Transcription factor Foxo3 controls the magnitude of T cell immune responses by modulating the function of dendritic cells

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1 Trnscription fctor Foxo3 controls the mgnitude of T cell immune responses by modulting the function of dendritic cells 9 Nture Americ, Inc. All rights reserved. Anne S Dejen, Dniel R Beisner,4, Irene L Ch en, Ynn M Kerdiles, Ann Bbour, Kren C Arden, Diego H Cstrillon 3,4, Ronld A DePinho 3 & Stephen M Hedrick Foxo trnscription fctors regulte cell cycle progression, cell survivl nd DNA-repir pthwys. Here we demonstrte tht deficiency in Foxo3 resulted in greter expnsion of T cell popultions fter virl infection. This exggerted expnsion ws not T cell intrinsic. Insted, it ws cused by the enhnced cpcity of Foxo3-deficient dendritic cells to sustin T cell vibility by producing more interleukin 6. Stimultion of dendritic cells medited by the coinhibitory molecule CTLA-4 induced nucler locliztion of Foxo3, which in turn inhibited the production of interleukin 6 nd tumor necrosis fctor. Thus, Foxo3 cts to constrin the production of key inflmmtory cytokines by dendritic cells nd to control T cell survivl. Foxo trnscription fctors guide the cellulr response to growth fctors, nutrients nd stress. This informtion is encoded s post-trnsltionl modifictions, referred to s the Foxo code, tht govern Foxo intrcellulr locliztion, cofctor ssocitions nd trnscriptionl ctivity. The resulting progrm of gene expression regultes cell cycle rrest, repir, poptosis nd utophgy nd mny other spects of cellulr homeostsis. In mmmls, four Foxo members hve been identified. Foxo (FKH nd FKHR), Foxo3 (FKHRL; A94) 3, nd Foxo4 (AFX) 4 re widely expressed nd similrly regulted, wheres expression of Foxo6 (ref. ) is confined to specific structures of the brin nd is subject to distinct regultory mechnisms. Insulin, insulin-like growth fctor nd other growth fctors induce ctivtion of phosphtidylinositol-3-oh kinse nd the kinse Akt, which phosphorylte three Foxo mino cids 6. These modifictions result in the ssocition of the Foxo protein with the dptor protein nd the nucler exclusion nd eventul degrdtion of Foxo3 (refs. 7 9). This process cn be ctively opposed by stress-induced signls tht ctivte the Jnk mitogen-ctivted protein kinse, which result in Foxo3 nucler locliztion.foxofctortrgetspecificitycn be further refined by the SIRT decetylse,. Given their involvement in coordinting cellulr growth, prolifertion nd survivl, we predicted tht Foxo fctors would be centrl to the highly dynmic, infection-medited expnsion nd contrction of ntigen-specific T cell popultions. Indeed, Foxo ctivity is regulted by signling by the T cell ntigen receptor (TCR) nd CD8, s well s by cytokines such s interleukin (IL-) 3, IL-3 (ref. 4) nd IL-7 (refs.,6). These stimuli result in the phosphoryltion of Foxo by Akt, the kinse Sgk or inhibitor of trnscription fctor NF-kB (IkB) kinse, nd in Foxo3 nucler exclusion 7. Withdrwl of growth fctor cuses dephosphoryltion of nucler Foxo3, binding of Foxo3 to the promoters of the genes encoding the propoptotic molecules Bim nd Pum, induction of trnscription of those genes, nd T cell poptosis 8. In contrst, enforced expression of constitutively nucler form of Foxo3 in T cell lines cuses cell cycle rrest 3. Furthermore, Foxo3 is involved in the persistence of CD4 + centrl memory T cells in mice nd humns 9. Published studies hve shown spontneous T cell ctivtion, lymphoprolifertive disese nd orgn infiltrtion in Foxo3 Trp mice,which hve mutted Foxo3 llele generted by gene-trp technology.such studies indicte n importnt function for Foxo3 in immune regultion, lthough the underlying moleculr mechnisms re not understood. In ddition, Foxo3 regultes superoxide dismutse in dendritic cells (DCs); this signling pthwy my ffect the suppressive or stimulting chrcteristics of plsmcytoid DCs,. In this study, we sought to determine whether Foxo3 is involved in the quiescence of cells of the immune system nd the dynmics of T cell popultion expnsion nd contrction. We found tht in response to LCMV infection, Foxo3 deficiency cused superbundnt expnsion of ntigen-specific T cell popultions. However, contrry to our expecttions, this phenotype ws not intrinsic to T cells but insted rose from ltered stimultory properties of Foxo3-deficient DCs. Foxo3 deficiency resulted in DC-specific higher production of Moleculr Biology Section, Division of Biologicl Sciences, nd Deprtment of Cellulr nd Moleculr Medicine, University of Cliforni, Sn Diego, Sn Diego, Cliforni, USA. Ludwig Institute for Cncer Reserch, University of Cliforni, Sn Diego School of Medicine, L Joll, Cliforni, USA. 3 Center for Applied Cncer Science, Belfer Institute for Innovtive Cncer Science, Deprtments of Adult Oncology, Medicine nd Genetics, Dn-Frber Cncer Institute, Hrvrd Medicl School, Boston, Msschusetts, USA. 4 Present ddresses: Genetics Institute of the Novrtis Foundtion, Sn Diego, Cliforni, USA (D.R.B.) nd Deprtment of Pthology, University of Texs, Southwestern Medicl Center t Dlls, Dlls, Texs, USA (D.H.C.). Correspondence should be ddressed to S.M.H. (shedrick@ucsd.edu). Received September 8; ccepted 9 Mrch 9; published online April 9; doi:.38/ni.79 4 VOLUME NUMBER MAY 9 NATURE IMMUNOLOGY

2 9 Nture Americ, Inc. All rights reserved. Tble Leukocyte subsets Spleen B6 B6 FVB FVB Foxo3 / CD4 +. ±. 7.3 ± ±. 3. ±.9 CD ± ±.3 3. ±. 4.9 ±.9 B ± ± ± ±. Gr- hi CDb hi.4 ±. 6. ±.*. ±..4 ±.3* Lymph node CD4 +.3 ± ±.. ±..8 ±.4 CD ±. 7.3 ±.7. ±. 7. ±.4 B ±.4.4 ±. 4. ±. 