The metabolic checkpoint kinase mtor is essential for IL-15 signaling during the development and activation of NK cells

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1 The metaoli hekpoint kinase mtor is essential for IL- signaling uring the evelopment an ativation of NK ells Antoine Marçais, Julien Cherfils-Viini 6,, Charlotte Viant 7,, Sophie Degouve, Séastien Viel,8, Aurore Fenis 7, Jessia Raillou, Katia Mayol, Armelle Tavares, Jaques Bienvenu 8, Yann-Gaël Gangloff,9, Eri Gilson 6,, Eri Vivier 7 & Thierry Walzer npg Nature Ameria, In. All rights reserve. Interleukin (IL-) ontrols oth the homeostasis an the peripheral ativation of natural killer (NK) ells. The moleular asis for this uality of ation remains unknown. Here we foun that the metaoli hekpoint kinase mtor was ativate an ooste ioenergeti metaolism after exposure of NK ells to high onentrations of IL-, whereas low oses of IL- triggere only phosphorylation of the transription fator STAT. mtor stimulate the growth an nutrient uptake of NK ells an positively fe ak on the reeptor for IL-. This proess was essential for sustaining NK ell proliferation uring evelopment an the aquisition of ytolyti potential uring inflammation or viral infetion. The mtorc inhiitor rapamyin inhiite NK ell ytotoxiity oth in mie an humans; this proaly ontriutes to the immunosuppressive ativity of this rug in ifferent linial settings. Natural killer (NK) ells are innate lymphoi ells (ILCs) involve in the immunosurveillane of aners an in the early ontrol of infetion y intraellular pathogens. They an kill ells reognize as targets through a attery of surfae reeptors an proue large amounts of interferon-γ (IFN-γ) upon ativation. The growing ILC family has een relassifie into three groups aoring to the pattern of ytokines they serete. In this lassifiation, NK ells are part of the group ILC suset (ILC). They express the ativating NK ell reeptor NKp6 (ref. ), a harateristi they share with the interleukin (IL-)-prouing suset ILC, whih is involve in gut innate immunity 6. In mie, NK ells evelop mainly in the one marrow (BM). Sequential evelopmental intermeiates, from immature to mature, an e efine on the asis of surfae expression of the tumor-nerosis fator superfamily memer CD7 an the integrin CD: CD lo CD7 hi NK ells (alle CD lo ells here), CD hi CD7 hi NK ells an CD hi CD7 lo NK ells (alle CD7 lo ells here) 7,8. Upon isruption of IL- signaling, the survival of NK ells is onsieraly reue an evelopment of the remaining ells is arreste at the CD lo immature stage, whih emonstrates a nonreunant role for this ytokine in the homeostasis an ifferentiation of NK ells 9. The trans-presentation of IL- y enriti ells ativate y ligans of Toll-like reeptors also ontrols the aquisition of effetor funtions y NK ells. How a single ytokine an have homeostati effets as well as inflammatory effets remains a hallenging question. It has een suggeste that ifferent quantities of IL- signaling inue grae responses y NK ells; this oul explain its funtional uality 6. In terms of signal transution, ligation of the reeptor for IL- is known to inue phosphorylation an ativation of the transription fator STAT. Deletion of STAT ompromises the survival of NK ells 7. Whether STAT is suffiient to inue the proliferation of NK ells an upregulate their ytotoxi potential, however, remains unknown. The link etween metaoli regulation an ativation of ells of the immune system has reeive onsierale attention 8. Following antigeni hallenge, T ells upregulate their metaolism to fae the iosyntheti emans, whih results in a hange from a quiesent state to a proliferative state. Conversely, the resolution of the response is aompanie y a shift of the T ells ak to a quiesent state. Metaoli regulation is also ouple to the aquisition of effetor funtions 9 an a migratory pattern of effetor ells. A entral moleule that integrates various metaoli, antigeni an inflammatory ues is the evolutionarily onserve serine-threonine kinase mtor ( mehanisti target of rapamyin ). mtor is part of two istint omplexes, mtorc an mtorc. mtorc ontrols translation mainly through phosphorylation of the translation-initiation fator eife ining protein EBP an the S6 riosomal kinase (S6K). S6K then phosphorylates the riosomal protein S6 an mtor itself (on Ser8). Moreover, mtorc also takes part in the ontrol of glyolysis y promoting expression of the transription fators HIF-α an -My, as well as y upregulating the expression of Centre International e Reherhe en Infetiologie International Center for Infetiology Researh, Lyon, Frane. IERM, U, Lyon, Frane. Eole Normale Supérieure e Lyon, Lyon, Frane. Université Lyon, Lyon, Frane. CNRS, UMR8, Lyon, Frane. 6 Institute for Researh on Caner an Aging, Nie, Nie University, CNRS UMR78 IERM U8, Faulty of Meiine, Nie, Frane. 7 Centre Immunologie e Marseille-Luminy, IERM U, CNRS UMR78, Aix Marseille Université, UM, Marseille, Frane. 8 Laoratoire Immunologie, Hospies Civils e Lyon, Centre Hospitalier Lyon Su, Lyon, Frane. 9 Laoratoire e Biologie Moléulaire et Cellulaire, CNRS, UMR 9, Lyon, Frane. Department of Meial Genetis, Arhet Hospital, Centre Hospitalier Universitaire of Nie, Nie, Frane. These authors ontriute equally to this work. Corresponene shoul e aresse to T.W. (thierry.walzer@inserm.fr). Reeive 9 August ; aepte June ; pulishe online 9 June ; oi:.8/ni.96 nature immunology VOLUME NUMBER 8 AUGUST 79

