The microrna mir-31 inhibits CD8 + T cell function in chronic viral infection

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1 A rt i l e s The mirorna mir-3 inhiits CD8 + T ell funtion in hroni virl infetion Howell F Moffett, Adm N R Crtwright, Hye-Jung Kim, Jernej Gode, Json Pyrdol, Trmo Äijö 3, Gustvo J Mrtinez,6, Anjn Ro, Jun Lu, Todd R Golu, Hrvey Cntor, Arlene H Shrpe, Crl D Novin & Ki W Wuherpfennig, During infetion, ntigen-speifi T ells undergo tightly regulted developmentl trnsitions ontrolled y trnsriptionl nd post-trnsriptionl regultion of gene expression. We found tht the mirorna mir-3 ws strongly indued y tivtion of the T ell ntigen reeptor (TCR) in pthwy involving lium nd tivtion of the trnsription ftor NFAT. During hroni infetion with lymphoyti horiomeningitis virus (LCMV) lone 3, mir-3-defient mie reovered from linil disese, while wild-type mie ontinued to show signs of disese. This disese phenotype ws explined y the presene of lrger numers of ytokine-sereting LCMV-speifi CD8 + T ells in mir-3-defient mie thn in wild-type mie. Mehnistilly, mir-3 inresed the sensitivity of T ells to type I interferons, whih interfered with effetor T ell funtion nd inresed the expression of severl proteins relted to T ell dysfuntion during hroni infetion. These studies identify mir-3 s n importnt regultor of T ell exhustion in hroni infetion. The ontrol of pthogens nd ners requires proper funtioning of the dptive immune system. CD8 + T ells re ruil omponent of suh immune responses; they re le to undergo mssive popultion expnsion to produe lrge pool of effetor ells tht eliminte virusinfeted or trnsformed ells. CD8 + T ells ontinuously integrte mny distint signls through the T ell ntigen reeptor (TCR) s well s through o-stimultory, o-inhiitory, ytokine nd hemokine reeptors. These environmentl signls either promote pthogen lerne nd long-term memory or n result in immune-system dysfuntion nd T ell exhustion. These disprte outomes re dependent on hnges in multiple networks of genes enoding proteins tht ffet the survivl, prolifertion nd effetor funtion of ells. MiroRNAs (mirnas) re lss of smll non-oding RNAs tht regulte gene expression y trnsltionl repression nd mrna destiliztion. Deletion of the key mirna-iosyntheti enzyme Dier during thymi T ell development mrkedly diminishes the undne of CD8 + T ells in the periphery,3, nd deletion of Dier in mture CD8 + T ells impirs the effetor response of CD8 + T ells in vivo. Individul mirnas hve importnt roles in the tivtion of CD8 + T ells. The mir-7-9 luster regultes the prolifertion of CD8 + T ells fter tivtion nd shpes effetor nd memory differentition. Other mirnas shpe signling from TCRs nd ytokine reeptors. mir-6 is prt of negtive feedk loop indued y the TCR nd pro-inflmmtory ytokines tht inhiits tivtion of the trnsription ftor NF-κB 6,7. In ontrst, mir- enhnes pro-inflmmtory ytokine signling y repressing the signling inhiitor SOCS nd promotes the popultion expnsion of nti-virl CD8 + T ells y loking the nti-prolifertive effet of type I interferons 8,9. Altertions in mirna expression n lso t downstrem of ytokine signling in CD8 + T ells. For exmple, signling vi interleukin (IL-) nd inflmmtory ytokines drives the differentition of CD8 + T ells y repressing the expression of mir- nd mir-39 (ref. ). However, the funtion of most mirnas expressed y CD8 + T ells remins unknown. Here we report tht the expression of mir-3 ws strongly indued following the tivtion of T ells vi TCR signling through pthwys dependent on lium nd the trnsription ftor NFAT. The expression of mir-3 promoted T ell dysfuntion y enhning the expression of multiple inhiitory moleules, prtiulrly during exposure to type I interferons. During hroni virl infetion, mir-3 promoted the dysfuntion of CD8 + T ells nd impired virl ontrol in T ell intrinsi mnner. These results identify role for mir-3 in promoting the exhustion of CD8 + T ells. RESULTS TCR triggering indues sustined expression of mir-3 To glolly define mirna expression during T ell tivtion nd differentition, we used ed-sed ssy to profile mirnas in CD8 + T ells efore nd fter tivtion vi omplex of the TCR nd its invrint hin CD3 with n ntiody to CD3 (nti-cd3) nd n ntiody Deprtment of Cner Immunology & Virology, Dn-Frer Cner Institute, Boston, Msshusetts, USA. Deprtment of Miroiology nd Immunoiology, Hrvrd Medil Shool, Boston, Msshusetts, USA. 3 Deprtment of Informtion nd Computer Siene, Alto University Shool of Siene, Alto, Finlnd. L Joll Institute for Allergy nd Immunology, L Joll, Cliforni, USA. Brod Institute of MIT nd Hrvrd, Cmridge, Msshusetts, USA. 6 Present ddress: Deprtment of Miroiology nd Immunology, Chigo Medil Shool, Roslind Frnklin University of Mediine nd Siene, North Chigo, Illinois, USA. Correspondene should e ddressed to K.W.W. (ki_wuherpfennig@dfi.hrvrd.edu). Reeived 8 Novemer ; epted 7 April 7; pulished online My 7; orreted online June 7 (detils online); doi:.38/ni.37 nture immunology VOLUME 8 NUMBER 7 JULY 7 79

