loxp/loxp nd Cre- Stt3 loxp/loxp mie with B16(F1) tumor ells. When tumors eme plple, we dministered poly(i:c) to oth groups. Alting the Stt3 lleles in hemtopoieti ells led to signifint growth inhiition of estlished B16(F1) tumors (Fig. d). Moreover, ltion of the Stt3 lleles in hemtopoieti ells indued regression nd, in most ses, erdition of estlished MB49 tumors (Fig. e). We oserved no effet on tumor growth fter poly(i:c) tretment of mie, onfirming the role of Stt3 signling in the oserved therpeuti ntitumor response. We further determined whether T-ell responses re involved in the oserved ntitumor effets medited y inhiiting Stt3 signling in immune ells. We injeted poly(i:c)-treted Cre-Stt3 loxp/loxp mie with either rt IgG or CD4- nd CD8-speifi ntiodies, whih depleted 99% of T ells s onfirmed y flow ytometry (dt not shown), nd hllenged mie with MB49 tumor ells. Although NATURE MEDICINE VOLUME 11 [ NUMBER 12 [ DECEMBER

5 2 Nture Pulishing Group Stt3 +/+ 3 3 Tumor size (mm 3 ) Tumor volume (fold inrese) ltion of the Stt3 lleles in one mrrow ells rogted MB49 tumor growth s expeted, the oserved ntitumor effets were essentilly lost in the sene of CD4 + nd CD8 + T ells (Fig. f). Previous studies indited tht long-term Stt3 ltion leds to utoimmunity. Our nlysis indited tht lthough Stt3 ltion indued ntitumor immunity is pprent within week, there ws virtully no evidene of systemi utoimmunity t 4 weeks fter indued depletion (Supplementry Fig. online), suggesting tht the ntitumor responses indued y Stt3 ltion re not simply mnifesttion of systemi utoimmunity in these mie, ut rther speifi effet on intrinsi tumor immune surveillne. Trgeting Stt3 indues ntitumor immune responses We next ssessed whether loking Stt3 with smll-moleule Stt3 inhiitor, CPA-7 (ref. 36), would tivte immune ells nd indue therpeuti ntitumor effets in vivo independent of diret effets on the tumor ells themselves. In ontrst to B16, MB49 tumor ells re insensitive to CPA-7 indued tumor-ell poptosis (Fig. 6). It hs een shown tht CPA-7 disrupts Stt3-DNA inding tivity, whih is followed, within hours, y redution of phosphorylted Stt3 in the treted ells in vitro 36. We showed tht CPA-7 inhiits Stt3 ut not Stt1 nd Stt in DC2.4 mouse DC line (Supplementry Fig. 6 online). Tumor-infiltrting DCs from the mie reeiving CPA-7 24 h erlier showed onsiderly redued phosphorylted Stt3 ompred to vehile-treted mie (Fig. 6). These tumor-infiltrting DCs lso hve inresed phosphorylted Stt1 (Fig. 6). An inrese in Stt1 tivity in ultured mrophges hs een reported, in Tumor size (mm 3 ) Tumor volume (fold inrese) d e f 2 2 Tumor size (mm 3 ) Tumor size (mm 3 ) keeping with opposing tivities often oserved etween these two Stt proteins 18,37,38. CPA-7 tretment in vivo lso led to signifint growth inhiition of estlished MB49 tumors (Fig. 6). In the sene of CD4 + nd CD8 + T ells, CPA-7 indued tumor growth inhiition ws rogted, suggesting diret role for immune ells in the in vivo ntitumor effets of CPA-7 (Fig. 6). NK ells my lso prtiipte in the ntitumor effets of CPA-7, s in vitro NK ell tivity is inresed from tumor-ering mie treted with CPA-7 (Fig. 6d) nd NK depletion prtilly rogtes the in vivo ntitumor effets of CPA-7 tretment (Fig. 6e). These dt suggest tht trgeting Stt3 with smll-moleule drugs hs the potentil to indue therpeuti ntitumor responses through tivting immune ells regrdless of the tumors