Immunity, Vol. 22, 493–505, April, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.immuni.2005.02.010

CCL19 and CCL21 Induce a PotentProinflammatory Differentiation Programin Licensed Dendritic Cells

Benjamin J. Marsland,2,4 Patrick Bättig,1,4

Monika Bauer,1 Christiane Ruedl,1

Ute Lässing,1 Roger R. Beerli,1 Klaus Dietmeier,1

Lidia Ivanova,1 Thomas Pfister,1 Lorenz Vogt,1

Hideki Nakano,3 Chiara Nembrini,2 Philippe Saudan,1

Manfred Kopf,2 and Martin F. Bachmann1,*1Cytos Biotechnology AGWagistrasse 258952 Zurich-SchlierenSwitzerland2Molecular BiomedicineDepartment of Environmental SciencesSwiss Federal Institute of TechnologyCH-8092 ZurichSwitzerland3Department of MedicineDuke University Medical CenterBox 3623Durham, North Carolina 27710

Summary

Dendritic cells (DCs) are key instigators of adaptiveimmune responses. Using an alphaviral expressioncloning technology, we have identified the chemokineCCL19 as a potent inducer of T cell proliferation in aDC-T cell coculture system. Subsequent studies showedthat CCL19 enhanced T cell proliferation by inducingmaturation of DCs, resulting in upregulation of costi-mulatory molecules and the production of proinflam-matory cytokines. Moreover, CCL19 programmed DCsfor the induction of T helper type (Th) 1 rather thanTh2 responses. Importantly, only activated DCs thatmigrated from the periphery to draining lymph nodes,but not resting steady-state DCs residing withinlymph nodes, expressed high levels of CCR7 in vivoand responded to CCL19 with the production of proin-flammatory cytokines. Migrating DCs isolated frommice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype,highlighting the importance of these chemokines forfull DC maturation in vivo. Our findings indicate thatCCL19 and CCL21 are potent natural adjuvants forterminal activation of DCs and suggest that chemo-kines not only orchestrate DC migration but also reg-ulate their immunogenic potential for the induction ofT cell responses.

Introduction

Dendritic cells (DCs) are key initiators of innate andadaptive immunity (for review, see Banchereau et al.,2000; Lanzavecchia and Sallusto, 2001; Mellman andSteinman, 2001). They are potent antigen-processingand -presenting cells with the unique ability to stimu-

*Correspondence: martin.bachmann@cytos.com

4 These authors contributed equally to this work.

late primary immune responses in addition to boostingsecondary immune responses. Immature DC precur-sors exit the bone marrow and circulate via the blood-stream to reach their target tissues, taking up residenceat sites of potential pathogen entry in a physiologicalstage that is specialized for antigen capture (Steinman,1991). After uptake of antigen by phagocytosis, viamacropinocytosis or via receptor-mediated endocyto-sis (Albert et al., 1998; Sallusto et al., 1995), the antigenis processed and peptides thereof are presented on thecell surface associated with the MHC class I or II mole-cules. After receiving maturation-inducing signals, eitherdirectly from pathogens or via inflammatory stimuli,DCs change the expression pattern of chemokine re-ceptors, allowing them to leave the peripheral tissueand to migrate to draining lymphoid organs (Rescignoet al., 1999). Upregulation of CCR7 during maturationrenders DCs sensitive to two chemoattractants, ELC/CCL19 and SLC/CCL21, which direct their migration toT cell regions of lymphoid organs (Cyster, 1999; Sal-lusto and Lanzavecchia, 2000). Within these regions,DCs can induce both activation and proliferation ofspecific CTLs and Th cells via presentation of immuno-genic peptides in association with MHC class I and IImolecules, respectively. In addition to protection againstpathogens, DCs also appear to be central to the regula-tion, maturation, and maintenance of cellular immuneresponses against cancer (Gunzer et al., 2001; Timmer-man and Levy, 1999). Recently, it has emerged that DCsnot only induce T cell responses but are equally impor-tant for the maintenance of peripheral T cell tolerancebecause resting DCs induce T cell tolerance rather thanimmunity (Bonifaz et al., 2002; Probst et al., 2003).Thus, regulation of DC activation is critical for the es-tablishment of protective T cell responses as well asfor the maintenance of T cell tolerance (for review, see(Bachmann and Kopf, 2002)).

Pathogen-mediated DC activation is usually trans-duced via the Toll-like receptor (TLR) family (Akira,2001; Beutler, 2002; Medzhitov and Janeway, 2000).TLR recognize invariable patterns associated withpathogens like peptidoglycans (TLR2) (Takeuchi et al.,1999), double-stranded RNA (TLR3) (Alexopoulou etal., 2001), LPS (TLR4) (Hoshino et al., 1999; Poltoraket al., 1998), flagellin (TLR5) (Hayashi et al., 2001), andbacterial RNA (TLR 7/8) (Diebold et al., 2004; Heil et al.,2004) or DNA (TLR9) (Hemmi et al., 2000; Schnare etal., 2000). Activation of dendritic cells is also inducedby proinflammatory cytokines and in particular bymembers of the TNF superfamily, including TNF-α,CD40L, and Trance/RANKL (Bachmann et al., 1999;Cella et al., 1996; Koch et al., 1996; Wong et al., 1999).Recently, chemokine receptors have also been shownto be directly involved in induction of DC maturation.Specifically, activation through CCR5 by T. gondii-derived cyclophilin led to production of high levels ofIL-12 in DCs (Aliberti et al., 2000; Aliberti et al., 2003).The importance of DC maturation for the generation ofprotective T cell responses is also highlighted by thefact that several viruses interfere with activation of DCs.