2 A rt i l e s to the o-stimultory reeptor CD8 (nti-cd8). These profiling dt reveled dynmi regultion of mirna expression during T ell tivtion (Fig., Supplementry Fig. nd Supplementry Tle ). Severl of the mirnas upregulted following tivtion, inluding mir-6, mir- nd mir-7 mir-8 from the mir-7-9 luster, hve estlished funtions in CD8 + T ells 9, while mny of the other upregulted mirnas do not hve nnotted funtions in T ells. Of prtiulr interest, mir-3 ws not expressed in totl thymoytes or nive CD8 + T ells, ut its expression ws sustntilly upregulted following the tivtion of CD8 + T ells (Fig. ). Consistent with the profiling dt, quntifition of mir-3 expression y quntittive PCR (qpcr) indited upregultion of >-fold in the expression of mir-3 in CD8 + T ells following stimultion with nti-cd3 plus nti-cd8 reltive to its expression in nive CD8 + T ells (Fig. ). We lso oserved upregultion of ~-fold in the expression of mir- 3 in CD + T ells following tivtion with nti-cd3 plus nti-cd8 nd in nturl killer (NK) ells following stimultion with the phorol ester PMA nd ionomyin, reltive to its expression in the orresponding unstimulted ell popultions (Fig. ). However, mir-3 expression ws not indued in CD9 + B ells following stimultion with n ntiody speifi for immunogloulin M nd CpG oligonuleotide (Fig. ). Kineti studies demonstrted rpid inrese in mir-3 expression in CD8 + T ells stimulted with nti-cd3 plus nti-cd8, with indution of ~-fold t 8 h fter tivtion reltive to the expression efore tivtion (Fig. ). Stimultion of nive OT-I CD8 + T ells (whih hve trnsgeni expression of n ovlumin (OVA)-speifi TCR) with dendriti ells (DCs) nd OVA peptide lso rpidly indued n inrese of >-fold in the expression of mir-3 reltive to its expression in unstimulted CD8 + T ells (Fig. d). These results indited tht mir-3 expression ws indued in tivted CD + nd CD8 + T ells, s well s in NK ells, with the highest expression in tivted CD8 + T ells. To investigte whether mir-3 expression ws mintined in tivted T ells, we quntified its expression nd weeks following stimultion. Nive CD8 + T ells isolted from OT-I mie were stimulted for 7 h with OVA peptide, followed y ulture with IL- or IL-. The expression of mir-3 remined high for weeks in CD8 + T ells ultured with IL- (Fig. e). In ontrst, its expression progressively deresed in CD8 + T ells ultured with IL-, lthough it remined higher t 7 d nd d in those ells thn in nive CD8 + T ells (Fig. e). Tretment of nive CD8 + T ells with the ytokines IL-, IL-7 or IL- in the sene of TCR stimultion did not indue the expression of mir-3 (Supplementry Fig. ), whih indited tht those ytokines were not suffiient to indue mir-3 expression. We lso investigted the expression of mir-3 ex vivo. Popultions of CD + or CD8 + effetor nd effetor-memory T ells, s well s entrl memory T ells, hd higher expression of mir-3 thn tht of their nive ounterprts; the highest expression ws oserved in tivted CD + CD8 + T ells (Fig. f). T ell tivtion indues degrdtion of Argonute proteins (whih re essentil for the ssemly nd funtion of the mirna-ontining RNA-indued silening omplex) nd remodeling of the mirna repertoire. We onfirmed indution of mir-3 expression in tivted T ells through the use of the smll nuleolr RNA sno3 s stndrd (Supplementry Fig. d f). These results indited tht inresed mir-3 expression ws feture of effetor CD + nd CD8 + T ell susets s well s memory CD + nd CD8 + T ell susets. The indution of mir-3 is dependent on lium signling The rpid indution of mir-3 fter T ell tivtion ples the gene enoding mir-3 (Mir3) mong the erly-response genes, mny of whih re trnsriptionlly tivted through the lium-nfat pthwy. Tretment of CD8 + T ells with ionomyin, lium ionophore, ws suffiient to indue mir-3 expression, leit t lower level thn tht indued y stimultion with nti-cd3 plus nti-cd8, while inhiition of lium signling with ylosporine A loked the indution of mir-3 following tretment with ionomyin or stimultion with nti-cd3 plus nti-cd8 (Fig. ). Expression (vi lentivirl vetor) of onstitutively tive NFAT ontining serine-to-lnine sustitutions of key residues within the regultory domin in CD8 + T ells indued expression of mir-3 to levels similr to those hieved with ionomyin tretment (Fig. ). Genome-wide nlysis of the inding of NFAT y hromtin immunopreipittion in CD8 + T ells stimulted with PMA nd ionomyin reveled three inding peks upstrem of the mir-3 hirpin on mouse hromosome (Fig. ), one of whih ws loted ner CpG islnd homologous to the promoter of the humn gene enoding mir-3 (MIR3HG) 3,. The inding of NFAT to these genomi elements ws not oserved in resting CD8 + T ells or in CD8 + T ells stimulted with PMA nd ionomyin nd lso treted with ylosporin A (Fig. ). These dt indited tht lium signling nd susequent NFAT trnsriptionl tivity were linked to the indution of mir-3 vi TCR tivtion in CD8 + T ells. Identifition of mir-3 trget genes in CD8 + T ells mirnas repress mny trget mrnas, ut urte predition of their trgets is hllenging due to the prtil omplementrity of mmmlin mirna mrna pirs nd limited understnding of the ftors tht regulte the inding of mirnas. Beuse regultion y mirna redues the undne of the mjority of trget mrnas, we used expression profiling 6,7 of OT-I CD8 + T ells trnsdued with lentivirl vetor expressing mir-3 nd enhned green fluoresent protein (egfp) (lenti-mir3), or ontrol vetor expressing egfp lone (lenti-egfp) to identify trgets of mir-3. Trnsdution of ells with lenti-mir3 resulted in signifint hnges in mrna expression reltive to tht of ells trnsdued with lenti-egfp (Fig. 3). The intertion etween mirnas nd repressed trget mrnas ours minly in the 3 untrnslted region (UTR) of the trget mrnas 8. The mrnas downregulted in CD8 + T ells trnsdued with lentimir3 reltive to their expression in CD8 + T ells trnsdued with lenti-egfp showed onsiderle enrihment for predited mir-3- inding sites (reltive to the undne of mrnas tht were expressed ut not regulted differentilly): 