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The αβ TCR consists of at least six different subunits (α, β, δ, γ, ε, ξ),which undergo rearrangement during activation by antigen-present-ing cells. These events lead to signal transduction and activation. Uponbinding of the antigen to the TCR, CD3 molecules are recruited to theTCR and associate with the αβ subunit, thus activating a signal trans-duction cascade driving T-cell proliferation1,2. The interactionbetween CD3 and the TCR is mediated by the transmembranesequence of the α-subunit. This region consists of an 8-mer peptidethat contains arginine and lysine, which serve as an anchor to themembrane3. A synthetically produced peptide that resembles thetransmembrane sequence of the TCR α-chain is able to block T-cellactivation by preventing the interactions of the CD3 molecules withthe TCR αβ complex4. In vivo application of this TCRpep leads todownregulation of T-cell activation in different autoimmune diseasessuch as rheumatoid arthritis, allergic contact dermatitis and diabetes5.However, we observed not only autoreactive T-cell suppression afterthe TCRpep treatment, but also a generalized suppression of T-cellfunction after systemic application. In light of these results, our aimwas to devise a system that allows local in vivo secretion of the TCRpepthereby preventing a generalized immunosuppression. We thereforetransduced bone marrow–derived DCs with a vector expressing thecDNA of the TCRpep, and to assess the function of these ‘suppressor’DCs in different inflammatory and autoimmune settings in vivo.

RESULTSTCRpep-transduced DCs suppress T-cell proliferation in vitroDCs are most efficiently transduced by adenoviral gene transfer. To testthe efficiency of adenoviral transduction and its effects on DC matura-tion, we infected DCs with adenovirus that expresses an enhancedgreen fluorescent protein (EGFP)-TCRpep fusion protein and assessed

the cells’ maturation status by fluorescence-activated cell sorting(FACS). Expression of major histocompatibility complex (MHC) classII and B7-2 molecules was high and reached levels comparable to thatin nontransduced DCs (Fig. 1a and data not shown). The transductionefficiency, as determined by EGFP expression, averaged 70%.

To test whether the transduced DCs actively express the TCRpepand suppress T-cell proliferation in vitro, mixed lymphocyte reactions(MLRs) were performed using DCs transduced with TCRpep or a con-trol peptide (CTRpep) as stimulator cells. DCs induced substantiallyless T-cell proliferation in MLRs after TCRpep transduction (Fig. 1b).This reduction was comparable to that obtained when the TCR pep-tide itself was added to the cultures. In contrast, DCs transduced withCTRpep stimulated T-cell proliferation normally. Likewise, additionof the control peptide had no effect on T-cell proliferation.

To test whether secreted TCRpep mediates the T cell–inhibitoryeffect, the tissue culture supernatants of transduced DCs were trans-ferred to freshly isolated CD4+ T cells (Fig. 1c) and allogeneic DCswere added to stimulate T-cell proliferation. Transfer of the tissue cul-ture supernatant of TCRpep-transduced DCs blocks T-cell prolifera-tion in a MLR (Fig. 1c). The corresponding control DCs stimulatedT-cell proliferation normally. Addition of anti-CD3 antibodies to thecultures restored T-cell proliferation, showing that the T cells areviable and that antibody crosslinking is able to overcome the peptide-induced inhibition of T-cell proliferation.

To test the effect of TCRpep transduction in an antigen-specific sys-tem, we employed ovalbumin (OVA)-pulsed DCs and used those asaccessory cells for OVA-specific TCR transgenic DO11.10 T cells. HereT-cell proliferation was blocked by expression of the TCRpep in DCs(Fig. 2a). Like allogeneic T cells, CTRpep-transfected DCs stimulatedT cells normally. To exclude the possibility that the T cells have become

Department of Dermatology, University of Mainz, Langenbeckstrasse 1, D-55101 Mainz, Germany. Correspondence should be addressed to K.M. (mahnke@hautklinik.klinik.uni-mainz.de).

