Alternative cross-priming through CCL17-CCR4- mediated attraction of CTLs toward NKT cell licensed DCs

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1 Alterntive cross-priming through CCL17-CCR4- medited ttrction of CTLs towrd NKT cell licensed DCs 21 Nture Americ, Inc. All rights reserved. Veren Semmling 1,1, Veronik Lukcs-Kornek 1,9,1, Christoph A Thiss 1, Thoms Qust 2, Kthrin Hochheiser 1, Ulf Pnzer 3, Jmie Rossjohn 4, Ptrick Perlmutter 5, Ji Co 5, Dle I Godfrey 6, Pul B Svge 7, Percy A Knolle 1, Wldemr Kolnus 2, Irmgrd Förster 8 & Christin Kurts 1 Cross-priming llows dendritic cells (DCs) to induce cytotoxic T cell (CTL) responses to extrcellulr ntigens. DCs require cognte licensing for cross-priming, clssiclly y helper T cells. Here we demonstrte n lterntive mechnism for cognte licensing y nturl killer T (NKT) cells recognizing microil or synthetic glycolipid ntigens. Such licensing cused cross-priming CD8 + DCs to produce the chemokine CCL17, which ttrcted nive CTLs expressing the chemokine receptor CCR4. In contrst, DCs licensed y helper T cells recruited CTLs using CCR5 lignds. Thus, depending on the type of ntigen they encounter, DCs cn e licensed for cross-priming y NKT cells or helper T cells nd use t lest two independent chemokine pthwys to ttrct nive CTLs. Becuse these chemokines cted synergisticlly, this cn potentilly e exploited to improve vccintions. Cross-presenttion permits specilized dendritic cells (DCs) to present extrcellulr ntigens to cytotoxic CD8 + T cells (cytotoxic T lymphocytes (CTLs)) 1 3. Cross-presenttion of utontigens or innocuous circulting ntigens usully results in cross-tolernce 4,5. Immunogenic cross-presenttion, lso known s cross-priming, requires the presence of pthogen-ssocited moleculr ptterns, such s Toll-like receptor (TLR) lignds, nd/or of specific CD4 + helper T cells 6,7 tht license the cross-presenting DC for crosspriming 1,8. The underlying moleculr mechnisms tht result from such licensing re under intense investigtion nd include enhnced costimultory signls nd diminished propoptotic signls y the DCs 9,1. The necessity of DC licensing in cross-priming indictes tht severl rre cells of the immune response, tht is, ntigen-specific CTLs, specific helper T cells nd cross-presenting DCs, must physiclly interct. Such interctions re usully governed y chemokines nd their receptors 11, which suggests tht these molecules might e involved in cross-priming. In support of this ide, it hs een shown tht the interction of helper T cells with TLR lignd stimulted DCs cuses the production of lignds for the chemokine receptor CCR5, which ttrct nive CTLs nd increse the likelihood of their encounter with licensed DCs 12,13. Activted CTLs themselves cn then produce CCR5 lignds tht ttrct further CTLs to DCs tht hve successfully cross-primed 14. Also, nturl killer T cells (NKT cells) cn ctivte DCs for cross-priming 15 17, ut the underlying mechnisms re uncler. As NKT cell dependent cross-priming y DCs lso requires the encounter of three immune cell types, role for chemokines is likely ut hs not yet een studied. NKT cells re thought to link innte nd dptive immunity of oth the T helper type 1 (T H 1) nd T H 2 type nd hve een ssocited with ntimicroil, utoimmune nd ntitumor immunity Most NKT cells express semi-invrint T cell ntigen receptor repertoire through which they recognize glycolipid ntigens presented y the nonpolymorphic mjor histocomptiility complex (MHC) clss I like protein CD1d 21. Bcteril lignds include α- glctosyldicylglycerol (α-gldag) from Borreli urgdorferi nd α-glycuronosylcermides such s GSL-1 from Sphingomons species Isoglootrihexosylcermide (ig3) is n endogenous lignd tht hs een linked to NKT cell ctivtion fter infection with Grm-negtive cteri 23. The glycosphingolipid α-glctosylcermide () hs een widely used s model NKT cell ntigen 15,18,19. OCH is synthetic derivtive with truncted sphingosine chin tht induces T H 2-like responses 25. Mutgenesis studies indicte tht CD1d inds nd other lignds, including ig3 nd α-gldag, in similr mnner 26,27. NKT cell ctivtion cuses rpid production of T H 1, T H 2 nd T H 17 cytokines nd vrious chemokines nd chemokine 1 Institutes of Moleculr Medicine nd Experimentl Immunology nd 2 Life nd Medicl Sciences Institute, Friedrich-Wilhelms-Universität, Bonn, Germny. 3 III Medizinische Klinik, Universitätsklinikum Hmurg-Eppendorf, Hmurg, Germny. 4 Deprtment of Biochemistry nd Moleculr Biology nd 5 Deprtment of Chemistry, Monsh University, Clyton, Austrli. 6 Deprtment of Microiology nd Immunology, University of Melourne, Prkville, Austrli. 7 Deprtment of Chemistry nd Biochemistry, Brighm Young University, Provo, Uth, USA. 8 Moleculr Immunology, Institut für Umweltmedizinische Forschung n der Heinrich- Heine-Universität Düsseldorf, Düsseldorf, Germny. 9 Present ddress: Dn-Frer Cncer Institute, Boston, Msschusetts, USA. 1 These uthors contriuted eqully to this work. Correspondence should e ddressed to I.F. (irmgrd.foerster@uni-duesseldorf.de) or C.K. (ckurts@we.de). Received 22 Octoer 29; ccepted 2 Ferury 21; pulished online 28 Ferury 21; doi:1.138/ni.1848 nture immunology VOLUME 11 NUMBER 4 APRIL