7% of mrnas with redued undne (68 of 99) ontined predited inding site in the 3 UTR (Fig. 3). We further vlidted trgets of mir-3 y loning their 3 UTRs downstrem of luiferse reporter vetor; 3 of these 3 UTRs resulted in lower luiferse tivity in the presene of exogenous mir-3 thn tht of ells trnsdued with luiferse vetor without n inserted 3 UTR (Fig. 3). Severl of these (Ppp6, Lts, Oxsr, Elvl (ref. 9) nd Stk) 3 hve een reported s trgets of mir-3, while eight mrnas (Psd, Shd, Ilf3, Coro7, R, Str3, Cdkn nd Ifi3) represented newly identified trgets of mir- 3 (Fig. 3). These results identified previously unknown trgets of mir-3 in tivted mouse CD8 + T ells. mir-3 promotes CD8 + T ell dysfuntion To investigte the funtion of mir-3 in vivo, we deleted Mir3 in the germline of C7BL/6 mie y rossing mie with loxpflnked Mir3 lleles (Mir3 fl/fl ) to mie expressing Cre reominse under the ontrol of the promoter of the gene enoding the glyoprotein Zp3 (Zp3-Cre) to generte Mir3 fl/fl Zp3-Cre mie (lled Mir3 / mie here; Supplementry Fig. 3). We lso deleted Mir3 onditionlly in T ells y rossing Mir3 fl/fl mie with mie in whih 79 VOLUME 8 NUMBER 7 JULY 7 nture immunology

3 A rt i l e s e Ativted CD8 + T ells mir-3 (reltive) mir-3 (reltive) mir- mir-3 mir-93 mir-3 mir-7 mir-98 mir-8 mir- mir-3 mir-6 mir-7 mir Expression (log ),. 8 CD3+CD8 (h) 3 Dy 7 Dy IL- IL- Thy B ell T ell DC DC-LPS f mir-3 (reltive) mir-3 (reltive) d mir-3 (reltive) 3 Cre is expressed under the ontrol of the T ell speifi Cd promoter (Cd Cre ; Mir3 fl/fl Cd Cre ). Ativtion of CD8 + ells from Mir3 / nd Mir3 fl/fl Cd Cre mie with nti-cd3 plus nti-cd8 demonstrted omplete loss of mir-3 expression reltive to tht of tivted wildtype or Mir3 fl/fl CD8 + T ells (Supplementry Fig. 3,). Mir3 / mie were orn t the expeted Mendelin rtios, displyed no ntomil ltertions nd hd ellulrity nd mture T ell suset omposition in the thymus, spleen nd peripherl lymph nodes similr to tht of wild-type mie (dt not shown). 3,. US CD + T ells CD + T ells At CD8 + T ells CD3+CD8 OVA DC NK ells B ells 6 8 Stimultion (h) N T T reg CD + m T em N T m T em CD8 + T ells Figure mir-3 is indued during T ell tivtion. () mirnas (middle) with n inrese in expression of more thn twofold following tivtion of CD8 + T ells for d with nti-cd3 plus nti-cd8, reltive to tht of unstimulted ells (dy ) (left), nd expression of those mirnas in totl thymoytes (Thy), B ells, T ells, one-mrrow-derived DCs (DC) nd LPS-treted DCs (DC-LPS) (right), presented s row-normlized reltive expression (log ). () qpcr nlysis of mir-3 in CD + or CD8 + T ells tivted (At) for h with nti-cd3 plus nti-cd8, NK ells tivted for h with PMA nd ionomyin, nd B ells tivted for h with n ntiody to immunogloulin M nd CpG; results re presented reltive to those of the unstimulted ounterprts (US). () qpcr nlysis of mir-3 in CD8 + T ells tivted for 8 h (horizontl xis) with nti-cd3 plus nti-cd8 (CD3+CD8); results re presented reltive to those t h. (d) qpcr nlysis of mir-3 in OT-I T ells tivted for 8 h (horizontl xis) with nti-cd3 plus nti-cd8 (CD3+CD8) or y o-ulture with OVA-peptide-pulsed DCs (OVA DC) (T ell/dc rtio, :) (key); results presented s in. (e) qpcr nlysis of mir-3 in OT-I CD8 + T ells tivted for 8 h y OVA-peptide-pulsed splenoytes, then treted for 7 or d (key) with IL- or IL- (horizontl xis); results re presented reltive to those of untreted ells. (f) qpcr nlysis of mir-3 in nive (CD lo CD6L + ) T ells (N), effetor memory (CD hi CD6L ) T ells (T EM ), entrl memory (CD hi CD6L + ) T ells (T CM ), regultory (CD + CD + GITR + ) T ells (T reg ), nd tivted CD8 + (CD8 + CD + ) T ells (CD + ), with ll CD + or CD8 + susets (horizontl xis) sorted ex vivo; results re presented reltive to those of nive T ells. Dt re representtive of one experiment (), three experiments (; error rs, s.d.) or two experiments ( f; error rs (d f), s.d.). mir-3 (reltive) Resting 6.8 Restim 6.8 Resting 6.8 Restim DMSO CyA US Iono CD3+ CD8 mir-3 hr:88,, 88,9, k CpG Islnd Ingenuity-pthwy nlysis of ll mrnas expressed differentilly in CD8 + T ells trnsdued with lenti-mir3 reltive to their expression in CD8 + T ells trnsdued with lenti-egfp reveled orreltions with pthwys linked to type I interferons, inluding ltertions in the expression of Ifn mrna (whih enodes the interferon IFN-α), nd Irf3 mrna nd Irf7 mrna (whih enode the trnsription ftors IRF3 nd IRF7, respetively) (Supplementry Tle ). We therefore nlyzed CD8 + T ells from wild-type nd Mir3 / mie y RNA-sed next-genertion sequening (RNA-seq) on dy 7 following in vitro stimultion with nti-cd3 plus nti-cd8 nd ulture in IL-, with or without stimultion with IFN-β for 8 h efore nlysis. Mir3 / CD8 + T ells hd signifintly higher expression of mrnas enoding T ell effetor moleules, inluding perforin (Prf) nd severl grnzymes (Gzmd, Gzm, Gzme, Gzmg nd Gzm), s well s osteopontin (Spp), thn tht of wild-type ells, with these hnges in gene expression eing more prominent mir-3 (reltive). TSS. k. k. k UT CA-NFAT Ctrl ve Figure mir-3 is indued y signling vi lium nd NFAT. () qpcr nlysis of mir-3 in CD8 + T ells stimulted for h with ionomyin (Iono) or with nti-cd3 plus nti-cd8 (horizontl xis) nd treted with vehile (DMSO) or ylosporin A (CyA) (key); results re presented reltive to those of unstimulted ells. () qpcr nlysis of mr-3 in T ells infeted with lentivirl vetor driving expression of onstitutively tive NFAT (CA-NFAT) or ontrol lentivirl vetor (Ctrl ve); results re presented reltive to those of untrnsdued CD8 + T ells (UT). () Chromtin-immunopreipittion nlysis of NFAT in CD8 + T ells tivted with nti-cd3 plus nti-cd8, followed y popultion expnsion for 6 d in medium ontining IL-, then left untreted (Resting) or re-stimulted for h with PMA nd ionomyin (Restim) (left mrgin). Below, enlrgement of three res of enrihment for NFAT- inding deteted in re-stimulted ells y the HOMER suite of tools for motif disovery. Top, positions of the pre-mir-3 hirpin (lue rrow, lotion nd diretion of trnsription), the CpG islnd, nd the predited trnsription strt site (TSS) of Mir3. Dt re representtive of two experiments (error rs (,), s.d.). nture immunology VOLUME 8 NUMBER 7 JULY 7 793