Dendritic cells, engineered to secrete a T-cell receptormimic peptide, induce antigen-specificimmunosuppression in vivoKarsten Mahnke, Yingjie Qian, Jürgen Knop & Alexander H Enk

A T-cell receptor mimic peptide (TCRpep) consisting of an 8-amino-acid peptide, homologous to the transmembrane region of theT-cell receptor (TCR) α chain, blocks T-cell activation after systemic application. When dendritic cells (DCs) were transduced tosecrete the TCRpep and injected into mice, evidence of immunosuppression was observed. In a CD8-driven allergy model, theinjection of DCs transduced with the TCRpep reduced inflammation markedly and in a CD4+ T cell–dependent model of multiplesclerosis (experimental autoimmune encephalitis, EAE), injection of TCRpep-secreting DCs abrogated EAE symptoms andprolonged survival. These effects were antigen specific, because transduced DCs that did not express the respective antigen failedto convey protection in the allergy model as well as in the EAE model. Thus these data show that DCs expressing the TCRpep areable to suppress T-cell activation and might be a useful tool for inducing antigen-specific immune suppression in vivo.

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anergic after contact with the TCRpep-transduced DCs, T cells wererecovered 24 h after initial coculture with TCRpep-transduced DCsand restimulated with OVA-pulsed DCs at day 2, 4 or 6 thereafter. Inthese experiments (Fig. 2b) T cells proliferated normally after a 6-dresting period, indicating that they had not become anergic.

To test whether antiproliferative effects could also be observed invivo, we injected mice with OVA-specific DO11.10 T cells labeled withthe fluorescent dye CFSE, and injected transduced, OVA-pulsed DCs.T-cell proliferation, as indicated by dilution of the CFSE dye, wasassessed by FACS. In these experiments TCRpep-transduced DCsfailed to induce T-cell proliferation, whereas injection of CTRpep-transduced—as well as untransduced—DCs led to vigorous T-cellproliferation (Fig. 2c). To investigate whether CD25+ regulatory T cellswere induced by injection of TCRpep-transduced DCs, we analyzedthe phenotype of the CD4+ T cells 5 d after injection of transducedDCs. The number of CD4+CD25+ T cells remained unchanged in allgroups, indicating that TCRpep-transduced DCs did not induce regu-latory T cells in vivo (Fig. 2d).

TCRpep-transduced DCs suppress hypersensitivity reactionsTo assess the potential use of TCRpep-transduced DCs in a delayed-typehypersensitivity (DTH) reaction, mice were reconstituted with DO11.10T cells and injected with OVA-pulsed, TCRpep-transduced DCs andcorresponding controls (Fig. 3a). A DTH reaction was induced by injec-

tion of OVA into the footpads, and footpad swelling was measured 24 hlater. In this CD4-driven disease model, we could suppress the footpadswelling by injection of TCRpep-transduced DCs. In animals thatreceived either CTRpep-transduced or untransduced DCs, or whenOVA was omitted from the DC preparation, no reduction of the footpadswelling was detected. These data suggest that the specific antigen isrequired for effective suppression of T-cell proliferation.

We next asked whether TCRpep-transduced DCs also affect CD8-driven immune responses. We chose a contact hypersensitivity (CHS)model using 2,4,6-trinitro-1-chlorobenzene (TNCB) as a contact sen-sitizer. In initial experiments TCRpep-transduced and control DCswere haptenated using the water-soluble TNCB analog trinitrobenzenesulfonic acid (TNBS) and injected into mice. After 6 d, mice were chal-lenged by application of the hapten on one ear and ear swelling was

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Figure 1 Transduction of DCs with cDNA encoding a TCRpep suppresses T-cell stimulatory capacity. (a) DCs were left untreated or transduced withadenovirus expressing a TCRpep-EGFP fusion protein and stained with PE-labeled anti-MHC class II or anti-CD86 antibodies as indicated. (b) Normal DCs (DC) or DCs transduced with virus encoding for TCRpep(DC/TCRpep) or control virus (DC/CTRpep), respectively, were coculturedwith allogeneic T cells. To some aliquots a recombinant TCR-peptide (DC +TCRpep) and the respective control peptide (DC + CTRpep) was added. T-cell proliferation was determined after 3 d. (c) DCs were transduced asindicated and cultured for 2 d. Thereafter the tissue culture supernatantswere harvested and added to freshly prepared MLRs. T-cell proliferation wasdetermined after 3 d. BMDC, bone marrow–derived DCs. Sup, supernatant.