2 Figure 1 Cognte NKT cell licensing of splenic DCs for cross-priming. ( c) In vivo -specific cytotoxic response in the spleen (,c) or the lood () 5 d fter priming of wild-type () mice with solule with or without (), 5 d fter priming of wild-type mice with plus t 1 d or 3 d fter shm opertion or splenectomy (splx) () or fter priming of MHC clss II deficient mice (MHCII-KO) or CD1d-deficient mice (Cd1d / ) with plus (c). (d) Flow cytometry nlysis of OT-I cells mong splenic CD8 + cells from m1 mice reconstituted with 5% m1 one mrrow nd 5% CD1d-deficient or wild-type one mrrow c d -specific weeks efore, nlyzed 3 d fter doptive trnsfer of OT-I cells nd priming with plus. Tet +, OT-I cells identified y stining with specific tetrmers. Dt re representtive of two individul experiments with four to five mice per group in ech (men nd s.d.). -specific (shm) (splx dy 1) (splx dy 3) -specific MHCII-KO Cd1d / Tet + cells mong CD8 + cells (%) m1 Cd1d / + m1 21 Nture Americ, Inc. All rights reserved. receptors, s well CD4L upregultion nd cytotoxic ctivity 19,28,29. In turn, DCs re ctivted y NKT cells nd respond y upregulting MHC clss II nd costimultory molecules nd y secreting interleukin 12 (IL-12) 19,2,3,31. One chemokine receptor expressed y NKT cells is CCR4 (refs ). This receptor nd its two lignds, CCL17 (lso clled thymus- nd ctivtion-regulted chemokine) nd CCL22 (lso clled mcrophge-derived chemokine) hve een linked to memory CD4 + helper T cell responses 35,36, especilly of the T H 2 type 37, nd to llergic conditions of the irwys nd the skin CCL17 expression is highly restricted to DCs, which suggests role in the unique ility of DCs to prime T cells. Little is known out CCR4 nd its lignds in immune responses in the spleen or out their roles in CTL responses. Here we report n unexpected role for CCR4 nd CCL17 in NKT cell licensed cross-priming. RESULTS Cognte NKT cell licensing of splenic DCs NKT cells cn enhnce CTL cross-priming y DCs 17,3,31. We studied the underlying mechnism in mouse model of cross-priming ginst the model ntigen ovlumin () in mice lso injected with CD1d-restricted NKT cell lignd. Consistent with pulished findings 3, ugmented CTL ctivtion in the spleen, s shown y greter cytotoxic ctivity (Fig. 1), production of interferon-γ (IFN-γ) nd more -specific CTLs (Supplementry Fig. 1). In mice lcking spleens, did not enhnce cytotoxicity (Fig. 1), c -specific -specific Ccr5 / CcI17 / Untreted CcI17 / Anti-CD3 + nti-cd28 Figure 2 NKT cell licensed cross-priming requires CCL17 nd CCR4. (,) -specific cytotoxicity 5 d fter priming of wild-type mice, CCR5- deficient mice (Ccr5 / ) or CCR4-deficient mice (Ccr4 / ) with solule plus () or wild-type mice, CCL17-deficient mice (Ccl17 / ) or CCR4-deficient mice with solule with or without (). (c) Division indices of endogenous CTLs, leled with the cytosolic dye CFSE, otined from wild-type, CCL17-deficient or CCR4-deficient mice nd left untreted or stimulted with nti-cd3 plus nti-cd28. Dt re representtive of two individul experiments with four mice (,) or four individul smples (c) per group in ech (men nd s.d.). which identified the spleen s the site where enhnced CTL cross-priming. We found tht ws lso opertive in MHC clss II deficient mice ut not in CD1d-deficient mice (Fig. 1c), which confirmed the dependence on NKT cells nd independence of helper T cells in this system. Clssicl cross-priming requires tht the sme DC tht cross-primes CTLs receives stimulting signls from helper T cells, which hs een clled cognte licensing 6,8. We determined whether this is lso necessry for NKT cell licensing of DCs y using mixed one mrrow chimers tht received m1 nd CD1d-deficient one mrrow t rtio of 1:1. DCs from m1 mice intercted vi CD1d with NKT cells ut not with -specific CTLs, wheres DCs from CD1ddeficient mice intercted with those CTLs ut not with NKT cells (Supplementry Fig. 2). In such mixed one mrrow chimers, did not enhnce the cross-priming of doptively trnsferred -specific CTLs from OT-I trnsgenic mice, in contrst to results otined with control chimers tht received m1 plus wild-type one mrrow, which hd DCs tht were le to interct with oth NKT cells nd CTLs (Fig. 1d). Thus, NKT cell licensing requires tht the sme DC physiclly intercts with NKT cells nd CTLs. NKT cell licensed cross-priming requires CCL17 nd CCR4 Helper T cells enhnce cross-priming y inducing CCR5 lignds tht ttrct nive CTLs towrd cross-presenting DCs 12. To determine whether CCR5 ws lso importnt in NKT cell licensed cross-priming, we chllenged CCR5-deficient mice with plus. We found tht -induced enhncement of cross-priming ws unchnged in these mice, wheres it ws 65% lower in CCR4-deficient mice (Fig. 2,) nd ws 5% lower in mice deficient in CCL17 (Fig. 2), DC-restricted CCR4 lignd 4. As CCL17-deficient mice, we used homozygous CCL17- egfp knock-in mice in which oth Ccl17 genes hd een replced with gene encoding enhnced green fluorescent protein (egfp), leving the Ccl17 promoter in plce 4. We lso found such dependency on CCR4 nd CCL17 when we counted -specific CTLs nd IFN-γ-producing cells s lterntive redouts (Supplementry Fig. 3). This result ws not due to intrinsic CTL defects in CCR4 nd CCL17-deficient mice, ecuse nive polyclonl CTLs from CCR4- nd CCL17-deficient mice responded like wild-type cells to stimultion in vitro with ntiody to CD3 (nti-cd3) nd nti-cd28 (Fig. 2c). These dt showed tht NKT cell licensed cross-priming did not depend on CCR5 ut insted depended on CCR4. Of the two CCR4 lignds, CCL17 medited most of the CCR4-dependent enhncement. NKT cells induce CCL17 in splenic DCs The role of CCL17 in -enhnced cross-priming in the spleen ws unexpected, ecuse splenic DCs hve een shown to e unle 314 VOLUME 11 NUMBER 4 APRIL 21 nture immunology