4 A rt i l e s Sore (oserved) Down (9) Luiferse tivity (reltive) Up (3) Sore (expeted) UTR Frequeny (reltive) following stimultion with IFN-β (Fig.,). In ddition, severl genes enoding produts known to promote T ell dysfuntion, inluding intrellulr metllothioneins (Mt nd Mt), whih ontriute to T ell dysfuntion in mouse tumors, s well s the trnsription ftor -Mf (Mf) nd the reeptor for prostglndin E (Ptger), whih ontriute to CD8 + T ell dysfuntion in model of hroni infetion with lymphoyti horiomeningitis virus (LCMV) lone 3 (refs.,,), hd higher expression in wild-type CD8 + T ells thn in Mir3 / CD8 + T ells (Fig.,). Consistent with those findings, gene-set enrihment nlysis identified LCMV Armstrong versus lone 3 s well s Effetor versus exhusted CD8 + T ell s mjor gene-expression signtures t h nd 8 h, respetively, fter stimultion with IFN-β (Fig. ). Vlidtion of those RNA-seq dt y qpcr indited tht Mf mrna nd Ptger mrna hd lower expression in Mir3 / CD8 + T ells thn in wild-type CD8 + T ells following tretment with IFN-β (Fig. d). The expression of mrna enoding perforin (Prf mrna) ws higher in Mir3 / CD8 + T ells thn in wild-type CD8 + T ells only following stimultion with IFN-β (Fig. d). Stimultion with IFN-β lso inresed the differenes in 3 6 mir-3 trgets (% predited) Control Stk Lts Psd Shd Ilf3 Coro7 R Ppp6 Str3 Cdkn Ifi3 Oxsr Elvl Ov Ef Down Null (9) Up Null (3) Figure 3 Mirorry profiling revels trgets of mir-3 in primry CD8 + T ells. () Mirorry nlysis of gene expression in CD8 + T ells trnsdued with lenti-mir3 reltive to tht of ells trnsdued with lenti-egfp (T sores): digonl lines indite expeted null distriution (middle line) nd signifine utoffs (top nd ottom lines); olors indite proes signifintly downregulted (lue) or upregulted (red), s determined y the SAM (signifine nlysis of mirorrys) tehnique; in prentheses, numer of upregulted proes (Up; top right) or downregulted proes (Down; ottom left). () Frequeny of predited mir-3 trget genes mong the 9 downregulted proes nd 3 upregulted proes in (key), nd mthing null distriutions generted from proes not expressed differentilly (Null). P <., up- or downregulted proe versus the orresponding null distriution (onesmple t-test). () Luiferin ioluminesene ssy of ells trnsdued with luiferse reporter gene ontining no 3 UTR (Control) or the 3 UTR of predited mir-3 trget genes (horizontl xis), in the presene of exogenous mir-3; results re presented reltive to those otined in the sene of exogenous mir-3. Eh symol represents n individul experiment. P =.8,.,.7,.,.,.3,.,.3..3,.3,.,. or. (left to right), versus ontrol reporter (unpired one-tiled Student s t-test). Dt re representtive of one experiment (,) or three experiments (; men + s.d.) d mrna (reltive) Mt Mf Vldlr Reds (fold) 6 8 P-vlue Spp R Gzme Sd Wdfy Mt Mt Mf Ptger e WT Mir3 / Grnzyme C (MFI) Il7r Spp Gzmd Prf 3,,, Mf Ptger Mt Il7r Mt Reds (fold) 6 8 p-vlue 8 IFN-β (h) Gzm Gzmd Spp 8 IFN-β (h) the expression of grnzyme-enoding mrnas s well osteopontinenoding mrna (Spp) in Mir3 / CD8 + T ells reltive to their expression in wild-type CD8 + T ells (Fig. d). Flow ytometry showed tht tretment with IFN-β lso resulted in greter undne of perforin nd grnzyme C in Mir3 / CD8 + T ells thn in wild-type CD8 + T ells (Fig. e). Immunolot nlysis indited only smll differenes etween Mir3 / CD8 + T ells nd wild-type CD8 + T ells in phosphoryltion of the trnsription ftor STAT following tretment with IFN-β (Supplementry Fig. d). Also, there were only smll differenes etween those ells in their expression of the trnsription ftors STAT, STAT nd IRF9 nd of SOCS (Supplementry Fig. e). Type interferons re importnt for the initil tivtion of CD8 + Prf mrna (reltive) Perforin (MFI) Gzme Gzm Enrihment sore Enrihment profile WT Mir3 / WT Mir3 /. Hits,,, Rnk in ordered dt set Mt Mt Mf Ptger Il7r Spp Gzmd Prf Figure mir-3 limits CD8 + T ell effetor progrms nd enhnes exhustion following stimultion with IFN-β. (,) RNA-seq nlysis of wild-type nd Mir3 / CD8 + T ells tivted with eds oted with nti-cd3 plus nti-cd8 nd then ultured for 7 d in IL-, then left unstimulted () or stimulted for 8 h with IFN-β (); results for Mir3 / ells re presented reltive to those of wild-type ells (red nd lue dots indite relevnt genes or genes with the most differentil expression (lels djent)). () Gene-set enrihment nlysis of ells t h (top) nd 8 h (ottom) fter stimultion with IFN-β, highlighting the LCMV Armstrong versus lone 3 progrm (top; normlized enrihment sore =.78; FDR q vlue, <.) nd Effetor versus Exhusted T ell progrm (ottom; normlized enrihment sore = 3.9; FDR q vlue, <.). (d) qpcr nlysis of genes enoding produts ssoited with T ell dysfuntion nd effetor-memory progrms (horizontl xes), ssessed in wild-type (WT) nd Mir3 / T ells (key) t h (left) nd 8 h (right) fter stimultion with IFN-β; results re presented reltive to those of the ontrol gene Gpdh. (e) Flow-ytometry nlysis of grnzyme C (left) nd perforin (right) in wild-type nd Mir3 / ells T ells (key) t h nd 8 h fter stimultion with IFN-β (horizontl xes); results re presented s men fluoresene intensity (MFI). P <., P <. nd P <. (unpired two-tiled Student s t-test). Dt re from one experiment ( ) or two independent experiments (d,e) with n = 3 ultures from three mie (men ± s.d. in d,e). 79 VOLUME 8 NUMBER 7 JULY 7 nture immunology