2 npg Nature Ameria, In. All rights reserve. nutrient transporters, an also takes part in the ontrol of lipi synthesis y ativating the transription fator SREBP, an in the ontrol of autophagy. mtorc phosphorylates the kinase Akt at Ser7, whih ompletes ativation initiate y phosphorylation of Akt at Thr8 y the phosphoinositie-epenent kinase PDK an allows export of memers of the Foxo family of transription fators from the nuleus. mtorc also ontrols ytoskeletal organization. Moreover, mtor phosphorylates itself at Ser8. Many stuies have explore the role of mtor an ownstream effetor moleules in T ell ifferentiation. In ontrast, there is a earth of information on the metaoli regulation of NK ells an the role of mtor in their physiology. We thus set out to haraterize the asi metaoli nees of NK ells an how they are linke to ifferentiation an priming following stimulation with IL-. We foun that as NK ells mature, they progresse to quiesene. That state was reverse upon stimulation with viruses or ytokines. These hanges were ontrolle y mtor; eletion of mtor reveale its ritial nonreunant role in the regulation of two key hekpoints of NK ell iology: proliferation in the BM an ativation in the periphery. Moreover, mtor was an essential omponent of signaling via IL- an was ativate upon exposure of NK ells to high onentrations of IL-. RESULTS Development an ativation regulate NK ell metaolism We monitore metaoli hanges uring the ifferentiation an ativation of NK ells. We etete sustantial ontration of ell size an granularity as the ells terminally ifferentiate (Supplementary Fig. a). Conversely, when NK ells were ativate in vitro with IL- (Supplementary Fig. ) or were ativate in vivo in mie given injetion of the syntheti RNA uplex poly(i:c) (the ligan of Toll-like reeptor an RIG-I) (Supplementary Fig. ) or in mie infete with influenza virus strain A/WSN/ HN (Supplementary Fig. ), the size an granularity of NK ells inrease sustantially. In metazoans, a ell s aility to aess nutrients epens on the expression of eiate transporters 8. Expression of the transferrin reeptor CD7 an amino-ai transporter CD98 (the heavy hain of the system L transporter), as well as gluose uptake (estimate y measurement of uptake of the fluoresent gluose analog -NBDG), erease y two- to threefol following the transition from CD hi CD7 hi to CD7 lo in the BM (Fig. a). There was lower expression of those markers (CD7 an CD98) in the spleen, ut their expression also erease upon ifferentiation (Fig. a). In vivo ativation in mie following injetion of poly(i:c) resulte in higher expression of CD98 an CD7, as well as in more uptake of gluose, Figure NK ell metaolism is regulate evelopmentally an after ativation. (a) Expression of CD7 an CD98 an inorporation of -NBDG y CD lo an CD7 lo NK ell susets from the BM or spleen, assesse y flow ytometry. Right, results for CD hi CD7 hi (DP) an CD7 lo NK ells, presente as mean fluoresent intensity (MFI) relative to that of the CD lo suset in the BM, set as %., not signifiant; P <., P <. an P <. (t-test). () Expression of CD7 an CD98 an inorporation of -NBDG y spleni NK ells otaine from mie 8 h after injetion of saline () or poly(i:c). (,) Real-time analysis of oxiative phosphorylation (OCR) () an aeroi glyolysis (ECAR) () in primary NK ells left unstimulate () or stimulate for 8 h or h with IL-, followe y injetion of (ashe vertial line) gluose (Glu; mm), oligomyin (Oligo; µm), FCCP (. M) plus pyruvate ( mm) (FCCP + pyr) an antimyin A ( µm) plus rotenone (. µm) (Ati + rot). Data are from four inepenent experiments (a; average an s.. of four mie), one experiment representative of three experiments () or two inepenent experiments (,; average an s.. of three mie assesse in tripliate). in spleni NK ells than that in resting NK ells from uninjete mie (Fig. ). To gain a more preise view of the metaoli hanges that our uring ifferentiation, we searhe for unique gene-expression signatures that were ifferent in CD lo NK ells an CD7 lo NK ells y using previously generate miroarrays of these susets 7 an software for gene-set enrihment analysis (Supplementary Tale ). Genes enoing moleules in pathways assoiate with ell growth ( ell yle an riosome ) were ownregulate in the CD7 lo suset. In ontrast, genes enoing moleules in pathways assoiate with the evelopment of a quiesent state ( aeroi sugar or fatty ai ataolism an autophagy ) were upregulate in the CD7 lo suset. Notaly, the CD7 lo suset showe higher expression of genes enoing several negative regulators of the mtor signaling pathway (ata not shown). We unertook a similar analysis to investigate the metaoli regulation assoiate with the ativation of NK ells. We use ata sets generate with NK ells ativate at. after infetion with mouse ytomegalovirus (MCMV) or after h of ativation in vitro with IL- (ref. ). The MCMV-eliite genes enoing moleules ientifie y metaoli terms were all also inue y treatment with IL- (Supplementary Tale ). Ativate NK ells ha higher expression of genes enoing moleules ientifie y terms assoiate with the ell yle, protein, lipi iosynthesis an arohyrate ataolism, onsistent with the oserve enhane ell growth an proliferation (Supplementary Fig. an ata not shown). To further haraterize the metaoli ativity of NK ells, we took avantage of Seahorse tehnology to analyze the oxygen-onsumption rate (OCR) an extraellular aiifiation rate (ECAR), whih are proportional to oxiative phosphorylation an aeroi glyolysis, respetively. The asal metaolism of spleni NK ells was very low for oth parameters (Fig.,). However, stimulation with IL- enhane the metaolism of spleni NK ells, sustantially inreasing the asal oxiative phosphorylation an aeroi glyolysis (as reveale y the aition of gluose). Aition of the mitohonrial unoupler FCCP to assess potential spare respiratory apaity reveale that stimulate NK ells were alreay eveloping their maximal respiratory ativity. Similarly, the glyolyti reserve (the ifferene etween the ECAR after the injetion of gluose an the ECAR after the injetion of oligomyin) was nearly zero, whih iniate that the maximal glyolyti a OCR (pmol O /min) CD7 CD98 -NBDG BM CD lo CD7 lo CD7 MFI (%) CD98 MFI (%) -NBDG MFI (%) Glu Oligo FCCP + pyr Ati + rot Time (min) ECAR (mph/min) CD lo BM DP CD7 lo CD lo DP CD7 lo CD7 CD98 -NBDG Glu Oligo FCCP + pyr Ati + rot Time (min) IL- (8h) IL- (h) 7 VOLUME NUMBER 8 AUGUST nature immunology