3 21 Nture Americ, Inc. All rights reserved. Figure 3 NKT cells induce CCL17 in splenic DCs. () RT-PCR nlysis of CCL17 RNA in splenic CD11c + cells from mice t 1, 3 or 5 h fter injection of or vehicle (). β-ctin, loding control. () CCL17 mrna expression in wild-type or CD1d-deficient mice 5 h fter injection of or vehicle lone, presented reltive to 18S RNA. (c) Immunofluorescence stining of B cell zones (lue; B22) in heterozygous CCL17-eGFP reporter mice (Ccl17 +/ ) nd CCL17-deficient mice injected with, presented with the CCL17-eGFP signl (green). Scle rs, 2 µm. (d,e) Flow cytometry of spleen cells from CCL17-eGFP reporter mice 5 h fter injection of vehicle or, with gting of CD11c + CCL17 + cells (red ox, d) followed y nlysis of the expression of CD8 nd CCL17 (e). Numers djcent to outlined res (e) indicte percent CD8 + CCL17 + cells (top) or CD8 CCL17 + cells (ottom). (f) Flow cytometry nlysis of CCL17-eGFP in CD11c + splenocytes 2 h fter injection of NKT cell lignds or vehicle (right mrgin). Numers djcent to outlined res indicte percent CCL17 + cells. SSC, side sctter. (g) Flow cytometry nlysis of the proportion of OT-I cells in splenic CD8 + cells of m1 mice reconstituted with 5% m1 one mrrow nd 5% CCL17-deficient or wild-type one mrrow 8 weeks efore, nlyzed on dy 3 fter doptive trnsfer of OT-I cells nd priming with plus. Dt re representtive of two experiments (men nd s.d. of three to four mice per group in ech). CCL17 to express this chemokine, even fter systemic TLR stimultion 4. To determine whether cn induce CCL17 expression, we used RT-PCR to nlyze splenic CD11c + DCs isolted from mice injected with this gent. Indeed, CCL17 mrna ws detectle fter 3 h nd continued to increse until 5 h fter injection (Fig. 3,). We did not otin this result with CD1d-deficient mice (Fig. 3), which confirmed tht NKT cells were required for CCL17 upregultion. To identify the cell tht expressed CCL17, we used heterozygous CCL17-eGFP knock-in mice in which egfp fluorescence represents reporter of CCL17 protein expression (clled CCL17-eGFP reporter mice here) 4. Histologicl nlysis of splenic tissue locted mny CCL17 + cells in the T cell DC res nd sprse presence of these cells in B cell zones nd red pulp (Fig. 3c). The lck of CCL17 in homozygous CCL17-eGFP knock-in mice did not lter this pttern (Fig. 3c). Flow cytometry showed tht only CD11c + cells expressed CCL17- egfp (Fig. 3d) nd tht these cells lso expressed MHC clss II nd CD8 (dt not shown), which identified them s DCs. CCL17 ws produced minly y CD8α + DCs (Fig. 3e), which re le to crosspresent 3. Notly, other NKT cell lignds (including more physiologicl lignds) induced CCL17-eGFP expression in splenic DCs in the following order of potency: > OCH > ig3 > α-gldag > GSL-1 (Fig. 3f). To determine whether the DCs tht cross-primed CTLs needed to produce CCL17, we creted mixed one mrrow chimers tht received m1 nd CCL17-deficient one mrrow t rtio of 1:1 (Supplementry Fig. 4). NKT cells did not enhnce cross-priming of OT-I cells doptively trnsferred into these mixed one mrrow chimers, wheres they did so in control chimers tht hd received m1 plus wild-type one mrrow nd thus contined DCs tht could oth produce CCL17 nd interct with CTLs (Fig. 3g). This indicted tht CCL17 ws effective CCL17 (/18s) β-ctin CD11c Cd1d / CCL17-eGFP 1 h 3 h 5 h d e f g CD8 Tet + cells mong CD8 + cells (%) c CCL17-eGFP Ccl17 +/ Ccl17 / + m1 Ccl17 / + m only when produced y the sme DC tht ctivted CTLs. Given tht CD1d expression y this DC ws lso required (Fig. 1d), it follows tht only CD1d-expressing DCs produce CCL17, which confirms tht NKT cells induce CCL17 only in those DCs they physiclly interct with. CCL17 effects on DCs nd NKT cells re not responsile We next sought to identify the cell types tht responded to CCL17 in cross-priming. The CCL17 receptor CCR4 is expressed y ctivted CD4 + T cells, Lngerhns cells, NK cells nd NKT cells 32 34,37,43. We first focused on DCs, ecuse the oosting effect of is usully scried to DC ctivtion fter contct with NKT cells 17,3. We hypothesized tht utocrine stimultion of DCs vi CCL17 might upregulte costimultory molecules, resulting in improved CTL ctivtion (the possiility of prcrine stimultion ws excluded y the experiment in Fig. 3g). Injection of indeed upregulted the expression of CD8, CD86, CD4 nd CD7 y DCs, ut this ws dependent on neither CCR4 nor CCL17 (Fig. 4 nd Supplementry Fig. 5), which indicted tht CCL17 did not ugment DC ctivtion. Nevertheless, such CCR4-independent effect of NKT cells on DC ctivtion my explin the CCR4-independent, quntittively smller component of NKT cell enhnced cross-priming (Fig. 2,). Although induced CCR4-dependent production of IL-12 in vivo (Supplementry Fig. 6), this cytokine ws irrelevnt in our system, ecuse the -enhnced CTL response to ws similr in IL-12p35-deficient nd wild-type mice (Supplementry Fig. 7), which confirmed pulished studies of mice lcking oth IL-12 nd IL-23 (ref. 17). To directly investigte whether CCL17 incresed the stimultory cpcity of DCs, we loded splenic DCs from -injected mice with the peptide SIINFEKL nd cultured them together with OT-I cells. Both prolifertion nd IFN-γ production were similr SSC 5K CCL17-GFP 4.16 GSL-1 (25 nmol) GSL-1 α-gldag α-gldag ig3 OCH nture immunology VOLUME 11 NUMBER 4 APRIL

4 21 Nture Americ, Inc. All rights reserved. Figure 4 CCL17 enhnces cross-priming neither y ctivting DCs nor y recruiting NKT cells. () Flow cytometry nlysis of CD86 expression on splenic DCs from wild-type, CCL17-deficient nd CCR4-deficient mice injected 14 h efore with or vehicle. MFI, men fluorescent intensity. () (left) nd IFN-γ concentrtion in superntnts (right) of OT-I cells cultured for 2 d together with wild-type or CCR4-deficient splenic DCs coted with peptide (SIINFEKL). (c) CCL17 mrna expression in wild-type nd CCR4-deficient DCs 5 h fter injection of, presented reltive to 18S RNA expression. (d) Division indices of splenic CD8 + T cells otined from -injected wild-type mice, then stimulted with nti-cd3 plus nti-cd28 nd cultured in the presence or sence of splenic NKT cells from mice injected with 5 h efore. (e) CCL22 mrna expression in wild-type nd CCR4-deficient NKT cells (left) nd DCs (right) 5 h fter injection of, presented reltive to 18S RNA expression. Dt re