5 A rt i l e s T ells 6. However, mir-3 is expressed only following TCR tivtion nd might therefore regulte the effets of seondry or hroni exposure to type I interferons. In models of hroni virl infetion, ontinued exposure to type I interferons impirs T ell responses nd drives disese progression 7 9. To ddress whih trgets of mir-3 might regulte sensitivity to type I interferons, we trnsdued the 7678 mouse T ell hyridom with lentivirl vetors ontining short hirpin RNA (shrna) trgeting eh of four mir-3-regulted genes (Lts, Stk, Ppp6 nd Shd) or ontrol lentivirl vetor nd ssessed expression of the tivtion mrker CD69, whih is indued y type I interferons. Trnsdution of ells with lenti-mir3 or with shrna for silening the mir-3 trget Ppp6 enhned their sensitivity to IFN-β reltive to tht of ells trnsdued with the respetive ontrol lentivirl vetor (Supplementry Fig. ). Ppp6 is phosphtse tht regultes ytokine signling through the kinse Mp3k7 nd hs een linked to inhiition of type I interferon signling in high-throughput sreen 3 3. In ontrst, trnsdution of the 7678 T ell hyridom with shrna trgeting Lts, Stk or Shd hd no effet on responsiveness to IFN-β (Supplementry Fig. ). To onfirm those findings in primry T ells, we trnsdued Mir3 / OT-I T ells with lenti-mir3, shrna trgeting Ppp6 or the respetive ontrol lentivirl vetor. Trnsdution with lenti-mir3 or shrna trgeting Ppp6 inresed the sensitivity of CD8 + T ells to IFN-β reltive to tht of CD8 + T ells trnsdued with the respetive ontrol lentivirl vetor (Supplementry Fig. ). We lso mesured Ppp6 y immunolot nlysis of OT-I CD8 + T ells tivted with OVA peptide nd oserved pproximtely four-fold lower expression of Ppp6 in wildtype CD8 + T ells thn in Mir3 / T ells (Supplementry Fig.,d). These results suggested role for mir-3 in tuning the sensitivity of CD8 + T ells to type I interferon signling. mir-3 inhiits T ell responses to hroni infetion In dult mie, LCMV Armstrong strin uses n ute infetion tht is lered y dy 8 fter infetion, while LCMV lone 3 uses hroni infetion tht persists >9 d fter infetion. Both expression of the inhiitory reeptor PD- nd signling vi type I interferons re ritil for the hroni infetion of mie with LCMV lone 3 (refs. 33 3), while lokde of hroni signling vi type I interferons results in ontrol of infetion with LCMV lone 3 (refs. 3,3). We next investigted if mir-3 hs role in ontrolling the hroniity of infetion with LCMV lone 3. The initil disese ourse ws similr in wild-type mie nd Mir3 / mie infeted with LCMV lone 3, with ll mie displying disese symptoms t dys 9 fter infetion (Fig.,). Wild-type mie exhiited signs of hroni disese nd wsting, while Mir3 / mie showed no signs of disese nd hd reovered ~9% of their initil ody weight y dy fter infetion (Fig.,). We next sought to determine whether disese resolution ws ssoited with ontrol of the virus. During the ute phse of the infetion (dy 9 fter infetion), wild-type mie nd Mir3 / mie hd similr titers of irulting virus (Fig. ). However, y dy fter infetion, virl titers were signifintly (>.-log) lower in the serum of Mir3 / mie thn in tht of wild-type mie (Fig. ). We lso deteted lower levels of virl RNA in tissue smples from Mir3 / mie thn in those from wild-type mie t dy 3 fter infetion (Supplementry Fig. 6). These results suggested tht mir-3 impired the ontrol of infetion with LCMV lone 3. We next investigted whether erly disese resolution nd improved virl ontrol were ssoited with enhned nti-virl CD8 + T ell responses. The frequeny of CD8 + T ells positive for tetrmer (Tet) of LCMV glyoprotein (mino ids 33 ) in lood ws similr in Body weight (reltive) WT Mir3 / WT Mir3 / WT Mir3 / Dy 9 Dy Time fter infetion (d) Time fter infetion (d) Disese sore Figure Mir3 / mie undergo fster reovery nd show etter virl ontrol in hroni-lmcv-infetion model. () Body weight of wild-type mie (n = ) nd Mir3 / mie (n = 6) (key) infeted intrvenously with LCMV lone 3 ( 6 plque-forming units), ssessed dily efore (dy ) nd fter infetion (time, horizontl xis); results re presented reltive to originl ody weight (dy ). P =. (dy 3) nd P =.3 (dy 7) (unpired two-tiled Student s t-test). () Disese sores of wildtype mie (n = ) nd Mir3 / mie (n = 6) s in. P =. (dy 3) nd P =. 