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Poxviruses inhibit DC maturation by lowering the ex- blpression of CD86, leading to reduced T cell costim-

ulation (Engelmayer et al., 1999). Along a similar line, miherpes simplex virus inhibits maturation of DCs (Salio

et al., 1999), and measles virus induces an aberrant pimaturation program resulting in inhibition rather than

activation of T cells (Schneider-Schaulies et al., 2003).To identify novel proteins capable of regulating T cell C

responses through the activation of DCs, we performed Tan alphavirus based expression cloning campaign. We tused Sindbis virus as a carrier of a normalized cDNA clibrary from activated splenocytes and generated a pro- vtein library by infection of baby hamster kidney (BHK) fcells (Koller et al., 2001). Sindbis virus belongs to the aalphavirus genus and contains a single-stranded RNA sgenome with positive polarity. Using a bipartite system, 2where one RNA molecule encodes the structural pro- Tteins and the other encodes the protein of interest plus pthe viral replicase, it is possible to generate replication wcompetent viruses encoding genes or even libraries of tinterest (Koller et al., 2001). d

We report here the identification of mouse CCL19 Tand CCL21 as potent maturation factors for DCs and oindirect regulators of Th cell differentiation. Our findings findicate that induction of DC maturation is an important Aproperty of CCL19/CCL21 and suggest that chemo- okines may not only organize the migration of DCs but Salso directly regulate their ability to prime T cell re- tsponses. i

cwResultsdCIdentification of CCL19 by Expression Cloning

Because DCs are crucial for the induction of immunePresponses, we set out to identify novel DC maturationifactors using an alphaviral screening technology. In or-Tder to generate a Sindbis virus-based cDNA library,CRNA was extracted from spleens of LCMV-infectedtBALB/c mice, transcribed into cDNA and normalized aspdescribed in the Experimental Procedures. The cDNAFlibrary was transformed into a viral library by in vitroutranscription and electroporation together with a viralphelper construct (Koller et al., 2001).wFollowing the strategy shown in Figure 1A, BHK cellsAwere infected at a multiplicity of infection (MOI) of 0.1Oand single infected cells were sorted on subconfluentpBHKs in 96-well plates. This led to the generation of auprotein library within 3 days, where each well corres-aponded to the expression of one cDNA-derived protein.uThe protein-containing supernatants were harvested,aand remaining virus was inactivated by UV light.aSpleen-derived DCs were pulsed with OVA peptide andbtreated with the supernatants overnight. The next day,tOVA-specific CD4+ T cells were added, and prolifera-Ction was assessed 2.5 days later by 3H-thymidine incor-

poration.The average counts per plate were calculated and C

Mevery supernatant that induced a count/min value twostandard deviations above the average was retested by T

rthe same assay. From those wells that scored positivea second time, the virus-encoded cDNA was recovered t

pby RT PCR, and the cDNA sequence was determined(Figure 1B). In total 10,000 genes derived from the li- c

rary were tested for their ability to enhance T cell pro-iferation in the assay. Besides known inducers of DC

aturation, like hsp70, the chemokine CCL19 wasdentified twice independently in this screening cam-aign. One of the plates in which CCL19 was identified

s shown in Figure 1B.

CL19 Induces Maturation of Dendritic Cellso confirm that CCL19 induces T cell proliferation inhe DC-T cell coculture system, the CCL19 cDNA wasloned into the alphaviral vector, and the recombinantirus was generated (SR5-CCL19). BHK cells were in-ected, and the supernatant was retested in the prolifer-tion assay. In these experiments, the proliferation-timulating activity of CCL19 was confirmed (FigureA). In order to assess whether CCL19 directly inducescell proliferation, naïve T cells were incubated in the

resence or absence of CCL19. No T cell proliferationas induced by CCL19 (data not shown), suggesting

hat the observed induction of T cell proliferation is me-iated solely through enhancement of DC maturation.o test this further, spleen-derived DCs were treatedvernight with the supernatants and analyzed for sur-ace expression of CD40 and CD86 by flow cytometry.

significantly higher proportion of mature DCs werebserved in cultures treated with supernatants fromR5-CCL19-infected BHK cells compared to DC cul-

ures treated with supernatants from control virus-nfected BHK cells (Figures 2B and 2C). As a positiveontrol, we included Sindbis virus-expressing TNF-α,hich enhanced T cell proliferation (Figure 2A) and in-uced DC maturation in a manner comparable toCL19 (Figures 2B and 2C).

urified CCL19 Enhances Proliferation of T Cellsn the DC-T Cell Coculture Systemo test whether the effects found where specific forCL19 and not due to the use of a viral expression sys-

em, an N-terminal FLAG-tagged CCL19 protein wasroduced in HEK 293 cells and purified over an anti-LAG column (Figure 3A). This purified protein wassed for all further experiments. First, the proliferationromoting quality in the DC-T cell coculture systemas retested. Bone marrow-derived DCs were taken asPCs, and proliferation was tested at three differentVA peptide concentrations. DCs were pulsed with theeptide for 1 hr and then treated with the different stim-li for 4 hr. OVA-specific T cells were subsequentlydded and proliferation was measured 3 days later (Fig-re 3B). In a similar experiment, T cell proliferation wasssessed by CSFE staining. DCs were treated as abovend incubated for 4 days with CSFE-labeled T cells. Inoth experiments, CCL19 enhanced T cell proliferationo the same extent as treatment with LPS plus anti-D40 antibodies or TNF-α (Figure 3C).

CL19 Induces Upregulation of Costimulatoryolecules and the Production of Cytokines in DCshe maturation of DCs is associated with the loss ofeceptors involved in antigen uptake and the upregula-ion of costimulatory molecules that are important forroductive interaction with T cells. Through the con-omitant production of cytokines, DCs can further pro-

CCL19 Is a Potent Activator of DCs495

Figure 1. Outline of the Screening Strategy with CCL19 Highlighted as One of the Results of the Screening Campaign

(A) BHK cells were infected at a MOI of 0.1 with Sindbis virus encoding genes from a normalized cDNA library derived from activated mousespleens. Infected BHK cells (stained with anti-Sindbis-specific antibody) were sorted one cell/well on BHK cells seeded in 96-well plates.After 3 days propagation, the supernatant of the infected wells were harvested, treated with UV-light to inactivate virus, and transferred ontoOVA peptide-pulsed spleen-derived DCs. Following overnight treatment, purified CD4+ DO11.10 T cells were added to the wells, and prolifera-tion of T cells was measured by incorporation of 3H-thymidine.(B) A 96-well plate is shown where one well showed substantially increased proliferation of OVA-specific CD4+ T cells (encircled point). RNAfrom the positive well was extracted, transcribed by RT-PCR into cDNA, and sequenced. The virus-encoded gene was CCL19. Dotted lineindicates the average counts/min of the plate.

mote the induction of an immunological response fol-lowing pathogen exposure. In order to analyze thematuration program triggered in DCs by CCL19, we ex-amined the surface expression of CD40, CD80, CD86,MHC II, and, additionally, the levels of the proinflamma-tory cytokines interleukin-1 beta (IL-1β), interleukin-12(IL-12), and tumor necrosis factor alpha (TNF-α).