3 npg Nature Ameria, In. All rights reserve. a MFI (%) BM p-akt(s7) p-akt(s7) p-akt(t8) p-mtor(s8) p-akt(t8) p-mtor(s8) p-mtor(s8) p-ebp p-stat p-mtor(s8) p-ebp p-stat p-akt(s7) p-akt(t8) p-mtor(s8) p-mtor(s8) ativity was alreay reahe. Together these ata showe that the asal metaolism of spleni NK ells was very low ut was inuile y stimulation with IL-. mtor regulation upon ifferentiation an ativation The oservation of metaoli hanges uring NK ell ifferentiation prompte us to assess the phosphorylation status of mtor targets. We analyze the phosphorylation status of mtor itself (at Ser8 an Ser8), of ownstream targets of mtorc (EBP, at Thr7 an Thr6; an S6, at Ser an Ser6) an of a target of mtorc (Akt, at Ser7). In parallel, we assesse the phosphorylation of STAT at Tyr69 y the kinase Jak, ownstream of the reeptor for IL-, an the phosphorylation of Akt at Thr8 y the kinase PDK. We first measure the phosphorylation status of these proteins in BM an spleni NK ells at steay state an orrelate it to the expression of CD an CD7. Phosphorylation erease in a oorinate fashion as the ells mature (Fig. a), iniative of a progressive shutown in mtor ativity oth in the BM an in the spleen. We oserve a similar pattern for the phosphorylation of STAT (Fig. a). In ontrast, the phosphorylation of Akt at Thr8 erease only in the BM (Fig. a). Diret omparison of phosphorylation in BM an spleni NK ells showe signifiantly greater phosphorylation of Akt at Ser7 an of mtor at Ser8 an Ser8 on BM NK ells, espeially in the CD lo suset (Supplementary Fig. ). We then set out to measure phosphorylation events inue in vivo after stimulation with poly(i:c). The extent of the various phosphorylation events was upregulate in a oorinate fashion (Fig. ), whih iniate an overall inrease in the ativity of the pathway. As expete, sine injetion of poly(i:c) inreases the availaility of IL-, phosphorylation of STAT was signifiantly upregulate (Fig. ). In ontrast, phosphorylation of Akt at Thr8, whih is epenent on the ativity of phosphatiylinositol--oh kinase (PI()K) via PDK, was not signifiantly hange (Fig. ). Together these results emonstrate that mtor ativity was uner the ontrol of evelopmental an inflammatory signals in NK ells. Moreover, there was a lose parallel etween the metaoli status of NK ells an mtor ativity. MFI (%) p-ebp Isotype CD lo DP CD7 lo CD lo DP CD7 lo p-stat p-akt(s7) MFI (fol) p-akt(t8) p-ebp p-stat p-akt(s7) p-akt(t8) p-mtor(s8) p-mtor(s8) p-ebp p-stat p-mtor(s8) p-mtor(s8) mtor ativity is mainly uner the ontrol of IL- To ientify the signals ale to regulate mtor ativity in NK ells, we treate spleni NK ells for h in vitro with a wie range of stimuli. Among all the signals teste, IL- was the only one to eliit a sustantial inrease in phosphorylate S6 (Fig. a). In partiular, we oserve no effet on phosphorylate S6 after triggering of ativating or inhiitory reeptors on NK ells (Fig. a). We also note slight ut reprouile effets after exposure of ells to IL-8, alone or in omination with IL- (Fig. a). To etter haraterize the response to IL-, we expose splenoytes for h to inreasing onentrations of this ytokine. We measure in parallel the phosphorylation of S6 an that of STAT. Larger amounts of IL- were neee to ativate mtor (meian effetive ose of. ng/ml or. ng/ml for phosphorylate STAT or phosphorylate S6, respetively; Fig. ). To etermine whether IL- signaling is neessary in vivo to maintain physiologial mtor ativity, we treate wil-type mie with an F(a ) fragment that loks signaling via CD (the β-hain of the reeptor for IL-). Treatment with antioy to CD (anti-cd) le to a rapi erease in the steay-state phosphorylation of S6 in BM NK ells (Fig., left). We onfirme an overall erease in the ativity of this pathway y analysis of the phosphorylation of other moleules (Fig., right). As expete, we otaine similar results for phosphorylate STAT (Fig. ). To etermine if the inrease ioavailaility of IL- onsequent to the injetion of poly(i:c) was responsile for the inrease mtor ativity, we injete loking antioy to CD together with poly(i:c). We measure phosphorylation in spleni NK ells h later. The inrease in phosphorylate STAT was ompletely arogate (Fig. ), whih iniate omplete inhiition of signaling via IL-. The inrease in phosphorylate Akt an phosphorylate S6 was also signifiantly ampene y the antioy treatment (Fig. ). However, phosphorylation of EBP was not affete (Fig. ), whih suggeste that other signals in vivo might e ale to ompensate for the asene of IL-. Overall, these results showe that IL- was suffiient to ativate mtor in NK ells. It was also neessary for the maintenane of steay-state ativity uring the evelopment of NK ells. Finally, this MFI (fol) Figure mtor ativity ereases as ifferentiation progresses an is upregulate upon ativation. (a) Phosphorylation (p-) of Akt at Ser7 (p-akt(s7) or Thr8 (p-akt(t8)) an of mtor at Ser8 (p-mtor(s8)) or Ser8 (p-mtor(s8)), an of EBP, S6 an STAT, in CD lo, CD hi CD7 hi an CD7 lo BM NK ell susets (top); Isotype, isotype-mathe ontrol antioy. Below, umulative results (presente as in Fig. a). () Phosphorylation (as in a) in spleni NK ells otaine from mie 8 h after injetion of saline or poly(i:c) (top). Below, umulative results, presente relative to those of the resting population, set as. P <., P <. an P <. (t-test). Data are from one experiment representative of four (a, top) or three (, top) experiments or are from four (a, ottom) or three (, ottom) inepenent experiments (average an s.. of four (a) mie or three () mie). nature immunology VOLUME NUMBER 8 AUGUST 7