representtive of two experiments (men nd s.d. of three to four mice per group in ech). when we used wild-type nd CCR4-deficient DCs (Fig. 4). Moreover, neither the solute numer of splenic DCs nor the rtio of crosspresenting CD8α + DCs to non cross-presenting CD8 DCs differed in wild-type, CCL17- nd CCR4-deficient mice (Supplementry Fig. 8), which demonstrted tht CCL17 did not render cross-priming DCs more numerous or immunostimultory. Notly, messge for CCL17 itself ws lower in DCs from injected CCR4-deficient mice (Fig. 4c), which indicted tht CCL17 positively regulted its own production y CCR4-expressing DCs either in n utocrine wy or indirectly y recruiting NKT cells. Hence, we next focused our ttention on NKT cells, hypothesizing tht CCL17 in our system might ttrct dditionl NKT cells to DCs to stimulte CTLs. This did not occur indirectly y further ctivtion of DCs, s this possiility ws excluded y the experiments descried ove (Fig. 4, nd Supplementry Figs. 5 nd 8). To test whether hypotheticlly recruited NKT cells directly stimulted CTLs, we ctivted CTLs with nti-cd3 nd nti-cd28 nd cultured them in the presence or sence of NKT cells isolted from -injected wildtype or CCR4-deficient mice. Neither CTL prolifertion (Fig. 4d) nor IFN-γ-production (dt not shown) ws greter in the presence of NKT cells. Thus, even if CCL17 recruited dditionl NKT cells in our system, these cells were unlikely to further stimulte CTLs. Messge for CCR5 lignds ws not more undnt in sorted NKT cells from -injected mice (dt not shown), which confirmed tht NKT cells did not recruit CTLs similr to helper T cells in clssicl cross-priming 12 (Fig. 2). Likewise, CCL17 messge ws not more undnt, which confirmed our finding tht mong splenocytes, only DCs expressed CCL17 (Fig. 3d,e). For completeness, we lso exmined CD86 (MFI 1 3 ) Ccl17 / c d e CCL17 expression (reltive) DC DC Anti-CD3 + nti-cd28 NKT the other CCR4 lignd, CCL22. Although the undnce of CCL22 messge in DCs ws very low, sorted NKT cells from -injected mice unexpectedly expressed CCL22 messge in CCR4-dependent wy (Fig. 4e). Together these experiments chllenge the ide of CCL17 ction on DCs nd NKT cells. The only relevnt meditors produced y these cells in CCR4-dependent wy were the CCR4 lignds themselves. This rises the possiility tht these lignds trgeted the CTLs directly, despite the lck of pulished evidence of function for CCR4 in CTL responses. DC-derived CCL17 cts directly on CTLs To investigte whether CCR4 lignds cted directly on CTLs in our system, we crossed OT-I mice with CCR4-deficient mice. We injected CCR4-sufficient or CCR4-deficient OT-I cells into recipient mice, followed y plus. CCR4-deficient OT-I cells dded little to the ckground cytotoxicity on dy 4, which resulted from endogenous polyclonl CTLs, wheres CCR4-sufficient OT-I cells cused sustntilly more cytotoxicity (Fig. 5), which demonstrted tht CCR4 expression y CTLs ws necessry for CCL17-medited enhnced cytotoxicity. Trnsfer of CCR4-sufficient OT-I cells into CCR4-deficient mice restored cytotoxicity to mounts oserved in wild-type mice (Fig. 5), which demonstrted tht CCR4 expression y CTLs ws sufficient for enhncing cytotoxicity. CCR4-expressing OT-I cells DC DC CCL22 expression (reltive) IFN-γ (ng/ml) NKT DC DC DC c d e -specific OT-l Ccr4 / OT-l Ccr4 / -specific MHCII-KO OT-I Ccr4 / OT-I IFN-γ (ng/ml) OT-I Ccr4 / OT-I Anti-CD3 + nti-cd28 CCL IFN-γ (ng/ml) Tet + cells mong CD8 + cells (%) Ccr4 / + Ccr4 / Ccr4 / Cd8 / + Ccr4 / Figure 5 Splenic DC derived CCL17 cts directly on CTLs. (,) In vivo cytotoxicity on dy 4 in wild-type mice, CCR4-deficient mice () nd MHC clss II deficient mice () given CCR4-sufficient or CCR4-deficient OT-I cells nd primed with plus 1 d lter. Horizontl lines, verge ckground cytotoxicity in wild-type mice on dy 4 due to endogenous CTLs. (c,d) (left) nd IFN-γ content in superntnts (right) of OT-I cells stimulted for 2 d with nti-cd3 plus nti-cd28 (c) or stimulted with nti-cd3 plus nti-cd28 with or without recominnt CCL17 (6 ng/ml; d). (e) Flow cytometry nlysis of endogenous -specific CTLs mong splenic CD8 + cells of wild-type mice reconstituted with 5% CCR4-deficient one mrrow nd 5% wild-type, CCR4-deficient or CD8-deficient one mrrow, ssessed 2 d fter priming with plus or lone. Dt re representtive of two experiments (men nd s.d. of four to five mice per group in ech). 316 VOLUME 11 NUMBER 4 APRIL 21 nture immunology

5 21 Nture Americ, Inc. All rights reserved. Figure 6 DC-derived CCL17 recruits CTLs into the splenic T cell DC zone. () Immunofluorescence stining of spleen cryosections from CCL17-eGFP reporter mice or CCL17- deficient mice injected with 1 6 fr red fluorochrome leled CCR4-sufficient OT-I cells on dy 1 nd then injected with with or without on dy. Blue stining indictes B22 + cells (defines B cell zones). Scle rs, 2 µm. (,c) Asolute numer of CCR4-sufficent or CCR4-deficient OT-I cells in CCL17-deficient or CCL17-eGFP reporter mice 1 h fter injection with, presented s cells per mm 2 of the T cell DC zone enclosed y the (lue) B cell zone. (d) Proportion of OT-I cells in c positioned djcent to GFP + DCs. (e) Asolute numer of CCR4-sufficent or CCR4-deficient OT-I cells in CCL17-deficient or CCL17-sufficient mice 1 h fter injection of ig3, presented s descried in,c. (f) Asolute numer of fr red fluorochrome leled polyclonl wild-type or OT-l in Ccl17 +/ were lso effective when trnsferred into MHC clss II deficient mice injected with plus (Fig. 5), which confirmed tht the CCR4 effect ws helper T cell independent. We ruled out the possiility of intrinsic deficiencies in CCR4-deficient OT-I cells, ecuse their prolifertive response to stimultion with nti-cd3 nd nti-cd28 ws equl to tht of CCR4-sufficient