Local IL-21 Promotes the Therapeutic Activity of Effector T cells by Decreasing Regulatory T Cells Within the Tumor Microenvironment

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1 originl rticle Locl IL- Promotes the Therpeutic Activity of Effector T cells y Decresing Regultory T Cells Within the Tumor Microenvironment Seunghee Kim-Schulze, Hong Sung Kim, Qing Fn, De Won Kim nd Howrd L Kufmn The Tumor Immunology Lortory, Division of Surgicl Oncology, Columi University, New York, New York, USA The erdiction of tumors y the immune system depends on the genertion of ntigen-specific T cells which cn migrte to sites of tumor growth nd mintin their effector functions despite locl tumor-derived T-cell inhiitory fctors. Interleukin- (IL-) is n IL--relted cytokine tht hs shown limited evidence of ntitumor ctivity in murine models nd erly phse clinicl trils. Effect of locl IL- on T-cell responses within the tumor microenvironment, however, hs not een extensively evluted. Thus, we developed stly trnsfected IL--secreting B melnom cell line to test the effects of locl IL- on endogenous nd doptively trnsferred T-cell responses. Tumors expressing IL- exhiited delyed growth in vivo, which ws ssocited with n increse in ctivted systemic effector nd memory CD8 + T cell responses. Locl IL- lso enhnced the therpeutic effects of doptively trnsferred gp-specific T cells nd ws synergistic with IL-. The effect ws lso ssocited with n incresed prolifertion of locl CD8 + T cells nd decresed ccumultion of regultory CD + FOXP3 + T cells within the tumor microenvironment. These dt suggest tht locl IL- enhnces endogenous nd doptively trnsferred T-cell immunity through incresed effector CD8 + T cells nd decresed CD + regultory T cells in the tumor microenvironment. Received August 8; ccepted Septemer 8; pulished online Novemer 8. doi:.38/mt.8.9 Introduction The role of the immune system in the rejection of solid tumors hs een well demonstrted in oth crcinogen-induced nd spontneous experimentl tumor models., In these models, immune-medited erdiction of estlished tumors depends on the presence of tumor ntigen-primed CD8 + effector T cells, the ility of such T cells to trffic to sites of tumor growth, the persistence of cells in sufficient concentrtions t the tumor site nd the cpcity of the T cells to mintin cytotoxic effector functions in the fce of locl immunosuppressive mechnisms. 3 The identifiction of trgeted T-cell ntigens nd considerle erly phse clinicl testing of vccines designed to elicit tumor-specific T-cell immunity supported the concept tht T cells could e primed, t lest in the peripherl lood, in cncer ptients ut therpeutic responses were disppointing. More recently, reports hve suggested etter results with doptive trnsfer of lrge numers of ntigen-specific CD8 + or CD + T cells in ptients with metsttic melnom., While these clinicl studies support the concept tht significnt concentrtion of effector T cells t sites of tumor growth cn medite tumor rejection, the loss of therpeutic ctivity hs een ssocited with chnges in the tumor or locl environment, such s loss of tumor ntigen nd/or MHC expression, incresed concentrtions of inhiitory cytokines (i.e., IL- nd TGF-β), ccumultion of regultory nd suppressor immune cells (i.e., CD + CD + FOXP3 + regultory T cells (Tregs) nd CD8 + mcrophges) nd presence of other nti-inflmmtory meditors (i.e., rginse, VEGF, PGE) in the tumor microenvironment. 7 Thus, strtegies imed t mintining effector T cells within the tumor microenvironment following vccintion or doptive trnsfer re high priority. Interleukin- (IL-) is four-undle α-helicl cytokine produced primrily y ctivted T cells nd functions to regulte T-cell homeostsis. Recominnt humn IL- hs een used s single-gent therpy for the tretment of metsttic melnom nd renl cell crcinom, s n djuvnt to tumor-ntigen vccintion nd to support doptively trnsferred T cells in vivo. 3 IL-, however, lso functions to promote CD + CD + FOXP3 + Tregs in vitro nd in vivo, suggesting potentil negtive influence on T-cell immunity. This effect on Tregs my hve importnt implictions s shown in recent murine tumor model where regression ws directly relted to the rtio of proliferting CD8 + T cells to CD + Tregs within the tumor microenvironment. 7 Thus, while IL- my promote effector CD8 + T cells, the effect on Tregs my inhiit potentil therpeutic ctivity of effector T cells, especilly if the tumor microenvironment is permissive to the ccumultion of Tregs. IL- is type I cytokine tht shres common cytokine receptor γ chin with other memers of the IL- fmily, including IL-, IL-7, nd IL- (ref. 8). IL- is produced y Th cells nd exerts regultory effects on virtully ll cells of the immune system, including B, T, nd NK cells. In T cells, IL- cn lock IL--induced poptosis nd promotes the differentition nd long-term survivl of CD8 + T cells. 9 IL- hs lso shown limited Correspondence: Howrd L. Kufmn, The Tumor Immunology Lortory, Columi University, 77 Fort Wshington Avenue, MHB-7SK, New York, New York 3, USA. E-mil: hlk3@columi.edu 38 vol. 7 no., fe. 9

