Autocrine IL-2 is required for secondary population expansion of CD8 + memory T cells

Transcription

1 Autorine IL-2 is required for seondry popultion expnsion of CD8 + memory T ells Soni Feu, Rmon Arens,2, Susn Togher & Stephen P Shoenerger 2 Nture Ameri, In. All rights reserved. Two ompeting theories hve een put forwrd to explin the role of CD4 + T ells in priming CD8 + memory T ells: one proposes prrine seretion of interleukin 2 (IL-2); the other proposes the tivtion of ntigen-presenting ells (APCs) vi the ostimultory moleule CD4 nd its lignd CD4L. We investigted the requirement for IL-2 y the relevnt three ell types in vivo nd found tht CD8 + T ells, rther thn CD4 + T ells or dendriti ells (DCs), produed the IL-2 neessry for CD8 + T ell memory. Il2 / CD4 + T ells were le to provide help only if their ility to trnsmit signls vi CD4L ws intt. Our findings reonile ontrditory elements impliit in eh model noted ove y showing tht CD4 + T ells tivte APCs through CD4L-dependent mehnism to enle utorine prodution of IL-2 in CD8 + memory T ells. The initil enounter with ntigen-presenting ells (APCs) onstitutes the primry fore ehind the popultion expnsion, differentition nd survivl of nive T ells. One set into motion y indutive signls reeived during this priming event, the development of oth effetor nd memory CD8 + T ells n e guided y n instrutionl progrm exeuted without the need for further ntigeni stimultion 6. CD4 + helper T ells hve n importnt role in estlishing the omponents of this progrm, nd their prtiiption n endow CD8 + T ells with key funtion of immune memory: the ility to undergo seondry prolifertive responses fter reenountering ntigen 7. A ruil spet of this mehnism involves ltertion of the developmentl progrm in helped CD8 + T ells to prevent the tivtion-indued ell deth medited y TRAIL tht ours fter restimultion of CD8 + T ells tht hve not reeived help ( helpless ; those primed in the sene of CD4 + T ells),2. A longstnding ontroversy in this proess hs foused on the sequene nd nture of the ellulr intertions through whih help is provided. Helper T ells hve een shown to hve ruil role in liensing APCs, through sequentil two-ell intertions involving the ostimultory moleule CD4 nd its lignd CD4L, from resting to n tivted stte in whih they re susequently le to diretly prime CD8 + T ells 3 6 An lterntive model suggests tht CD4 + T ell helps CD8 + T ell vi prrine seretion of the growth nd differentition ftor interleukin 2 (IL-2) when oth re in three-ell luster with n APC 7 9. Although the three-ell luster model ws initilly sed on demonstrtion of the epitope linkge tht would e required for CD4 + T ell CD8 + T ell ollortion nd in vitro restimultion studies, more diret in vivo evidene hs demonstrted tht CD8 + T ells require IL-2 signls during priming to enle their lonl progeny to mount optiml seondry prolifertive responses 2. Prolonged IL-2 signling hs een shown to promote the development of (short-lived) effetor CD8 + T ells, whih suggests tht the mgnitude or durtion of IL-2 signls n hve profound influene on the funtionl differentition of the ells 2,22. Suh studies hve eluidted the effet of IL-2 on CD8 + T ells ut hve not provided informtion out its relevnt physiologil soure in vivo. Here we investigted the ell types known to produe IL-2 in vivo (CD4 + T ells, CD8 + T ells nd dendriti ells (DCs)) to identify whih re ruil for progrmming the pity for the seondry popultion expnsion of memory CD8 + T ells. Our results indite tht CD4 + T ells required the ility to trnsmit CD4L signls ut did not need to produe IL-2 to provide the help needed for CD8 + T ell memory, nd tht utorine IL-2 produed y the helped memory CD8 + T ells enled their optiml seondry popultion expnsion. RESULTS Helper T ells require CD4L ut not IL-2 The CD4 + T ell dependent priming of CD8 + T ells involves three distint types of ells: CD4 + T ell, CD8 + T ell nd n APC ering the surfe peptide mjor histoomptiility omplex (MHC) lignds reognized y eh. As eh of these ell types hs the ility to produe IL-2, we sought to estlish whih funtions s the relevnt soure of this ytokine in vivo to enle the seondry prolifertive response of CD8 + T ells. Studies of mie lking expression of IL-2 or its reeptor CD25 (IL-2 reeptor α-hin) hve een omplited y the ft tht these mie suffer from rnge of utoimmune nd lymphoprolifertive T ell disorders presumly due to the lk of IL-2-dependent CD4 + regultory T ells 23,24. To overome this, we used strtegy in whih we speifilly ontrolled the prodution of IL-2 in vivo in eh of the relevnt ell types le to produe this ytokine during CD8 + T ell priming. This involved the doptive trnsfer of wild-type or Il2 / CD4 + () or CD8 + (OT-I) T ells speifi for the model Lortory of Cellulr Immunology, L Joll Institute for Allergy nd Immunology, L Joll, Cliforni, USA. 2 Present ddress: Deprtment of Immunohemtology nd Blood Trnsfusion, Leiden University Medil Center, Leiden, The Netherlnds. Correspondene should e ddressed to S.P.S. (sps@lii.org). Reeived 2 April; epted 23 June; pulished online 3 July 2; doi:.38/ni VOLUME 2 NUMBER 9 SEPTEMBER 2 nture immunology