In the first instance, DCs were treated overnight andthe surface expression of CD40, CD80, CD86, and MHCII was assessed by flow cytometry (Figures 4A and 4Band Figure S1 in the Supplemental Data available withthis article on line). To exclude that the observed effectwas due to LPS contamination, the samples weretreated with proteinase K and subsequent heat dena-turation. CCL19 was found to induce upregulation of

CD86 (Figure 4A) and CD40 (Figure 4B) in an LPS-inde-pendent fashion, shown by a loss of function followingproteinase K treatment. Similar upregulation of CD86and CD40 was found for CCL19 as for TNF-α and LPStreatment (Figures 4A and 4B). To further ensure thatthe DC maturation effect was mediated by CCL19, wetested its activity on DCs genetically deficient in theCCL19 receptor, CCR7. The CCL19-induced DC matu-ration was indeed mediated through binding to CCR7because production of IL-12 (Figure 4C) and upregula-tion of CD86, CD80, and CD40 surface expression wasnot evident after stimulation of CCR7−/− DC by CCL19(Figure 4D). Of note, the activity of CCL19 was Pertus-sis toxin sensitive and also independent of MyD88(data not shown). We next performed titration experi-

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Figure 2. Cloned CCL19 Expressed by Sindbis Virus Enhances T Cell Proliferation and Induces Maturation of DCs

The cDNA encoding CCL19 was cloned into a Sindbis virus expression vector, and recombinant viruses expressing CCL19 were generated(SR5-CCL19).(A) UV-inactivated supernatants of control, SR5-TNF-α, or SR5-CCL19 virus-infected BHK cells were tested in the DC-T cell coculture systemas described in Figure 1. Error bars represent the standard deviation of triplicates.(B and C) Spleen derived DCs were treated overnight with UV light-inactivated control Sindbis virus (SR5), Sindbis virus-expressing TNF-α(SR5-TNF-α), or CCL19 (SR5-CCL19). Following overnight incubation, DCs were stained with anti-CD40 and anti-CD86 antibodies and ana-lyzed by flow cytometry. The frequency of mature DCs (oval gate) was determined. In (C), the frequency (±SD) of mature DCs is shown fortriplicate cultures. One representative experiment of two similar experiments is shown in (A)–(C).

ments and found that CCL19 induced DC maturation at psconcentrations of 0.1 �g/ml and higher (Figures 4E and

4F), roughly corresponding to chemokine concentra- cttions used to induce cellular migration (Figure 4G) (see

also Kwan and Killeen, 2004; Moutaftsi et al., 2004). moTwo commercially available CCL19 batches pro-

duced in a prokaryotic expression system were also lttested and found to be less active. This limited activity

correlated with a roughly 100-fold reduced ability of the (scommercial CCL19 to induce migration of DCs (data

not shown). Thus, in our experimental system, commer- STcial bacterially expressed CCL19 appears to have an

overall limited potency. pdTo test whether CCL19 induced secretion of relevant

inflammatory cytokines, supernatants of the stimulatedDCs were analyzed for the presence of IL-1β and IL-12 C

T(p40) by ELISA. CCL19 induced strong production ofboth cytokines (Figure 5A). Induction of IL-12 pro- a

pduction was similar to LPS-treated DCs, whereas IL-1β

roduction was even higher than that seen after LPStimulation. In comparison to TNF-α, CCL19 inducedlearly higher levels of both IL-12 and IL-1β. In addi-ional experiments, we compared induction of inflam-atory cytokines by CCL19 with combined stimulationf DCs with LPS and anti-CD40 antibodies. This stimu-

us has recently been shown to be sufficient for induc-ion of autoimmunity by using peptide-pulsed DCsEriksson et al., 2003). Cytokine production was as-essed by intracellular cytokine staining (Figure 5B).urprisingly, CCL19 induced similar levels of IL-1β,NF-α, and IL-12 as compared to stimulation with LPSlus anti-CD40 antibodies, confirming that CCL19 is in-eed a strong maturation trigger for DCs (Figure 5B).

CL21 Also Induces Maturation of DCso test whether the second ligand of CCR7, CCL21,lso induces maturation of DCs, we expressed andurified a FLAG-tagged version of CCL21 as described

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Figure 3. Purified Recombinant CCL19 En-hances Peptide-Specific Proliferation in aDC-T Cell Coculture Assay

(A) CCL19 was subcloned into an eukaryoticexpression vector to produce an N-terminalFLAG-tagged CCL19 protein. 293-EBNAcells were transfected and FLAG-CCL19 waspurified from supernatants over an anti-FLAG column. Purified recombinant FLAG-tagged CCL19 was analyzed on a Coomas-sie-stained protein gel (left) and by Westernblot stained with an anti-FLAG antibody(right).(B) OVA-pulsed (0, 10−8 M, 10−7 M, or 10−6 M)bone marrow-derived DCs were treated withPBS, LPS and anti-CD40 antibodies (0.1 µg/ml/5 µg/ml), TNF-α (50 ng/ml), or CCL19(0.33 µg/ml) for 4 hr. OVA-specific transgenicCD4+ T cells were added and the prolifera-tion was assessed by 3H-Thymidine incorpo-ration. Error bars represent standard devia-tion of triplicate wells.(C) OVA-pulsed (10−8 M) bone marrow-derived DCs were activated as described in(B), then OVA-specific CSFE-labelled CD4+ Tcells were added, and after 4 days incuba-tion, analyzed for CSFE staining. Data in (B)and (C) are from independent experiments,and comparable data were found in a sim-ilar experiment.

for CCL19. Bone marrow-derived dendritic cells werethen stimulated with titrated amounts of FLAG-CCL21and analyzed for the expression of CD86 and CD40.As observed for CCL19, CCL21 was able to stronglystimulate DC maturation at concentrations above 0.1�g/ml (Figure S2).