2 IL- Induces Effective Antitumor Immunity clinicl enefit s single gent ginst melnom nd renl cell crcinom in Phse I clinicl tril. Furthermore, in n experimentl tumor model, depletion of Tregs efore IL- exposure resulted in optiml expnsion of ntigen-specific CTL in vitro nd in vivo., While the dose, schedule nd route of dministrtion significntly influence the effects of IL-, the net effect of these prmeters on IL- iologicl ctivity is not known. Furthermore, the role of IL- in the tumor microenvironment hs not een previously evluted. Thus, we hypothesized tht locl IL- would significntly enhnce the therpeutic ctivity of ntigen-specific T-cell responses ginst n estlished murine melnom. To test this, we first estlished stly trnsfected B cell line tht secretes functionl IL- nd found tht mice did exhiit delyed tumor growth fter chllenge with the tumor. The delyed tumor growth ws ssocited with induction of endogenous ctivted nd memory T-cell responses. Locl IL- lso improved the therpeutic ctivity of doptively trnsferred tumor-rective CD8 + T cells. Anlysis of the tumor microenvironment showed tht these effects were due, in prt, to decrese in the ccumultion of Tregs shifting the lnce of effector CD8 + T cells nd CD + Tregs. These dt suggest tht locl expression of IL- my e n pproch for improving the therpeutic effects of oth endogenous vccineprimed T cells nd doptively trnsferred T cells. Results B cells secreting functionl IL- disply delyed growth in vivo B melnom cells trnsfected with pcdna contining murine IL- trnscript were selected in G8-contining medium to otin stly trnsfected clones. Stle clones demonstrting IL- secretion y enzyme-linked immunosorent ssy (ELISA) (dt not shown) were tested for their growth kinetics in vitro. The growth rte of one such B clone expressing IL- (B/ mil-) ws similr to tht of B expressing n empty vector pcdna (B/pcDNA) or the prentl B cell line (Figure ), nd this clone ws selected for further study. To document the functionl property of secreted IL-, nive T cells (. ) derived from splenocytes were plted in n nti-cd3 precoted plte nd incresing doses of IL- ( to, pg/ml) in superntnt from B/mIL- clones grown on miniml growth medium were dded in the sence or presence of nti-cd8 ntiodies. Superntnt from B/mIL- cultures, ut not from B/pcDNA cultures, ugmented T-cell prolifertion in dose-dependent mnner (Figure ). Sucutneous (SC) injection of the B/mIL- in syngeneic CBL/ nimls resulted in delyed tumor growth compred to B/pcDNA or prentl B tumors (Figure ). B/ mil- tumors were significntly smller thn B/pcDNA or B t the indicted times (*P <., **P <.3). Locl IL- induces systemic CD37 +hi CD8 + T-cell expnsion nd T-cell memory To exmine the effect of IL- on tumor microenvironment, tumors were isolted t dy 3 nd TIL were nlyzed y flow cytometry. IL- secreting tumors showed significnt increse in frequency of CD3 + T cells (Figure 3, P <.9). The percentge (.%) nd solute numer (, cells) of tumor infiltrting Cell numer 3 H Thymidine incorportion 7,,,, 3,,, 8 B B/pcDNA B/IL- 8 Hours 7 9, 7, :IL- (pg/ml) :Anti-CD3 + :Anti-CD8 Figure B/mIL- melnom cells exhiit norml growth kinetics nd produce functionl IL-. () Growth curve of stly trnsfected B melnom secreting IL- in vitro. () IL- dose-dependent prolifertion of mouse nive splenocytes. Prolifertion ssy ws performed triplictes in n nti CD3 precoted plte, incresing doses of superntnt contining mil- (conditioned medium) were dded t the time of stimultion s descried in Mterils nd Methods. Anti CD3 nd CD8 ntiodies re used to stimulte cells. Brs, mens ± SD. Tumor size (cm ) B B/pcDNA B/mIL Time (dys) Figure Locl IL- results in delyed tumor growth. Tumor growth of B, B/pcDNA, nd B/mIL- in syngeneic mice is represented s verge tumor size (men ± SD) of ech experimentl group (n = ), s detiled in Mterils nd Methods. Tumor growth dt shown re representtive pttern of tumor growth in one of three experiments. Sttisticl nlysis with t-test showed tht the difference in tumor size etween the control group nd B/mIL- ws significnt. *P <., **P <.3. CD3 + T cells were significntly higher in IL--secreting tumors. A notle increse ws seen in CD8 + cells in B/mIL- tumor (3.% of TIL nd, cells versus % of TIL nd,39 cells), while there ws little chnge in the numer of CD + T cells (% nd 8,9 cells) compred to control group B/pcDNA (% nd,7 cells, Figure 3, P <.). The rtio of CD + T cells to CD8 + T cells ws.7 for the B/mIL while they were. nd.9 for B/pcDNA nd B, respectively. The frequency of NK popultion in the spleen nd tumor y surfce phenotype * ** ** Moleculr Therpy vol. 7 no. fe. 9 38