8 (dy 7) (unpired two-tiled Student s t-test). () Virl lod in the serum of mie s in (n = 3 per group), ssessed y plque ssy t dys 9 nd fter infetion (horizontl xis) nd presented s plque-forming units (PFU) per ml serum. Eh symol represents n individul mouse; smll horizontl lines indite the men (± s.d.). P =.8 (unpired two-tiled Student s t-test). Dt re representtive of two experiments (men ± s.d. in,). wild-type mie nd Mir3 / mie on dy 8 fter infetion with LCMV lone 3 (Fig. 6). However, on dy fter infetion, Mir3 / mie hd higher frequeny of Tet + CD8 + T ells in lood thn tht of wild-type mie (Fig. 6,). At dy fter infetion, more Tet + CD8 + T ells in the lood of Mir3 / mie thn in tht of wild-type mie expressed the effetor T ell mrker KLRG, nd fewer Tet + CD8 + ells in the lood of Mir3 / mie thn in tht of wild-type mie expressed PD- (Fig. 6 ). At dy 3 fter infetion, there ws greter frequeny of CD8 + T ells in the spleen of Mir3 / mie thn in tht of wild-type mie, n inrese driven y the popultion expnsion of CD + CD6L CD8 + effetor nd effetor-memory T ells (Fig. 6d). Tetrmer leling indited popultion expnsion of Tet + KLRG + CD8 + T ells nd Tet + KLRG CD7 CD8 + T ells in Mir3 / mie reltive to the size of those popultions in wild-type mie t 3 d fter infetion, while the frequeny of Tet + CD7 + CD8 + T ells in Mir3 / mie ws similr to tht of wild-type mie (Fig. 6e). We did not oserve differene etween wild-type mie nd Mir3 / mie in their CD + CD6L CD + effetor nd effetormemory T ells (Fig. 6f). Also, we did not detet differenes etween wild-type mie nd Mir3 / mie in their titers of LCMV-speifi ntiodies (Fig. 6g). CD + Foxp3 + regultory T ells undergo popultion expnsion during infetion with LCMV lone 3 nd inhiit the response of CD8 + T ells to LCMV 36,37. mir-3 is known to inhiit the differentition of CD + regultory T ells in humns through diret regultion of expression of the trnsription ftor Foxp3 nd inhiits the differentition of indued regultory T ells in mie through repression of the G-protein-oupled reeptor Gpr 38,39. We therefore investigted the role of mir-3 in regulting the expression of Foxp3 in mie. We found tht luiferse reporter linked to the mouse Foxp3 3 UTR ws not repressed y mir-3 (Supplementry Fig. 6). We lso did not oserve differenes in the frequeny of Foxp3 + CD + T ells isolted from the spleen of Mir3 / mie versus those isolted from tht of wild-type mie t seline or t dy 3 following infetion with LCMV lone 3 (Fig. 6h nd Supplementry Fig. 6). In mie hllenged with influenz virus (strin X3- gp33), weight loss, virl urden in the lungs nd the frequeny of influenz-virus-speifi CD8 + T ells were similr in Mir3 +/ mie nd Mir3 / mie on dy 8 following infetion (Supplementry Fig. 7); LCMV in serum (PFU/ml) nture immunology VOLUME 8 NUMBER 7 JULY 7 79

6 A rt i l e s WT Mir3 / PD- + Tet + CD8 + T ells (%) f CD + splenoytes (%) CD CD8 + Tet +.6 KLRG Dy 9 Dy E/EM CM N 7. 3 d CD8 + splenoytes (%) g Serum LCMV A (titer) this onfirmed tht mir-3 inhiited CD8 + T ell responses during hroni infetion ut not during ute infetion. T ell intrinsi effet of mir-3 in hroni LCMV infetion To isolte T ell intrinsi effets of mir-3, we infeted Mir3 fl/fl Cd Cre mie with LCMV lone 3. The disese ourse in Mir3 fl/fl Cd Cre mie ws similr to tht of Mir3 / mie following infetion with LCMV lone 3, with rpid resolution of disese, in ontrst to the WT Mir3 / WT Mir3 / Tet PD- E/EM N WT Mir3 / Tet + CD8 + T ells (%) 8 6 KLRG + KLRG Tet + CD8 + T ells/ spleen ( ) h Foxp3 + CD + ells (%) Dy 9 Dy WT Mir3 / WT Mir3 / 3 KLRG + CD7 + CD7 KLRG 8 6 WT Mir3 / WT Mir3 / PLN Spleen Figure 6 Enhned LCMV-speifi CD8 + T ell responses in mir-3 defiient mie. () Flow ytometry of CD8 + T ells from the lood of wild-type nd Mir3 / mie (left mrgin) on dy fter infetion with LCMV lone 3 (left), nd surfe expression of KLRG nd PD- on Tet + CD8 + T ells gted s t left (right). Numers djent to outlined res (left) indite perent CD + Tet + ells; numers in qudrnts (right) indite perent ells in eh. () Frequeny of KLRG + nd KLRG ells (key) mong Tet + CD8 + T ells in the lood of wild-type mie (n = ) nd Mir3 / mie (n = 6) (horizontl xis) on dys 9 nd (ove plots) fter infetion s in. P =. (unpired two-tiled Student s t-test). () Frequeny of PD- + Tet + CD8 + T ells in wild-type mie (n = 3) nd Mir3 / mie (n = 6) (key) on dys 9 nd (horizontl xis) fter infetion s in. P =. (unpired two-tiled Student s t- test). (d) Flow ytometry of effetor nd effetor-memory (CD + CD6L ) CD8 + T ells (E-EM), entrl memory (CD + CD6L + ) CD8 + T ells (CM) nd nive (CD CD6L + ) CD8 + T ells (N) (key) in the spleen of wildtype mie (n = ) nd Mir3 / mie (n = 6) (horizontl xis) on dy 3 fter infetion s in. P =.3 (unpired two-tiled Student s t-test). (e) Quntifition of totl KLRG +, CD7 KLRG nd CD7 + ells (key) mong Tet + CD8 + T ells in the spleen of mie s in d. P =. (unpired two-tiled Student s t-test). (f) Flow ytometry of CD + T ell popultions, gted s in d (key), in the spleen mie s in d. (g) ELISA of LCMV-speifi ntiodies (LCMV A) in the serum of wild-type nd Mir3 / mie (horizontl xis) on dy fter infetion with LCMV lone 3. (h) Frequeny of Foxp3 + CD + T ells in peripherl lymph nodes (PLN) nd spleen (horizontl xis) of wild-type nd Mir3 / mie (key) on dy 3 fter infetion with LCMV lone 3. Eh symol (,g,h) represents n individul mouse; smll horizontl lines (g) indite the men (± s.d.). Dt re representtive of two experiments (error rs ( f,h), s.d.). CM e Weight (reltive to d) PD- + Tet + CD8 + T ells Tet + CD8 + T ells in lood (%) WT 3 Time fter infetion (d) WT Dy 8 Mir3 fl/fl CD Cre Mir3 fl/fl CD Cre WT Mir3 fl/fl CD Cre 8 6 d Tet + CD8 + T ells per spleen ( ) Dy 3 3 hroni disese oserved in wild-type mie (Fig. 7). Mir3 fl/fl Cd Cre mie hd greter frequeny of irulting Tet + CD8 + T ells t dy fter infetion, nd lower expression of PD-, ompred with tht of wild-type mie (Fig. 7,). We lso oserved greter undne of totl Tet + KLRG + CD8 + T ells nd Tet + KLRG CD7 CD8 + T ells in the spleen of Mir3 fl/fl Cd Cre mie on 8 fter infetion reltive to the undne of suh ells in the spleen of wild-type mie (Fig. 7d). These results demonstrted tht mir-3 promoted CD8 + T ell dysfuntion nd the hroniity of