3.6 ±.7 Results re presented s the number of cells 6 (± s.e.m.). *, P o., wild-type versus Foxo3-deficient mice (two-smple t-test ssuming equl vrinces). Dt re representtive of two experiments with five to six mice per group. IL-6. Nucler locliztion of Foxo3 induced by signls initited by cytotoxic T lymphocyte ssocited ntigen 4 (CTLA-4; A76) suppressed Toll-like receptor induced production of IL-6. Our results collectively emphsize the importnce of Foxo3 in restrining the production of inflmmtory cytokines by DCs nd T cell vibility. RESULTS No spontneous ctivtion of Foxo3-deficient T cells To exmine the function of Foxo3 in the immune system, we used two independently derived Foxo3-null strins distinct from the Foxo3 Trp mutnt. One strin, clled here, ws bckcrossed to the C7BL/6 strin, wheres mice of the other strin, clled Foxo3 / here, were mintined s congenic FVB mice. Although we detected strinspecific differences, deletion of Foxo3 hd no effect on the proportion of T cells or B cells in the spleens nd lymph nodes of 6- to -weekold mice (Tble ). However, we noted greter proportion of Gr- hi CDb hi cells, which include grnulocytes nd mcrophges, in the spleens of both Foxo3-deficient strins. We detected neither lymphocytic orgn infiltrtion nor lymphdenopthy 3 (dt not shown) in 6- to -week-old Foxo3-deficient mice. However 3- to 6-month-old Foxo3-deficient mice hd enlrgement of the spleen CD44 CD69 B6 B6 FVB FVB Foxo3 / 7. ±.6 7 ±. 4. ± ±. CD6L 4. ± ±. ± ±.3 CD4 CD8 CD69 B6 B6 FVB FVB Foxo3 / 3. ± ±. 3.4 ±..8 ±.4 CD ± ±.3 8 ±.8 8. ±. CD8 nd more erythrocyte progenitors (Ter9 + ; dt not shown). Tht observtion probbly reltes to the shorter erythrocyte lifespn nd lower rte of erythrocyte mturtion in Foxo3-deficient mice reported before 4. Next we mesured the expression of CD69, CD44 nd CD6L (L-selectin) on T cells from Foxo3-deficient mice nd their wild-type littermtes. Although we gin detected differences between the C7BL/6 nd FVB strins, inctivtion of Foxo3 hd no effect on the proportion of ctivted (CD69 hi ) or memory-effector (CD44 hi CD6L lo )Tcells(Fig. ). Published studies hve shown constitutive ctivtion of the NF-kB pthwy in Foxo3 Trp mice, s reflected by the bsence of IkB. However, T cells purified from nd Foxo3 / mice showed no chnge in the expression of IkB, IkBb or IkBe protein reltive to tht of wild-type T cells (Supplementry Fig. online). To mesure the effect of Foxo3 deficiency on T cell prolifertion, we lbeled purified lymph node T cells with the cytosolic dye nd then cultured the cells with plte-bound ntibody to CD3 (nti-cd3) with or without nti-cd8. Neither the number of cell divisions nor the ccumultion of cells in ech division ws ltered by Foxo3 deficiency (Fig. b). Also, freshly explnted nd wild-type CD4 + nd CD8 + T cells produced similr mounts of cytokines (Fig. c). As FsL (CD9L), the lignd for the cell surfce receptor Fs, is known trget of Foxo3, we lso nlyzed FsL expression on unstimulted nd ntibody-stimulted T cells from wild-type nd mice; however we found no differences relted to Foxo3 sttus (dt not shown). These results suggest tht loss of Foxo3 lone is not sufficient to elicit mnifesttions of T cell ctivtion nd spontneous utoimmunity. mice show enhnced T cell ccumultion As Foxo3 is involved in cell cycle progression nd poptosis,we sought to investigte its function in the dynmics of T cell popultion expnsion nd contrction in response to virl infection. Historiclly, the C7BL/6 strin hs been used to study the progression of virl responses, nd s the two mutnt strins homozygous for Foxo3 deficiency hd identicl profiles here nd in published results 3,6,our further efforts t chrcteriztion focused on the C7BL/6 mice. We infected mice nd their wild-type littermtes with b B6 FVB Cells α-cd3 + Control α-cd3 α-cd8 4,, 4, Foxo3 / Figure Foxo3-deficient mice show no spontneous T cell ctivtion. () Flow cytometry of the expression of CD44, CD6L nd CD69 by T cells from C7BL/6 (B6) nd FVB Foxo / mice nd their wild-type (Foxo +/+ ) congenic littermtes (n ¼ 6 mice per genotype), gted on CD4 + T cells (left) or CD8 + T cells (right). Numbers djcent to outlined res indicte percent cells in gte (men ± s.e.m.). (b) Prolifertion of -lbeled CD4 + T cells from nd Foxo / mice nd their wild-type littermtes, ctivted in the presence of nti-cd3 (-CD3) lone or c CD4 + T cells.6 ±. IL-4 ±.7 ±. IFN-γ ± IL-7. ±. ±. ±. IFN-γ ± CD4 ± ± TNF CD8 + T cells 3 ± 3 ± 4 ± 7 ± 6 ± with nti-cd8 (-CD8) nd ssessed by dilution fter 7 h of stimultion. Numbers in plots indicte number of ccumulted + cells. (c) Cytokine secretion by splenocytes from mice nd their wild-type littermtes (n ¼ 6 mice per group), ctivted for 3 h in the presence of phorbol -myristte 3-cette nd ionomycin nd nlyzed by intrcellulr stining with gting on CD4 + T cells (left) or CD8 + T cells (right). Numbers in qudrnts nd djcent to outlined res indicte percent cells in ech (men ± s.e.m.). Similr results were obtined with lymph node T cells. Dt re from five seprte experiments () or re representtive of three independent experiments (b,c). IL- IFN-γ 6 ± CD8 IL- ± 3 ± 3 NATURE IMMUNOLOGY VOLUME NUMBER MAY 9

3 9 Nture Americ, Inc. All rights reserved. b LCMV No infection c T cells ( 6 ) gp6-specific CD4 + T cells Time (d) * T cells ( 6 ) gp33-specific CD8 + T cells 3 6 Time (d) Adoptive trnsfer Adoptive trnsfer lymphocytic choriomeningitis virus (LCMV) nd ssessed LCMVresponsive T cell popultions t vrious times fter infection. Wheres the Foxo3 genotype hd no effect on the kinetics of T cell popultion expnsion, mice developed threefold greter ccumultion of LCMV-specific T cells thn did their wild-type littermtes (Fig. ). Mesurement of virus in the liver t dy 8 fter infection showed complete virl clernce in both strins (dt not shown). The lck of n effect of Foxo3 deficiency on T cells stimulted in culture prompted us to test whether the enhnced T cell ccumultion during n LCMV response ws T cell intrinsic. We trnsferred T cells from LCMV-specific TCR-trnsgenic or wild-type mice into wild-type C7BL/6 mice nd mesured the ccumultion of T cells fter infection of the recipient mice with LCMV. We detected no significnt difference in the ccumultion of nd wild-type T cells (Fig. b). To further exmine this issue, we trnsferred wild-type T cells into either wild-type or mice nd infected recipients with LCMV. T cells trnsferred into mice ccumulted in greter numbers thn did T cells trnsferred into wild-type hosts (Fig. b). We reproduced tht result with the trnsfer of ovlbumin (OVA)-specific OT-I T cells into wildtype nd hosts tht we subsequently infected with OVAexpressing vesiculr stomtitis virus (Supplementry Fig. online). To further investigte the cellulr cuse of the enhnced T cell popultion expnsion, we reconstituted irrdited wild-type mice with bone mrrow from wild-type or mice. After 8 weeks, we infected the recipient mice with LCMV nd nlyzed expnsion of the LCMV-specific T cell popultion. Excessive T cell ccumultion