diret sensitivity to the inhiitors (Fig. 6f). Additionl experiments further indited tht loking Stt3 signling with smll moleule inhiits tumor metstsis nd prolongs survivl of tumor ering mie (Supplementry Fig. 7 online). As mentioned ove, long-term ltion of Stt3 in hemtopoieti ells is known to use Crohn disese like pthology (Supplementry Fig. online). To determine whether there is indeed therpeuti window for Stt3-sed ner tretment, we ssessed the effets of 3-week CPA-7 tretment on lymphoytes nd mrophge infiltrtion in olons. We oserved no suh infiltrtion in tumor-ering mie treted with CPA-7 over the 3-week tretment period neessry for effetive ntitumor responses (Supplementry Fig. online). Moreover, no signifint toxiity ws deteted in mie reeiving 4-week CPA-7 tretment (Supplementry Tle 1 online). These results 2 one mrrow one mrrow rt IgG + nti-cd4/8 Figure Alting Stt3 in hemtopoieti ells indues ntitumor effets. () B16 tumor development is signifintly inhiited in mie. Shown re the results representtive of three independent experiments; n ¼ 1 for eh experiment. P o.1; P o.1; P o.. () MB49 tumor growth is rogted in mie with hemtopoieti ells. Shown re the results representtive for two independent experiments; n ¼ 1 for eh experiment. () MB49 tumor growth is olished in mie engrfted with whole one mrrow. Tumor hllenge ws performed 2 months fter trnsplnttion of one mrrow from poly(i:c)-treted Stt3 loxp/loxp nd Cre-Stt3 loxp/loxp mie, n ¼ 1. (d) Alting Stt3 signifintly inhiits estlished B16 tumor growth. Poly(I:C) ws dministered to Stt3 loxp/loxp nd Cre-Stt3 loxp/loxp mie ering B16 tumors (verge dimeter, 4 mm) on dys indited y rrow, n ¼ 12. (e) Altion of the Stt3 indued omplete regression of estlished MB49 tumors. Averge tumor dimeter ws 6 mm on the dy of initil poly(i:c) tretment, n ¼ 9. Shown re the results representing two independent experiments. (f) Stt3 lokde-indued ntitumor effets require T ells. MB49 tumors were implnted in poly(i:c)-treted Stt3 loxp/loxp nd Cre-Stt3 loxp/loxp mie s in. mie were injeted with ontrol rt IgG or ntiodies to CD4 nd CD8, s indited. Sttistil signifine of the experiment ws tested y one-wy ANOVA, P ¼.196, n ¼. Dt re men numers ± s.e.m VOLUME 11 [ NUMBER 12 [ DECEMBER 2 NATURE MEDICINE

6 2 Nture Pulishing Group Perent of poptoti ells CPA (µm) B16 MB49 Tumor volume (mm 3 ) e Tumor volume (mm 3 ) Vehile CPA-7 + rt IgG CPA-7 + nti-cd4/ suggest tht trgeting Stt3 n indue immune ell medited ntitumor effets without systemi utoimmunity. DISCUSSION In our induile Stt3 knokout mouse model, Stt3 ltion ffeted multiple lineges of immune ells, mny of whih show enhned ntitumor tivity when ssyed in isoltion. In vivo, speifi ltertions in ellulr intertions tht our when Stt3 is phrmologilly loked or genetilly lted led to potent ntitumor responses, irrespetive of tumor sensitivity to Stt3 inhiition. Our previous ell-ulture studies showed tht tumor-derived ftors suh s VEGF nd IL-1 tivte Stt3 in DC progenitors, nd tht loking Stt3 y either Stt3 peptide inhiitor or gene ltion rogtes tumor-indued mturtion inhiition in vitro 11. Bsed on these dt, it is prole tht Stt3 signling diretly influenes DC mturtion nd tivtion. Whether Stt3 defiieny within T ells, neutrophils nd NK ells is solely responsile for the enhned ytolyti tivity remins to e further investigted. Nonetheless, our study shows tht Stt3 signling restrins multiple