CCL19 Primes DCs for the Inductionof Th1 ResponsesWe sought to determine whether CCL19 stimulation ofDCs would preferentially facilitate the induction ofeither Th1 or Th2 responses. Accordingly, we employedthe use of a DC-transgenic T cell coculture systemwhere T helper cell differentiation is controlled by pep-tide concentration (Marsland et al., 2004; Ruedl et al.,2000a). In this coculture system, high concentrations ofpeptide result in Th1 differentiation and IFN-γ pro-duction, while low concentrations of peptide result inTh2 differentiation and IL-4 production. Dendritic cellswere incubated for 4 hr in the presence or absence ofCCL19, washed twice, and used to stimulate CD4+

TCR-transgenic T cells from SMARTA mice (Oxenius etal., 1998) specific for peptide GP13 derived from LCMV.To ascertain whether CCL19 was capable of directly ac-tivating T cells, naïve T cells were also incubated for 4hr in the presence or absence of CCL19 before use inthe coculture. A peptide concentration of 100 nM was

used in these cultures because this induces differentia-tion of both Th1 and Th2 cells, allowing any additionalpolarizing effects mediated by CCL19 to be identified.After 3 days of culture, production of IL-4 and IFN-γ bythe transgenic CD4+ T cells was assessed by intracel-lular cytokine staining. As expected, untreated DCspulsed with 100 nM GP13 induced both Th1 and Th2cells, as shown by the presence of both IL-4- andIFN-γ-producing cells (Figure 6, left panel). In markedcontrast, DCs activated with CCL19 preferentially medi-ated Th1 development, increasing the proportion ofIFN-γ-producing cells and decreasing the proportion ofIL-4-producing cells (Figure 6, middle panel). Preincu-bating T cells with CCL19 did not influence T cell differ-entiation, indicating that the effect of CCL19 was lim-ited to the activation of DCs (Figure 6, right panel).Taken together, these data show that CCL19 selectivelymediates induction of Th1 responses.

TLR-Activated Migratory DCs Are Licensedfor CCL19-Mediated ActivationIn addition to the previously described chemoattractiveproperties of CCL19, we have shown that it can directlyactivate DCs and lead to the production of proinflam-matory cytokines and upregulation of costimulatorymolecules. However, the constitutive production ofCCL19 within lymphoid tissues would not be expected

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Figure 4. Purified Eukaryotic CCL19 Induces Maturation of BM-DCs

(A and B) BM-DCs were treated overnight with PBS, TNF-α (50 ng/ml), LPS (1µg/ml), or FLAG-CCL19 (0.33µg/ml), without proteinase Ktreatment (black bars) or after proteinase K treatment (white bars) and stained for the expression of CD86 and CD40. The error bars representthe standard deviation of quadruplicates.(C) CCR7−/− or wild-type DC were activated with CCL19, and the proportion of IL-12-producing cells was determined by flow cytometry.Numbers represent percentage of CD11c+ IL-12+ cells.(D) Surface expression of CD86, CD80, and CD40 was determined by flow cytometry for CCL19-stimulated control and CCR7-deficient DCs.(E and F) BM-DCs were treated overnight with titrated amounts of FLAG-CCL19 and expression of (E) CD86 and (F) CD40 was assessed byflow cytometry. Error bars represent the standard error of the mean for quadruplicate cultures. Horizontal lines indicate expression levels ofDCs cultured in the absence of FLAG-CCL19. GMFI indicates geometric mean fluorescence intensity.(G) Migration of DCs in response to the indicated concentrations of FLAG-CCL19 was examined as described in the Materials and Methods.In the presented assay, nonspecific migration was 3.2 × 103 ± 0.8 × 103 DCs. Error bars represent the standard deviation for triplicate cultures.Data in (A), (B), (E), and (F) represent combined values from two experiments. Data in (C), (D), and (G) are from independent experiments thatwere repeated with comparable results.

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Figure 5. CCL19 Induces Secretion of Inflammatory Cytokines

(A) Bone marrow-derived DCs were treated with PBS, TNF-α (50 ng/ml), LPS (1µg/ml), or FLAG-CCL19 (0.33µg/ml). 18 hr after addition ofstimuli, the supernatant of these cells were harvested and analyzed with a sandwich-ELISA for the presence of IL-1β and IL-12 (p40). Theerror bars represent the standard deviation of triplicates.(B) After stimulation with PBS, LPS/anti-CD40 antibodies (0.1 µg/ml/5 µg/ml), or FLAG-CCL19 (0.33 µg/ml) for 8 hr, bone marrow-derived DCswere analyzed for cytokine expression by intracellular staining for IL-1β, TNF-α, and IL-12 (p40). Data are from two independent experimentsand are representative of two (A) or four (B) similar experiments. The error bars represent the standard deviation of triplicates.

draining lymph node were semimature/steady-stateOVA protein (FITC-OVA) and LPS via the intranasal

Figure 6. CCL19 Primes DCs for the Induction of Th1 Cytokines

C57BL/6 primary DCs or SMARTA-2 TCR transgenic CD4+ T cells were stimulated with 1 µg/ml CCL19 or medium alone for 4 hr. After washingtwice with medium, naïve DCs together with naïve SMARTA-2 TCR transgenic CD4+ T cells; CCL19-activated DCs together with naïveSMARTA-2 TCR transgenic CD4+ T cells; or naïve DCs together with CCL19-activated SMARTA-2 TCR transgenic CD4+ T cells, were culturedin the presence of 100 nM GP13 peptide for 3 days. Following a 4 hr restimulation with PMA and ionomycin, IFN-γ and IL-4 producing TCR-transgenic T cells were identified by flow cytometry. Numbers represent percentage of CD4+ T cells positive for the indicated cytokine. Dataare from one representative experiment of two similar experiments.