OT-I cells (Fig. 5c). Without injection of plus, CCR4-sufficient nd CCR4- deficient OT-I cells were present in similr numers (Supplementry Fig. 9), which ruled out the possiility tht these cells entered the T cell pool differently fter intrvenous injection. CCL17 did not ct like growth cytokine, ecuse dding recominnt CCL17 did not enhnce OT-I cell ctivtion in vitro (Fig. 5d). To confirm the findings reported ove in n endogenous CTL repertoire, we creted mixed one mrrow chimers tht received CD8-deficient nd CCR4-deficient one mrrow t rtio of 1:1. The CD8-deficient one mrrow cn generte CCR4 + cells, including NKT cells nd DCs, wheres ll CD8 + cells re derived from CCR4- deficient one mrrow (Supplementry Fig. 1). In these chimers, the ddition of filed to result in more CD8 + -specific CTLs, in contrst to results otined with control chimers generted with wild-type plus CCR4-deficient one mrrow (Fig. 5e), which could generte CCR4 + CD8 + cells. Insted, CTL numers equled those in control chimers tht hd received only CCR4-deficient one mrrow (Fig. 5e). Thus, CCR4 hd to e expressed y endogenous CTLs, which confirmed tht CCL17 enhnced cross-priming y cting directly on CTLs. DC-derived CCL17 recruits CTLs As CCL17 did not ct like growth cytokine (Fig. 5d), we hypothesized tht it might ffect CTL migrtion. To test our hypothesis, we leled OT-I cells with fr red fluorochrome nd injected the cells into heterozygous or homozygous CCL17-eGFP knock-in mice, which we susequently chllenged with plus. After 1 h, we counted the leled OT-I cells y immunofluorescence microscopy (Fig. 6 nd Supplementry Fig. 11) in the splenic T cell DC zone, where CCL17 ws expressed (Fig. 3c). We distinguished this zone from the B cell zone y costining with nti-b22 nd y the GFP fluorescence of NKT cell licensed DCs (Fig. 3c). More OT-I cells were present in the T cell DC zone fter injection of plus OT-l in Ccl17 +/ B22-PE OT-l fr red CCL17-GFP OT-l in Ccl17 / c d e f Cells (per mm 2 ) OT-l Ccl17 +/ CTLs in contct with DCs (%) Cells (per mm 2 ) + ig3 thn fter injection of lone (Fig. 6,). Injection of lone ws sufficient to produce this result (Fig. 6), which demonstrted tht OT-I cell ctivtion ws not necessry. In other words, nive CTLs were recruited. This depended on CCL17-CCR4, ecuse fewer OT-I cells were present in the T cell DC zone when DCs could not produce CCL17 (in experiments with homozygous CCL17-eGFP knock-in mice), when DCs were not licensed for CCL17 production (in experiments with no injection) or when CTLs could not respond to CCL17 (in experiments with CCR4-deficient OT-I cells; Fig. 6 c nd Supplementry Fig. 11). In prticulr, more CTLs were locted djcent to CCL17-expressing DCs thn to CCL17-deficient DCs (Fig. 6d), which demonstrted tht CTL recruitment trgeted individul DCs. We lso sought to test one of the other NKT cell lignds tht induced splenic CCL17 expression (Fig. 3f). We chose ig3, ecuse OCH is too close mimic of nd ecuse the responses induced y α-gldag nd GSL-1 were too smll. Furthermore, ig3 is n exmple of n endogenous lignd tht NKT cells re likely to encounter. We found tht 1 nmol ig3 nd 1 nmol enhnced the crosspriming of endogenous CTLs eqully well (Supplementry Fig. 12). Indeed, ig3 lso resulted in more recruitment of OT-I cells into the T cell DC zone in strictly CCR4-dependent wy (Fig. 6e), which demonstrted tht this NKT cell lignd engged the sme CTLrecruitment mechnism. The ility of CCL17 to ttrct nive OT-I cells (Fig. 6) indicted tht CTL specificity ws irrelevnt for recruitment. If so, then our in vivo recruitment ssy should yield similr results with polyclonl CTLs. We tested this y trnsferring fr red fluorochrome leled endogenous CTLs from wild-type nd CCR4-deficient mice into CCL17 reporter mice or CCL17-deficient mice. Polyclonl CTLs were recruited more effectively into the T cell DC zone when DCs produced CCL17, DCs were licensed y NKT cells nd CTLs could respond to CCL17 (Fig. 6f nd Supplementry Fig. 11). These findings demonstrted tht NKT cell licensed DCs produced CCL17 to recruit nive CCR4 + CTLs to the splenic T cell DC zone. Directionl CTL migrtion nd more contct time As CCL17-medited recruitment of CCR4 + CTLs hs not een reported efore, to our knowledge, we used n in vitro Trnswell Cells (per mm 2 ) 25 Cells (per mm 2 ) OT-l CTL / +/ Ccl17 +/ / Ccl17 +/ / +/ CCR4-deficient CTLs trnsferred into -injected CCL17-deficient mice or CCL17-eGFP reporter mice s descried in c., not significnt; P <.5 (Kruskl-Wllis nd Dunn s post-test). Dt re representtive of two individul experiments with t lest 25 T cell DC zones per group derived from three to four nonconsecutive sections from three mice ech (men nd s.d.). Ccl17 / OT-l nture immunology VOLUME 11 NUMBER 4 APRIL

6 21 Nture Americ, Inc. All rights reserved. Figure 7 CCL17 improves the directionl migrtion of CTLs towrd CCL17-producing DCs nd increses their contct time. () Trnswell ssy of the migrtion of polyclonl CTLs towrd CCL17 (8 ng/ml); cells were from mice injected with 3, 6, 8 or 12 h efore nlysis. (,c) Flow cytometry of the inding of fluorescein isothiocynte leled CCL17 to CTLs from spleens of vehicle- or -injected wild-type mice fter 4, 8 nd 14 h () or CD1d-deficient mice fter 14 h (c), presented s men fluorescence intensity. Dshed lines, ckground fluorescence of control-stined CTLs. (d,e) In vitro migrtion of CTLs with or without CCR4 expression towrd DCs with or without CCL17 production, recorded y time-lpse videomicroscopy over 2 3 h nd presented s CTL directionlity efore physicl contct with DCs (d) or susequent durtion of CTL-DC contct (e). Below grphs: indictes DCs or CTLs from donor mice injected with 14 h efore (+) or not ( ). Numers djcent to verticl rckets (e) indicte