4 npg Nature Ameria, In. All rights reserve. Figure mtor ativity is mainly uner the ontrol of IL-. (a) Intraellular phosphorylate S6 in NK ells among splenoytes ulture for h on plates in meium alone (Me) or plates oate with rosslinking antioies to various reeptors (left group) or ytokines (right group). () Intraellular phosphorylate S6 an STAT in NK ells among splenoytes ulture for h with grae onentrations of IL- (horizontal axis); results are presente relative to the maximal response (%). () CD7 expression versus aunane of phosphorylate S6 in BM ells freshly isolate from mie given injetion of isotype-mathe ontrol antioy (Ctrl A) or anti-cd, analyze y flow ytometry (left); numers in quarants iniate perent ells in eah. Right, phosphorylate proteins in BM NK ells from mie treate as at left (presente as in Fig. a). () Phosphorylation of proteins in spleni NK ells otaine from mie h after injetion of saline or poly(i:c) with or without anti-cd (left). Right, phosphorylate proteins in spleni NK ells otaine from mie after injetion of saline or poly(i:c) along with isotype-mathe ontrol antioy or anti-cd (key); results are presente relative to those of the resting population treate with ontrol antioy, set as. P <., P <. an P <. (t-test). Data are from four (a) or three () inepenent experiments with one mouse in eah (average an s..) or are from one experiment representative of three experiments (,, left) or are from three inepenent experiments (,, right; average an s.. of three mie). ytokine was a hief ut not exlusive soure of extraellular signaling that le to hyperativation of mtor in an inflammatory ontext. mtor ontrols the maturation an numer of NK ells To estalish the physiologial relevane of mtor signaling in NK ells, we elete mtor in NK ells y rossing mie with loxp-flanke alleles enoing mtor (Mtor lox/lox ) 6 with mie expressing Cre reominase from the gene enoing the NK ell speifi ativating reeptor NKp6 (Nr; alle Nkp6-iCre here) 7. This ross resulte in Mtor lox/lox NKp6-iCre mie (alle NK-Mtor / mie here). We analyze the onsequenes of this eletion in NK ells in various organs (Supplementary Fig. ). We onfirme that in NK-Mtor / mie, mtor was inee asent from NK ells ut was present in normal amounts in the surrouning T ells (Supplementary Fig. a). In aition, NK ells were also smaller, an phosphorylation of mtor targets was reprouily iminishe (Supplementary Fig.,), whih emonstrate that mtor was funtionally asent. Despite that fining, the frequeny an numer of BM NK ells were normal in NK-Mtor / mie (Fig. a). In ontrast, NK ells almost ompletely isappeare from the peripheral organs of NK-Mtor / mie (Fig. a an Supplementary Fig. ). Phenotyping of the remaining NK ells in NK-Mtor / mie reveale a sustantial lok in ifferentiation in the BM at the CD lo -to-cd hi CD7 hi stage (Fig. an Supplementary Fig. a). This resulte in a shift in the istriution among the various NK ell susets in the spleen (Fig. an Supplementary Fig. a). Consistent with that oservation, expression of the maturation an senesene marker KLRG on NK-Mtor / NK ells almost ompletely isappeare (Fig. ). Next we onute a roa phenotypi analysis of spleni Mtor +/+ NKp6-iCre (alle here) NK ells an NK-Mtor / a ( MFI) Ctrl A Anti- CD CD7 7 8 p-akt(s7) p-stat < 8 6 < p-ebp Me NKp6 NK. NKGD Ly9D Ly9C, Ly9I NKGA IL- IL- IL-8 IL- + IL-8 IFN-β p-akt(s7) MFI (%) + anti-cd CD lo DP CD7 lo p-akt(s7) MFI (fol) p-ebp MFI (%) IL-7 TGF-β MFI (% of max) Ctrl A CD lo DP CD7 lo p-ebp MFI (fol) p-stat p-stat MFI (fol) NK ells (Fig. ). To ompensate for the ifferentiation ias that ourre in the asene of mtor, we fouse our analysis on the CD lo suset. The expression of markers suh as CXCR, CD7 an NK. was upregulate on spleni NK-Mtor / NK ells relative to their expression on NK ells (Fig. ). In ontrast, NK- Mtor / NK ells ha lower expression than i NK ells of ativating reeptors aquire uring maturation, suh as B, NKGD, Ly9D, Ly9G an Ly9H (Fig. ). The frequeny of ells expressing reeptors of the Ly9 family, however, was similar in the presene or asene of mtor, with the exeption of Ly9H (Supplementary Fig. ). Expression of the T-ox transription fators Eomes an T-et, whih are responsile for the maturation of NK ells 8, was also ownregulate in NK-Mtor / NK ells relative to suh expression in NK- Mtor WT/WT NK ells (Fig. ). As expete, expression of the nutrient reeptor CD7 an gluose uptake were ownregulate in the asene of mtor (Fig. ). Expression of CD an CD, moleules that onstitute the IL-Rβγ heteroimer of the reeptor for IL-, was halve in the asene of mtor (Fig. ). The funtional relevane of that last result is explore elow. Expression of the other markers analyze was not altere (Fig. ), whih iniate that eletion of mtor seletively affete part of the NK ell gene-expression program. We oserve a similar pattern for the BM (Supplementary Fig. ). The Nkp6 promoter also rives Cre expression in IL--prouing NCR + ILC ells in the gut. We thus investigate whether eletion of mtor affete this ell suset. Inee, NKp6 + ILC ells were asent from the gut of NK-Mtor / mie (Supplementary Fig. ), whih iniate a nonreunant role for mtor in the generation of this population. Overall, these results emonstrate that funtional mtor was require for the presene of mature NK ells in peripheral organs an seletively affete part of the NK ell gene-expression program. MFI (%) Anti-CD.. IL- (ng/ml) CD lo DP CD7 lo MFI (fol) p-stat MFI (%). CD lo DP CD7 lo Ctrl A Anti-CD 7 VOLUME NUMBER 8 AUGUST nature immunology

5 npg Nature Ameria, In. All rights reserve. a BM Liver Bloo NK-Mtor / CD + CD Optimal response to IL- epens on mtor We next investigate whether the asene of mature NK ells in the periphery of NK-Mtor / mie was a result of iminishe survival, erease generation of NK ells or oth. The viaility of NK- Mtor / spleni NK ells was iential to that of their ounterparts in the CD lo suset an CD hi CD7 hi suset an was only slightly lower than that of ells in the mature CD7 lo suset (Fig. a). Moreover, aute eletion of mtor in Mtor lox/lox NK ells treate in vitro with the ell-permeale Cre reominase fusion protein TATCre an then transferre ak into.7 KLRG + ells (%) NK ells ( 6 ) BM Liver Bloo NK-Mtor / MFI/ MFI NK-Mtor / CXCR CD7 NK-Mtor / wil-type mie in vivo i not impair their grafting apaity ompare with that Mtor +/+ ells treate with TATCre an transferre together into the mie (Supplementary Fig. 6a). Thus, iminishe viaility proaly ha only a minor role in the efet oserve. The CD lo -to-cd hi CD7 hi transition is preee in the BM y a proliferation phase 9. Deletion of mtor resulte in a threefol erease in the frequeny of proliferating ells, as etermine y inorporation of the thymiine analog BrU (Fig. ) or staining with the proliferation marker Ki67 (Supplementary Fig. 6). This proaly profounly affete the output from the BM an CD7 NK. DX Ly9EF CD98 KLRG CXCR NKGACE -NBDG CD7 CD69 BM Eomes EBP NKp6 CCR NKGD Tet 6 NK. CD Figure mtor ontrols the