ws pprent in recipients of bone mrrow (Fig. c), which indicted involvement of bone mrrow derived cell type in this phenotype. Thus, non T cell bone mrrow derived cell type ws responsible for the enhnced T cell prolifertion nd/or survivl in Foxo3-deficient mice. Stimultory cpcity of DCs from mice The efficiency of ntigen presenttion cn ffect the mgnitude of T cell response to virl infection 7. Therefore, we ssessed whether Foxo3 regultes the number, phenotype nd/or function of +/+ Foxo3 T cells ( 6 ) T cells ( 6 ) LCMV No infection ntigen-presenting DCs. Nive mice hd significntly more DCs thn did their wild-type littermtes (Fig. 3), nd there ws slightly greter proportion of DCs with high expression of the costimultory molecules B7- (CD8) nd B7- (CD86). We found no difference in the expression of mjor histocomptibility complex (MHC) clss I, MHC clss II or CD4 on DCs from these mice (Fig. 3b), which suggested tht smll number of Foxo3-deficient DCs hd more mture phenotype. Anlysis of vrious DC subsets showed tht Foxo3-deficient mice hd more CDc + CDb + CD8, CDc + CDb CD8 + nd CDc + B + DCs (Fig. 3c). All subsets showed moderte shift towrd high expression of B7- nd B7- (Fig. 3d), lthough the biologicl importnce of this shift is uncler. To further chrcterize the ctivtion sttus of DCs from nive mice, we cultured splenic DCs overnight nd then mesured secreted cytokines. DCs produced more IL-6, tumor necrosis fctor (TNF) nd chemokine CCL (MCP-) thn did wild-type DCs (Fig. 3e). Interferon-g (IFN-g), IL- nd IL- were undetectble (dt not shown). To determine whether DCs from mice hd enhnced ntigen presenttion, we infected mice with LCMV nd nlyzed DCs 3 d fter infection, when virus is still present nd T cell popultions re expnding exponentilly 8. Compred with wildtype DCs, DCs hd slightly higher expression of B7-, B7- nd MHC clss II (Fig. 4) nd more effectively stimulted the ccumultion of T cells, s mesured by dilution nd stining with nnexin V nd 7-mino-ctinomycin D (; Fig. 4b). This enhnced stimultory cpcity ws not due to difference in ntigen processing, s differences between cultures with nd wild-type DCs remined even fter DCs were pulsed with LCMV glycoprotein 33 (gp33) peptide (Fig. 4b). To rule out the possibility tht Foxo3-deficient DCs from LCMVinfected mice cn cuse prolifertion of bystnder T cells independently of the presence of ntigen, we lso cultured DCs from LCMV-infected mice with OT-I T cells with or without OVA peptide (mino cids 7 64; OVA (7 64)). In the bsence of OVA (7 64), there ws no detectble division of OT-I T cells (Fig. 4b), which ruled out the possibility of bystnder effects. Finlly, T cells ( 6 ) Rdition chimer gp6-specific CD4 + T cells gp33-specific CD8 + T cells. * Figure Foxo3 regultes the mgnitude of n LCMV-induced immune response. () TheCD4 + nd CD8 + T cell responses to LCMV in mice nd their wild-type littermtes infected with LCMV, Armstrong strin ( plque-forming units), nlyzed fter restimultion with gp6 or gp33 peptide, respectively, by intrcellulr stining for IFN-g. (b) T cells in recipient mice given CD4. congenic wild-type or T cells (trnsferred into (-) CD4. wild-type mice; top) nd CD4. congenic wild-type T cells (trnsferred into CD4. wild-type or CD4. mice; bottom) nd then left uninfected (right) or infected with LCMV (left) nd ssessed with congenic mrker 8 d lter. (c) Virus-specific T cells in lethlly irrdited (gry lightning bolt round) CD4. wild-type mice reconstituted with CD4. congenic wild-type or bone mrrow nd then infected with LCMV 8 weeks lter, ssessed by specific peptide restimultion nd intrcellulr IFN-g stining. *, P o., nd, P o. (unpired two-tiled Student s t-test). Dt re representtive of four () ortwo(b,c) independent experiments with t lest three mice per group (error brs, s.e.m.). 6 VOLUME NUMBER MAY 9 NATURE IMMUNOLOGY

4 9 Nture Americ, Inc. All rights reserved. CDc + cells/spleen ( ) 4 * b B7- hi (%) B7- B7- MHC II MHC I CD4 6 4 Figure 3 mice hve more DCs nd greter ctivtion of DCs. () Totl DCs mong splenocytes obtined from mice nd their wild-type littermtes (n ¼ mice per group) nd stined for CDc. (b) Flow cytometry of CDc + DCs obtined from nive mice nd their wild-type littermtes (n ¼ mice per group) nd stined with ntibodies specific for vrious mrkers (below plots). Bottom row, ccumulted dt. MHCII, MHC clss II; MHCI, MHC clss I. (c) Absolute numbers of CDc + CDb + CD8, CDc + CDb CD8 + nd CDc + B + DCs in spleens B7- hi (%) we mesured the cpcity of DCs from uninfected mice to stimulte T cells in the presence of gp33 peptide. We noted slightly greter ccumultion nd vibility of T cells from cultures contining DCs thn from those contining wild-type DCs (Fig. 4c). To determine if the enhnced function of Foxo3-deficient DCs ws cell utonomous, we generted DCs in vitro. For this, we cultured bone mrrow cells for 8 d with grnulocyte-mcrophge colonystimulting fctor. The number nd proportion of the resulting CDc + cells (clled BMDCs here) were unffected by Foxo3 sttus, nd none of the BMDCs hd higher expression of B7-, B7-, CD4, MHC clss I or MHC clss II (Supplementry Fig. 3 online). Consistent with published reports of cells stimulted with grnulocyte-mcrophge colony-stimulting fctor, wild-type BMDCs hd uniformly cytoplsmic Foxo3 locliztion 9 (Supplementry Fig. 3b). Next we used BMDCs to stimulte nd T cells t vrious rtios of T cells to DCs. BMDCs induced ccumultion of nd T cells more effectively thn did wild-type BMDCs (Fig. ). Moreover, the Foxo3-deficient BMDCs lso more effectively sustined T cell vibility, regrdless of cell division (Fig. b,c). To determine whether the differences in vibility correlted with chnges in Bcl- fmily members, we nlyzed expression of the prosurvivl fctors Bcl- nd Bcl-x L. Totl nd nive CD44 lo T cells hd higher expression of both when stimulted with Foxo3-deficient BMDCs (Fig. d). To ssess the cpcity of BMDCs to enhnce T cell survivl in vivo, we trnsferred -lbeled T cells into nive CD4. congenic hosts. Then, d lter, we trnsferred wild-type or Foxo3-deficient BMDCs, with or without preloded OVA peptide (mino cids ; OVA(33 339), into the footpds of these mice. After dditionl dys, BMDCs induced greter ccumultion of T cells in the drining lymph nodes but similr number of T cell