to result in the activation of steady-state DCs becauseautoimmunity would otherwise result. To examine thisdirectly, we compared CCR7 expression on semi-mature/steady-state DCs in the lymph node to that ofDCs that had been activated in the periphery by a TLRligand and had migrated to the lymph node to presentexogenous antigen. We administered FITC-conjugated

route, and 24 hr later, we removed the draining lymphnode and determined which cell populations containedFITC-OVA. The only cell population positive for FITC-OVA were CD11chigh MHC IIhigh DCs (Figure 7A), consis-tent with active transport of the antigen rather thanpassive drainage where additional cell populationswould be expected to take-up OVA. Also present in the

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Figure 7. Pathogen-Derived Signals Are Required to License DCs for CCL19-Mediated Activation

FITC-conjugated OVA protein and LPS were instilled via the intranasal route into wild-type (A–C) and plt/plt mice (D–E). After 24 hr, thedraining mediastinal lymph node was removed and DCs isolated.

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DCs that expressed CD11chigh/int and MHC IIint but werenegative for FITC-OVA (Figure 7A). We assessed thelevel of CCR7 surface expression on these two DC pop-ulations and found that the LPS-activated, OVA-pre-senting CD11chigh MHC IIhigh DCs expressed higherlevels of CCR7 than the semimature/steady-state DCs(Figure 7A). To determine whether CCR7high TLR ligand-activated OVA-presenting DCs exhibited a comparableresponse to CCL19 as the CCR7low DCs, we next sortedthe respective DC populations by FACS then activatedthem for 6 hr with either CCL19 or LPS, and we thenassessed the production of IL-12, IL-10, or the surfaceexpression of CD40. The TLR ligand-activated OVA-presenting DCs produced moderate levels of IL-12without further stimulation ex vivo, reflective of their ac-tivated in vivo phenotype and possibly an increasedsensitivity to activation during the isolation and sortingprocedure (Figure 7B). Comparatively, the proportion ofCCR7low DCs that produced IL-12 without further stim-ulation ex vivo was 6-fold lower (Figure 7B). UponCCL19 or LPS activation, the TLR ligand-activatedOVA-presenting DCs produced high levels of IL-12 andupregulated CD40 expression, while the CCR7low DCsexhibited no response to CCL19 and were slightly re-active to LPS stimulation (Figure 7B). Overall, thesedata indicate that steady-state DCs express lowerlevels of CCR7 than DCs activated in the periphery byantigen- and pathogen-derived signals and are less re-sponsive to CCL19-mediated activation. Thus, CCL19preferentially results in the activation of those DCs thathave previously encountered pathogen-associatedsignals.

Licensed Dendritic Cells Require CCL19and CCL21 for Full Maturation In VivoWe next investigated the role of CCL19 in activatingDCs within an in vivo setting. Mice homozygous for thepaucity of lymph node T cell (plt) mutation do not ex-press CCL19 or CCL21 protein in secondary lymphoidorgans, although some expression of CCL21 is detecta-ble in lymphatic endothelium. One consequence of thismutation is the reduced, but not abolished, migrationof DCs to lymph nodes (Gunn et al., 1999). Using thesame protocol described above, we exposed wild-type(Figure 7C) and plt/plt (Figure 7D) mice to FITC-OVA inthe presence of the TLR ligand LPS, via the intranasalroute, and then analyzed the migrating DCs in thedraining lymph node 24 hr later. The number of migrat-ing DCs was reduced in the plt/plt mice as compared towild-type mice, however a clear population of CD11chi

FITC+ cells was evident (Figures 7C and 7D), consistent

al., 2003). This induces a potent activation program in

(A) DCs were stained with antibodies specific for CD11c, MHC II, and additionally, CCL19-Ig-Cy5. The uptake of OVA protein was determinedby FITC florescence. Solid lines indicate CD11chi MHC IIhi cells; dashed lines indicate CD11chi/int and MHC IIint cells.(B) DCs expressing the indicated markers were sorted by FACS, then activated in the presence of either CCL19 or LPS for 6 hr. The proportionof cells producing IL-12 (p40) and IL-10 was determined by intracellular staining, and expression of CD40 determined by surface staining andflow cytometry. Numbers represent percentage of cells positive for IL-12, or geometric mean of CD40 expression, respectively. Data are frompooled lymph node cells from 5 mice.(C and D) FITC-conjugated OVA protein and LPS were instilled via the intranasal route into wild-type mice (C) and plt/plt mice (D), and uptakeof OVA by DCs was monitored 24 hr later by assessing the presence of FITC in the indicated gate (dashed lines) as compared to semimatureDCs (solid lines). In (E), FITC+ cells from the gates indicated in (C) and (D) were assessed for their surface expression of MHC II, CD80, CD86,and CD40. Numbers represent geometric mean of the respective cell populations.Data in (A) and (B) and (C)–(E) are from two independent experiments and are representative of two similar experiments.

with previous reports (Gunn et al., 1999). Immediatelyex vivo, we examined the surface expression of the ac-tivation markers MHC II, CD80, CD86, and CD40 on themigrating DCs by flow cytometry (Figure 7E). Irrespec-tive of the plt mutation, the migrating DCs exhibitedcomparable surface levels of MHC II, however strikingdifferences in the surface expression of CD80 and CD86,and a minor difference in CD40, were evident (Figure7E). This partially activated phenotype of the migratingDCs in the plt/plt mice highlights the dual role ofCCL19/21 in both the migration and the activation ofTLR-licensed DCs.

Discussion

Regulation of dendritic cell maturation is essential forthe balance between maximal protective T cell re-sponses and minimal immunopathology. Here, we showthat CCL19, a chemokine attracting DCs from the pe-riphery to T cell regions of lymphoid organs, is a strongmaturation signal for DCs and the subsequent induc-tion of a Th1 response.