3 IL- Induces Effective Antitumor Immunity c % of CD3 + T cells in TIL 3 3 B/PBS P <.9 B/pcDNA B/mIL- CD + % of TIL 3 P <. CD CD8 CD CD8 CD CD8 B/PBS B/pcDNA B/mIL- CD % 3.% CD37 # Cells CDL CCR7 d B B/pcDNA B/mIL- B B/pcDNA B/mIL- CD + CD8 + B/PBS B/pcDNA B/mIL- B/PBS B/pcDNA B/mIL- B/PBS B/pcDNA e B/mIL- Counts IL- Counts IL- Counts CD + CD8 + IFN-γ IFN-γ CFSE Figure 3 Locl IL- results in n increse in the frequency of effector nd memory CD8 + T cells. () IL- strongly enhnces infiltrtion of CD3 + T cells in tumor. The percentge of CD +, CD8 + T cells in tumor tissue ws determined y flow cytometry nd sed on CD + cells. () Specific increses in the frequency of CD8 + T cells in TIL. (c) Single-cell suspension of tumor isolted from ech group t dy 3 ws stined with CD37, CDL, nd CCR7 for ctivted memory T cells phenotypes followed y flow cytometric nlysis. (d) Splenocytes isolted from ech group t dy 3 were stimulted for hours with phorol myristte cette/ionomycin A followed y intrcellulr stining of IL- nd IFN-γ nd flow cytometric nlysis in CD + T cells nd CD8 + from the lymphocyte popultions. The unshded histogrm in the plot indictes the isotype ntiody control. (e) Splenocytes were croxyfluorescein succinimidyl ester (CFSE) leled nd stimulted with suoptiml dose of nti-cd3 ma (. µg/ml) for 7 hours followed y surfce stining of CD nd CD8 nd flow cytometry nlysis of CFSE dilution. CFSE profiles gted on CD + nd CD8 + in the plot re shded histogrm nd the dotted line indictes the unstimulted cells. CD9+NK.+CD3 stining reveled similr pttern cross the niml groups. ( % of CD + TIL). Phenotypic chrcteriztion of CD + nd CD8 + T cells in B/mIL- tumor showed up-regultion of CD37 expression nd CDL low CCR7 low phenotype suggesting tht IL- enhnced genertion of ctivted effector memory T cells (Figure 3c). Anlysis of ctivtion mrkers CD nd CD9 lso reveled tht T cells isolted from IL- producing tumors showed n increse compred to those isolted from control groups (dt not shown). To evlute the effect of locl IL- secretion on the induction of systemic T cells in vivo, the functionl cpcity of spleen-derived CD8 + T cells ws evluted for cytokine secretion in vitro. Both CD + nd CD8 + T cells enriched from splenocytes of mice ering B/mIL- tumors (3 dys) produced significntly higher level of IL- nd IFN-γ determined y intrcellulr cytokine stining (Figure 3d). Next, to determine whether CD8 + T cells were functionlly relted to memory cell phenotype, splenocytes were leled with croxyfluorescein succinimidyl ester (CFSE) nd stimulted with suoptiml nti-cd3 ntiody (. µg/ml) for 7 hours followed y CFSE dilution nlysis. Both CD + nd CD8 + T cells of B/mIL- injected mice initited prolifertion t. µg/ml while cells from control B/pcDNA showed no prolifertion (Figure 3e). Both CD + nd CD8 + T cells underwent roust prolifertion with minimum of three divisions. The initil sl level of prolifertion without nti-cd3 stimultion ws similr mong the niml groups suggesting tht the higher prolifertive rte of T cells from B/mIL- tumor ering mice is ecuse of induction of systemic memory T cells. This suggests tht locl IL- secretion enhnces prolifertion nd ccumultion of ctivted memory CD8 + T cells vol. 7 no. fe. 9

4 IL- Induces Effective Antitumor Immunity Foxp3 B/PBS B/pcDNA B/mIL CD CD % of Treg in TIL...8. P <. B/PBS B/pcDNA B/mIL- c CPM,,, 3,,, CD + CD + CD + CD B/PBS E:T rtio : B/pcDNA B/mIL- Figure Locl IL- prevents the ccumultion of Tregs in the tumor microenvironment. () Representtive stining for CD + FOXP3 + nd CD + FOXP3 + cells in B/mIL- tumor infiltrting cells. Gted CD + T cells stined with CD nd FOXP3. Percentge of CD + FOXP3 + cells is indicted in the upper right qudrnt. () Grph shows the proportion of CD + CD + FOXP3 + Tregs in tumor infiltrting CD + T cells of ech niml group. *P <.. (c) Whole splenocytes were isolted nd Treg cells were enriched y microed cell isoltion method nd purity of 9 9% ws determined y flow cytometry. CD + CD T cells were cocultured with CD + CD + Treg cells t : rtio for 3 dys in nti-cd3 ma precoted plte with nti-cd8 ma stimultion. Locl IL- secretion prevents ccumultion of CD + CD + FOXP3 + Tregs within the tumor microenvironment Becuse IL- elongs to the IL- fmily nd IL- tretment hs een shown to increse Tregs in melnom nd renl cell crcinom ptients, we nlyzed the effects of locl IL- on the frequency of Tregs. First, we evluted the frequency of Tregs, defined y expression of CD, CD, nd FOXP3, in the spleen nd found no difference etween mice ering B/m-IL-, B/pcDNA, or prentl B SC tumors (dt not shown) t dy 3. However, when cells were nlyzed from single-cell suspensions from the tumors, B/mIL- tumors showed sttisticlly significnt decrese in Treg frequency within dy 3 tumors compred to mice with non-il--secreting tumors (Figure,). Representtive Tregs cells defined s Foxp3 + CD + nd Foxp3 + CD + T cells in CD + CD3 + T cell popultions re shown in Figure. The frequency of Tregs in TIL of B/mIL- tumor ws significntly lower thn control tumors (Figure, P <.). Further the solute numer of Treg in the TIL popultion of B/mIL- decresed to 3 cells from control group with 337 cells. Becuse of the limited numer of Tregs in the TIL for the functionl ssy, Tregs were isolted from the spleen for suppression function in mixed coculture ssys using CD + CD T cells stimulted y nti-cd3 nd -CD8 ntiodies with or without Tregs derived from mice hroring different tumors. The level of suppression y Tregs ws similr mong ll groups (Figure c). Thus, fewer Tregs ccumulted in the tumor microenvironment of mice with estlished tumor secreting IL- without ffecting the Treg suppressive cpcity on per cell sis. Locl IL- enhnces the therpeutic response of doptively trnsferred T cells We next sought to determine whether locl IL- secretion could lso ugment doptively trnsferred ntigen-specific T cells in the murine melnom model. This ws ccomplished using the well-estlished Pmel- experimentl model with minor modifictions. 3 Tumors (B/mIL-, B/pcDNA or prentl B) were estlished y SC injection of.3 cells t dy. When tumors reched out mm (dy 7 ), the mice were sulethlly irrdited with Gy followed y doptive trnsfer of in vitro Tumor size (mm ) Tumor size (mm ) 3 3 B/PBS + pmel- + gp B/pcDNA + pmel- + gp B/pcDNA + pmel- + gp + IL- 3 8 Time (dys) 3 8 B/pcDNA + pmel- + gp + IL- B/mIL- + pmel- + gp B/mIL- + pmel- + gp + IL Time (dys) Figure Locl IL- enhnces therpeutic responses of doptively trnsferred T cells nd cts synergisticlly with IL-. () Tumor response to doptive trnsfer of ctivted gp-specific T cells (pmel-), gp peptide vccintion nd systemic IL- tretment s descried in Mterils nd Methods (n = ). () Enhnced tumor response to the doptive trnsfer of pmel- cell nd gp peptide vccintion in mice ering IL- secreting tumor (n = ). Error rs represents ± SDs. prectivted Pmel- cells ( CD8 + Vβ3 + T cells) y til vein injection. This ws followed y vccintion with μg of hgp 33 peptide SC lone or with IU of recominnt humn IL- intrperitonelly (IP) s descried in Mterils nd Methods. First, we vlidted previous reports showing tht mice hroring B melnom treted with doptively trnsferred ctivted Pmel- T cells followed y gp vccintion requires the ddition of IL- to reduce tumor growth (Figure ). When the B/mIL- tumors, which secrete locl IL-, were treted with doptively trnsferred ctivted pmel- T cells nd gp vccintion they exhiited similr degree of delyed growth s * * Moleculr Therpy vol. 7 no. fe

5 IL- Induces Effective Antitumor Immunity mock-trnsfected B tumors treted y doptive trnsfer, vccintion nd IL- (Figure ). The most significnt therpeutic response, however, ws seen in mice treted with locl IL-, Pmel- T cells, vccintion nd IL- (Figure ). Thus, locl IL- my sustitute for systemic IL- in doptive T-cell trnsfer therpy nd lso ppers to ct synergisticlly with IL- to induce tumor regression following doptive T-cell trnsfer. Locl IL- stimultes doptively trnsferred tumor-specific T cells in vivo In order to correlte the in vivo therpeutic response with tumor ntigen-specific T-cell responses, the effect of locl IL- on the gp-specific TCRvβ3 + Thy. + T cells (Pmel-) in the tumor site were nlyzed y flow cytometry. While tumors received Pmel- T-cell trnsfer, vccintion nd IL- injections demonstrted n increse in Thy. + T cells within the tumors, the numer of Thy. + T cells ws gretest in those tumors producing locl IL- (Figure ). We next tested gp-specific T-cell function y in vitro stimultion with gp peptide nd used IFN-γ production y intrcellulr cytokine stining s the redout in spleen. The level of IFN-γ producing Thy. T cells were similr in niml groups treted with IL-, ering IL- secreting tumor, nd group ering mil- tumor treted with IL-. All these three groups showed