infetion with LCMV lone 3 in T ell intrinsi mnner. Beuse CD8 + T ells progressively lose the ility to serete the ytokines IL- nd TNF nd, finlly, IFN-γ during hroni virl infetion, we ssessed the funtionlity of ntigen-speifi CD8 + T ells in wild-type, Mir3 / nd Mir3 fl/fl Cd Cre mie on dy 8 or 3 Disese sore WT Mir3 fl/fl WT Mir3 fl/fl CD Cre CD Cre Time fter infetion (d) Dy 8 WT Mir3 fl/fl WT Mir3 fl/fl CD Cre CD Cre KLRG + CD7-KLRG CD7 + KLRG + KLRG Figure 7 T ell intrinsi effet of mir-3 on nti-virl CD8 + T ell responses during hroni infetion with LCMV. () Body weight (left) nd disese sores (right) of wild-type nd Mir3 fl/fl Cd Cre mie (n = per group) infeted intrvenously with LCMV lone 3 ( 6 plque-forming units), ssessed dily efore (dy ) nd following infetion; weight is presented reltive to strting ody weight (dy ). P =.3 (dy ) nd P =. (dy ), ody weight; P =. (dy ) nd P =. (dy ), disese sore (unpired two-tiled Student s t-test). () Frequeny of KLRG + nd KLRG ells (key) mong Tet + CD8 + T ells in the lood of mie s in (n = per group), ssessed y flow ytometry t dys 8, nd 8 fter infetion. P =. (dy ) nd P =.3 (dy 8) (unpired two-tiled Student s t-test). () Frequeny of PD- + Tet + CD8 + T ells in the lood of mie s in (n = per group), ssessed y flow ytometry t dy fter infetion treted. P =. (unpired two-tiled Student s t-test). (d) Quntifition of KLRG +, CD7 KLRG nd CD7 + ells (key) mong Tet + CD8 + T ells in the spleen of mie treted s in (n = per group), ssessed y flow ytometry. Eh symol () represents n individul mouse. P =. (unpired two-tiled Student s t-test). Dt re representtive of one experiment (error rs, s.d.). 796 VOLUME 8 NUMBER 7 JULY 7 nture immunology

7 IFN-γ + ells per spleen ( ) IFN-γ + TNF + ells per spleen ( ) IFN-γ + ells per spleen ( ) IFN-γ + TNF + ells per spleen ( ) A rt i l e s 3 WT Mir3 / US GP33 GP76 NP396 fter infetion with LCMV lone 3. Splenoytes from infeted mie were pulsed ex vivo with the peptides GP33 nd GP76 (from LCMV glyoprotein) nd NP396 (from LCMV nuleoprotein), followed y intrellulr ytokine stining for IFN-γ, TNF nd IL- in CD8 + T ells. We found signifintly more IFN-γ + CD8 + T ells responsive to stimultion with the epitopes GP33 nd GP76 in the spleen of Mir3 / mie nd Mir3 fl/fl Cd Cre mie thn in tht of wild-type mie (Fig. 8,). We lso oserved more polyfuntionl IFN-γ + TNF + CD8 + T ells in the spleen of Mir3 / mie nd Mir3 fl/fl Cd Cre mie thn in tht of wild-type mie (Fig. 8,d). It hs een suggested tht mir-3 promotes the seretion of IL- y T ells,. However, we did not oserve differenes in the seretion of IL- from wild-type, Mir3 / or Mir3 fl/fl Cd Cre CD8 + T ells in response to stimultion with LCMV peptide (dt not shown), nd stimultion with PMA plus ionomyin indued similr frequeny of IL- + CD + T ells nd IL- + CD8 + T ells in ll mouse strins (Supplementry Fig. 8). Comprison of ytokine seretion in response to GP33 peptide versus totl Tet + T ells indited tht Mir3 / mie hd more funtionl effetor pool thn tht of wild-type mie: Mir3 / mie hd.9-fold more IFN-γ + CD8 + T ells thn wild-type mie hd, even fter orretion for lrger popultion of Tet + CD8 + T ells (Supplementry Fig. 8). These results indited tht mir-3 impired the funtion of CD8 + T ells in T ell intrinsi mnner during hroni infetion with LCMV lone 3. DISCUSSION Here we hve shown sustntil indution of mir-3 following T ell tivtion vi proess involving lium signling nd inding of NFAT to the lous enoding mir-3. During hroni infetion with LCMV lone 3, Mir3 / mie hd lrger nd d WT Mir3 / WT Mir3 fl/fl CD Cre WT Mir3 fl/fl CD Cre 6 US GP33 GP76 NP US GP33 GP76 NP396 US GP33 GP76 NP396 Figure 8 Enhned prodution of ytokines y LCMV-speifi CD8 + T ells in the sene of mir-3. Quntifition of IFN-γ + CD8 + T ells (,) or IFN-γ + TNF + CD8 + T ells (,d) mong splenoytes otined from wild-type mie (n = ) or Mir3 / mie (n = 6) t dy 3 fter infetion with LCMV lone 3 (,) or otined from wild-type nd Mir3 fl/fl Cd Cre mie (n = per group) t dy 8 fter infetion with LCMV lone 3 (,d), then left unstimulted (US) or pulsed for h with GP33, GP76 or NP396 peptide ( µg/ml) (horizontl xes) in the presene of refeldin A nd ssessed y intrellulr stining; results re presented s totl positive ells per spleen. Eh symol represents n individul mouse; smll horizontl lines the men (± s.d.). P =.8 (GP33) nd P =.6 (GP76) (); P =. (GP33) or P =.9 (GP76) (); P =.(GP33), P =.7 (GP76) or P =.8 (NP396) (); nd P =. (GP33) or P =.3 (GP76) (d) (unpired two-tiled Student s t-test). Dt re representtive of two experiments (,) or one experiment (,d). more funtionl ntigen-speifi CD8 + T ell response thn tht of wild-type mie, whih ws ssoited with reovery from infetion nd ontrol of virl replition. Gene-expression studies reveled tht the sene of mir-3 diminished the expression of genes enoding produts ssoited with T ell dysfuntion, inluding -Mf, the prostglndin E reeptor nd metllothioneins, nd lso enhned T ell effetor progrms. Those differenes ppered in prtiulr fter tretment with type I interferons. Our dt demonstrted tht mir-3 promoted CD8 + T ell exhustion during the hroni stge of infetion. Erly during virl infetion, type I interferons promote the priming of nti-virl CD8 + T ells. Tretment of mie with exogenous type I interferons t very erly time points following infetion with LCMV enhnes nti-virl CD8 + T ell responses nd improves virl ontrol. However, sustined prodution of type I interferons during infetion with LCMV lone 3 inhiits T ell medited