2 H-2K OVA A rt i l e s 2 Nture Ameri, In. All rights reserved. ntigen hiken ovlumin (OVA) into wild-type isogeni hosts, or of OVA-expressing wild-type or Il2 / DCs tht re the only APC le to present the omplex of OVA mino ids (OVA( )) nd H-2K reognized in vivo y OT-I CD8 + T ells in H-2K m hosts ( strin unle to present this peptide to H-2K -restrited CD8 + T ells) 25. We first investigted whether IL-2 prodution y CD4 + T ells ws required for the priming of CD8 + T ells le to undergo seondry popultion expnsion. To hieve this, we doptively trnsferred 5 4 wild-type or Il2 / T ells into dult mie tht hd undergone thymetomy nd hd een depleted of endogenous CD4 + T ells y tretment with monolonl ntiody 5 d efore to ensure tht ny CD4 + T ell help would derive from the trnsferred ells (Supplementry Fig. ). After immuniztion nd susequent rehllenge, ontrol mie depleted of CD4 + T ells mounted only wek primry nd seondry CD8 + T ell responses (s determined y nlysis of the OVA( ) H-2K tetrmer nd prodution of interferon-γ (IFN-γ)), in ontrst to the strong responses in those tht reeived either wild-type or Il2 / CD4 + T ells (Fig. ), whih onfirmed the dependene of CD8 + T ell responses on CD4 + T ells in this experimentl system. Notly, we found no gret differene in the mgnitude of the primry nd seondry endogenous ntigen-speifi CD8 + T ell responses, either y perentge or solute numers, in mie tht reeived either wild-type or Il2 / CD4 + T ells (Fig. ). The response nonetheless did inlude n IL-2-produing omponent (Supplementry Fig. ), nd the help provided y CD4 + T ells Figure CD4 + T ells require CD4L, not IL-2, to provide help to CD8 + T ells. () Flow ytometry of spleni CD8 + T ells from C57BL/6J mie tht hd undergone thymetomy nd were first depleted of CD4 + T ells, then given injetion of PBS lone (No ) or 5 4 ells () or Il2 / ells ( Il2 / ), then primed d lter with 5 6 At-mOVA H-2K -defiient splenoytes nd hllenged 3 d lter with 5 6 plque-forming units (PFU) of vini virus OVA. Numers djent to outlined res indite perent H-2K OVA tetrmer positive CD8 + T ells (men ± s.e.m. of six mie). (,) Frequeny () nd solute numer () of CD8 + T ells mong totl splenoytes from the mie in (n = 6 per group), ssessed y intrellulr stining t dy 7 (primry) or t dy 35 in mie tht hd (seondry) or hd not (memory) een hllenged with vini virus OVA 5 d erlier. Eh symol represents n individul mouse; smll horizontl lines indite the men (). (d) Flow ytometry of CD8 + T ells in lood from the mie in tht were given no ntiody tretment (fr left) or treted with ontrol immunogloulin (ontrol Ig) or loking ntiody to CD4L (α-cd4l), ssessed t dy 7 (primry), dy 3 (memory) or dy 35 (seondry) fter mie were hllenged with vini virus OVA 5 d erlier. Numers djent to outlined res indite perent OVA( ) H-2K tetrmer speifi CD8 + T ells (men ± s.e.m. of six mie). NS, not signifint; *P <.5 (two-tiled unpired t-test). Dt re representtive of three (,) or two (,d) independent experiments (men ± s.e.m.). H-2K OVA d No.9 ±..5 ± ± CD No CD44 ws lerly ntigen speifi (Supplementry Fig. ). In ontrst, ntiody lokde of CD4L prevented oth wild-type nd Il2 / ells from providing help to the sme endogenous CD8 + T ells (Fig. d). These dt demonstrte tht lthough CD4 + T ells re ritil in priming roust primry nd seondry CD8 + T ell response from endogenous polylonl preursors, their ility to produe IL-2 is irrelevnt to this proess. The ility of CD4 + T ells to trnsmit the help messge, however, is ompletely dependent on intt CD4-CD4L intertions, in support of the sequentil two-ell intertion model 6. DC-derived IL-2 is not essentil for CD8 + memory To determine if IL-2 prodution y APCs is neessry for CD8 + T ell memory, we used system in whih popultion of doptively trnsferred DCs serves s the only APCs le to prime CD8 + T ells speifi for OVA( ) H-2K in vivo. This involved the genertion of DCs from wild-type (Il2 +/+ ) or Il2 / At-mOVA mie, whih express memrne-ound form of OVA t their ell surfe nd present OVA-derived peptides on their surfe MHC lss I nd lss II moleules 26. To ensure tht only the trnsferred DCs were le to present OVA nd to void ross-presenttion, we used H-2K m mie s reipients euse endogenous APCs from this mouse strin re unle to present the OVA( ) peptide to H-2K -restrited CD8 + T ells suh s OT-I ells vi the K m vrint form of the H-2K MHC moleule they express 27. We first gve H-2K m mie OT-I K m CD8 + T ells (from mie krossed to the H-2K m strin to prevent rejetion in the H-2K m host) nd then immunized 8.2 ± ± 2.6 ± ±.4 6 ± ± 3 Control Ig CD α-cd4l Control Ig α-cd4l.8 ± ±.8 ±. 4.5 ±.5.8 ±..6 ±.9.8 ±..5 ± ±.9.5 ±.6.8 ±.4 9. ± 2.3. ± ±.5.7 ±.2 CD8 + ells ( 4 ) No 2 No 5 5 NS * nture immunology VOLUME 2 NUMBER 9 SEPTEMBER 2 99