increse in Thy. + CD8 + T cells n verge of tenfold increse in IFN-γ positive T cells compred to control groups upon peptide stimultion in vitro (Figure, ll P <.). This suggested ut did not prove tht locl IL- promoted the prolifertion of T cells within the tumor microenvironment nd systemic comprtment. To confirm whether IL- induced expnsion of gp- specific TCRvβ3 + Thy. + T cells in vivo, mice were injected IP with BrDu for hours. Splenocyte-derived T cells were nlyzed y flow cytometry for BrDu incorportion with ntiodies ginst CD8, Vβ3 nd BrdU. T cells from mice with locl IL--secreting tumors induced gp-specific T-cell prolifertion in vivo nd further increse in BrdU positive T cells ws oserved y dditionl IL- tretment (Figure c). Thus, locl IL- induces expnsion of ctivted Pmel- cells in vivo. Locl IL- reduced Treg ccumultion following doptive T-cell trnsfer Becuse peptide vccintion nd systemic IL- could increse Treg cells, we crefully nlyzed the frequency of Tregs in the spleen nd tumor t different times fter doptive T-cell trnsfer in our model (Figure 7). The frequency of Tregs in the spleen of B/mIL- mice ws significntly lower thn control groups t dy (P <.7; solute numer of cells,.3 versus.9 ), ut this difference ws no longer pprent t dy (Figure 7). In fct, the frequency of Treg ws higher in IL-- treted group t dy compred to control groups consisted with studies of IL- medited upregultion of IL- (solute numer of cells,.9 versus.7 ). B/mIL- tumor ering mice treted with dditionl IL- showed significnt decrese in Tregs t dy (P <.) ut not t dy (P <.77) spleen. In contrst, Tregs within the tumor microenvironment of B/ mil- tumor ering mice showed sustined reduction on dys nd (Figure 7, P <.8 for dy,.3 for dy ). Thy. IFNγ-APC c # Cells PBS PBS TCRvβ3Thy. + cells in tumor (%) pcdna IL- mil- mil- + IL- TCRvβ3-FITC PBS pcdna pcdna + IL- mil- mil- + IL- pcdna IL- mil- mil- + IL PBS % of IFNγ producing pmel- cells 3 Thy.-PE PBS pcdna pcdna + IL- pcdna P <.7 P <.39 P <.3 mil- mil- + IL Further, the frequency of Tregs in B/mIL- tumor ering mice treted with dditionl IL- showed significnt decrese t dys nd. The solute numer of Tregs lso decresed to 7 cells in mil- tumor compred to pcdna tumor with 97 cells t dy. Similrly, solute numer of Treg ws mintined low in IL- BrDu-FITC mil- mil- + IL- Figure Locl IL- enhnces therpeutic responses of doptively trnsferred T cells through expnsion of CD8 + T cells. () IL- induced ccumultion of Thy. + TCRvβ3 + T cells in tumor. The percentge of Thy. + TCRvβ3 + T cells is indicted in the ovl gte of ech dot plot. The plots shown were gted on smll lymphocytes infiltrting tumor. Br grph on the right shows the verge percentges of Thy. + TCRvβ3 + T cells nd the error r represents ± SDs. The r grph shows the % of verge IFN-γ producing cells. () Effector cell function ws nlyzed y production of IFN-γ y whole splenocytes y stimultion with gp peptide in vitro. The percentge of IFN-γ + Thy. + T cells is indicted in the ox gte of ech plot determined y intrcellulr cytokine stining nlyzed y flow cytometry. Br grph on the right shows the verge percentges of Thy. + IFN-γ + T cells nd the error r represents ± SDs. (c) IL- nd IL- induced tumor ntigen specific-t cells prolifertion in vivo. All Vβ3 + T cells were nlyzed for BrdU stining in lymphocyte popultions of whole splenocytes vol. 7 no. fe. 9

6 IL- Induces Effective Antitumor Immunity % of CD + CD + FOXP3 + T cells 7 3 P <. P <.7 PBS pcdna pcdna mil- +IL- Dy mil- +IL- % of CD + CD + FOXP3 + T cells Spleen 7 3 PBS P <.77 P <.7 pcdna pcdna +IL- Dy mil- mil- +IL- % of CD + CD + Foxp3 + T cells 8 P <. P <.8 PBS pcdna pcdna mil- +IL- Dy mil- +IL- % of CD + CD + Foxp3 + T cells Tumor 8 P <. P <.3 PBS pcdna pcdna mil- +IL- Dy mil- +IL- Figure 7 Locl IL- prevents the ccumultion of Tregs in the tumor microenvironment following doptive T-cell trnsfer. () Tretment of IL- nd comintion with IL- induced decrese in the frequency of Treg cells in spleen t dy while no difference ws oserved t dy. The percentge of Treg cells were from CD + T cells. () In contrst, Treg cells in the tumor displyed low frequency t dy nd mintined low t dy. The percentge of Treg is sed on CD + T cells of the smll lymphocytes in tumor single-cell suspension. the mil- tumor t dy to control pcdna tumor ( versus 89 cells). Consistent with untreted B/mIL--ering mouse dt (Figure c), the suppressive cpcity of the Tregs ws similr mong ll niml groups (dt not shown). These dt support the conclusion tht peptide vccintion nd systemic IL- did not increse the Treg frequency in vivo of mice hroring tumors engineered to secrete IL- nd lso suggest tht locl IL- my lock ccumultion of Tregs within the tumor microenvironment ltering the rtio of doptively trnsferred T cells nd Tregs. Discussion Immune recognition of tumors occurs in cncer ptients is documented y the frequent oservtion of T-cell infiltrtion into tumors nd hs the impct on prognosis. 