lerne of the virus y suppressing prolifertion nd inresing the expression of PD- y T ells 3. Antiody lokde of the reeptor for IFN-α nd IFN-β during hroni infetion with LCMV hs een shown to promote virl lerne 3,3. Mehnistilly, lokde of tht reeptor is ssoited with redued expression of the PD- lignd PD-L. We showed tht shrna-medited knokdown of the mir-3 trget Ppp6 inresed the sensitivity of CD8 + T ells to type I interferons in vitro nd thus ws phenoopy of the effets of mir-3 expression in these ells. The phosphtse Ppp6 hs een identified s negtive regultor of interferon signling in mouse emryoni firolsts 9. Given tht mirnas typilly trget mny different mrnas, it is possile tht mir-3 trgets other mrnas (in ddition to Ppp6) tht ontriute to the regultion of interferon signling in T ells. Also, the ontriution of Ppp6 to the in vivo phenotype in Mir3 / mie remins to e determined. We did not oserve diminished CD8 + T ell response to ute infetion with influenz virus or LCMV in mir-3-defiient mie. While the indution of mir-3 ourred rpidly fter T ell tivtion, the in vivo effets of mir-3 defiieny were evident only fter sustntil dely. Tht pttern is onsistent with wht hs een oserved for other tivtion-indued mirnas, suh s mir- (ref. 8). In prt, this dely is proly due to the kinetis of mirna expression nd the reltive stility of the proteins enoded y the trgets of mirna. Argonute proteins, whih re the effetors of the mirnaindued silening omplex, re post-trnsriptionlly downregulted following T ell tivtion through uiquitintion nd protesomemedited degrdtion. This mehnism might sustntilly dely the mirna-medited regultion of mrna trgets. The expression of mir-3 inreses the undne of proteins tht ontriute to T ell dysfuntion. The undne of mrna enoding prostglndin E reeptor (Ptger) inresed in CD8 + T ells during the hroni stge of infetion with LCMV lone 3, prtiulrly for T ells with high expression of PD-. In vitro studies hve shown tht prostglndin E inhiits the funtion of ytotoxi T lymphoytes nd strongly suppresses the prodution of ytokines. Inhiition of the prodution of prostglndin E with n inhiitor of the ylooxygense COX- hs demonstrted synergy with lokde of PD- nd improved T ell funtion. Our dt showed tht expression of mir-3 ws le to inrese expression of the reeptor for prostglndin E in CD8 + T ells following stimultion with type I interferons. Delinetion of the dysfuntion signture of tumor-infiltrting CD8 + T ells t the single-ell level hs demonstrted tht metllothioneins sustntilly ontriute to T ell dysfuntion. Metllothionein-defiient mie hve signifintly lower tumor urden nd stronger CD8 + T ell funtion thn tht nture immunology VOLUME 8 NUMBER 7 JULY 7 797

8 A rt i l e s of metllothionein-suffiient mie. Both Mt nd Mt (whih enode metllothioneins) hd higher expression in Mir3 +/+ T ells thn in Mir3 / T ells. Mf (whih enodes the proto-onoprotein -Mf) hs lso een shown to e overexpressed y T ells during the hroni stge of infetion with LCMV lone 3 (ref. ). However, the expression of severl T ell effetor moleules ws higher in Mir3 / T ells thn in Mir3 +/+ T ells, prtiulrly following exposure to type I interferons. Suh dt explin the enhned funtionlity of CD8 + T ells in Mir3 / mie nd the improved linil ourse following infetion with LCMV lone 3. Consistent with our findings for mie, stimultion vi the TCR indues mir-3 expression in humn T ells 6. Bloking mir-3 expression in humn T ells might prove therpeutilly useful for oosting immune responses during hroni virl infetion. Moreover, mir-3 might lso ontriute to humn utoimmune onditions hrterized y strong signling vi type I interferons, suh s systemi lupus erythemtosus 7. Low expression of mir-3 y irulting T ells hs een ssoited with systemi lupus erythemtosus 8, whih suggests tht T ells might espe exhustion nd promote utoimmunity through low expression of mir-3. Finlly, the funtion of mir-3 might lso e relevnt to ner, in whih T ell dysfuntion represents sustntil lok to nti-tumor immunity. Methods Methods, inluding sttements of dt vilility nd ny ssoited ession odes nd referenes, re ville in the online version of the pper. Note: Any Supplementry Informtion nd Soure Dt files re ville in the online version of the pper. Aknowledgments We thnk R. Wey (St. Jude Children s Reserh Hospitl) for reominnt influenz virus ontining the LCMV GP33 epitope inserted into the neurminidse protein stlk region; J. Mirn for tehnil ssistne; nd the Dn Frer Cner Institute Moleulr Biology Core Fility for RNA sequening. Supported y the US Ntionl Institutes of Helth (RCA737 to K.W.W.; RDK6 nd RCA986 to C.N.; PAI699 to A.S.; nd RAI7386 to H.C.) nd the Cner Reserh Institute (A.N.R.C.). AUTHOR CONTRIBUTIONS H.F.M. nd K.W.W. designed the study nd disussed nd nlyzed dt; H.F.M., A.N.R.C. nd K.W.W. wrote the pper; H.F.M. nd A.N.R.C. performed experiments nd nlyzed dt; H.-J.K. nd J.G. helped with infetion experiments; J.P. performed immunolot nlysis; T.A., G.J.M. nd A.R. provided NFAT hromtin-immunopreipittion dt; J.L. nd T.R.G. performed mirna profiling; H.C., A.H.S. nd C.D.N. provided dvie on experimentl design nd dt interprettion; nd K.W.W. supervised the study. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Reprints nd permissions informtion is ville online t reprints/index.html. Pulisher s note: Springer Nture remins neutrl with regrd to jurisditionl lims in pulished mps nd institutionl ffilitions.. Wherry, E.J. et l. 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