hemtopoietilly derived elements of innte nd dptive immunity. Moreover, our results show tht Stt3 is persistently tivted in tumor-infiltrting immune ells nd tht trgeting Stt3 in the hemtopoieti system eliits multiomponent therpeuti ntitumor immunity. Stt3 regultes multiple genes ruil for tumor-ell survivl nd prolifertion 39,4. Trgeting Stt3 in tumors with onstitutive Stt3 tivtion uses diret tumor-ell deth 4 nd growth inhiition in vivo 41,42. Our findings show tht inhiiting Stt3 in the hemtopoieti system n result in immune ell medited ntitumor d Stt3P Vehile CPA-7 Vehile CPA :1 2:1 :1 Vehile CPA-7 + rit IgG CPA-7 + GM1-speifi ntiody f NK Reltive ell numer Perent of ytotoxiity DC NPH phospho-stts Vehile CPA-7 MHC II CD8 CD86 IL-12 Stt1P IFN-γ T reg CD8 + T ell Figure 6 Trgeting Stt3 with smll-moleule inhiitor tivtes ntitumor immunity. () Sensitivity of tumor ells to CPA-7 indued poptosis in vitro s determined y nnexin V stining. B16 ells, sensitive to CPA-7 indued poptosis, served s positive ontrol. () Stt3 is tivted in tumorinfiltrting DCs, nd dministering CPA-7 to MB49 tumor-ering mie redues Stt3 wheres Stt1 tivity ws inresed. Top pnel: Phospho- Stt3 (Stt3P) levels in tumor-infiltrting DCs were ssessed 24 h fter intrvenous injetion with vehile (red line) or CPA-7 (lue line). ws spleni CD11 + ells from tumor-free mie. Lower pnel: Stt1YP levels in tumor-infiltrting DCs from the sme mie s in top pnel. () Bloking Stt3 with CPA-7 inhiits the growth of estlished MB49 tumors, whih is T ell dependent. Mie with tumors (out 6 mm in dimeter) reeived intrvenous injetions of either vehile or CPA-7; P ¼.88 (y one-wy ANOVA), n ¼ 6. (d) Trgeting Stt3 with CPA-7 tivtes NK ells in tumor-ering hosts. Shown is 1 Cr-relese ssy using YAC-1 trget ells; n ¼ 3. (e) Effets of depleting NK ells with ntiody to silo GM1. Mie with estlished MB49 tumors were treted with vehile or CPA-7 in omintion with ontrol or silo GM1-speifi ntiody; P o.1 (y one-wy ANOVA), n ¼ 11. (f) Shemti overview on Stt3 s negtive regultor of ntitumor immunity nd potentil trget for immunotherpy. Tumor miroenvironment indues Stt3 tivity in vrious immune ells. Stt3 tivity in tumor-ssoited NK ells nd neutrophils inhiits diret ell ytotoxiity nd ntitumor innte immune responses. Ativtion of Stt3 in tumor-infiltrting DCs impedes CD8 + T-ell funtion, nd my ontriute to umultion of tolerogeni T reg ells inside tumors. immunity ginst tumors otherwise insensitive to diret Stt3 inhiition. Stt3 ltion leds to utoimmune mnifesttion in the gut 3,43. We oserved mrginl immune ell infiltrtion in olon 4 weeks fter hemtopoieti Stt3 ltion, ut the ntitumor effets of hemtopoieti Stt3 ltion re more rpid, eoming ovious within week. Thus, there is onsiderle therpeuti window of ntitumor immunity versus utoimmunity. This therpeuti window is emphsized y our studies showing CPA-7 tretment uses ntitumor immune responses without detetle mnifesttion of utoimmunity within the gut. Inhiiting Stt3 y mens other thn gene deletion is inomplete nd/or intermittent s result of limited hlf-life of drugs 11,36, suggesting tht moleulr trgeting of Stt3 llows t lest some norml funtion of Stt3. Trgeting Stt3 is expeted to ffet Stt3 tivity most mrkedly in tumor-infiltrting immune ells, tumor ells nd tumor stroml ells, euse Stt3 is preferentilly tivted in these ells ompred to their ounterprts in norml tissue. We propose tht trgeting Stt3 