9 The presence of tumor-infiltrting T cells lone, however, does not gurntee rejection of estlished tumors. Thus, the induction of tumorrective T cells nd their migrtion to sites of tumor growth is not sufficient for tumor rejection. Effector T cells must lso overcome fctors in the tumor microenvironment tht prevent immunologicl destruction of ntigenic tumors These fctors include the ccumultion of regultory cells (i.e., CD + Tregs, myeloidderived suppressor cells nd CD8 + tumor-ssocited mcrophges), high levels of suppressive cytokines nd growth fctors (i.e., TGF-β, IL-, nd VEGF) nd errnt expression of tumor ntigens, MHC complexes, costimultory (i.e., B7.) nd coinhiitory (i.e., B7-H) molecules y tumor nd/or strom cells. 9,3 38 The consequence of these fctors is tht infiltrting T cells my not e le to mintin effector functions, s evidenced y recent reports showing loss of the T-cell receptor (TCR)-zet signl-trnsducing chin in tumor-infiltrting T cells compred to peripherl lood T cells in cncer ptients. 39, Collectively, these studies imply tht tumors hror the ility to neutrlize effector T cells through diverse cellulr nd moleculr mechnisms even if such T cells enter the tumor microenvironment. In this study, we exmined the ntitumor effects of locl IL- using stly trnsfected B clone fter showing tht the tumor cells secrete functionl IL- ut does not ffect tumor growth kinetics in vitro. In fct, these tumor cells grew in vivo s well, ut locl production of IL- did result in significnt dely in tumor growth of the otherwise reltively nonimmunogenic B melnom (see Figure ). Although we do not know which ntigens re responsile for the delyed growth, the fct tht these responses were T cell medited is supported y the phenotypic chrcteriztion of the tumor-infiltrting T cells, which reveled n increse in CD37 + CD8 + T cells. These cells were lso considered true effector cells sed on intrcellulr cytokine stining for IL- nd IFN-γ (Figure 3). We lso oserved n increse in memory CD + nd CD8 + T cells in the spleen of mice hroring IL- secreting tumors, which further supports the notion tht tumor growth ws rrested through tumor-rective T-cell response. Similr effects on doptively trnsferred T cells ws seen with mice expressing locl IL- hving the gretest dely in tumor growth nd highest numer of tumor infiltrting effector CD8 + T cells. Significnt interest hs recently een lso focused on the potentil of IL- to ctivte NK cells nd dendritic cells therey promoting ntitumor ctivity. In contrst IL- downregultes NKGD expression in primry humn NK cells. Further studies focused on effect of locl production of IL- on NK nd NKT cell popultions nd Moleculr Therpy vol. 7 no. fe. 9 38

7 IL- Induces Effective Antitumor Immunity their contriution to tumor rejection is in progress. The effect of IL- on dendritic cells is lso somewht controversil tht its opposing effects on dendritic cell mturtion nd function hve een reported oth in humn nd murine models., A prior Phse I clinicl tril suggested tht IL- could induce ojective clinicl responses in smll suset of ptients ut lso reported tht the optiml dosing, schedule, nd routes of IL- dministrtion ws unknown. Our dt would support locl delivery or expression of IL- s possile strtegy for promoting CD8 + T cell immunity while minimizing toxicity nd the suppressive influence of the tumor microenvironment. A mjor ostcle to successful tumor immunotherpy is the presence of lrge numers of CD + CD + FOXP3 + Treg cells, especilly within the tumor microenvironment where such cells my directly nd indirectly inhiit tumor-specific CD8 + effector T cells. In this study, we found tht locl IL- secretion resulted in decresed ccumultion of Tregs y s much s % compred to tumors in control mice (Figure ). We lso oserved decrese in Tregs within the tumor ut not the spleen of mice hroring IL- secreting tumors treted y doptive trnsfer of ctivted Pmel T cells suggesting this my e more generl phenomenon of locl IL- secretion. An importnt oservtion ws tht the lowest numer of Tregs ws found in mice with tumors secreting IL- nd lso receiving systemic IL- fter T-cell trnsfer ecuse IL- is known to promote Treg ctivity in ptients with melnom nd renl cell crcinom, 9, locl delivery of IL- my e strtegy for locking the inhiitory