divisions, compred with wild-type DCs (Fig. e). Notbly, DCs fcilitted greter T cell survivl, even MHC II hi (%) d MHC I hi (%) CD4 hi (%) CDc + CDb + CDc + B + CDc + CD8 + B7- B7- B7- hi B7- hi MHCII hi MHCI hi CD4 hi ccdc+cdb+ cells/spleen ( ) e IL-6 (pg/ml) * 6 4 CDc + B + cells/spleen ( ) CDc + CD8 + cells/spleen ( ) in the bsence of OVA(33 339). These dt collectively estblish tht Foxo3-deficient DCs produce greter ntigen-induced T cell popultion expnsion by enhncing survivl. Enhnced IL-6 secretion by Foxo3-deficient DCs To determine if the enhnced T cell survivl induced by Foxo3- deficient DCs ws due to ltered cytokine secretion, we infected wild-type nd mice with LCMV nd mesured cytokine concentrtions in blood plsm t vrious times fter infection. The concentrtions of IL-, IL-7, CCL (MCP-), IL-b, IL-, IL- were similr in wild-type nd mice (Supplementry Fig. 4 online), but the concentrtions of IL-6 nd TNF were higher in Foxo3- deficient mice (Fig. 6 nd Supplementry Fig. 4). To determine if DCs were the source of this bundnt IL-6 nd TNF, we collected splenic DCs from mice on dy 3 fter infection nd cultured the cells for 4 h. Superntnts of DC cultures hd significntly higher concentrtions of IL-6, TNF, CCL (MCP-) nd IFN-g (Fig. 6b nd Supplementry Fig. 4b), wheres we detected no difference in the concentrtion of IL- or IL-. We lso found higher expression of IL-6 mrna in DCs (Fig. 6b). To determine whether cells other thn DCs could be responsible for the greter IL-6 production, we collected T cells, B cells nd mcrophges from mice 3 d fter LCMV infection nd cultured the cells for 4 h. Neither T cells nor B cells hd detectble production of IL-6 (dt not shown). Mcrophges did produce IL-6, lthough there ws no difference relted to Foxo3 sttus (dt not shown). However, given the finding of more mcrophges in Foxo3-deficient mice, these cells my hve contributed to the excess IL-6 production in vivo. We next determined whether BMDCs lso produced excess IL-6 in the bsence of Foxo3. We detected significntly more IL-6 in cultures contining CD4 + T cells or OT-I CD8 + T cells nd BMDCs thn in those contining T cells nd wild-type DCs (Fig. 6c). Confirming the DC-specific nture of this bundnt IL-6, RT-PCR TNF (pg/ml) 6 3 IL-6 * * * 6 of mice nd their wild-type littermtes (n ¼ mice per group). (d) Flow cytometry of the expression of B7- nd B7- on DC subsets (bove plots) from mice nd their wild-type littermtes. (e) Cytokine bed rry nlysis of the concentrtion of IL-6, TNF nd CCL (MCP-) in superntnts of splenic DCs (CDc + ) purified from wild-type or mice nd cultured for 4 h (4 cells). In,c, ech symbol represents n individul mouse; smll horizontl lines indicte the men. *, P o.;, P o.; nd *, P o. (unpired two-tiled Student s t-test). Dt re representtive of three independent experiments (,b) or two independent experiments with t lest three mice per group (c e; error brs (b,e), s.e.m.). MCP- (pg/ml) 3 4 NATURE IMMUNOLOGY VOLUME NUMBER MAY 9 7

5 9 Nture Americ, Inc. All rights reserved. b Annexin V B7- B7- MHCII MHCI, no peptide, gp33p OTI, no peptide OTI, OVAp showed tht BMDCs hd twofold more IL-6 mrna thn did wild-type BMDCs; IL-6 mrna ws t lest tenfold greter in BMDCs thn in CD4 + T cells, nd IL-6 mrna ws undetectble in CD8 + T cells (Fig. 6d). In combintion with trnsforming growth fctor-b, IL-6induces the differentition of IL-7-producing T cells 3. Given the results presented bove, we nlyzed the blnce between IL-7-producing T cells nd Foxp3 + regultory T cells in Foxo3-deficient mice. d Cells 3: 9: 7: 3, c Uninfected mice, no peptide, gp33p Annexin V b The proportion of Foxp3 + nd IL-7 + CD4 + T cells in nive LCMV-infected mice ws similr to tht of their wild-type littermtes (Supplementry Fig. online). We did not detect IL-7-producing cells in the CD8 + linege (dt not shown). To evlute whether the excess IL-6 produced by Foxo3-deficient DCs ws required for their bility to induce enhnced T cell survivl, we cultured nd T cells with BMDCs in the presence of n IL-6-specific blocking ntibody or n immunoglobulin G (IgG) isotype-mtched control ntibody. IL-6 blockde diminished the expnsion of T cell popultions cultured with BMDCs to n mount resembling tht of T cells cultured with wild-type DCs (Fig. 6e). Counting of ded cells stined with demonstrted OVAp No peptide gp33p No peptide Figure 4 Greter immunogenicity of LCMV-infected Foxo3-deficient DCs. () Flow cytometry of the expression of ctivtion mrkers on wild-type nd DCs on dy 3 fter LCMV infection. (b) Prolifertion nd vibility of -lbeled wild-type CD8 + T cells stimulted by DCs isolted from LCMV-infected mice or their wild-type littermtes (dy 3) in the presence or bsence of gp33 peptide (gp33p; left), or of -lbeled wild-type OT-I CD8 + T cells stimulted in the presence or bsence of OVA (33 339) (OVAp; bystnder prolifertion; right), ssessed by dilution (prolifertion; top row) or stining with nnexin V nd fter 3 d of culture (vibility; middle nd bottom rows). Numbers in dot plots indicte percent live CD8 + Tcells.(c) Prolifertion nd vibility of T cells from mice stimulted by DCs purified from the spleens of uninfected mice, cultured nd nlyzed s described in b. Dt re representtive of two independent experiments with t lest three mice per group (,c) or three independent experiments (b) e CD4. mice 3 c Annexin V Cells Bcl-, CD44 lo, CD44 lo, CD44 lo, CD44 lo Bcl-x L -lbeled CD4. T cells BMDC OVAppulsed BMDC footpd BMDC + OVAp CD4 + CD4. + cells ( ) 4 3 Anlysis of T cells in drining LN * BMDC BMDC + OVAp Figure Enhnced T cell responses induced by BMDCs. () -dilution nlysis of the ccumultion of wild-type CD4 + T cells or wild-type CD8 + T cells stimulted for 3 d t vrious rtios (bove plots) with wild-type or BMDCs in the presence of OVA(33 339) (for T cells) or gp33 peptide (for T cells). Numbers in plots indicte number of ccumulted CSFE + cells. (b) Deth of CD4 + T cells or CD8 + Tcells ( ) cultured with wild-type or BMDCs (3.3 ) in the presence or bsence of the pproprite peptide, ssessed by stining. Numbers djcent to outlined res indicte percent + Tcells.(c) Vibility of CD4 + TcellsorCD8 + T cells cultured for 3 d with BMDCs in the presence or bsence of the pproprite peptide nd stined with nnexin V nd. Numbers in outlined res indicte percent vible CD4 + or CD8 + Tcells.(d) Flow cytometry of the expression of Bcl- nd Bcl-x L in totl T cells or gted CD44 lo T cells mong or T cells cultured s described in b. Dshed lines, isotype-mtched control ntibody. (e) Prolifertion (left) nd number (right) of CD4 + T cells purified from wild-type CD4. mice, lbeled with nd injected intrvenously into CD4. recipient mice, followed by subcutneous injection of unpulsed or OVA(33 339)-pulsed (+ OVAp) BMDCs from mice or their wild-type littermtes (n ¼ 4 mice per group) into the footpds of the sme recipient mice nd nlysis 3 d lter. Left, -dilution nlysis; right, CD4. cells in drining lymph nodes (LN). Dt re representtive of t lest six independent experiments (,b) or two (c,d) orthree(e) independent experiments (error brs (e), s.e.m.). 8 VOLUME NUMBER MAY 9 NATURE IMMUNOLOGY