NK-Mtor maturation an homeostasis of WT/WT NK-Mtor / NK ells. (a) Expression of CD an NK-Mtor / 6 CD9 versus NK. y single-ell suspensions of BM, spleen, liver an loo (aove plots) from an NK-Mtor / mie, assesse y flow ytometry (left), an quantifiation KLRG of NK ells in those organs an loo BM (right). Numers in plots (left) iniate perent CD CD9 NK. + NK ells (outline areas). () Expression of CD7 an CD on gate NK ells from the BM an spleen of an NK-Mtor / mie, assesse y flow ytometry. Numers in quarants iniate perent ells in eah. () KLRG expression on spleni NK ells from an NK-Mtor / mie (left), an frequeny of KLRG + ells among NK ells from the BM an spleen (right). () Aunane of various markers (horizontal axis) in spleni CD NK ells, presente as the ratio of MFI for NK-Mtor / NK ells to that of NK ells (NK-Mtor / MFI/ MFI); gray horizontal line iniates a ratio of. P <., P <. an P <. (t-test). Data are from one of three experiments (a, left) or are from three inepenent experiments (a, right; average an s.. of three mie), are from one experiment of more than ten experiments with similar results () or are from three inepenent experiments (,; average an s.. of four () or three () mie). Figure mtor is neessary for the optimal fitness of NK ells, their proliferation in the BM an their maximal response to IL-. (a) Staining of an NK-Mtor / spleni NK ells with annexin V (Ann V) an the DNA-interalating ye 7-AAD (left), an frequeny of viale ells (negative for annexin V an 7-AAD) in CD lo, CD hi CD7 hi an CD7 lo NK ell susets from an NK-Mtor / mie (right). Numers in quarants (left) iniate perent ells in eah. () BrU staining of the CD lo suset of BM NK ells from an NK-Mtor / mie, assesse y flow ytometry (left), an frequeny of BrU + ells among those ells (right). Numers in plots (left) iniate perent BrU + ells. () Expression of CD an CD an aunane of phosphorylate STAT in CD lo BM NK ells from an NK-Mtor / mie; numers in plots iniate mean fluoresene intensity. () Aunane of a NK-Mtor /,7,69 CD 7-AAD 8 Ann V CD Viale ells (%) 8 6 NK-Mtor / CD lo p-stat 9 DP CD7 lo NK-Mtor / NK-Mtor / p-stats MFI (%) CD7 BrU. BrU + ells (%). IL- (ng/ml) CD Ly9D Ly9G CD Ly9H B NK-Mtor / phosphorylate STAT in NK ells among an NK-Mtor / splenoytes ulture for h with grae onentrations of IL-, presente relative to the maximal response (%). P <., P <. an P <. (t-test). Data are from one experiment representative of three experiments (a,, left) or are from three inepenent experiments (a,, right; average an s.. of three (a) or six () mie), are from one experiment representative of four experiments with one mouse in eah () or are from three inepenent experiments with one mouse in eah (; average an s..)... nature immunology VOLUME NUMBER 8 AUGUST 7

6 npg Nature Ameria, In. All rights reserve. Figure 6 Defetive ativation of NK ells in the asene of mtor. (a) Phosphorylation of Akt at Ser7 an of S6 in spleni ells from CD. + CD. + host mie given CD. + Mtor +/+ an CD. + Mtor lox/lox splenoytes pretreate for in vitro with TATCre, analyze y flow ytometry 8 h after injetion of saline or poly(i:c) (left), an quantifiation of results of ells from mie given injetion of poly(i:c) as at left, presente relative to those of the resting population, with lines linking the Mtor +/+ an Mtor / NK ells transferre together into the same host. Numers in plots (left) iniate mean fluoresene intensity. () Forwar satter (FSC) of NK ells from mie given injetion of poly(i:c) as in a (presente as in a, right). () Expression of CD7, CD98 an CD69 an staining of -NBDG in spleni NK ells from mie treate as in a (top; presente as in a, left), an quantifiation of results aove (ottom; presente as in a, right). () Intraellular expression of granzyme B (GzmB) in spleni NK ells from mie treate as in a (top), an quantifiation of results aove (elow; presente as in a, right). (e) Expression of intraellular Ki67, granzyme B an IFN-γ in Ly9H + an Ly9H spleni NK ell susets from an NK-Mtor / mie at ay, or 6. after infetion with MCMV, presente as quantifiation of Ki67 + NK ells, aunane of granzyme B (relative to that of uninfete mie) an frequeny of IFN-γ + NK ells. Numers aove olumns (left) iniate the population expansion of Ki67 + NK ells after infetion relative to that of NK ells in uninfete mie. Eah symol represents an iniviual mouse; small horizontal lines iniate 9 CD. + Mtor +/+ CD. + Mtor lox/lox the average (± s..). P <., P <. an P <. (t-test). Data are from one experiment representative of three inepenent experiments with one mouse in eah (a, left) or from three inepenent experiments with five mie (a, right), are from three inepenent experiments with six mie (), are from one experiment representative of three experiments (, left) or from three inepenent experiments with six mie (, right), are from one experiment representative of three experiments (, top) or from three inepenent experiments with five mie (, ottom) or are from four inepenent experiments with at least six mie (e). loke susequent ifferentiation of NK ells. NK ell proliferation is ontrolle y IL-. As note aove, NK-Mtor / NK ells ha aroun half the surfae expression of the β-hain (CD) an γ-hain (CD) of the reeptor for IL- that ells ha (Fig. an Supplementary Fig. 6). That translate into a lower steay-state amount of phosphorylate STAT in the BM of NK-Mtor / mie than in that of mie (Fig. an Supplementary Fig. 6). In support of the proposal of a role for mtor in the maintenane of optimal IL-R expression, aute eletion of mtor inue y treatment with TATCre le to a reprouile erease in the surfae expression of CD (Supplementary Fig. 6). Quantifiation of the phosphorylation of STAT in response to in vitro treatment with inreasing onentrations of IL- le to the fining that NK-Mtor / ells properly sense low onentrations of IL- an eome efetive only at onentrations ale to inue proliferation (Fig. an ata not shown). This suggeste that IL-R was limiting when NK-Mtor / NK ells were expose to an IL--rih environment that itate NK ell proliferation. Overall, these results showe that mtor efiieny ha a