percent contcts lsting longer thn 4 min. (f,g) CTL directionlity (f) nd contct durtion (g) of mixed popultions of DCs with or without CCL17 production, recorded y time-lpse videomicroscopy over 2 3 h. (h,i) CTL directionlity (h) nd contct durtion (i) of mixed popultions of CTLs with or without CCR4 expression, recorded y time-lpse videomicroscopy over 2 3 h. In d i, ech symol represents n individul cell (n = 3 4 cells (directionlity) or n = 1 3 cells (contct durtion)); smll horizontl lines indicte the men. P <.5 nd P <.5 (Kruskl-Wllis nd Dunn s post-test (d,e) or Mnn-Whitney (f i)). Dt re representtive of three experiments with three to four mice per group ech (men nd s.d.). ssy to demonstrte this directly. Although polyclonl CTLs from untreted donor mice filed to migrte towrd recominnt CCL17 (dt not shown), some CTLs from donor mice injected with 8 12 h erlier did migrte (Fig. 7). This indicted tht NKT cells not only cted y inducing CCL17 production in splenic DCs ut lso mde nive CTLs responsive to this chemokine. In confirmtion of tht interprettion, CTLs from -injected wild-type mice ound fluorescence-leled recominnt CCL17 with kinetics similr to those in Trnswell migrtion (Fig. 7,). We did not oserve such inding in CD1d-deficient mice (Fig. 7c), which indicted tht NKT cells induced CCR4 expression on CTLs. Notly, we oserved such expression on only 15 2% of the CTLs (Supplementry Fig. 13), which mtched the proportion of CTLs tht migrted in the Trnswell ssys (Fig. 7). We next did live-cell imging of splenic DCs from -injected wild-type mice cultured together with polyclonl splenic CTLs from -injected or uninjected donor mice. CTLs from -injected wild-type mice showed more directionlity towrd DCs (Fig. 7d,e nd Supplementry Fig. 14) nd longer contct time with DCs (Fig. 7e nd Supplementry Movies 1 nd 2) compred with CTLs from CD8 + T cells (per mm 2 ) CpG CD8 + T cells (per mm 2 ) CpG c -specific CpG + CpG d CD8 + T cells (per mm 2 ) Trnsmigrted CTLs (%) d Directionlity 2 Medium +CCL f g h i Directionlity DC CTL Time (h) Contct durtion (min) CCL17 inding (MFl 1 3 ) Ccl17 / e Contct durtion (min) 4 h 8 h 12 1 uninjected donor mice, unless we used CCL17-deficient DCs or CCR4-deficient CTLs (Fig. 7d,e nd Supplementry Movies 3 nd 4). We confirmed those results when we exmined CTL migrtion in three-prt mixed cultures with CCL17-producing or CCL17-deficient DCs (Fig. 7f,g) nd in three-prt cultures of CCL17-competent DCs with CCR4-deficient or CCR4-sufficient CTLs (Fig. 7h,i nd Supplementry Movie 5). These findings directly demonstrted tht DC-derived CCL17 ttrcted CTLs tht hd een conditioned y NKT cells to express CCR4. Synergy of helper T cell nd NKT cell licensed cross-priming CCL17 nd CCR4 seem to regulte -enhnced cross-priming, in contrst to the CCR5-medited regultion of clssicl crosspriming in the presence of TLR lignds 12. To confirm tht different chemokines were involved, we trnsferred CCR4- or CCR5-deficient CTLs into -primed mice lso injected with either or the TLR9 lignd CpG. Only CpG enhnced the recruitment of CCR4- deficient CTLs (Fig. 8), wheres DC CTL c CCL17 inding (MFl 1 2 ) enhnced only the recruitment of CCR5- deficient CTLs (Fig. 8). If helper T cells nd NKT cells use distinct chemokine mechnisms to regulte cross-priming, then ctivting oth cell types should e synergistic. Indeed, the use of mximlly effective doses of CpG nd enhnced cytotoxicity eyond mounts chieved y these gents lone (Fig. 8c). CpG nd together lso enhnced the recruitment of doptively trnsferred fr red fluorochrome leled endogenous CTLs into the splenic T cell DC zone (Fig. 8d). Mice primed sucutneously with together with oth nd CpG hd mny more specific CTLs in the drining lymph node thn did mice primed with plus DC Ccl17 / DC Ccl17 / DC DC CTL CTL CTL CTL + CpG + CpG 2 15 Figure 8 Helper T cell nd NKT cell licensed cross-priming re synergisticlly regulted y distinct chemokines. (,) Asolute numer of doptively trnsferred CCR4-deficient T cells () or CCR5-deficient T cells () in -primed wild-type mice injected with or CpG, ssessed y histology s descried in Figure 6 (presented s cells per mm 2 of the T cell DC zone). (c) In vivo cytotoxicity on dy 5 in -primed wild-type mice injected with, CpG or oth gents. (d) Asolute numer of doptively trnsferred polyclonl T cells, ssessed s descried in,. P <.5 nd P <.5 (Kruskl-Wllis nd Dunn s selected columns). 1 5 Directionlity 14 h veh Ccl17 / Contct durtion (min) Cd1d / 318 VOLUME 11 NUMBER 4 APRIL 21 nture immunology

7 21 Nture Americ, Inc. All rights reserved. or CpG lone (Supplementry Fig. 15). These findings verified tht CCR4 nd CCR5 medited distinct nd synergistic CTL-recruitment mechnisms tht were opertive in cross-priming licensed y NKT cells nd in cross-priming licensed y helper T cells nd enhnced y TLR lignds, respectively (Supplementry Fig. 16). DISCUSSION Cross-priming requires physicl contct mong t lest three rre cells of the immune response: ntigen-specific CTLs, crosspresenting DCs nd ntigen-specific helper T cells tht license the DCs for cross-priming. Alterntively, NKT cells cn license DCs, ut little is known out the underlying mechnisms, except tht DCs mture nd upregulte costimultory molecules 15,17,31. Here we hve reported n dditionl, quntittively stronger effect tht NKT cells exert on DCs, medited y the induction of CCL17 tht cts on CTLs expressing CCR4. CCL17 is n inflmmtory chemokine with highly orgn- nd DC-restricted expression profile 4,44 nd hs een linked to llergic diseses of the skin nd lung Notly, splenic DCs normlly completely lck CCL17 expression, even fter systemic injection of vrious TLR lignds 4,44. However, we found here tht NKT cells stimulted cross-priming CD8α + splenic DCs