Figure 2 DCs transduced with TCRpep suppresses proliferation of OVA-specific T cells in vitro and in vivo. (a) Normal DCs (DC) or transduced DCsas indicated were left untreated or were pulsed with ovalbumin (+ OVA) andwere cocultured with DO11.10 T cells. Proliferation was assayed after 3 d.Some aliquots received (1 µM) recombinant TCR peptide (TCRpep) and/oranti-CD3 antibodies added to overcome the TCRpep-induced T-cellunresponsiveness. (b) DCs were transduced and cocultured with DO11.10 T cells as in a. Twenty-four hours later, T cells were retrieved, cultivated andrestimulated with OVA-pulsed DCs at day 2 or day 6, respectively. T-cellproliferation was determined by [3H]thymidine incorporation. (c) Mice werereconstituted with CFSE-labeled DO11.10 T cells and injected withtransduced and/or OVA-pulsed DCs. At day 2, lymph node T cells werestained with clonotypic antibody KJ1-26 to detect DO11.10 T cells. CFSEstaining was further analyzed by FACS with gating on the KJ1-26+ cells.Proliferation is indicated by dilution of the CFSE fluorescence. (d) Mice wereinjected with DO11.10 T cells followed by DCs as indicated. Five days later,lymph node T cells were purified and stained with anti-CD4 FITC and anti-CD25-PE. Samples were analyzed by FACS and the percentage ofCD4+CD25+ T cells is shown.

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determined 24 h later. As expected, the injection of TNBS-haptenizedDCs into mice caused sensitization, as indicated by a strong increase inear thickness after challenge (Fig. 3b). In contrast, if the DCs expressedthe TCRpep, sensitization was suppressed. Injection of the CTRpep-transduced DCs had no effect on the ear swelling reactions. Thus,expression of the TCRpep by DCs suppresses activation of TNCB-specific T cells in vivo.

In preliminary experiments we observed that haptenized or anti-gen-pulsed DCs were able to block T-cell proliferation more effec-tively than their untreated counterparts. We therefore tested whetherthe injection of TCRpep-transduced DCs suppresses activation ofT cells in an antigen-specific manner. We reconstituted mice withOVA-specific DO11.10 T cells and injected TCRpep-transduced,TNBS-pulsed DCs 24 h later. The ear swelling reaction, as well as thein vitro T-cell proliferation data, clearly demonstrate that these recon-stituted mice were effectively sensitized against TNBS (Fig. 3c,d),whereas injection of TCRpep-transduced haptenated DCs failed tosensitize mice as expected. However, when T cells from those animalswere restimulated with OVA-pulsed DCs in vitro, the OVA-specific T cells proliferated normally and a suppressive effect after injection ofTCRpep-transduced DCs could not be detected (Fig. 3e). Thereforewe conclude that injection of TCRpep-expressing DCs does not resultin a generalized immunosuppression; instead it suppresses T-cell acti-vation in an antigen-specific manner.

The suppressive effect of transduced DCs is antigen-specificThe results presented in Figure 3c,d indicate that the induced T-cellunresponsiveness is antigen specific. However, the OVA-specificDTH reaction and the TNCB-induced CHS reaction rely on differ-ent mechanisms, that is, CD4+ versus CD8+ T cells. We thereforecompared how CHS reactions to different haptens were affected byinjection of TCRpep-transduced DCs. In these experiments (Fig. 4a),mice were injected with a mixture of DCs that were transduced byTCRpep and CTRpep and that had been pulsed with the allergensTNBS and 2,4-dinitrofluorobenzene (DNFB), respectively. Animalswere then challenged with the respective allergen and ear swellingwas measured 24 h later. As demonstrated, injection of a mixture ofTCRpep-transduced, TNCB-loaded DCs and CTRpep-transduced,DNFB-loaded DCs, only results in the abrogation of TNCB-induced ear swelling reactions. In contrast, the DNFB swellingreactions were not reduced. Similar results were obtained with areciprocal experimental design (Fig. 4a). Here, ear-swelling reac-tions after challenge with DNFB were affected only by injection ofDNFB-loaded TCRpep-transduced DCs, whereas the TNCB reac-tion was not substantially reduced. The CHS reaction against a par-ticular antigen was reduced only if DCs that presented the