6 9 Nture Americ, Inc. All rights reserved. Serum IL-6 (pg/ml) * b Foxo3 c Foxo3 d +/+ +/+ IL-6 (pg/ml) 7 3 tht this reversion ws due to lower T cell survivl (Fig. 6f). In ddition, we determined whether exogenous IL-6 ws sufficient to enhnce the survivl of T cells cultured with wild-type BMDCs. Indeed, we noted direct correltion between the mount of IL-6 dded nd the proportion of live T cells in ech culture 3 (Fig. 6g). To directly scertin if IL-6 ws responsible for the greter expnsion of T cell popultions in response to LCMV, we treted mice on dys nd +4 (reltive to LCMV infection t dy ) with mg of blocking ntibody to IL-6 receptor -chin. Blockde of this receptor resulted in LCMV-specific T cell responses of substntilly smller mgnitude in Foxo3-deficient mice but did not ffect the number of LCMV-specific T cells in wild-type mice (Fig. 6h). These experiments collectively suggest tht wild-type DCs re restrined from bundnt IL-6 production by Foxo3 nd thus tht the fctors tht ffect the locliztion nd trnscriptionl ctivity of Foxo3 will influence the mgnitude of T cell medited immune response. Foxo3 cts downstrem of CTLA-4-induced signls Stimultion of B7 receptors by CTLA-4 promotes nucler locliztion of Foxo3 nd ctivtion of DCs 3. As cell surfce CTLA-4 expression is induced on ctivted T cells, we nlyzed whether signling through the B7 receptor inhibits IL-6 production in Foxo3-dependent wy. We stimulted splenic DCs with loxoribine, Toll-like receptor 7 gonist, in the presence of vrying mounts of fusion of CTLA-4 nd immunoglobulin (CTLA-4 Ig). The production of IL-6 nd TNF stimulted by loxoribine ws strongly inhibited by CTLA-4 Ig in wild-type DCs but not in * Il6 mrna (reltive expression) f 3 Time (d) Time (d) Time (d) Figure 6 IL-6 synthesis by Foxo3-deficient DCs is involved in enhnced T cell survivl. () Cytokine bed rry nlysis of IL-6 in plsm from wild-type nd mice (n ¼ 4 mice per group) t vrious times fter LCMV infection. (b) Cytokine bed rry nlysis (left) of IL-6 secreted by DCs (CDc + ) purified from wild-type nd mice without infection ( d) or 3 d fter LCMV infection (n ¼ 4 mice per group) nd then cultured for 4 h ( cells). Right, RT-PCR nlysis of Il6 mrna..... Isotype IL-6 (pg/ml) Anti-IL-6 Il6 mrna (reltive expression) g Live cells (%) 6 4 DC α-il-6 ril-6 (ng/ml) h T cells 6 IgG α-il-6r Foxo3-deficient DCs (Fig. 7). Although tretment with CTLA-4 Ig resulted in slightly greter proportion of ded cells, its toxic effects were equivlent in wild-type nd splenic DCs showed the sme toxicity (Fig. 7). To confirm the involvement of Foxo3 in the inhibition of cytokine production induced by CTLA-4 Ig, we nlyzed the intrcellulr locliztion of Foxo3 fter stimultion with loxoribine or CTLA-4 Ig. Foxo3 remined in the cytosol of unstimulted nd loxoribine-treted BMDCs; however, CTLA-4 Ig stimultion strongly promoted the nucler ccumultion of Foxo3 (ref. 3; Fig. 7b). Notbly, signling through B7 ws dominnt over Toll-like receptor 7 signls, s DCs treted with both loxoribine nd CTLA-4 Ig showed Foxo3 nucler locliztion. These experiments collectively show tht CTLA-4 Ig tretment profoundly suppressed Toll-like receptor induced production of IL-6 nd TNF in wild-type DCs by promoting the nucler ccumultion of Foxo3. If CTLA-4 signls DCs through B7- nd B7-, then blocking CTLA-4 should llow wild-type DCs, like Foxo3-deficient DCs, to enhnce T cell survivl. To investigte this issue, we cultured TCR-trnsgenic T cells in the presence of wild-type or DCs nd specific peptides with or without the ddition of blocking ntibody to CTLA-4. As expected, cultures with BMDCs included more vible T cells thn did cultures with wild-type BMDCs (Fig. 7c). However, the ddition of nti-ctla-4 essentilly eliminted the difference in T cell ccumultion nd vibility in these cultures. The results of these experiments re consistent with the ide tht CTLA-4 is involved in promoting the nucler locliztion of e 3 Isotype gp33-specific CD8 + T cells 4 IgG α-il-6r Anti-IL-6 gp6-specific CD4 + T cells expression, presented reltive to the expression in uninfected wild-type cells, set s. (c) Enzyme-linked immunosorbent ssy of IL-6 in superntnts of wild-type or T cells cultured for 3 d in the presence of wild-type or BMDCs loded with OVA(33 339) or gp33 peptide. (d) RT-PCR nlysis of Il6 mrna in BMDCs (DC), T cells nd T cells purified fter d of culture s described in c, presented reltive to Gpdh mrna (encoding glycerldehyde phosphte dehydrogense). (e,f) Accumultion (e) nd deth(f) of wild-type or T cells ctivted by BMDCs generted from wild-type or mice pulsed with OVA(33 339) (for CD4 + T cells) or gp33 peptide (for CD8 + T cells), in the presence of n IgG isotype-mtched control ntibody or IL6-specific blocking ntibody ( mg/ml), ssessed fter 3 d of culture by dilution (ccumultion) or stining (deth). Numbers djcent to outlined res (f) indicte percent ded + + cells. (g) Deth of T cells stimulted by wild-type BMDCs in the presence of incresing mounts of recombinnt IL-6 (ril-6) or IL-6-specific blocking ntibody (-IL-6), ssessed fter 3 d of culture by stining. (h) LCMV-specific T cell responses of mice nd their wild-type littermtes (n ¼ 4 mice per group) treted on dys nd +4 with mg of ntibody to IL-6 receptor -chin (-IL-6R) or isotype-mtched control ntibody (IgG) nd infected on dy with LCMV, Armstrong strin, nd then nlyzed on dy +8 by counting of CD8 + T cells nd CD4 + T cells producing IFN-g fter restimultion with OVA(33 339) (for CD4 + Tcells) or gp33 peptide (for CD8 + T cells). Dt re representtive of three ( f) ortwo(g,h) independent experiments (error brs ( d,g,h), s.e.m.). NATURE IMMUNOLOGY VOLUME NUMBER MAY 9 9