3 2 Nture Ameri, In. All rights reserved. Figure 2 DC-derived IL-2 is not required for the seondry popultion expnsion of CD8 + T ells. () Flow ytometry of splenoytes from H-2K m mie given injetion of 5 4 OT-I H-2K m CD45. + ells, then primed d lter with 7 4 wild-type (WT) or Il2 / At-mOVA DCs, followed y nlysis of the primry CD8 + T ell response fter 7 d (top row) or oosted 39 d lter with 7 5 wild-type or Il2 / At-mOVA DCs, followed y nlysis of the OT-I response in oth primed nd oosted groups 5 d lter. Numers djent to outlined res indite perent OT-I CD8 + T ells mong totl splenoytes (men ± s.e.m.). () Asolute numer of OT-I CD8 + T ells mong totl splenoytes during the memory nd seondry response in. Dt re representtive of two experiments (men ± s.e.m. of three to four mie in ). the mie d lter with wild-type or IL-2-defiient At-mOVA DCs tht hd een exposed to the Toll-like reeptor 9 lignd CpG to indue IL-2 seretion 28. We susequently monitored the primry nd seondry popultion expnsion of OVA-speifi CD8 + T ells t dy 7 fter immuniztion nd then gin over 5 months lter fter hllenge with wild-type or IL-2-defiient At-mOVA DCs tht hd een tivted y CpG. Although the At-mOVA DCs trnsferred into the H-2K m hosts in this system would e expeted to e rejeted y host T ells euse of their expression of foreign MHC (H-2K ), this did not prelude their ility to trnsmit the signls neessry to generte oth primry nd memory CD8 + T ells (Fig. 2). Notly, we found tht the sene of IL-2 on either the priming or oosting APC hd no disernle effet on the mgnitude of the primry or seondry prolifertive response of the OT-I CD8 + T ells. These results demonstrte tht DC-derived IL-2 is not required for the progrmming of CD8 + T ell memory. CD8 + T ells require utorine IL-2 To determine whether the ility to produe utorine IL-2 is importnt for CD8 + T ell memory, we monitored the response of physiologil numer (~5) of wild-type versus Il2 / OT-I CD8 + T ells fter doptive trnsfer into wild-type mie. This pproh llowed us to simultneously trk oth wild-type endogenous CD8 + T ells nd trnsferred Il2 / OT-I CD8 + T ells t eh phse (primry, memory nd seondry) of CD4 + T ell dependent CD8 + T ell response indued y heterologous priming nd oosting with infetious pthogens (vini virus OVA or Listeri monoytogenes OVA) 29. We found tht OT-I ells lking IL-2 expression underwent more modest primry popultion expnsion thn did wild-type OT-I ells, s reported efore 3, lthough we reproduily deteted popultion of primry memory ells efore rehllenge (Fig. 3). After hllenge of intt reipient mie, however, the Il2 / OT-I CD8 + T ells hd profound defet in their seondry popultion expnsion similr in mgnitude to tht of either endogenous or OT-I CD8 + T ells tht hd not reeived help (Fig. 3 ). The endogenous IL-2-suffiient CD8 + T ells, in ontrst, underwent roust seondry popultion expnsion in the intt reipients (Fig. 3), whih showed tht dequte help ws indeed provided y host CD4 + T ells in this setting. Despite the vilility of CD4 + T ell help, only the CD8 + T ells lking IL-2 hd defet in their seondry popultion expnsion. Together these dt show tht under onditions of heterologous priming nd oosting with infetious pthogens, the ility to produe utorine IL-2 is ritil for CD8 + T ells to mount optiml seondry prolifertive responses. To investigte whether those results were lso otined in response to nonrepliting, noninflmmtory immunogens, we immunized C57BL/6 mie ontining wild-type or Il2 / OT-I CD8 + T ells with Prime: Dy 7 Dy 44 Boost (dy ) WT DCs Il2 / DCs CD CD8 + CD45. + ells ( 4 ) WT DCs Il2 / DCs.85 ±..7 ±.3.24 ±..4 ±..28 ±.4.3 ±.6.2 ±..7 ± CD WT DCs + WT DCs WT DCs + Il2 / DCs Il2 / DCs + WT DCs Il2 / DCs + Il2 / DCs ell-sed immunogen: At-mOVA splenoytes with homozygous deletion of the gene enoding H-2K. In this setting, the OVA ntigen must e ross-presented y host APCs euse of the sene of the H-2K MHC lss I moleule required for diret presenttion of the relevnt OVA( ) peptide to CD8 + T ells 3. Although the OVAspeifi responses to the ell-sed immunogen were, s rule, smller thn those indued y infetious pthogens, we nonetheless lerly oserved the speifi defet in the seondry popultion expnsion of Il2 / CD8 + T ells fter rehllenge (Fig. 4). Together these results show tht IL-2 produed y CD8 + T ells is essentil for their optiml seondry popultion expnsion in response to oth repliting nd nonrepliting immunogens, even under onditions in whih dequte help is ville from CD4 + T ells le to produe prrine IL-2. Funtion nd phenotype of Il2 / CD8 + T ells Phenotypi exmintion of wild-type nd IL-2-defiient OT-I ells indited tht those lking IL-2 produed fewer multifuntionl ytokine-produing ells (positive for IFN-γ nd tumor nerosis ftor) during ll mesured phses of the response to infetious pthogens, with the most profound defiit eing pprent in the memory popultion (Fig. 5). Monitoring of surfe mrkers showed no sustntil differene in the frequeny of short-lived effetor ells (KLRG- + CD27 ), memory preursor effetor ells (KLRG- CD27 + ), entrl memory ells (CD27 + CD62L + ) or effetor memory ells (CD62L CD27 + ) mong wild-type or IL-2-defiient OT-I ells t eh phse of the response (Fig. 5,). 9 VOLUME 2 NUMBER 9 SEPTEMBER 2 nture immunology