A key function of chemokines and their receptors isthe orchestration of leukocyte migration (Cyster, 1999;Sallusto and Lanzavecchia, 2000). Chemokines regu-late segregation of T areas from B areas in lymphoidorgans and the traffic of lymphocytes between the twoareas. Chemokines are also instrumental for inductionof local inflammation. Leukocytes traverse the endo-thelium of blood vessels and migrate into inflamed tis-sues in a three-step process: recognition of endothe-lium by selectins, activation by chemokines, followedby firm attachment by integrins (Campbell et al., 2003).Naïve resting T cells, and a subset of memory T cells,express the chemokine receptor CCR7, which bindsCCL19 and CCL21 (Ravkov et al., 2003; Sallusto et al.,1999; Unsoeld et al., 2002). These two chemokines areconstitutively produced in lymphoid organs and attractT cells to the T regions of these organs. Intriguingly,DCs, upon activation, also express CCR7 and becomeresponsive to CCL19/CCL21, which attracts them tothe T regions for optimal T cell priming (Sallusto andLanzavecchia, 2000). It is thought that this mechanismis responsible for the migration of Langerhans cellsfrom the skin to lymphoid organs upon activation. How-ever, chemokine receptors may not only be responsiblefor cellular migration but also for activation of dendriticcells. Indeed, it has recently been shown that the C-Cchemokine receptor CCR5 is triggered by cyclophilinderived from T. gondii (Aliberti et al., 2000; Aliberti et

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DCs leading to massive production of cytokines, in par- fticular IL-12. This CCR5-mediated activation of DCs re- ssults in induction of a protective Th1 response and wclearance of the pathogen. However, if over exagger- gated, this response may also result in immunopathol- Cogy (Aliberti et al., 2000). (

The present study extends this concept by showing sthat maturation of DCs, and the production of proin- lflammatory cytokines, is an important component of wchemokine biology in general. Specifically, CCL19 wasidentified by a global screen for proteins that activate LDCs as a strong inducer of dendritic cell maturation. In sfact, CCL19 was more potent than TNF-α or anti-CD40 aantibodies at inducing the release of proinflammatory ucytokines such as IL-1. Interestingly, CCL19 alone was lable to induce high levels of proinflammatory cytokines lin DCs. However, we cannot exclude at present time Cthat trace amounts of LPS in our CCL19 preparations tor the in vitro conditions per se (such as contact with stissue culture plastic) may have primed the DCs for re- ksponding to the chemokine. From a physiological point cof view, it seems likely that CCL19 alone may not be aable to induce the full maturation program in DCs. cOtherwise it remains difficult to explain why all DCs are tnot mature given they reside in a CCL19 rich lymphoid rorgan. Indeed, resting, immature/steady-state DCs in slymph nodes (Ruedl et al., 2000b; Salomon et al., 1998;Ohl et al., 2004) expressed low levels of CCR7 and did Enot respond with rapid production of inflammatory

Mcytokines upon stimulation with CCL19. Only activatedBDCs that had immigrated from the periphery upon stim-Nulation with LPS expressed high levels of CCR7 andT

produced IL-12 upon stimulation with CCL19. Hence, bDCs apparently need to first be licensed by a microbial rstimulus before they respond to CCL19 by producing a

ocytokines and upregulating expression of costimula-otory molecules.fInterestingly, DCs isolated from plt/plt mice, which doa

not express CCL19 or CCL21 protein in secondary lym- uphoid organs, displayed a partially activated phenotypeas compared to wild-type controls. These data suggest Cthat whilst the antigen/ TLR-ligand stimulation in the Rperiphery was sufficient to induce migration and upreg- L

uulation of MHC II, CCL19/CCL21 was required for fullyactivation. However, we cannot rule out the possibilitytthat other maturation cues might be absent in thesetmice. Junt et al. have previously shown that the im- R

mune response mounted against LCMV is only slightly treduced in plt/plt mice (Junt et al., 2002). It is important pto note that LCMV efficiently replicates in lymphoid or- r

hgans, and in addition, has multiple ways to activate thebinnate immune system. In such a case, taking away justAone component of the normal DC-activation process,a

i.e. CCL19/CCL21, may not be sufficient to significantly cimpair antiviral immune responses. Of note, functions hother than chemotactic activity have been attributed to dCCL19 and CCL21 including inducing endocytosis (Ya- (

Tnagawa and Onoe, 2003), dendritic extension (Yana-pgawa and Onoe, 2002) and inhibition of apoptosisw(Sanchez-Sanchez et al., 2004). Our data now extends

these findings and reveal an important, physiologically Rrelevant, role for CCL19 and CCL21 in programming the Rnature of ensuing T cell immune responses. Taken to- p

tgether, these findings may have important implications

or rational vaccine design and therapy. Lee et al. havehown that CD4+ T cell responses are enhanced in vivohen mice are given plasmid DNA encoding CCR7 li-ands (Lee et al., 2003), and intratumoral injection ofCL19 has been shown to increase tumor clearance

Hillinger et al., 2003). The underlying mechanism foruch CCL19-mediated enhanced immune responses isikely to be 2-fold, increasing DC and T cell recruitmenthile also directly activating the DCs.In summary, our data suggest the following model:

angerhans cells and other DCs in peripheral tissuescavenge antigens and, upon encountering pathogensnd receiving a stimulus, such as via toll-like receptors,ndergo an initial activation step. This leads to upregu-

ation of CCR7, and consequent recruitment of DCs toymphoid organs. During the process of migration,CL19/CCL21 induces full maturation of DCs rendering

hem capable of effectively triggering potent T cell re-ponses once they reach lymphoid organs. Chemo-ines, such as CCL19/CCL21, therefore not only or-hestrate immunity through guiding the leukocyteslong the complex pathways of the lymph and bloodirculation but also through regulation of DC matura-ion. Thus, CCL19/CCL21 ensures both that antigenseach lymphoid organs and that these antigens are pre-ented in a maximally immunogenic context.