effects of these cells on effector CD8 + T cells within the tumor microenvironment. In fct, mice with locl IL- secreting tumors treted with doptively trnsferred T cells nd IL- hd the highest numer of IFNγ-producing TCRvβ3 + Thy. + CD8 + T cells within the tumors. A recent in vitro study showing IL- selectively ntgonizes suppression of humn Tregs on CD + CD T cells ut not on CD8 + T cells suggest n dditionl mechnism y which IL- could hve direct effect on responding CD + CD T cells rther thn on Tregs themselves. Another importnt oservtion in our dt is the differentil effect of loclly produced IL- on Tregs in the spleen nd tumor microenvironment. Tregs were decresed erly (dy ) fter T-cell trnsfer in oth the spleen nd tumor of mice with B tumors secreting IL-. The frequency of Tregs returned to seline levels in the spleen y dy ut re-ccumultion in the tumor ws locked in mice whose tumors secreted IL- (Figure 7). Thus, it ppers tht Tregs re systemiclly repopulted while loclly produced IL- prevents ccumultion within the tumor microenvironment for longer periods of time, which my llow the expnsion of CD8 + effector T cells nd susequently ltering the effector to Treg rtio in the tumor microenvironment. In fct, the rtio of effector-to-tregs in the tumor of metsttic melnom ptients is linerly correlted with tumor necrosis underscoring the importnce of this rtio in determining therpeutic outcomes with immunotherpy. 7,8 Becuse IL- my promote Treg expnsion nd given recent evidence showing tht IL- nd IL- confer opposing effects on CD8 + T cells in doptive immunotherpy, it is noteworthy tht our dt suggest potentil synergistic effect etween locl IL- nd systemic IL- (ref. 9). This my not e surprising given tht IL- promotes erly CD8 + T cell ctivtion nd IL- supports secondry expnsion T cells nd prevents IL--induced poptosis. Thus, locl IL- secretion, through oth expnsion of effector CD8 + T cells nd reduced ccumultion of CD + Tregs, my e useful for overcoming the limittions of IL- s single-gent therpy or s djuvnt in doptive T-cell trnsfer regimens. In summry, we hve shown tht locl IL- secretion enhnces the therpeutic response of B melnom through promoting oth endogenous nd doptively trnsferred T-cell immunity. This likely occurs through prolifertion nd expnsion of effector nd memory CD8 + T cells nd y locking ccumultion of CD + Tregs within the tumor microenvironment. The delivery of IL- into estlished tumors my e novel strtegy for improving responses to IL-, vccintion nd doptive T-cell trnsfer pproches in the tretment of ptients with cncer. Mterils And Methods Animls. Pmel- TCR trnsgenic mice were otined from the Jckson Lortory (strin 3, Br Hror, ME) nd C7BL/ mice were otined from Chrles River Lortory (Wilmington, MA). These mice were housed in pthogen-free conditions t the Institute for Comprtive Medicine of Columi University ccording to pproved institutionl protocols. The B murine melnom cell line ws kindly provided y Dr Rphel Clynes, Columi University. Cloning of Murine IL-. The murine IL- gene ws cloned from PHActivted splenocytes y RT-PCR nd integrted into the mmmlin expression vector pcdna3 (Invitrogen, Crlsd, CA) y using primers for the PCR; forwrd primer 3 -ATGGAGAGGACCCTTGTCT- nd ckwrd primer 3 -CTAGGAGAGATGCTGATGAATC-. PCR conditions were s follows: min 9 C; 3 cycles (3 s 9 C, min 8 C, min 7 C); 7 min 7 C. The PCR product ws purified using PCR purifiction kit (Qigen, Vlenci, CA) nd cloned into the KpnI nd XhoI sites of the expression vector pcdna3 nd ws verified y DNA sequencing. Expression vector pcdna3/mil- ws linerized using BglII for trnsfection into B cells. A stle trnsfected B cell line expressing IL- ws generted y trnsfecting 3 cells with µg of liner plsmid y Lipofectmine. Trnsfected cells were selected y ntiiotic geneticin t the concentrtion of, µg/ml (Invitrogen) nd screened for expression of IL- y ELISA nd selected line ws denoted s B/mIL- nd control B cell line contining only pcdna vector is denoted s B/pcDNA. Detection of IL- y ELISA. ELISA ws performed to detect nd quntify the concentrtion of solule IL- produced y B/mIL- following the mnufcturer s recommendtion (Mouse IL- DuoSet, DY9, R & D system, Minnepolis, MN). Briefly, micro 9-well pltes were coted with highly purified cpture nti-il- nd incuted overnight t C. Pltes were wshed nd filtered superntnt of B/mIL- cells ws pplied nd incuted for hours. Bound IL- ws detected with iotin-conjugted detection nti-il- ntiodies followed y n enzyme-leled streptvidin, nd color development ws red y ELISA-plte reder t OD nm. Homogenous cell popultions were otined y limiting dilution nd clones with high expression of IL- were selected. The stle mil-- trnsfected cell line is referred to herefter s B/mIL-, nd control cell line B/pcDNA3. Prolifertion ssy y 3 [H]-thymidine incorportion nd CFSE leling. Primry 3-dy prolifertion ssy ws performed with mouse splenocytes in 9-well nti-cd3 precoted plte (Biocot; BD Biosciences, Sn Jose, CA). Antimouse CD8 ntiody ws dded t concentrtion of µg/ml to the pproprite wells. 3 [H]Thymidine ws dded to the cultures 8 hours efore hrvesting nd incorportion ws determined y scintilltion spectrometry using LKB Betplte counter 38 vol. 7 no. fe. 9