4 2 Nture Ameri, In. All rights reserved. Figure 3 CD8 + T ells require utorine IL-2 for memory response to repliting immunogen. (,) Frequeny () nd solute numer () of OT-I (CD45. + ) CD8 + T ells mong totl splenoytes from intt C57BL/6J (CD ) mie (+ help) or C57BL/6J (CD ) mie depleted of CD4 + T ells ( help; n = 3 4 per group) given 5 wild-type or Il2 / (CD45. + ) OT-I CD8 + T ells, then infeted d lter with 6 PFU vini virus OVA nd, for some groups (seondry), hllenged 4 d lter with.6 hlf-mximl lethl dose of L. monoytogenes OVA, followed y nlysis t dy 7 (primry), or t dy 45 for mie tht hd (seondry) or hd not (memory) een hllenged with L. monoytogenes OVA 5 d erlier. Eh symol represents n individul mouse; smll horizontl lines indite the verge (). *P <.5 (two-tiled unpired t-test). Dt re representtive of three experiments (error rs (), s.e.m.). () IFN-γ prodution y the endogenous (CD45. ) nd OT-I (CD45. + ) CD8 + T ells during the primry, memory nd seondry responses in,. Numers in qudrnts indite perent ells in eh (men ± s.e.m.). Dt re representtive of three experiments with 8 2 mie. (d) Popultion expnsion of the OT-I CD8 + T ells in,. Dt re representtive of three experiments. Both wild-type nd IL-2-defiient OT-I ells hd in vivo ytotoxi ility (Fig. 5d), lthough the ltter ells did show dely in the prodution of grnzyme B (Fig. 5e). The defet in seondry popultion expnsion in IL-2-defiient OT-I ells ws not due to ltered migrtion tivity reltive to tht of wild-type ells, s we found similr rtios of the two popultions in every orgn ssessed 3 2 OT-I Il2 / help ells ( 4 ) IFN-γ OT-I Il2 / help OT-I Il2 / help.7 ±.2.3 ±..2 ±.. ±.2.8 ±..3 ±..4 ±.. ± ±.2.8 ±.4.7 ±.2.4 ±.. ±.2.3 ±.4 3 ± ±.7 2 ± CD45..2 ±.. ±.5.4 ±.9.4 ±.6.4 ±..4 ±..8 ± ±.3.4 ±.2 ells ( 4 ) ±..2 ±.9.7 ±.3.4 ±.7.8 ±.2.3 ± ±.4 2. ±.4.2 ±.2 OT-I Il2 / help.4 ±.2.2 ±.7.8 ±.3.4 ±.6.4 ±.2.2 ±.2 ±.6.7 ±.4 (Supplementry Fig. 2). These dt show tht utorine IL-2 is not stritly required for ytotoxi funtion y CD8 + T ells or for their ility to quire the surfe phenotype ssoited with either entrl nd effetor memory ells. DISCUSSION The findings reported here hve demonstrted tht CD8 + T ells, rther thn CD4 + T ells, onstitute the ellulr soure of IL-2 required for the genertion of helper T ell dependent CD8 + T ell memory. The ontriution of CD4 + T ells to this proess ws medited through their ility to tivte APCs vi CD4L-CD4 intertions to stte in whih they eme le to endow the CD8 + T ells they primed with the ility to produe their own utorine IL-2. Our findings re inonsistent with severl tenets of the three-ell model of helper T ell dependent CD8 + T ell priming, whih envisions the nerly ontemporneous intertion of n ntigen-speifi CD4 + T ell nd CD8 + T ell t the surfe of the sme APC so tht prrine seretion of IL-2 (y the CD4 + T ell for the CD8 + T ell) n tke ple, nd insted support the ide tht, when properly stimulted y the pproprite signls emnting from CD4-tivted APC, CD8 + ± d Expnsion (fold) * IFN-γ.2 ±.2.2 ±.5. ±.7.7 ± ±.6. ±.2.7 ±.4.2 ±.7.4 ±.2.7 ±.2.4 ±..3 ± ± 2.5 ± CD45..2 ±.4.2 ±.2.9 ±.2.7 ±.2.7 ±.2.5 ±. 2.7 ±.3. ±.2. ±.6. ±.6.7 ±.2.3 ±.4.2 ±.3.4 ±.2 Figure 4 CD8 + T ells require utorine IL-2 for memory response to nonrepliting immunogen. (,) Frequeny () nd solute numer () of OT-I (CD45. + ) CD8 + T ells mong totl splenoytes from C57BL/6 (CD ) mie (n = 3 4 per group) given 5 wild-type or Il2 / (CD45. + ) OT-I CD8 + T ells, then immunized d lter with 5 6 At-mOVA H-2K -defiient splenoytes nd, for some groups (seondry), hllenged 3 d lter with 5 6 PFU vini virus OVA, followed y nlysis t dy 7 (primry), or t dy 45 in mie tht hd (seondry) or hd not (memory) een hllenged with vini virus OVA 5 d (presented s in Fig. 3,). () IFN-γ prodution y endogenous (CD45. ) nd OT-I (CD45. + ) CD8 + T ells during the primry, memory nd seondry responses in (presented s in Fig. 3; n = 8 2 mie). Dt re representtive of two experiments (men ± s.e.m. in,). nture immunology VOLUME 2 NUMBER 9 SEPTEMBER 2 9

5 H-2K OVA Events Events A rt i l e s 2 Nture Ameri, In. All rights reserved. Figure 5 Funtionl nd phenotypi omprison of wild-type nd IL-2-defiient OT-I CD8 + T ells. () Frequeny of OT-I CD8 + T ells produing IFN-γ lone ( ) or oth IFN-γ nd tumor nerosis ftor ( TNF + ) mong totl splenoytes from C57BL/6J (CD ) mie (n = 3 4 per group) given wild-type (OT-I) or Il2 / (OT-I Il2 / ) CD45. + OT-I CD8 + T ells nd infeted d lter with 6 PFU vini virus OVA, nlyzed y intrellulr stining of CD45. + ells t dy 7 (primry), or t dy 45 in mie tht hd (seondry) or hd not (memory) een hllenged with.6 hlfmximl lethl dose of L. monoytogenes OVA 5 d erlier. *P <.5 nd **P <.5 (two-tiled unpired t-test). () Frequeny of OT-I ells with positive or negtive expression of the tivtion nd memory mrkers KLRG- nd CD27 (IL-7 reeptor α-hin) on CD45. + ells in the lood from the mie in t dy 7 (primry). () Frequeny of OT-I CD8 + T ells with the surfe phenotype of entrl memory ells (CD27 + CD62L + ) or effetor memory ells (CD27 + CD62L ) t dy 4 (memory) in the lood of the mie in. (d) In vivo ytotoxiity of wild-type nd Il2 / OT-I ells 6 d fter immuniztion with vini virus OVA, presented s the frequeny of tetrmerpositive OT-I ells in wild-type mie (numers ove outlined res; left) nd frequeny of trget ells loded with vrious onentrtions of the ytosoli dye CSFE nd pulsed with the peptide epitope of denovirus erly B protein mino ids 92 2 (EB) or OVA( ) (OVA) 6 h fter doptive