sustantial effet on NK ell proliferation in the BM ue to iret an iniret effets through the regulation of IL-R expression. The maturation lok oserve in NK-Mtor / NK ells was proaly a onsequene of this proliferation efet. a p-akt(s7) CD. + Mtor +/+ CD. + Mtor lox/lox e Ki67 + Ly9H + NK ells (log ) Ki67 + Ly9H NK ells (log ), 7,97,7 CD7 (MFI)......,8, 9,98 8 x x CD98 ( MFI) 8 6,,,7, p-akt(s78) MFI (fol) Time after infetion () x x.... -NBDG ( MFI) GzmB MFI Ly9H + NK ells (fol) GzmB MFI Ly9H NK ells (fol) p-6 MFI (fol) CD69 ( MFI) 7,76 P(I:C) 8,7 P(I:C) 6 CD. + Mtor +/+ CD. + Mtor lox/lox Time after infetion () Defetive ativation of NK ells in the asene of mtor To assess the role of mtor in NK ell ativation, we treate CD. + Mtor lox/lox splenoytes an CD. + Mtor +/+ splenoytes in vitro with TATCre an transferre them in vivo into CD. + CD. + host mie. Then later, we gave the host mie an injetion of poly(i:c) or saline an ollete spleens 8 h later. Flow ytometry with phosphorylation-speifi antioies emonstrate a onsequent erease in the atalyti ativity of mtor resulting from eletion of mtor (Fig. 6a). The inrease in ell size that results from ativation of NK ells was suppresse in mtor-efiient ells (Fig. 6), whih suggeste these ells were unale to upregulate their metaolism. Consistent with that iea, upregulation of expression of the nutrient reeptors CD7 an CD98, as well as uptake of -NBDG, was lunte, as was upregulation of expression of the ativation marker CD69 (Fig. 6). This was onsistent with the known role of mtor in metaoli regulation an nutrient uptake. As for effetor funtions, poly(i:c)-inue expression of granzyme B was halve in NK ells renere efiient in mtor (Fig. 6). We otaine similar results when we ompare the responses of NK- Mtor WT/WT mie an NK-Mtor / mie to poly(i:c) (Supplementary Fig. 7a ). We also monitore the egranulation of CD lo NK ells an seretion of IFN-γ from NK ells following stimulation in vitro with a omination of IL- an IL-8 or plate-oun agonisti antioies speifi for the ativating NK ell reeptors NKp6, NK. an MFI (fol) CD. + Mtor +/+ CD. + Mtor lox/lox IFN-γ + Ly9H + NK ells (%) IFN-γ + Ly9H NK ells (%) FSC GzmB,,6 MFI (fol) CD. + Mtor +/+ CD. + Mtor lox/lox P(I:C) P(I:C) GzmB CD. + Mtor +/+ CD. Mtor lox/lox CD. + Mtor +/+ CD. + Mtor lox/lox Time after infetion () NK- Mtor WT/WT NK- Mtor / 7 VOLUME NUMBER 8 AUGUST nature immunology

7 a MFI (fol) Ctrl IL- IL- + rapa IL- + Ku IL- + PP GzmB MFI (fol) 8 6 Ctrl IL- IL- + rapa IL- + Ku IL- + PP MFI (fol) GzmB MFI (fol) Ctrl Rapa + poly(i:c) Remaining ells (%) Ctrl Rapa + poly(i:c) MFI (fol) Me IL- IL- + rap ( nm) IL- + rap ( nm) IL- + Ku IL- + PP GzmB MFI (fol) Me IL- IL- + rap ( nm) IL- + rap ( nm) IL- + Ku IL- + PP npg Nature Ameria, In. All rights reserve. Figure 7 Aute inhiition of mtor arogates inflammation-inue priming. (a) Phosphorylate S6 an aunane of granzyme B in gate NK ells among splenoytes ulture for h in meium alone (ontrol (Ctrl)) or with IL- ( ng/ml) alone or IL- plus the mtor inhiitors rapamyin (IL- + rapa), Ku-679 (IL- + Ku) or PP (IL- + PP), analyze y flow ytometry. () Phosphorylate S6 an aunane of granzyme B in gate NK ells among splenoytes from untreate ontrol mie (Ctrl) or from untreate mie () or rapamyin-treate mie (Rap + poly(i:c)) given injetion of poly(i:c), analyze 8 h later y flow ytometry; results are presente relative to those of ontrol mie. () In vivo ytotoxiity of NK ells, from mie treate as in, towar missing-self target ells, presente as perentage of remaining target ells. () Phosphorylate S6 an granzyme B in gate NK ells among human peripheral loo mononulear ells ulture for 6 h in meium alone (Me) or with IL- alone or IL- plus or nm rapamyin, or Ku-679 or PP, analyze y flow ytometry. Eah symol (,) represents an iniviual mouse (n = 9 (ontrol), n = 9 (poly(i:c) or n = 8 (rapamyin plus poly(i:c))); small horizontal lines iniate the average (± s..). P <., P <. an P <. (paire t-test). Data are from five inepenent experiments with one mouse in eah (a; average an s..), four inepenent experiments (,) or three inepenent experiments (; average an s.. of nine iniviual onors). Ly9D. For these analyses, we use resting spleni NK ells or spleni NK ells preativate with poly(i:c). As expete, preativation with poly(i:c) le to a threefol inrease in the proportion of ells that egranulate in response to ytokines or antioies (Supplementary Fig. 7). In ontrast, although asal egranulation was fairly normal in the asene of mtor, stimulation with poly(i:c) was unale to enhane it (Supplementary Fig. 7). IFN-γ proution followe a similar pattern when we assesse the response to stimulation of ativating reeptors on NK ells (Supplementary Fig. 7). However, stimulation with IL- plus IL-8 rove normal IFN-γ responses, in terms of the frequeny of IFN-γ + ells (Supplementary Fig. 7) an quantity of IFN-γ per ell (ata not shown). The last fining emonstrate that NK-Mtor / ells were reative to IL- plus IL-8 an were not generally insensitive to stimuli. To assess the effet of mtor efiieny on a more physiologial NK ell response, we infete mie an NK-Mtor / mie with MCMV. During MCMV infetion, it is well esrie that early ativation of NK ells is meiate y a omination of various ytokines, while later uring the infetion, the interation etween Ly9H an the miroe-enoe protein m7 rives the proliferation an sustains the ativation of Ly9H + NK ells. We measure the proliferation of Ly9H + an Ly9H NK ell susets an the expression of granzyme B, IFN-γ, KLRG an the hemokine CCL y these ells, as well as egranulation of these ells, uring the ytokine-riven (early) an m7- an Ly9H-epenent (late) phases of the response. The ytokine-riven phase of the response was severely impaire in terms of expression of granzyme B (Fig. 6e, mile) an CCL (Supplementary Fig. 7e, mile) as well as egranulation (Supplementary Fig. 7e, right), in agreement with ata reporte aove (Fig. 6 an Supplementary Fig. 7,). In ontrast, we onfirme that IFN-γ seretion was unaffete y mtor efiieny (Fig. 6e, right). Unexpetely, at ay 6., the mtor-efiient Ly9H + NK