7 IL-6 (pg/ml) 6 4 TNF (pg/ml) cells (%) 4 3 c Anti-CTLA-4 Anti-CTLA Loxo CTLA-4 Ig Loxo CTLA-4 Ig 4 Loxo CTLA-4 Ig 4 b Control Ig CTLA-4 Ig Medi Nture Americ, Inc. All rights reserved. Loxo Foxo3 nd the resulting inhibition of inflmmtory cytokines produced by DCs. Published experiments hve shown tht ntibody specific for CTLA-4 cn enhnce n immune response in vivo 33,34, lthough, to our knowledge, this hs not been demonstrted before in n immune response to LCMV. Nonetheless, we sought to determine whether we could detect Foxo3-dependent enhnced T cell popultion expnsion. We inoculted mice with mg of CTLA-4-specific ntibody nd, 4 h lter, infected the mice with LCMV. We further inoculted mice with CTLA-4-specific ntibody every other dy nd ssessed the results on dy 8. We did not find enhncement of T cell ccumultion in either wild-type or Foxo3-deficient mice (Supplementry Fig. 6 online). DISCUSSION In this report, we hve estblished n essentil function for Foxo3 in modulting the mgnitude of ntigen-specific T cell immune responses. Foxo3-deficient mice showed enhnced T cell prolifertion in response to virl infection, n enhncement tht ws not T cell intrinsic but insted depended on the ugmented cpcity of DCs to sustin T cell vibility. Despite the mny potentil trgets of Foxo3, including regultors of cell cycle, poptosis nd rective oxygen detoxifiction, Foxo3 loss-of-function muttion lone hd no effect on the stedy-stte homeostsis, survivl or prolifertion of T cells. Tht finding is consistent with studies showing tht Foxo is essentil for norml T cell homeostsis nd self tolernce 6 (dt not shown). Insted, we found criticl DC-intrinsic function for Foxo3 in the control of T cell responses. Foxo3 cted downstrem of CTLA-4-induced signls to constrin IL-6 production by DCs.The nturl conditions tht would override the CTLA-4-medited nucler locliztion of Foxo3 re not known; however, new immunostimultory cncer tretment regimen tht involves blocking CTLA-4 my ct in prt by this mechnism 3. Annexin V Figure 7 Production of IL-6 nd TNF by stimulted DCs is inhibited by CTLA-4 Ig stimultion in Foxo3-dependent wy. () Immunossy of the concentrtion of IL-6 (left) nd TNF (middle) in superntnts of DCs ( ) purified from the spleens of mice nd their wild-type littermtes nd stimulted for 8 h with (+) or without ( ) loxoribine (Loxo) plus incresing mounts of CTLA-4 Ig (below grphs, in mg/ml). Right, DC vibility, ssessed by stining. (b) Immunofluorescence nlysis of Foxo3 locliztion (green) fter 8 h of stimultion of BMDCs from wild-type nd mice with loxoribine (bottom) or without loxoribine (Medi; top) in presence of control immunoglobulin or CTLA-4 Ig. Blue, DAPI stining of nuclei. Originl mgnifiction, 63. (c) Accumultion (top) nd deth (below) of CD4 + T cells or CD8 + Tcells( ) cultured for 3 d with wild-type or BMDCs (3.3 ) in the presence of the pproprite peptide plus nti-ctla-4 ( mg/ml; 9D9), ssessed s dilution (ccumultion) nd stining with nnexin V nd (deth). Numbers in plots indicte number of ccumulted cells (top) or percent live CD4 + T cells or CD8 + T cells (middle nd bottom). Dt re representtive of t lest two independent experiments (error brs (), s.e.m.). Our results differ substntilly from published chrcteriztion of Foxo3 Trp mice. Foxo3 Trp mutnt mice were derived from n independent insertionl mutnt embryonic stem line obtined from ByGenomics nd were bckcrossed to the 9 strin for three genertions. Foxo3 Trp mice show spontneous T cell ctivtion, lymphoprolifertion nd orgn infiltrtion ssocited with constitutive ctivtion of NF-kB. In contrst, we found none of those chrcteristics in Foxo3 / or mice. T cells isolted from these two strins of mice seemed to be indistinguishble from wildtype T cells in terms of NF-kB ctivtion, expression of ctivtion mrkers nd response to mitogenic stimultion. Our results re consistent with published nlyses of nd Foxo3 / mice in tht extensive histologicl nlysis hs not shown lymphocytic infiltrtion of orgns 3,6. Differences in utoimmunity re not uncommon in mixed C7BL/6 nd 9 mouse strins. The 9 mice hve utoimmunitysusceptibility loci, one of which (Sle6) induces humorl utoimmunity in congenic C7BL/6 mice. Severl studies hve noted modifier genes in 9 strins tht confer enhnced utoimmune disese to 9 C7BL/6 mice Foxo Trp mice re pprently inbred 9 mice without contribution from the C7BL/6 strin, but perhps 9 susceptibility locus tht modifies the Foxo3 deficiency is present. IL-6 is pleiotropic cytokine tht regultes mny spects of the immune system, including ntibody production, hemtopoiesis, inflmmtion nd, most relevnt to our study, T cell survivl 39.IL-6 rescues resting T cells from poptosis by inhibiting the downregultion of Bcl- in dose-dependent wy 4,ndIL-6incresesthesurvivl of ntigen-stimulted T cells 3. Consistent with tht observtion, we noted higher expression of Bcl- nd Bcl-x L in T cells from LCMVinfected mice thn in those from wild-type mice. In ddition, the division rte of T cells stimulted with DCs ws not VOLUME NUMBER MAY 9 NATURE IMMUNOLOGY

8 9 Nture Americ, Inc. All rights reserved. different from tht of T cells stimulted with wild-type DCs; insted, the effect ws mnifested s improved T cell survivl. Moreover, DCs sustined T cell vibility both in vitro nd in vivo even in the bsence of specific peptide, which suggests tht the enhnced T cell survivl induced by IL-6 is not restricted to TCR-engged T cells. Notbly, IL-6-specific blocking ntibody did not ffect the vibility of wild-type T cells in vitro, nor did it result in lower T cell ccumultion in vivo. Tht observtion indictes tht in the conditions tested, the mount of IL-6 produced by wild-type DCs ws not sufficient to ffect T cell vibility. It would be useful to determine whether immune responses to other gents re sensitive to IL-6 concentrtion nd, in those infections, whether CTLA-4, regultory T cells nd Foxo3 ffect the mgnitude nd dynmics of the T cell response. CTLA-4 cts s counterblnce to costimultion of CD8 by ntgonizing TCR signls in ctivted T cells nd