trnsfer into the immunized mie (right; numers in plots indite perent CSFE + ells). (e) Expression of grnzyme B y wild-type nd Il2 / OT-I ells t dys 5 fter immuniztion of the mie in with vini virus OVA. IgG (key), stining with isotype-mthed ontrol ntiody. Dt re representtive of three (,) or two (,d,e) experiments (error rs ( ), s.e.m.). T ell eomes ompetent to mke its own IL-2. Our findings reonile disrepnies regrding the speifi role of CD4 + T ells nd the soure of IL-2 required for optiml seondry popultion expnsion inherent in the three-ell model versus the sequentil intertion model nd llow meningful extension of the ltter y sriing IL-2 prodution to the CD8 + T ell rther thn the CD4 + T ell. Our results re onsistent with pulished findings showing tht CD8 + memory T ells (whih hve low expression of the CD25 (IL-2 reeptor α-hin)) hve greter pity to produe this ytokine 2. Furthermore, our results hve the potentil to inform the finding tht Il2 / CD8 + T ells n undergo seondry prolifertion in one mrrow himer if provided with prrine IL-2 (ref. 2). Given tht, it is oneivle tht in ertin model systems, immunogens tht indue T ell response of suffiient mgnitude will generte enough prrine IL-2 t sites of tivtion to overome the utorine requirement demonstrted y our studies. Similrly, the ellulr soures for IL-2 required for the popultion expnsion of CD8 + memory T ells my differ depending on the nture of the priming stimulus, the numer of eh ell type reruited into the response nd the miroenvironmentl loliztion. Our findings ontrst with those of pulished study supporting the possiility of role for prrine seretion of IL-2 y CD4 + T ells, whih might e explined y the nture of the priming stimulus (peptide-loded DC versus ross-presented ntigen) or perhps in the different numers of ntigen-speifi CD4 + T ells used in the two experimentl systems 32. It is possile tht the experimentl system used in tht study 32, whih fetured the presene of up to 2-fold more ells during priming thn used in our experiments here, my hve influened the quntity of IL-2 produed nd thus the distint results we otined. We lso knowledge tht the struture of seondry lymphoid orgns in our system my hve een ltered s onsequene of dult thymetomy nd ntiody-medited depletion of mture CD4 + T ells. Whtever the degree of potentil struturl ltertion, however, it did not prelude the genertion of roust CD ** CD27 + CD62L + TNF + OT-I OT-I Il2 / KLRG- + CD27 KLRG- CD27 + CD27 + CD62L d Nive OT-I OT-I Il2 / 3 2 * TNF + 3 ± EB T ell dependent OVA-speifi memory CD8 + T ells from endogenous responders. Notly, our study hs provided evidene tht CD4 + T ells do not need to produe IL-2 to provide the help needed for CD8 + T ell memory nd onfirm in seond model of CD4 + T ell dependent CD8 + T ell priming tht lokde of CD4L prevents trnsmission of the help signl from CD4 + T ells to APCs 3,5,6. Although IL-2 produed y DCs is importnt for the initition of innte immune responses, our results hve demonstrted tht this did not influene the outome of the dptive CD8 + T ell response during either priming or seondry ntigeni enounter 33. Under ertin onditions, suh s fter immuniztion with virus or live vetor or djuvnt known to reruit DCs nd to enhne their persistene t the site of immune-response initition, the requirement for DC-derived IL-2 my e ltered. The H-2K m model system used in our experiments ould prove useful in other settings, s it llows the in vivo ntigen-presenting ility of mny different ell types to e evluted. Although the H-2K m moleule poorly selets the OT-I T ell ntigen reeptor, we were nonetheless le to isolte fully funtionl mture OT-I ells from the OT-I.Km strin for use in doptive trnsfer experiments 34. Our studies hve identified dditionl spets of the mehnism underlying the role of helper T ells in the formtion of CD8 + memory T ells, whih hve een shown to t from the erliest stges of this proess in direting the hemokine-medited ttrtion of nive CD8 + T ells to sites of intertion etween DCs nd CD4 + T ells, nd, s reinfored y the findings of our study, y tivting DCs vi CD4L- CD4 intertions so they n trnsmit the signls neessry to prime CD8 + memory T ells 3,5,6,35. Although our studies did not speify role for helper T ell derived IL-2 in this proess, we must point out tht prrine IL-2 ws nonetheless ville from either CD4 + T ells or DCs in our experimentl systems. Despite tht, prrine IL-2 ws not suffiient to resue the seondry popultion expnsion defet of the Il2 / CD8 + T ells. Our finding tht suh ells funtioned s ytotoxi effetors in vivo is onsistent with results otined with Il2 / mie, TNF + OVA 99 ± CD45. CFSE e Dy Dy 3 Dy 4 Dy ± ±.7 IgG OT-I OT-I Il2 / Grnzyme B 92 VOLUME 2 NUMBER 9 SEPTEMBER 2 nture immunology