xperimental Procedures

iceALB/c and C57BL/6 mice were purchased from Harlan (Horst, Theetherlands) and used between the ages of 8–14 weeks. DO11.10CR-transgenic mice (Murphy et al., 1990) were obtained from thereeding colony at the Institut fur Labortierkunde (University of Zu-ich, Zurich, Switzerland). SMARTA-2 transgenic mice (Oxenius etl., 1998) were bred at the Cytos breeding colony. Plt/plt mice werebtained from Dr T Junt, University of Zurich. CCR7−/− cells werebtained with the permission of Dr M Lipp (Max-Delbruck Center

or Molecular Medicine). All experiments were performed with thepproval of the Zurich animal ethics committee. Animals were keptnder SPF conditions.

onstruction of Normalized cDNA LibrariesNA was isolated from splenocytes of C57BL/6 mice infected withCMV 8 days earlier. Tester cDNA was produced from poly(A)+ RNAsing the template switch protocol (Zhu et al., 2001). Photobiotin-lated poly(A)+ RNA from naïve splenocytes was used as driver forhe normalization. Therefore 10 �g RNA was mixed with 30 �g pho-obiotin acetate and irradiated for 20 min using a 300 W Phillipseflector Floodlamp. Free photobiotin was removed. For hybridiza-

ion, 2 �g tester cDNA was mixed with 2 �g biotinylated driveroly(A)+ RNA and 0.2 �g oligo-dT (20mer), ethanol precipitated, andesuspended in formaldehyde hybridization buffer (80% formalde-yde, 25 mM Hepes [pH 7.5], 250 mM NaCl, and 5 mM EDTA). Hy-ridization was carried out in an Eppendorf Mastercycler personal.fter an initial denaturation step, nucleic acid hybridization wasllowed to proceed for 16 hr at 42°C. Unhybridized single-strandedDNA was purified by streptavidin-mediated removal of RNA/DNAybrids and excess driver RNA. Double-stranded cDNA was pro-uced by PCR, digested with the restriction endonuclease Sfi1

Roche), and cloned into the alphaviral expression vector pDelSfi.he library plasmid, linearized with NotI or PacI, and the helperlasmid pDHEB (Bredenbeek et al., 1993), linearized with EcoRI,ere subjected to SP6 RNA polymerase-mediated in vitro tran-cription using the mMessage mMachineTM kit (Ambion). LibraryNA was coelectroporated with an equimolar amount of helperNA into 107 baby hamster kidney (BHK) cells. Eighteen hoursosttransfection, cell supernatants were harvested and the viral ti-

ers determined.

CCL19 Is a Potent Activator of DCs503

Generation of Protein LibraryBHK cells were infected with the normalized viral library at a multi-plicity of infection of 0.1. Two hours later, cells were washed andfurther incubated in the presence of a neutralizing anti-Sindbis anti-body to avoid superinfection. Eight hours postinfection, cells weredetached and stained for the expression of Sindbis virus proteinsand single-cell sorted into a 96-well plate containing BHK cells.Plates were then incubated for 3 days at 37°C until full cytopathiceffect had developed. A master plate with 50 µl supernatant con-taining replication competent viruses was made and stored at−80°C. The remaining supernatants were UV irradiated in order toinactivate viruses and then used for the DC-T cell proliferation as-say. The procedure is schematically shown in Figure 1A.

Preparation of Dendritic Cells and T CellsImmature mouse dendritic cells (DC) were isolated from spleensand mesenteric lymph nodes of naïve BALB/c mice as described(Ruedl and Bachmann, 1999). Purified DCs were pulsed with 4 nMof OVA-peptide (Ovalbumin [OVA] 323–339; ISQAVHAAHAEINEAGR).OVA-peptide-specific CD4+ T cells were isolated from spleens ofnaïve DO11.10 mice using MACS beads according to the instruc-tions of the supplier (Miltenyi Biotec). Bone marrow-derived den-dritic cells (BM-DCs) were prepared from bone marrow of naïveBALB/c mice as described elsewhere (Lutz et al., 1999).

For DC maturation, 50,000 cells in a final volume of 100 �l werepulsed for 4 hr or 18 hr with TNF-α (50 ng/ml; R&D systems) or LPS(0.1 µg/ml; Sigma-Aldrich; E. coli, 026:B6) with or without anti-CD40(clone3/23, 5 µg/ml; BD PharMingen) or FLAG-CCL19 (0.33 µg/mlor 0.99 µg/ml). Alternatively, these samples were treated with 10µg/ml proteinase K (Sigma-Alrich) for 45 min at 37°C in DPBS (con-taining Ca2+ and Mg2+) and subsequently boiled at 95°C for 10 min.

The following antibodies were used for staining: FITC-conjugatedanti-CD86, FITC-conjugated anti-CD80, PE-conjugated anti-CD11c,allophycocyanin-conjugated anti-TNF-α, biotin-conjugated anti-IL-1β, biotin-conjugated anti-IL-12-p40 (all BD Pharmingen), andALEXA633-conjugated Streptavidin (IBA). Dead cells were alwaysstained with PI and gated out. Intracellular cytokine stainings wereperformed according to standard protocols.

For detection of IL-1β and IL-12 produced by BM-DCs, 1 × 105

cells in 100 �l media were cultured for 18 hr with the indicatedstimuli. Then the supernatants were analyzed by sandwich ELISAfor IL-1β (R&D Systems) or IL-12 p40 (BD PharMingen). As stan-dards, recombinant IL-1β and IL-12 (both R&D systems) were used.

To test for T cell polarization, naïve SMARTA-2 mice (Oxenius etal., 1998) were sacrificed and spleens removed. CD4+ T cells wereisolated by MACS bead separation (Miltenyi, Germany) and werefound to be 90%–95% CD4+ CD62Lhigh by subsequent FACS analy-sis. Dendritic cells were isolated from naïve wild-type C57BL/6spleens as previously described (Ruedl and Bachmann, 1999). Iso-lated T cells (5 × 104 cells/well) and DCs (1 × 104 cells/well) werecultured in the presence of 100 nM GP13 peptide in 96-well plates(Corning Incorporated, USA). On day 3 of culture, cells were acti-vated in the presence of PMA and ionomycin for 4 hr and IFN-γ andIL-4 production determined by flow cytometry.