by promoting tolernce 4. CTLA-4 is lso expressed on regultory T cells nd is essentil for their effectiveness 4. CTLA-4 cn lso modify function of cells of the immune system by triggering reverse signling through B7 receptors; these signls promote the DC production of stimultory nd/or suppressive meditors. Reverse signls through B7 lso ctivte the immunosuppressive pthwy of tryptophn ctbolism in plsmcytoid DCs involving indolemine,3-dioxygense (IDO) 43. Foxo3 is involved in the IDO pthwy in plsmcytoid DCs, s CTLA- 4 Ig leds to nucler ccumultion of Foxo3 nd expression of superoxide dismutse (ref. 3). As IDO induces T cell deth through the genertion of kinurenin nd tryptophn privtion 43,wespeculted tht potentil IDO defect in DCs could explin the improved T cell survivl; however, ddition of the IDO inhibitor -MT did not enhnce T cell survivl in cultures contining wild-type or BMDCs (dt not shown). Moreover, IDO is expressed minly by plsmcytoid nd CD8 + DC subsets,ndlowornoido protein is detected in resting DCs derived from bone mrrow 44. Presumbly CTLA-4 expressed on regultory T cells constitutes the min B7-stimultory lignd, but the possibility tht ctivted T cells or other cells contribute to B7 ligtion in vivo hs not been investigted. In ddition, the reltive contribution of ech CTLA-4-medited suppressive mechnism to the regultion of immune responses to vrious infectious gents is not well understood. Our experiments collectively indicte tht nucleus-loclized Foxo3 functions in DCs to inhibit the production of inflmmtory cytokines tht would otherwise be ctivted in response to infection. We deduce tht the IL-6-induced T cell survivl in mice resulted from n inbility of Foxo3-deficient DCs to shut down cytokine production in response to CTLA-4 signls. Conditions tht lter Foxo3 ctivity, such s stress, growth fctors or nutrients, would be expected to substntilly influence the ctivity of DCs nd perhps mcrophges, nd such conditions might be expected to lter the outcome of responses to infectious gents. METHODS Mice. C7BL/6, C7BL/6-CD4.,, OT-I nd mice were mintined in specific pthogen free conditions t the University of Cliforni, Sn Diego. C7BL/6 mice lcking Foxo3 were generted from embryonic stem cell clones from the OmniBnk embryonic stem cell librry of rndomly trgeted cell lines (Lexicon Genetics) 3. These mice (Foxo3 Gt(VICTR)Kc ; clled here) were bckcrossed to the C7BL/6J strin for genertions nd were then intercrossed to generte congenic C7BL/6 mice. Foxo L/L mice,with conditionl trgeting of Foxo3, were produced in FVB embryonic stem cells, nd the loxp-flnked llele ws deleted by breeding with mle EII-Cre trnsgenic mice 6. Offspring with complete excision were bred to exclude EII-Cre; these mice re clled Foxo3 / here 6. TCR-trnsgenic mice with Foxo3-null genotype were produced by breeding of C7BL/6 mice with either or mice. All procedures were pproved by the Institutionl Animl Cre nd Use Committee of the University of Cliforni, Sn Diego. Virus infection nd nlysis. mice nd their wild-type littermtes 6 weeks of ge were infected by intrperitonel injection of plque-forming units of LCMV, Armstrong strin, in. ml PBS. Alterntively, mice were infected with plque-forming units of vesiculr stomtitis virus 4 expressing OVA (provided by L. Lefrnçois). At vrious times, spleens were collected from LCMV-infected mice nd uninfected control mice nd splenocytes were stimulted with gp33 peptide, n MHC clss I restricted LCMV epitope (gp33-4), or gp6 peptide, n MHC clss II restricted LCMV epitope (gp6-8; Genemed Synthesis) nd brefeldin A ( mg/ml; Golgistop; BD PhrMingen). After h of stimultion, cells were stined for intrcellulr IFN-g (XMG.; ebioscience). For trnsfer experiments, CD4. congenic wild-type or T cells ( 4 ) were injected intrvenously into CD4. wild-type hosts, which were infected with LCMV 4 h lter. Conversely, CD4. congenic wild-type T cells were trnsferred into congenic CD4. wild-type littermte or mice. T cells in the spleen were counted t dy 8 fter LCMV infection. Infection with vesiculr stomtitis virus ws done in n identicl wy except tht T cells from CD4. OT-I mice were trnsferred. For bone mrrow chimer experiments, congenic CD4. recipient mice were lethlly irrdited (, rds) nd were injected intrvenously with 6 bone mrrow cells from or wild-type littermte donor mice (both CD4.). After months, the reconstitution of peripherl blood lymphocytes ws over 9%. These rdition chimers were then infected with LCMV nd the mgnitude of the LCMV response ws ssessed t dy 8 fter infection. For ntibody blockde of IL-6 receptor -chin, wild-type or Foxo3-deficient mice were inoculted intrperitonelly with mg of ntibody to IL-6 receptor -chin (purified ntibody to mouse nd rt CD6; D77A7; BioLegend) on dy nd dy +4 nd were infected with LCMV, Armstrong strin, on dy. Cell isoltion nd purifiction. Bone mrrow cells were isolted by flushing of the femur nd tibi with RPMI medium (contining % (vol/vol) FBS, mm L-glutmine, U/ml of penicillin, mg/ml of streptomycin nd mm b-mercptoethnol). Spleens were removed nd were incubted for min t 37 C with collgense D ( mg/ml; Roche), nd splenocytes were collected by homogeniztion through -mm tissue striner. Cells were resuspended in Tris mmonium chloride buffer for lysis of red blood cells. For purifiction of splenic DCs, cells were first incubted for min with superntnts of.4g hybridom cultures (nti mouse FcgRII-III), then were incubted for min with nti mouse pn-dc microbeds (Miltenyi Biotec), followed by positive selection. The positive frction ws typiclly over 9% CDc +. For T cell purifiction, splenocytes from or mice were incubted with mixture of biotinylted nti-b (RA3-6B), nti-cd9, nti-gr- (RB6-8C), nti MHC clss II, nti-dx nd nti-cdb (M/7; ll from ebioscience) nd were negtively selected to over 9% purity with strepvidincoupled microbeds (Miltenyi Biotec). BMDC cultures. Bone mrrow cells were isolted nd were cultured t density of 6 cells/ml in RPMI medium contining recombinnt mouse grnulocyte-mcrophge colony-stimulting fctor ( ng/ml; Peprotech). On dys, 4 nd 6, hlf the culture ws removed nd centrifuged nd the cell pellet ws resuspended in ml fresh medi contining recombinnt mouse grnulocyte-mcrophge colony-stimulting fctor ( ng/ml). Cultures were collected on dy 8 nd CDc + cells were selected with microbeds (Miltenyi Biotec). The finl popultion ws 98% CDc + CDb + B DCs. Flow cytometry. Cells were incubted for min with nti mouse FcgRII-III nd then were stined with primry ntibodies for min on ice. The following mouse monoclonl ntibodies were used (ll from ebioscience): llophycocynin- or phycoerythrin-conjugted nti-cdc (N48); fluorescein isothiocynte or phycoerythrin-conjugted nti-b (RA3-6B); phycoerythrin-indotricrbocynine or peridin chlorophyll protein conjugted nti-cdb (M/7); llophycocynin-conjugted nti-cd8 (3-6.7); fluorescein isothiocynte conjugted nti MHC clss I nd nti-gr- (RB6-8C); peridin chlorophyll protein conjugted nti-cd3; nd phycoerythrinconjugted nti-cd86, nti-cd8, nti MHC clss II, nti-ly6c, nti-cd9, nti-cd3 nd nti-nk.. NATURE IMMUNOLOGY VOLUME NUMBER MAY 9