ell suset expane -fol (Fig. 6e). However, that proliferation was less than the proliferation of mtor-suffiient NK ells (-fol), an the numer of mtor-efiient Ly9H + NK ells never reahe that of mtor-suffiient Ly9H + NK ells (ata not shown). Notaly, the inution of KLRG + NK ells following infetion with MCMV was impaire in the asene of mtor in oth the Ly9H + suset an the Ly9H suset (Supplementary Fig. 7e, left). Overall these results emonstrate that mtor efiieny profounly impaire the early ytokine-riven ativation of NK ells at multiple levels. In ontrast, proliferation inue y m7 an Ly9H was partly inepenent of mtor. The last result orrelate with the reporte inepenene of IL- of Ly9H-meiate NK ell proliferation uring MCMV infetion. Inhiition of mtor arogates inflammation-inue priming The results reporte aove suggeste that mtor efiieny might affet the ytotoxiity of NK ells. To test this hypothesis further an to eliminate onfouning evelopmental effets, we first stimulate wil-type NK ells with IL- in vitro an autely inhiite mtor with three pharmaologial inhiitors: rapamyin, an inhiitor of mtorc that is use linially ; an two ompetitors of ATP, Ku-679 an PP, that inhiit mtorc an mtorc (these inhiitors have een teste prelinially ),. To evaluate inhiitor effiay, we measure phosphorylation of S6. All three inhiitors onsistently impaire the inrease in the phosphorylation of S6 an granzyme B in response to IL-, with Ku-679 an PP showing higher poteny (Fig. 7a). As rapamyin is an inhiitor that is alreay in use linially, we fe mie an orally aministere form of rapamyin for efore treatment with poly(i:c) to test whether in vivo inhiition oul e ahieve. Inee, treatment with rapamyin resulte in a erease in the aunane of phosphorylate S6 an granzyme B (Fig. 7). Treatment with rapamyin i not seem to affet the trans-presentation of IL- to NK ells, sine the aunane of Akt phosphorylate at Ser7 an upregulation of CD69 expression were normal after suh treatment (Supplementary Fig. 8a,). We then iretly investigate whether treatment with rapamyin impaire the reativity of NK ells to missing-self targets. For this we transferre a mixture of wil-type target ells an target ells sensitive to NK ells an laking major histoompatiility omplex lass I (efiient in the gene enoing β -mirogloulin) into untreate (ontrol) mie or mie given injetion of poly(i:c), previously fe rapamyin orally or not given rapamyin. The killing of β -mirogloulin efiient target ells was signifiantly less effiient in rapamyin-treate mie than in their ontrol ounterparts (Fig. 7), whih emphasize the importane of mtor ativity in NK ell funtion. nature immunology VOLUME NUMBER 8 AUGUST 7

8 npg Nature Ameria, In. All rights reserve. Rapamyin is use in various therapeuti settings. We thus thought it important to investigate whether the priming of human NK ells was sensitive to inhiitors of mtor. To aress this, we stimulate human NK ells in vitro with IL- in the presene of mtor inhiitors an measure phosphorylation of S6 an the aunane of granzyme B. IL- inue an inrease in phosphorylate S6, whih, as expete, was suppresse y pharmaologial inhiitors of mtor (Fig. 7). The inhiitors of mtor also prevente IL--inue expression of granzyme B (Fig. 7), with little effet on the expression of perforin (Supplementary Fig. 8). These results showe that, as in the mouse, the mtor pathway ontrolle the ytotoxiity of human NK ells ownstream of IL-R. Overall, these results showe that mtor ativity in NK ells was inhiite in vitro an in vivo y pharmaologial inhiitors an that this inhiition resulte in iminishe priming an ytolyti funtionality of NK ells. DISCUSSION mtor is an integrator of various extraellular ues. Among all the signals we teste in vitro, we foun that IL- an IL-8, with or without IL-, were the only inuers of mtorc an mtorc ativity in NK ells. In ontrast, none of the other homeostati ytokines or agonisti antioies known to stimulate ativating reeptors on NK ells ha any effet on mtor ativity. This was unexpete, sine ligation of the T ell antigen reeptor plus the oreeptor CD8 is a potent inuer of mtor ativity 6 8, an the pathways inue y ligation of ativating reeptors on NK ells or ligation of the T ell antigen reeptor plus CD8 are similar 9. Our in vitro finings were onfirme y the fining that following infetion with MCMV, Ly9H-epenent responses were affete less than were ytokineepenent responses of the Ly9H NK ell suset. It thus seems that the Ly9H-epenent signal is ale to ypass the nee for mtor signaling. Proliferation in partiular was still important in the asene of mtor. Ly9H-meiate proliferation an e inepenent of IL- (ref. ); this may explain the nonessential role of mtor in this ontext. We hypothesize that Ly9H-epenent signals ompensate in part for the lak of IL- responsiveness in the inution of NK ell proliferation. In CD8 + T ells, several pathways onverge at the level of the phosphorylation of S6 to ontrol metaoli signaling. In partiular, pathways involving the mitogen-ativate protein kinases MEK an Erk ontriute to the phosphorylation of S6, an this might ompensate for mtor efiieny in NK ells. How mtor is ativate ownstream of IL- reeptor in NK ells remains also unsolve. So far, mtorc s moe of ativation remains elusive ut involves PI()K. As for mtorc, the exat sequene of events that lea to its ativation in T ells an in partiular the involvement of PI()K signal are still eate. However, a relevant fat is the oservation that NK ells ouly efiient in oth the pγ an pδ atalyti suunits of PI()K or efiient in pδ alone, have a phenotype partially onvergent with that of NK-Mtor / NK ells, aleit miler. Inee, a efet in PI()K signaling prevents the final maturation of NK ells an impairs their response to ativating reeptors on NK ells. The importane of PI()K in the responsiveness of NK ells to IL- was not teste in those stuies. This suggests that mtor ativity is ontrolle partly ut not exlusively y PI()K in NK ells. IL- is a pivotal ytokine that ontrols various aspets of NK ell iology. We foun that IL- ontrolle oth steay-state mtor ativity an ativation-inue