Functional Expression Screening of DC Maturation FactorsMaturation of dendritic cells was determined via their capacity toinduce T cell proliferation in the presence of specific antigen.Briefly, 30,000 naïve OVA-peptide pulsed dendritic cells/well wereseeded in 96-well round-bottom plates. Fifty microliters of UV-irra-diated viral supernatant containing one protein of the normalizedactivated mouse spleen library was added, and the plates wereincubated at 37°C. As controls, supernatants containing virally ex-pressed TNF-α or supernatants from SR5-infected BHK cells wereused. After 18–20 hr, 50,000 naïve OVA-specific CD4+ cells wereadded to each well, and the plates were further incubated for 2.5days. 3H-thymidine (1mCi/well) was added for the final 14 hr ofculture. Cells were harvested using a multiple-well harvester and3H-thymidine incorporation was determined in a scintillationcounter. Viral supernatants leading to maturation of dendritic cellsresulting in high 3H-thymidine incorporation into CD4+ cells werereanalyzed in a second proliferation assay for confirmation.

RT-PCR Rescue of Positive SupernatantsRT-PCR was performed on supernatants from wells that showedincreased proliferation rates. Viral RNA was isolated from superna-tant using the QIAmp Viral RNA Kit (Qiagen). RNA was transcribedinto cDNA using SUPERSCRIPTTM II RNase H-reverse transcrip-tase (Invitrogen Life Technologies). Complementary RNA was re-moved by RNase H treatment. The inserts were amplified by PCRusing Expand High Fidelity PCR System (Roche), separated on anagarose gel, purified, and sequenced.

In order to generate an expression vector for the recombinantproduction, CCL19 was amplified by PCR using gene-specificprimers bearing a BamHI-site at N-terminus (5#-atcgcgGATCCCGGTGCTAATGATGCGGAAGA-3#) and a NheI-site at the C-terminus(5#-ctatactagctagctcaAGACACAGGGCTCCTTCTGG-3#). This PCRproduct was then cloned into pCEP-N-FLAG leading to constructpCEP-N-FLAG-CCL19. This construct was used for the eukaryoticproduction of N-termianlly FLAG-tagged CCL19.

Expression and Purification of the Recombinant Proteins293-EBNA cells were seeded at a density of 4 × 106 cells/90 mmplate in DMEM (GIBCO BRL) containing 10% FCS (GIBCO BRL) 1day prior to transfection. The cells were then transfected withpCEP-N-FLAG-CCL19 using lipofectamine 2000 (Invitrogen) ac-cording to the manufacturer’s recommendations. One day aftertransfection cells were passaged under puromycin selection. Theresistant population was then further expanded. Serum-free super-natants were collected and recombinant N-terminal, FLAG taggedCCL19 was affinity purified using ANTI-FLAG M2-Agarose-sepha-rose (SIGMA) according to the manufactures instructions. Afterextensive dialysis against PBS, the protein was stored in PBS at4°C. The protein concentration was determined by Bradford assay(Biorad).

CFSE In Vitro Proliferation AssayCFSE was purchased from Molecular Probes (Eugene, OR). BM-DCs were activated as described above for 4 hr. Then DO11.10transgenic CD4+ T cells were added, obtained by positive MACSMicroBeads isolation (Miltenyi Biotec) with a purity of at least 90%.The T cells were labelled with 0.5 µM CSFE by incubation at 37°Cfor 10 min. After labelling, the cells were subsequently washed withPBS at 4°C. After 4 days of incubation, cells were harvested,stained with PE labelled anti-CD4 mab (0.1 µg/ml; BD PharMingen),and the CD4+ T cells were analyzed for CSFE staining by FACSanalysis.

DC Chemotaxis AssayThe chemotactic activity of CCL19 was assessed using theChemoTx system according to the manufacturer’s instructions(NeuroProbe, Inc). In brief, aliquots of 20,000 DCs in 25 �l mediawere placed on a 3 �m pore-size filter above wells containing 30�l of CCL19 at the indicated concentrations. The cells were incu-bated at 37°C for 4 hr and the total number of cells that had mi-grated through the filter determined by cell counts. Nonspecificmigration was determined by counting wells containing no chemo-kine. Each assay was performed in triplicate, and values representthe mean ± SD of total migrating cells.

Activation and Isolation of OVA-Presentingand Steady-State DCsMice were exposed to 100 �g FITC-conjugated OVA (MolecularProbes, Eugene, OR) and 5 �g LPS (Sigma-Aldrich; E. coli, 026:B6)in a total volume of 50 �l PBS by intranasal inoculation. After 24 hr,draining lymph nodes were removed, digested with collagenaseand DCs isolated. Semimature/steady-state DCs were defined ascells expressing CD11chi/int MHC IIint FITC– and mature/TLR-acti-vated DCs were defined as cells expressing CD11chi MHC IIhi

FITC+. The distinct cell populations were sorted by FACS and 1 ×105 total sorted cells were incubated in either medium or mediumsupplemented with CCL19 (1 �g/ml) or LPS (1 �g/ml) for 6 hr (forcytokine analysis) or 16 hr (for CD40 expression) in round-bottom96-well plates. Production of IL-12 and IL-10 was determined by

Immunity504

intracellular cytokine staining, and surface expression of CD40 was (cdetermined by flow cytometry, as described.

EW

Supplemental Data iSupplemental Data include two additional figures and can be found iwith this article online at http://www.immunity.com/cgi/content/full/

E22/4/493/DC1/.T(oAcknowledgments1

GWe would like to thank Annette Oxenius and Roman Spörri for help-lful discussions and Bettina Ernst for her cell-sorting expertise.ohReceived: November 8, 2004GRevised: February 10, 2005cAccepted: February 23, 2005

Published: April 19, 2005 HD

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