6 2 Nture Ameri, In. All rights reserved. lthough our dditionl informtion out the presene of onsiderly fewer ells produing oth IFN-γ nd tumor nerosis ftor my provide new insight into the role of utorine IL-2 in the genertion of the multifuntionl suset tht ers further investigtion 36. The oservtion tht CD4 + T ell derived IL-2 ws irrelevnt for the priming of memory CD8 + T ells removes entrl funtion sried to these ells in the three-ell model, s well s the requirement for the physil proximity of CD4 + T ells nd CD8 + T ells invoked y the prrine seretion model. By enling CD8 + T ells to produe nd onsume their own IL-2, the rhiteture of the immune system onfers onsiderle degree of independene on these ntigen-experiened killer T ells nd enles them to undergo oth ytolyti nd prolifertive responses without requiring their intertion with either ntigen-speifi CD4 + T ells or professionl IL-2-expressing tivted APCs, oth of whih my e either rre or trnsitory under physiologil onditions of re-enountering ntigen t peripherl sites of infetion. The findings reported here provide new insight into the mehnism through whih helper T ells enle CD8 + T ells to hieve the funtionl utonomy essentil for their tsk s rmed sentinels ptrolling oth lymphoid nd peripherl environments. Future efforts should e imed t identifying the APCderived signls tht imprint CD8 + T ells with the ility to produe utorine IL-2, determining the point(s) during the development of CD8 + T ell memory t whih utorine IL-2 ts to enle memory ell formtion nd eluidting whether its min effet is relted to its role in promoting metolism or new ptterns of gene expression 37. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Immunology wesite. Aknowledgments We thnk S. Slek-Ardkni (L Joll Institute for Allergy nd Immunology) for vini virus OVA; M.K. Jenkins (University of Minnesot Medil Shool) for At-mOVA-trnsgeni mie; M. Rfii-El-Idrissi for demonstrting how to produe nd purify the virus; F. Lmolez for the protool for purifying T ells from the liver; S. Trifri nd M. Pipkin for dvie on grnzyme B expression; nd C. Kim nd K. Vn Gunst for tehnil dvie nd ssistne. Supported y the US Ntionl Institutes of Helth (R AI76972 nd RCA826 to S.P.S.), the Leukemi nd Lymphom Soiety (63-6 to S.P.S.) nd the Kurz Fmily Foundtion. AUTHOR CONTRIBUTIONS S.F., R.A. nd S.P.S. designed the experiments; S.F. nd R.A. did the experiments with ssistne from S.T.; nd S.F. nd S.P.S. nlyzed the dt nd wrote the mnusript with ontriutions from R.A. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprints/index.html.. Ahmed, R. & Gry, D. Immunologil memory nd protetive immunity: understnding their reltion. Siene 272, 54 6 (996). 2. vn Stipdonk, M.J. et l. Dynmi progrmming of CD8 + T lymphoyte responses. Nt. Immunol. 4, (23). 3. vn Stipdonk, M.J., Lemmens, E.E. & Shoenerger, S.P. Nive CTLs require single rief period of ntigeni stimultion for lonl expnsion nd differentition. Nt. Immunol. 2, (2). 4. Keh, S.M. & Ahmed, R. CD8 + T ell differentition: initil ntigen enounter triggers developmentl progrm in nive ells. Nt. Immunol. 2, (2). 5. Bdovin, V.P., Porter, B.B. & Hrty, J.T. Progrmmed ontrtion of CD8 + T ells fter infetion. Nt. Immunol. 3, (22). 6. Msopust, D., Keh, S.M., Wherry, E.J. & Ahmed, R. The role of progrmming in memory T-ell development. Curr. Opin. Immunol. 6, (24). 7. Sun, J.C. & Bevn, M.J. Defetive CD8 T ell memory following ute infetion without CD4 T ell help. Siene 3, (23). 8. Shedlok, D.J. & Shen, H. Requirement for CD4 T ell help in generting funtionl CD8 T ell memory. Siene 3, (23). 9. Bourgeois, C., Roh, B. & Tnhot, C. A role for CD4 expression on CD8 + T ells in the genertion of CD8 + T ell memory. Siene 297, (22).. Jnssen, E.M. et l. CD4 + T ells re required for seondry expnsion nd memory in CD8 + T lymphoytes. Nture 42, (23).. Jnssen, E.M. et l. CD4 + T-ell help ontrols CD8 + T-ell memory vi TRAILmedited tivtion-indued ell deth. Nture 434, (25). 2. Bdovin, V.P., Messinghm, K.A., Griffith, T.S. & Hrty, J.T. TRAIL defiieny delys, ut does not prevent, erosion in the qulity of helpless memory CD8 T ells. J. Immunol. 77, (26). 3. Bennett, S.R. et l. Help for ytotoxi-t-ell responses is medited y CD4 signlling. Nture 393, (998). 4. Lnzvehi, A. Immunology. Liene to kill. Nture 393, (998). 5. Ridge, J.P., Di Ros, F. & Mtzinger, P. A onditioned dendriti ell n e temporl ridge etween CD4 + T-helper nd T-killer ell. Nture 393, (998). 6. Shoenerger, S.P., Toes, R.E., vn der Voort, E.I., Offring, R. & Melief, C.J. T-ell help for ytotoxi T lymphoytes is medited y CD4 CD4L intertions. Nture 393, (998). 7. Hork, I., Lohler, J., M, A. & Smith, K.A. Interleukin-2 defiient mie: new model to study utoimmunity nd self-tolerne. Immunol. Rev. 48, (995). 8. Keene, J.A. & Formn, J. Helper tivity is required for the in vivo genertion of ytotoxi T lymphoytes. J. Exp. Med. 55, (982). 9. Mithison, N.A. & O Mlley, C. Three-ell-type lusters of T ells with ntigenpresenting ells est explin the epitope linkge nd nonognte requirements of the in vivo ytolyti response. Eur. J. Immunol. 7, (987). 2. Willims, M.A., Tyznik, A.J. & Bevn, M.J. Interleukin-2 signls during priming re required for seondry expnsion of CD8 + memory T ells. Nture 44, (26). 2. Kli, V. et l. Prolonged interleukin-2rα expression on virus-speifi CD8 + T ells fvors terminl-effetor differentition in vivo. Immunity 32, 9 3 (2). 22. Pipkin, M.E. et l. Interleukin-2 nd inflmmtion indue distint trnsriptionl progrms tht promote the differentition of effetor ytolyti T ells. Immunity 32, 79 9 (2). 