mtor ativity in NK ells. Several groups have reporte that ifferent egrees of ioavailaility of IL- trigger istint effets on NK ells, ranging from the inution of ell survival at low onentrations to the inution of ativation an proliferation at higher onentrations,6,. The moleular asis of this phenomenon, however, remains unlear. Here we foun that the Jak-STAT an mtor pathways were ativate y ifferent oses of IL-. We thus hypothesize that the ifferent outomes resulting from varying strengths of IL- signaling are ue to the relative involvement of these two pathways. When triggere alone, STAT woul e expete to ontrol ell viaility 7,6, while ativation of oth pathways together woul e responsile for proliferation an ativation. This moel is supporte y the fining that eletion of mtor marginally affete the viaility of NK ells ut impaire their proliferation an ativation. The phenotype of NK-Mtor / NK ells orrelates well with the metaoli requirements of NK ells uring evelopment an ativation. Inee, NK ell ifferentiation was assoiate with the onset of ell quiesene, as shown y the erease in ell size an uptake of gluose onomitant with loss of the transmemrane nutrient reeptors CD7 an CD98. These hanges were regulate transriptionally, sine we oserve higher expression of genes enoing moleules in ategories of metaoli terms assoiate with erease ell yling an inrease ataolism along ifferentiation. We propose that eletion of mtor loks susequent ifferentiation y loking early steps of NK ell evelopment assoiate with proliferation. In this ontext, the evelopmental efet oserve in NK ells otaine from mie uner alori restrition might e ue to partial inhiition of mtor 7. Moreover, it has eome evient that metaoli regulators ontrol effetor T ell funtion 8. We mae a similar oservation with NK ells, sine eletion of mtor greatly iminishe their aility to e ativate in response to treatment with poly(i:c). Inee, espite their normal upregulation of the expression of early markers suh as CD69, NK-Mtor / NK ells ha lower expression of granzyme A an granzyme B. Moreover, the responses of mtor-efiient NK ells to the triggering of ativating reeptors on NK ells an in partiular egranulation were ompletely insensitive to injetion of poly(i:c). Therefore, mtor ontrols a key hekpoint for the ativation of NK ells. How mtor meiates these effets remains a fiel for future investigation. We foun that IL--meiate ativation of NK ells inue a sustantial inrease in glyolysis an respiration. These essential ioenergeti pathways proaly support the enhane energeti emans assoiate with ellular ativation an proliferation. Moreover, mtor is known to inrease mrna translation oth qualitatively an quantitatively through inativation of EBP (ref. 8). This oul e partiularly relevant for NK ells that ontain large amounts of untranslate mrna enoing effetor moleules suh as granzyme B or perforin. Finally, mtor also regulates rearrangements of the ytoskeleton through the kinase PKC-α (ref. 9), whih oul e important for ell-ell interations require for proper ativation of NK ells. For example, the role of the trans-presentation of IL- y enriti ells to NK ells uring inflammation is well esrie in the literature. Rapamyin is a linially approve inhiitor of mtorc that is use mainly in renal transplantation, in the treatment of renal an reast aners an in the treatment of afflitions suh as tuerous slerosis. Several erivatives of rapamyin an ATP-ompetitive inhiitors of the ative site of mtor have also een evelope an teste linially. mtor is also viewe as a potential target for antiaging therapy, an some of the aforementione inhiitors oul e use on healthy patients. Given our results otaine with mie, we teste the effets of some of these inhiitors on the ytotoxiity of NK ells. Suh treatment resulte in inhiition of the inution of granzyme B following stimulation with IL- in vitro. In vivo treatment with Sirolimus (a linial form of rapamyin) notiealy 76 VOLUME NUMBER 8 AUGUST nature immunology

9 npg Nature Ameria, In. All rights reserve. erease the ytotoxiity of NK ells towar missing-self target ells. Notaly, these results oul e translate to humans, sine ativation of NK ells from healthy onors was inhiite y inhiitors of mtor; this emphasizes the evolutionarily onserve role of this kinase in the ontrol of NK ell ytotoxiity. These results oul have roa impliations in the esign of therapies that target mtor. In summary, our finings have reveale mtor as an essential part of the IL- signaling pathway in NK ells that ontrols two key hekpoints of their iology: evelopment in the one marrow an ativation in the periphery. Moreover, we have expane to NK ells the well-oumente role of rapamyin as an immunosuppressant of aaptive immunity. Given the frequent therapeuti use of inhiitors of mtor, our finings might have iret linial appliations. Methos Methos an any assoiate referenes are availale in the online version of the paper. Note: Any Supplementary Information an Soure Data files are availale in the online version of the paper. Aknowlegments We thank the Plateau e Biologie Expérimentale e la Souris, an the flow ytometry faility of the SFR Biosienes Gerlan an of Institute for Researh on Caner an Aging, Nie (the Cytome platform fune y the CG6, IERM an FEDER). Supporte y the FINOVI founation (T.W. laoratory), Agene Nationale e la Reherhe (for the T.W. laoratory), the European Researh Counil (ERC-Stg 8 for the T.W. laoratory an a THINK Avane Grant for the E.V. laoratory), Institut National e la Santé et e la Reherhe Méiale (T.W. an E.V. laoratories), Centre National e la Reherhe Sientifique (T.W. an E.V. laoratories), Université Claue Bernar Lyon (T.W. laoratory), Eole Normale Superieure e Lyon (T.W. laoratory), the Ligue ontre le Caner ( équipe laelisée ; E.G. an E.V. laoratories) an Aix-Marseille University to Centre Immunologie e Marseille Luminy (E.V. laoratory). 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Marçais, A. et al. Regulation of mouse NK ell evelopment an funtion y ytokines. Front. Immunol., (). nature immunology VOLUME NUMBER 8 AUGUST 77