in tumor-infiltrting immune ells my lok expression of mny tumor-ssoited ftors nd their orresponding reeptor signling, neutrlizing the tumor-indued immunosuppressive miroenvironment nd therey ontriuting to ntitumor immunity. In light of immune impirment in individuls with ner, short-term lokde of Stt3 in ontrolled mnner using smll-moleule drugs or RNA interferene my reverse immune suppression nd tivte ntitumor immune responses, whih ould e hrnessed to improve the effiy of immunotherpeuti pprohes. METHODS Cells lines. We purhsed mouse B16 melnom nd YAC-1, P81 trget ells, from Amerin Type Culture Colletion; MB49 ldder rinom nd C4 mouse melnom lines were gifts from T. Rtliff (University of Iow) nd J. Fidler (M.D. Anderson Cner Center), respetively. In vivo experiments. Mouse re nd experimentl proedures were performed under pthogen-free onditions in ordne with estlished institutionl guidne nd pproved protools from Institutionl Animl Cre nd Use Committees of University of South Florid nd John Hopkins University. We otined Mx1-Cre mie from the Jkson Lortory nd Stt3 loxp/loxp mie NATURE MEDICINE VOLUME 11 [ NUMBER 12 [ DECEMBER

7 2 Nture Pulishing Group from S. Akir nd K. Tked (Osk University). Genertion of mie with hemtopoieti ells y n induile Mx1-Cre reominse system hs een reported 17,3. Briefly, we verified deletion of Stt3 y PCR, using primer sets tht distinguish Stt3, Stt3 loxp nd Stt3-deleted lleles, nd y EMSA. For EMSA, we prepred nuler extrts from nd one mrrow ells stimulted for 2 min with IL-1 (1 ng/ml) nd/or GM-CSF (2 ng/ml). We performed one mrrow trnsplnttions with whole one mrrow otined from nd donor mie s desried previously 44. For suutneous tumor hllenge, we injeted 1 4 B16 or 1 MB49 tumor ells into 7 8-week-old wild-type mie t d fter poly(i:c) tretment, or 2 months fter one mrrow trnsplnt. After 3 weeks, mie were killed nd spleens s well s tumor speimens were hrvested. For therpeuti studies, we hllenged Stt3 loxp/loxp or Cre-Stt3 loxp/loxp mie with either B16 or MB49 tumor ells, followed y poly(i:c) tretments when tumors were t lest 3 mm in dimeter. Beuse of mle ntigen speifi response in femle mie y tumor hllenge, we rried out MB49 tumor experiments using mle mie only. For CPA-7 tretment, we implnted MB49 tumors into mle C7BL/6 mie nd llowed the tumors to grow until 7 mm in dimeter; we gve mie intrvenous injetions of either vehile (1% DMSO/PBS) or mg/kg CPA-7 one every 3 d. Flow ytometry. We prepred single-ell suspensions y mehni dispersion of spleen or tumor tissues. We preinuted freshly prepred ells suspended in mixture of PBS, 2% FCS nd.1% (wt/vol) sodium zide with FgIII/IIR-speifi ntiody to lok nonspeifi inding nd stined the ells with different omintions of fluorohrome-oupled ntiodies to CD11, I-A (MHC lss II), CD86, CD11, Gr1, CD49, CD3, CD2 or Lg-3, or with nnexin V (BD Biosienes). We olleted fluoresene dt on FACSCliur (Bekton Dikinson) nd nlyzed them using FlowJo softwre (Tree Str). Isoltion of tumor-infiltrting immune ell susets nd intrellulr stining of signling moleules. We gently mined freshly exised tumor tissues into smll piees, nd inuted them in 4 U/ml of ollgense D (Rohe) solution for 3 min t 37 1C. We used ell suspensions filtered through mesh filter for isoltion of vrious immune ell susets using speifi ntiodies in omintion with EsySep mgneti nnoprtiles from StemCell Tehnologies. We fixed isolted