23. Mlek, T.R. & Byer, A.L. Tolerne, not immunity, ruilly depends on IL-2. Nt. Rev. Immunol. 4, (24). 24. Sdlk, B. et l. Generlized utoimmune disese in interleukin-2-defiient mie is triggered y n unontrolled tivtion nd prolifertion of CD4 + T ells. Eur. J. Immunol. 25, (995). 25. Kurts, C. et l. Constitutive lss I-restrited exogenous presenttion of self ntigens in vivo. J. Exp. Med. 84, (996). 26. Ehst, B.D., Ingulli, E. & Jenkins, M.K. Development of novel trnsgeni mouse for the study of intertions etween CD4 nd CD8 T ells during grft rejetion. Am. J. Trnsplnt. 3, (23). 27. Nikolić-Zugić, J. & Bevn, M.J. Role of self-peptides in positively seleting the T-ell repertoire. Nture 344, (99). 28. Grnui, F., Feu, S., Angeli, V., Trottein, F. & Riirdi-Cstgnoli, P. Erly IL-2 prodution y mouse dendriti ells is the result of miroil-indued priming. J. Immunol. 7, (23). 29. Bdovin, V.P., Hring, J.S. & Hrty, J.T. Initil T ell reeptor trnsgeni ell preursor frequeny dittes ritil spets of the CD8 + T ell response to infetion. Immunity 26, (27). 3. D Souz, W.N. & Lefrnois, L. IL-2 is not required for the initition of CD8 T ell yling ut sustins expnsion. J. Immunol. 7, (23). 3. Kurts, C. et l. CD4 + T ell help impirs CD8 + T ell deletion indued y ross-presenttion of self-ntigens nd fvors utoimmunity. J. Exp. Med. 86, (997). 32. Wilson, E.B. & Livingstone, A.M. Cutting edge: CD4 + T ell-derived IL-2 is essentil for help-dependent primry CD8+ T ell responses. J. Immunol. 8, (28). 33. Grnui, F. et l. A ontriution of mouse dendriti ell-derived IL-2 for NK ell tivtion. J. Exp. Med. 2, (24). 34. Clrke, S.R. et l. Chrteriztion of the ovlumin-speifi TCR trnsgeni line OT-I: MHC elements for positive nd negtive seletion. Immunol. Cell Biol. 78, 7 (2). 35. Cstellino, F. et l. Chemokines enhne immunity y guiding nive CD8 + T ells to sites of CD4 + T ell-dendriti ell intertion. Nture 44, (26). 36. Kündig, T.M. et l. Immune responses in interleukin-2-defiient mie. Siene 262, 59 6 (993). 37. Mintyre, A.N. et l. Protein kinse B ontrols trnsriptionl progrms tht diret ytotoxi T ell fte ut is dispensle for T ell metolism. Immunity 34, (2). nture immunology VOLUME 2 NUMBER 9 SEPTEMBER 2 93

7 2 Nture Ameri, In. All rights reserved. ONLINE METHODS Mie. C57BL/6 mie, B6.C-H2 m /ByJ (H-2K m ) mie, C57BL/6J mie nd dult C57BL/6J mie tht hd undergone thymetomy were from The Jkson Lortory. At-mOVA-trnsgeni mie were gift from M.K. Jenkins. Il2 / mie, OT-I reomintion-tivting gene defiient (Rg / ) CD45. + mie nd At-mOVA-K -defiient mie on the C57BL/6J kground hve een desried,38. The OT-I Rg / Il2 / (CD45. + ), Rg / Il2 / nd AtmOVA Il2 / strins were generted y interrossing. Mie were mintined nd red in the L Joll Institute for Allergy nd Immunology vivrium under speifi pthogen free onditions in ordne with guidelines of the Assoition for Assessment nd Aredittion of Lortory Animl Cre Interntionl. Antiody tretment. For OT-I T ell responses, helpless groups reeived intrperitonel injetion of 2 µg ntiody to CD4 (nti-cd4; GK.5; produed in-house ) 3 d nd d efore immuniztion, whih resulted in the depletion of >99% of CD4 + T ells, onfirmed y stining with phyoerythrinleled nonompeting nti-cd4 (RM4.4; ebiosiene). Mie in oth the helped nd helpless groups reeived 2 µg nti-cd4 (GK.5) 4 d fter primry immuniztion. For endogenous T ell responses, C57BL/6J dult mie tht hd undergone thymetomy reeived n initil dose of 2 µg nti-cd4 (GK.5) nd seond dose of µg 2 d lter. Then, 2 weeks fter tht finl dose, ells were stined with nti-cd4 for onfirmtion of the sene of CD4 + ells (Supplementry Fig. ). For CD4L lokde, mie reeived 5 µg ntiody to mouse CD4L (MR-; produed in-house ) or isotypemthed polylonl ontrol hmster immunogloulin G (7--3; Jkson ImmunoReserh) on dys, nd +2 reltive to immuniztion. T ell preprtion. OT-I (CD45. + ) or OT-I Il2 / (CD45. + ) ells were olleted from lood. Mie reeived 5 OT-I ells y intrvenous injetion mixed with 6 P4 CD9. + splenoytes used s n internl ontrol for quisition y flow ytometry. OT-I K m ells were olleted from the spleen nd 5 4 ells were injeted. or Il2 / CD4 + T ells or CD4 + T ells from SMARTA mie (with trnsgeni expression of T ell ntigen reeptor speifi for lymphoyti horiomeningitis virus glyoprotein) were isolted y negtive seletion (MACS; Miltenyi), nd 5 4 ells were injeted intrvenously. OT-I K m CD45. + ells were isolted y positive seletion (MACS; Miltenyi). The numer of eh trnsferred ell type ws determined y diret ounting nd y flow ytometry stining for the speifi omintion of T ell ntigen reeptor α-hin nd β-hin vrile region they express. DC preprtion. Offspring produed y interrossing of At-mOVA Il2 +/ mie were injeted etween dy 5 nd dy fter irth with one mrrow nd splenoytes from wild-type CD45. + mie to inhiit the lymphoprolifertion due to the lk of regultory T ells in the Il2 / mie produed in the F genertion. At 8 weeks of ge, mie were implnted suutneously with B6 mouse melnom ells expressing the ytokine Flt3L to inrese the numer of spleni DCs olleted 5 d lter 39. Spleens from wild-type or Il2 / At-mOVA mie were perfused with ollgense D ( mg/ml) in