to secrete lrge mounts of CCL17. Moreover, interction with DCs induced NKT cells to upregulte mrna for CCL22, second CCR4 lignd tht is known minly s mcrophge-derived chemokine. Its role in our system, however, ws smll. CCR4 is expressed y ctivted T H 2 cells 37, especilly in the lung nd skin 45,46, nd hs een linked to llergic diseses of these orgns 38,39,42. However, the ssocition with T H 2 responses is not strict, nd it hs een proposed tht CCR4 is expressed y ll memory CD4 + helper T cell types, except those homing to the intestine 35,36,38. CCR4 is expressed lso y ntigen-presenting cells nd NKT cells 32 34,37,43. In our model, we found no evidence tht CCL17 improved crosspriming y stimulting CCR4-expressing DCs or NKT cells. Even if this were the cse, these cells would hve to stimulte CTLs y nother signl, for exmple, y more costimultory molecules on DCs. However, expression of such molecules ws independent of oth CCR4 nd CCL17. We found only two DC-derived signls tht resulted from interction with NKT cells tht depended on CCL17 nd its receptor: IL-12 nd CCL17. Using mice deficient in IL-12p35, we ruled out the possiility tht NKT cell licensed DCs used IL-12 to stimulte CTLs, consistent with pulished study of mice deficient in oth IL-12 nd IL-23 (ref. 17). Initilly, involvement of CCL17 seemed unlikely ecuse neither this chemokine nor its receptor, CCR4, hs een linked to CTL responses. A suset of circulting CCR4-expressing humn memory CTLs hs een chrcterized tht migrte towrd CCR4 lignds in vitro, ut the functionl relevnce of this is still uncler 47. Nevertheless, CCL17 ttrcted nive CCR4 + CTLs oth in vitro nd in vivo. It guided nive CCR4 + CTLs into the splenic T cell DC zone, in which the cross-priming CCL17 + DCs resided, therey loclly incresing the numer of CTL precursors ville for ctivtion. This ws true not only for OT-I cells ut lso for endogenous polyclonl CTLs. Moreover, CCL17 guided CTLs into the splenic T cell DC zone precisely towrd those DCs tht hd een licensed y NKT cells. Histologicl nlysis showed tht there were more CTLs djcent to CCL17-producing DCs. Moreover, this conclusion cn e extrpolted from our experiments with mixed one mrrow chimer, which showed tht the sme DC tht cross-primed CTLs lso intercted with NKT cells in CD1d-restricted mnner nd produced CCL17. In other words, CCL17 produced y neighoring DCs ws ineffective, which indictes tht CCL17 operted not only on tissue-specific level ut lso on DC-specific level. Selective recruitment towrd relevnt DCs proly ccelertes CTL priming etter thn mere ttrction into lrger re such s the T cell DC zone, ecuse CTLs will not lose time exmining unlicensed DCs. Furthermore, CCL17 incresed the DC-CTL contct time, which my e importnt for crosspriming, s sustined T cell ntigen receptor signling is necessry for immunogenicity 48,49. The dependency of CCL17 production on its receptor CCR4 indicted tht this chemokine ws regulted y positive feedck loop. Although this loop did not increse the stimultory ctivity of DCs for CTLs per se, it my nevertheless e relevnt in our system, ecuse CCL17-expressing DCs might ttrct further CCR4-expressing NKT cells to upregulte CCL17 to mximl mounts, therey permitting fster ttrction of CCR4 + CTLs. Our findings hve identified CCR4-CCL17 s the second chemokine chemokine receptor pir tht regultes cross-priming. It differs from CCR5 nd its three lignds, which fcilitte helper T cell licensed clssicl cross-priming 12,14. Our dt hve therefore demonstrted tht t lest two independent mechnisms exist tht recruit nive CTLs for cross-priming nd tht these discriminte etween helper T cell nd NKT cell licensed DCs. This might permit n dditionl level of selectivity in CTL ttrction for cross-priming. For exmple, if during n infection DCs presented microil ntigen such s GSL-1 or α-gldag or self ntigen such s ig3 (ref. 23), then NKT cells would induce CCL17 expression nd therey render the DCs especilly ttrctive for nive CTLs, enhncing the likelihood tht these DCs would e scnned erlier thn unlicensed DCs or DCs licensed y helper T cells. In contrst, CTLs my locte DCs licensed y helper T cells nd TLR lignds fster y following CCR5 lignds. When we engged oth pthwys simultneously, the recruitment of nive CTLs ws even fster nd their resulting cytotoxic response ws higher. This synergism my serve to preferentilly scn DCs tht hve successfully presented ntigenic epitopes to oth helper T cells nd NKT cells, s such DCs evidently contin relevnt foreign ntigen nd moreover will provide optiml costimultion ecuse they hve een licensed y two lymphocyte types. In our studies, only CTLs from -injected mice expressed CCR4, reminiscent of pulished findings showing tht injection of lipopolyscchride or CpG is necessry to render nive CTLs responsive to CCR5 lignds 12. Hence, signls must exist tht induce expression of these chemokine receptors, which oviously must operte in trns (tht is, efore CTLs encounter DCs); this suggests tht solule cytokines my e cndidtes. Further studies re needed to identify the signls tht condition nive CTLs to express CCR4 nd CCR5. In summry, we hve descried n unexpected role for CCL17 nd CCR4: they fcilitte n NKT cell dependent wy of licensing DCs for cross-priming, which my e importnt for cross-priming ginst microes ering NKT cell glycolipid ntigens. Our findings support the theory tht DC T cell encounters re not rndom ut re highly coordinted through chemokines 12 nd extend tht theory y showing tht different chemokines coordinte the recruitment of nive CTLs towrd the relevnt DCs. The synergism etween these two mechnisms my e exploited to improve vccintions imed t inducing CTLs, for exmple, y comining djuvnts tht elicit oth helper T cell nd NKT cell licensing. Methods Methods nd ny ssocited references re ville in the online version of the pper t nture immunology VOLUME 11 NUMBER 4 APRIL