9 9 Nture Americ, Inc. All rights reserved. T cell prolifertion nd deth ssys. For in vitro experiments, DCs were purified from wild-type or mice on dy 3 fter LCMV infection. DCs were lso purified from uninfected mice or were derived from the bone mrrow of wild-type or mice. These DCs were used to stimulted CD8 + T cells, CD4 + T cells or OT-I CD8 + T cells ( cells per well) lbeled with mm (crboxyfluorescein dicette succinimidyl ester; Moleculr Probes) in the presence of gp33 peptide (. mg/ml) or OVA(33 339); ( mg/ml). Prolifertion ws nlyzed by flow cytometry by mesurement of dilution, nd T cell deth ws mesured by stining with nnexin V nd. For in vivo trnsfer experiments, -lbeled CD4 + T cells purified from mice were injected retro-orbitlly into congenic CD4. C7BL/6 mice. Wild-type or BMDCs were pulsed with OVA(33 339) nd were injected intrdermlly fter 4 h. Then, 3 d lter, drining lymph nodes were collected, were stined with llophycocynin-conjugted nti-cd4 (GK.) nd phycoerythrin-conjugted nti-cd4. (A; both from ebioscience) nd were nlyzed by flow cytometry. In vitro stimultion of DCs nd quntifiction of cytokine production. Sorted DCs ( ) were cultured in 96-well round-bottomed culture pltes (Nunc) nd were stimulted with medium lone or.3 mm loxoribine (InvivoGen) lone or in the presence of recombinnt mouse fusion protein consisting of the ectodomin of CTLA-4 linked to the Fc portion of IgG (CTLA-4 Ig) t concentrtion of or 4 mg/ml (R&D Systems). After culture for 8 h, superntnts were collected nd cytokine concentrtions were determined by immunossy. Enzyme-linked immunosorbent ssy kits were used for the detection of TNF or IL-6 (Redy-Set-Go; ebioscience) unless otherwise specified. Cytokines were lso mesured by cytokine bed rry (BD Biosciences) or Luminex technology (Invitrogen). Fluorescence microscopy. DCs derived from bone mrrow of wild-type or mice were cultivted for h on sterile poly-l-lysine-coted coverslips (BioCot; BD). Cells were fixed in 4% (vol/vol) formldehyde, then were mde permeble with.% (vol/vol) Triton X-. Coverslips were blocked for h in PBS contining % (wt/vol) BSA nd nti mouse FcgRII-III. Anti-Foxo3 (FKHRL; Cell Signling) ws dded for h, followed by secondry ntibody conjugted to fluorescein isothiocynte (got nti rbbit IgG; 6-6; Zymex). For stining of nuclei, DAPI (4-6-dimidino--phenylindole) ws dded t concentrtion of.4 mg/ml. All cells were visulized with microscope (Axiovert M; Crl Zeiss MicroImging) with 63 objective. Imges were cptured with n Axiocm monochrome digitl cmer nd were nlyzed with Axiovision softwre (Crl Zeiss MicroImging). Semiquntittive RT PCR. Totl RNA ws extrcted from DCs with Trizol regent ccording to the mnufcturer s instructions (Invitrogen), then cdna ws synthesized with SuperScript First-Strnd synthesis System for RT-PCR (Invitrogen). Il6 nd the gene encoding cyclophilin A (Ppi) were mplified by PCR with the following primers: Il6 forwrd, -ACCTGGAGTACATGAAGAA CAACTT-3 nd reverse, -GGAAGCACTCACCTCTTGGT-3 ; Ppi forwrd, -CACCGTGTTCTTCGACATC-3 nd reverse, -ATTCTGTGAAAGGAG GAACC-3. Immunoblot nlysis. Equl mounts of protein from whole-cell extrcts were resolved by 4 % SDS-PAGE (Invitrogen) nd were trnsferred to polyvinylidene difluoride membrne (Millipore) by semidry trnsfer (Bio-Rd). Blots were blocked nd incubted with primry ntibody overnight t 4 C, followed by h of incubtion t C with the pproprite horserdish peroxidse conjugted secondry ntibody. Rbbit polyclonl nti-foxo3 ws provided by A. Brunet (Stnford University). Antibodies specific for IkB proteins were from Snt Cruz Biotechnology. Accession codes. UCSD-Nture Signling Gtewy ( A94 nd A76. Note: Supplementry informtion is vilble on the Nture Immunology website. ACKNOWLEDGMENTS We thnk J.P. Allison (Slon-Kettering Memoril Hospitl) nd J.A. Bluestone (University of Cliforni t Sn Diego) for CTLA-4-specific blocking ntibodies; A. Brunet (Stnford University) for Foxo3-specific ntibody; L. Lefrnçois (University of Connecticut Helth Center) for OVA-expressing vesiculr stomtitis virus; L. Mck nd E. Zunig for ssistnce with virus titers; nd M. Niw for microscope fcility use. Supported by the Americn Cncer Society (R.A.D.), the Robert A. nd Renee E. Belfer Institute for Innovtive Cncer Reserch (R.A.D.), the US Ntionl Cncer Institute (R.A.D.), the Division of Biologicl Sciences of the University of Cliforni, Sn Diego (S.M.H.) nd the Fondtion pour l Recherche Médicle (A.S.D.). AUTHOR CONTRIBUTIONS D.R.B. initited the project nd did lymphocyte chrcteriztion nd LCMV infection under the supervision of S.M.H.; A.S.D. designed nd did the remining experiments in collbortion with D.R.B., I.L.C., Y.M.K. nd S.M.H.; A.B. provided expertise in fluorescence microscopy nlysis; R.A.D. nd D.H.C. produced Foxo3 / mice nd provided intellectul input on the dt; K.C.A. provided mice; S.M.H. initited the project with input from R.A.D. nd K.C.A. nd supervised the experiments; nd A.S.D. nd S.M.H. wrote the mnuscript with editoril nd intellectul contributions from the other uthors. Published online t Reprints nd permissions informtion is vilble online t reprintsndpermissions/. Clnn, D.R. & Brunet, A. The FoxO code. Oncogene 7, (8).. Glili, N. et l. Fusion of fork hed domin gene to PAX3 in the solid tumour lveolr rhbdomyosrcom. Nt. Genet., 3 3 (993). 3. Hillion, J., Le Conit, M., Jonveux, P., Berger, R. & Bernrd, O.A. 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