presene of DNAse I nd isolted s desried 4. DCs were tivted for 3 min t 37 C with mm CpG nd stined with llophyoynin-onjugted nti-cd (HL3; BD Phrmingen), phyoerythrin-onjugted nti-cd45.2 (4; ebiosiene) nd 7-mino-tinomyin D efore sorting to otin CD + CD ells negtive for 7-mino-tinomyin D. In vivo experiments. OT-I, or SMARTA ells were trnsferred into reipient mie d efore immuniztion. Mie were immunized intrperitonelly with 5 6 At-mOVA H-2K -defiient splenoytes (priming) nd were hllenged intrperitonelly with 5 6 PFU vini virus OVA, or were primed with 6 PFU vini virus OVA nd oosted intrvenously with 3 3 olony-forming units of Listeri monoytogenes OVA. For DC experiments, groups of H-2K m (CD ) mie initilly reeived 5 4 OT-I H-2K m (CD45. + ) ells nd were injeted intrvenously d lter with 5 4 At-mOVA Il2 +/+ or Il2 / DCs. Then, 7 d lter, spleens were isolted nd the response of the OT-I K m (CD45. + ) T ells ws nlyzed y mesurement of the frequeny of CD45. + ytokine-produing ells. Some groups were oosted intrvenously with 5 5 At-mOVA Il2 +/+ or Il2 / DC t dy 39 fter infetion nd the seondry responses were mesured 5 d lter. Ex vivo restimultion nd ntiody stining. Cytokine prodution in spleni CD8 + T ells ws ssessed with the Fixtion/Permeiliztion Solution Kit with BD GolgiPlug ording to the mnufturer s instrutions (BD Phrmingen) fter in vitro restimultion for 5 h t 37 C in 96-well round-ottomed pltes (for At-mOVA H-2K -defiient ells with vini virus OVA priming nd oosting) or in 96-well flt-ottomed pltes for oosting with Listeri monoytogenes OVA, in 2 µl Isove s modified Duleo s medium plus OVA( ) (SIINFEKL) peptide) t onentrtion of µg/ml in the presene of GolgiPlug (BD Biosienes). Cells were stined with leled phyoerythrin Texs Red onjugted nti-cd8 (MCD87; Invitrogen), llophyoynin efluor 78 onjugted nti-cd62l (MEL-4; ebiosiene), Alex Fluor 7 onjugted nti- CD44 (IM7; ebiosiene), Pifi Blue onjugted nti-cd45. (A2; BioLegend) nd peridinin hlorophyll protein ynine 5.5 onjugted nti-cd45.2 (4; ebiosiene) followed y fixtion for 2 min t 4 C. Fixed ells were sujeted to intrellulr ytokine with phyoerythrin-indotriroynine onjugted ntiody to tumor nerosis ftor (MP6-XT22; BD Phrmingen), llophyoyninonjugted nti-ifn-γ (XMG.2; ebiosiene) nd phyoerythrin-onjugted nti-il-2 (JES6-5H4; BioLegend). Smples were quired on LSR II (Beton Dikinson) nd dt were nlyzed with FlowJo softwre. Cytokine prodution in ells ws ssessed with the Fixtion/ Permeiliztion Solution Kit with BD GolgiPlug ording to the mnufturer s instrutions (BD Phrmingen) fter in vitro restimultion for 7 h t 37 C in 96-well round-ottomed pltes in 2 µl medium plus the I-A - inding peptide onsisting of OVA mino ids ( µg/ml) efore the ddition of GolgiPlug for the finl 5 h (BD Biosienes). Cells were stined for 2 min t 4 C with leled fluoresein isothioynte onjugted nti-cd4 (GK.5; ebiosiene), nti-cd45., nti-cd44 nd llophyoynin efluor 78 onjugted nti-tcrβ (H57-597; ebiosiene), followed y fixtion with Cytofix/Cytoperm (BD Biosienes). Fixed ells were sujeted to intrellulr ytokine stining with nti-ifn-γ nd nti-il-2. Smples were quired on LSR II (Beton Dikinson) nd dt were nlyzed with FlowJo softwre. Tetrmer stining nd flow ytometry. Cells were stined for 2 min t 25 C with phyoerythrin-onjugted OVA( ) H-2K tetrmer (Bekmn Coulter), followed y stining with nti-cd8, nti-cd62l, nti- CD44 nd llophyoynin-onjugted nti-cd27 (A7R34; ebiosiene). Smples were quired on n LSR II (Beton Dikinson) nd dt were nlyzed with FlowJo softwre. Grnzyme B stining nd flow ytometry. Mie reeived 5 5 OT-I or OT-I Il2 / (CD45. + ) ells d efore intrvenous immuniztion t dy with 5 6 vini virus OVA. Blood smples were olleted t dys 3, 4 nd 5 fter immuniztion nd ells were stined with nti-cd8 nd nti-cd45., followed y fixtion for 2 min t 4 C with Cytofix/Cytoperm (BD Biosienes). Fixed ells were sujeted to intrellulr stining for 3 min t 4 C in Perm/Wsh uffer (BD Biosienes) with mouse immunogloulin G isotype-mthed ontrol ntiody (MG4; Invitrogen) or ntiody to grnzyme B (GRB4; Invitrogen). Smples were quired on LSR II (Beton Dikinson) nd dt were nlyzed with FlowJo softwre. In vivo ytotoxi ssy. Mie were injeted with 5 3 OT-I or OT-I Il2 / (CD45. + ) ells d efore intrvenous immuniztion t dy with 6 PFU vini virus OVA. At dy 5, 5 6 CD45. + splenoytes loded with irrelevnt peptide (denovirus type 5 EB, mino ids 92 2) nd 5 6 CD45. + splenoytes loded with speifi peptide (OVA( )) stined with.2 µm or.2 µm CFSE (roxyfluoresein diette suinimidyl ester), respetively, were injeted intrvenously. Then, 5 h lter, spleens were removed nd smples quired on n LSR II (Beton Dikinson) nd susequently nlyzed with FlowJo softwre s reported. 38. Shorle, H., Holtshke, T., Hunig, T., Shimpl, A. & Hork, I. Development nd funtion of T ells in mie rendered interleukin-2 defiient y gene trgeting. Nture 352, (99). 39. Mh, N. et l. Differenes in dendriti ells stimulted in vivo y tumors engineered to serete grnuloyte-mrophge olony-stimulting ftor or Flt3-lignd. Cner Res. 6, (2). 4. Henri, S. et l. The dendriti ell popultions of mouse lymph nodes. J. Immunol. 67, (2). nture immunology doi:.38/ni.279