8 21 Nture Americ, Inc. All rights reserved. Note: Supplementry informtion is ville on the Nture Immunology wesite. Acknowledgments We thnk W. Keßler (University of Greifswld) for CCR4-deficient mice; F. Tcke (University of Achen) for CD1d- nd MHC clss II deficient mice; R. Goldszmid (Ntionl Institute of Allergy nd Infectious Diseses, Ntionl Institutes of Helth) for CD8-deficient mice; J. Alferink (University of Bonn) for CCL17-eGFP knock-in mice; A. Peters for technicl ssistnce; C. Coch nd Rolf Fimmers for dvice on sttistics; nd the Centrl Animl Fcilities nd the Flow Cytometry Core Fcility t the Institutes of Moleculr Medicine nd Experimentl Immunology for support. Supported y the Deutsche Forschungsgemeinschft (Sonderforschungsereich 74 grnts A1 (I.F.), A2 (C.K.), A5 (P.A.K.) nd A8 (W.K.), nd Klinische Forschergruppe 228 grnts P1 (U.P.) nd P5 (C.K.)), the Austrlin Ntionl Helth nd Medicl Reserch Council (D.I.G. nd J.R.) nd the Austrlin Reserch Council (J.R.). AUTHOR CONTRIBUTIO V.S. nd V.L.-K. designed nd did most experiments, nlyzed nd interpreted dt nd contriuted to the writing of the mnuscript; C.A.T., T.Q. nd K.H. designed, did nd nlyzed individul experiments; U.P., J.R., P.P., J.C., D.I.G., P.B.S., P.A.K., W.K. nd I.F. contriuted tools, discussed nd interpreted results nd edited the mnuscript; nd C.K. conceived the project, designed nd interpreted experiments nd wrote the mnuscript. COMPETING finncil INTERESTS The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Crone, F.R., Kurts, C., Bennett, S.R., Miller, J.F. & Heth, W.R. Cross-presenttion: generl mechnism for CTL immunity nd tolernce. Immunol. Tody 19, (1998). 2. den Hn, J.M., Lehr, S.M. & Bevn, M.J. CD8 + ut not CD8 dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192, (2). 3. Shortmn, K. & Nik, S.H. Stedy-stte nd inflmmtory dendritic-cell development. Nt. Rev. 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Exogenous nd endogenous glycolipid ntigens ctivte NKT cells during microil infections. Nture 434, (25). 24. Kinjo, Y. et l. Nturl killer T cells recognize dicylglycerol ntigens from pthogenic cteri. Nt. Immunol. 7, (26). 25. Miymoto, K., Miyke, S. & Ymmur, T. A synthetic glycolipid prevents utoimmune encephlomyelitis y inducing T H 2 is of nturl killer T cells. Nture 413, (21). 26. Florence, W.C. et l. Adptility of the semi-invrint nturl killer T-cell receptor towrds structurlly diverse CD1d-restricted lignds. EMBO J. 28, (29). 27. Scott-Browne, J.P. et l. Germline-encoded recognition of diverse glycolipids y nturl killer T cells. Nt. Immunol. 8, (27). 28. Yoshimoto, T. & Pul, W.E. CD4 pos, NK1.1 pos T cells promptly produce interleukin 4 in response to in vivo chllenge with nti-cd3. J. Exp. Med. 179, (1994). 29. Kitmur, H. et l. The nturl killer T (NKT) cell lignd α-glctosylcermide demonstrtes its immunopotentiting effect y inducing interleukin (IL)-12 production y dendritic cells nd IL-12 receptor expression on NKT cells. J. Exp. Med. 189, (1999). 3. Fujii, S., Shimizu, K., Smith, C., Bonifz, L. & Steinmn, R.M. Activtion of nturl killer T cells y α-glctosylcermide rpidly induces the full mturtion of dendritic cells in vivo nd therey cts s n djuvnt for comined CD4 nd CD8 T cell immunity to codministered protein. J. Exp. Med. 198, (23). 31. Hermns, I.F. et l. NKT cells enhnce CD4 + nd CD8 + T cell responses to solule ntigen in vivo through direct interction with dendritic cells. J. Immunol. 171, (23). 32. Kim, C.H., Johnston, B. & Butcher, E.C. Trfficking mchinery of NKT cells: shred nd differentil chemokine receptor expression mong V α 24 + V β 11 + NKT cell susets with distinct cytokine-producing cpcity. Blood 1, (22). 33. Thoms, S.Y. et l. 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Intervention of thymus nd ctivtion-regulted chemokine ttenutes the development of llergic irwy inflmmtion nd hyperresponsiveness in mice. J. Immunol. 166, (21). 4. Alferink, J. et l. Comprtmentlized production of CCL17 in vivo: strong induciility in peripherl dendritic cells contrsts selective sence from the spleen. J. Exp. Med. 197, (23). 41. Horikw, T. et l. IFN-γ-inducile expression of thymus nd ctivtion-regulted chemokine/ccl17 nd mcrophge-derived chemokine/ccl22 in epiderml kertinocytes nd their roles in topic dermtitis. Int. Immunol. 14, (22). 42. Cmpell, J.J., O Connell, D.J. & Wurel, M.A. Cutting edge: chemokine receptor CCR4 is necessry for ntigen-driven cutneous ccumultion of CD4 T cells under physiologicl conditions. J. Immunol. 178, (27). 43. Ktou, F. et l. Mcrophge-derived chemokine (MDC/CCL22) nd CCR4 re involved in the formtion of T lymphocyte-dendritic cell clusters in humn inflmed skin nd secondry lymphoid tissue. Am. J. Pthol. 158, (21). 44. Lieerm, I. & Forster, I. The murine β-chemokine TARC is expressed y susets of dendritic cells nd ttrcts primed CD4 + T cells. Eur. J. Immunol. 29, (1999). 45. Gonzlo, J.A. et l. Mouse monocyte-derived chemokine is involved in irwy hyperrectivity nd lung inflmmtion. J. Immunol. 163, (1999). 46. de Lvreille, A. et l. Clonl Th2 cells ssocited with chronic hypereosinophili: TARC-induced CCR4 down-regultion in vivo. Eur. J. Immunol. 31, (21). 47. Kondo, T. & Tkiguchi, M. Humn memory CCR4 + CD8 + T cell suset hs the ility to produce multiple cytokines. Int. Immunol. 21, (29). 48. Kech, S.M. & Ahmed, R. Memory CD8 + T cell differentition: initil ntigen encounter triggers developmentl progrm in nive cells. Nt. Immunol. 2, (21). 49. vn Stipdonk, M.J. et l. Dynmic progrmming of CD8 + T lymphocyte responses. Nt. Immunol. 4, (23). 32 VOLUME 11 NUMBER 4 APRIL 21 nture immunology