tumor-infiltrting ells in prformldehyde nd permeted them in methnol efore intrellulr stining with ntiodies to phosphotyrosine-stt3 nd Stt1 (BD Biosienes) or with Foxp3-speifi ntiody (ebiosienes). We olleted fluoresene dt on FACSliur (Bekton Dikinson) nd nlyzed them using FlowJo softwre (Tree Str). Isoltion nd funtionl nlysis of spleni DCs. We isolted DCs from mouse spleens essentilly s desried 4 nd purified them using CD11 MACS eds (Miltenyi Biote). For IL-12 mesurement y ELISA (Endogen), we ultured DCs with 1 ng/ml lipopolyshride for 18 h to ollet superntnts. T-ell prolifertion ssy with CD4 + T ells purified from OTII mie stimulted ws performed s desried previously 11. Immunohistohemistry. We fixed -mm setions of the flsh-frozen tumor speimens in etone, stined them with ntiodies to CD4 nd CD8, nd deteted them with peroxidse- or lkline phosphtse oupled seondry ntiodies using NovRED nd Blue Chromogen from Vetor s previously desried 44. Cytotoxiity ssys. We performed NK ell nd neutrophil ytotoxiity ssys s previously desried 46. Briefly, we rried out 4-h 1 Cr-relese ssy using YAC-1 ells or the FgR + mouse mstoytom (P81) ells s trgets for enrihed NK ells nd neutrophils, respetively. ELISPOT ssy. We hrvested spleens from mie hllenged suutneously with 1 1 B16 tumor ells 2 weeks erlier. We seeded 1 splenoytes into eh well of 96-well filtrtion plte in the presene or sene of 1 mg/ml of p1e peptide nd inuted them t 37 1C for 24 h. We deteted peptidespeifi IFN-g positive spots ording to the mnufturer s proedure (Cell Sienes), nd snned nd quntified them using Immunospot Anlyzer from Cellulr Tehnology Ltd. Sttistil nlysis. To ompre tumor size or surfe mrker expression etween multiple test groups in mouse experiments, we performed onewy ANOVA followed y Newmn-Keuls test. We used unpired t-test to lulte two-tiled p vlue to estimte sttistil signifine of differenes etween two tretment groups. Sttistilly signifint p vlues were leled s follows: P o.1; P o.1 nd P o.. Dt were nlyzed using Prism softwre (GrphPd). Note: Supplementry informtion is ville on the Nture Mediine wesite. ACKNOWLEDGMENTS We would like to thnk G. Go for sttistil nlyses, C. Muro-Cho for evluting immunohistohemil dt, K. Nguyen nd T. Ghnsh for shring their expertise nd L. Lutz for tehnil ssistne. This work ws supported y US Ntionl Institutes of Helth grnts (to H.Y.), nd y the Dr. Tsi-fn Yu Cner Reserh Endowment. Work in D.P. s l ws supported y grnts from the Commonwelth Foundtion, Jney Fund, the Serph Foundtion, the Topeers nd D. Needle. W.G.K. is the Newmn Sholr of the Leukemi nd Lymphom Soiety. We would lso like to thnk S. Akir nd K. Tked for Stt3 loxp mie, nd the Pthology Core t the University of South Florid for tehnil ssistne. We lso knowledge dedition of stff memers t the niml filities nd flow ytometry ores t oth Moffitt Cner Center nd Johns Hopkins Cner Center. COMPETING INTERESTS STATEMENT The uthors delre tht they hve no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Dunn, G.P., Brue, A.T., Iked, H., Old, L.J. & Shreier, R.D. Cner immunoediting: from immunosurveillne to tumor espe. Nt. Immunol. 3, (22). 2. Prdoll, D. Does the immune system see tumors s foreign or self? Annu. Rev. Immunol. 21, (23). 3. Hwiger, D. et l. Dendriti ells indue peripherl T ell unresponsiveness under stedy stte onditions in vivo. J. 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