8 IL- Induces Effective Antitumor Immunity (Perkin-Elmer, Boston, MA). Men cpm of triplicte cultures nd the SD from the men were clculted. In some experiments, superntnts of B/mIL- cells were dded to responding cells t vrious concentrtions (mil- from to, pg/ml). Superntnt from control B/ pcdna3 ws used t the sme volume in prllel control cultures. For CFSE leling, cells t density of 7 cells per ml were stined for min t 37 C with µmol/l CFSE (croxyfluorescein dicette succinimidyl; Moleculr Proes, Crlsd, CA) in phosphte uffered sline with.% (vol/vol) ovine serum lumin. Rections were quenched y the ddition of n equl volume of % fetl clf serum followed y three wshes in phosphte uffered sline with.% (vol/vol) ovine serum lumin to ensure complete removl of free CFSE. Mixed lymphocyte prolifertion ssy. Regultory CD + CD + FOXP3 + T cells were enriched with Treg isoltion kit (Miltenyi Biotec, Auurn, CA) ccording to the mnufctures recommendtions. The purity of enriched Treg cells ws 8 9%. All ssys were performed in 9-well U-ottom pltes (BD Biosciences), precoted with ntihumn CD3 ( μg/ml) nd stimulted with solule ntihumn CD8 ( µg/ml) for 7 hours t 37 C nd % CO. Tregs were cocultured with CD + CD T cells t. 3 cells/well t : rtio. Cultures were then pulsed with 3 [H]Thymidine (Perkin-Elmer) for the lst 8 hours of culture, hrvested on glss fier filters, nd incorported rdioctivity mesured with liquid scintilltion counter (Wllc, Wlthm, MA). All experiments were done in triplicte. In vitro ctivtion of Pmel- cells. Pmel- splenocytes from trnsgenic mice were depleted of erythrocytes y hypotonic lysis nd cultured in RPMI supplemented with % het-inctivted FBS (complete medium) with 3 IU/ml rhil- (R&D systems, Minnepolis, MN) in the presence of μmol/l hgp 33 peptide, nd used on dys fter strt of the culture. The synthetic, H-D-restricted peptides hgp 33 were synthesized y New Englnd peptide LLG (Grdner, MA) to purity >99% y HPLC nd mino cid nlysis. Adoptive cell trnsfer, vccintion, nd cytokine dministrtion. C7BL/ mice t 8 week of ge were implnted SC with 3 B melnom cells secreting functionl IL-. To deplete endogenous T-cell popultions, C7BL/ tumor ering mice were sulethlly irrdited with Gy efore doptive trnsfer. When tumor size reches mm, in vitro-ctivted Pmel- splenocytes ( CD8 + Vβ3 + T cells) were trnsferred y til vein injection. Following doptive trnsfer of Pmel- T cells, mice (n = for ech group) were vccinted y SC injection with μl phosphte uffered sline/ifa emulsion contining μg of hgp 33 peptide nd injected IP with IU of recominnt humn IL- in phosphte uffered sline twice dily for totl of five doses. Tumor sizes were mesured every second dy with cliper s the product of two perpendiculr dimeters. BrdU leling in vivo nd detection. Mice were injected IP with mg BrdU (-romodeoxyuridine). Mice were killed fter hours nd BrdU incorportion ws nlyzed ccording to the mnufcturer s instructions (BD Biosciences). Cell surfce ntigens were stined with pproprite ntiodies, followed y fixtion nd permeiliztion. BrdU epitopes were exposed y digestion for hour t 37 C with DNAse I nd were detected with monoclonl ntiody to BrdU. Flow cytometry nd intrcellulr IFN-γ detection. On the dys indicted fter vccintion, mice were killed, nd spleen nd tumor were hrvested nd homogenized into single-cell suspension. Cells were leled with the following mas (BD Biosciences): FITC-conjugted nti-vβ3 (MR-3), PE-conjugted nti-thy. (HIS), PE-conjugted nti-cd (IM7), PE-conjugted nti-cdl (MEL-), nd PerCP-conjugted nti-cd8 (3-.7). Smples were nlyzed using FACSCliur flow cytometry nd CellQuest softwre. For intrcellulr stining, tumors were homogenized nd pulsed with μmol/l humn gp 33 nd Golgi-Stop (BD Biosciences) for 3. hours. Cells were then first stined for surfce mrkers followed y permeiliztion using the Cytofix/Cytoperm kit (BD Biosciences), nd stined with llophycocynin-conjugted nti- IFN-γ mas (XMG.). Sttisticl nlysis. 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