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Figure 3 Injection of TCRpep-transduced DCs specifically suppresses T-cellactivation in CHS and DTH reactions. (a) Control DCs or transduced DCs asindicated were left untreated or were pulsed with ovalbumin (+ OVA) and were injected into Balb/c mice previously seeded with DO11.10 cells. Aftertreatment with OVA + CFA to boost the OVA response, OVA peptide wasinjected into the left hind footpad. Footpad swelling was measured 24 h later. Results are displayed as mean swelling ± s.d. of the left versus right foot (*, significant difference compared to DC + OVA, P < 0.05, n = 5). (b) Transduced DCs as indicated were left untreated or were haptenized withTNBS (+TNBS). Cells were injected and mice were challenged by applicationof the hapten TNCB onto their left ear. Ear swelling reaction was measured24 h later. Results are displayed as mean swelling ± s.d. (*, significantdifference compared to group that was immunized by injection of DC +TNBS, P < 0.05, n = 5). (c) Mice, seeded with OVA-specific DO11.10 T cellsreceived DNFB-haptenized TCRpep or CTRpep-transduced DCs. Thereaftermice were challenged and ear swelling reactions were measured as in b.Results are displayed as mean ear thickness ± s.d. (*, significant differencecompared to groups that were immunized by injection of DC + TNBS, P < 0.05, n = 5). (d,e) Lymph node cells were prepared from mice treated asdescribed in a and pulsed with the hapten TNBS or cultured with OVA-pulsedDCs. T-cell proliferation was measured after 2 d.

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particular antigen also expressed the TCRpep. The CHS reaction toother antigens was not affected. Therefore we conclude thatTCRpep-transduced DCs are able to suppress T-cell proliferationin an antigen-specific manner.

Injection of TCRpep-transduced DCs suppresses sensitizationWe designed a ‘prophylactic’ desensitization approach to testwhether injection of TCRpep-transduced DCs is able to prevent top-ical sensitization in a CHS model (Fig. 4b). We injected groups ofmice with haptenized TCRpep-transduced DCs and correspondingCTRpep- transduced DCs twice and sensitized these animals 2 dafter the last DC injection by topical application of TNCB to theirabdomens. Mice were challenged 6 d later and CHS reactions wereassayed. Injection of TCRpep-transduced DCs prevented sensitiza-tion as indicated by reduced ear swelling, whereas induction of CHSwas not affected in controls (Fig. 4b). We further tested how long theprotective effect of TCRpep-transduced DCs would last. We injectedgroups of mice with TCRpep-transduced and CTRpep-transducedDCs and sensitized these animals 4, 7 and 9 d after the final DC injec-tion. Upon challenge with TNCB on one ear, the ear swelling reac-tion was determined as an indicator of sensitization. Sensitizationwas still impaired up to 7 d after injection of TCRpep-transducedDCs (Fig. 4c), but animals showed a normal ear swelling reaction 9 dafter injection. Therefore, we conclude that the suppressive effectpersists for about 7 d.

Transduced DCs prevent CHS reactions in sensitized miceIn the experiments described above, TCRpep-transduced DCs wereinjected before or at the same time as sensitization occurred. Inmedical situations, however, the suppression of ongoing allergicand/or autoimmune responses would be beneficial. We thereforeinvestigated whether injection of TCRpep-transduced DCs couldalso suppress the ear swelling reaction of already sensitized mice. Ina CHS model (Fig. 5), mice were sensitized against TNCB and earswelling reactions were induced thereafter by application of TNCBto the ears. The ear swelling indicated successful sensitization of allmice. Thereafter TCRpep-transduced DCs and control DCs wereinjected twice, followed by a rechallenge. In this approach, injec-tion of TCRpep-transduced DCs substantially decreased the earswelling reaction upon rechallenge. In contrast, neither CTRpep-transduced DCs, nor DCs that lacked the specific antigen TNBS,had an effect on ear swelling, indicating antigen specificity.

Efficiency of TCRpep-transduced DCs in an EAE modelTo further assess the capability of TCRpep-transduced DCs to sup-press autoimmune diseases, we used transduced DCs in mice withEAE, a mouse model of multiple sclerosis6. In an ‘immunization’approach, we injected TCRpep-transduced DCs and correspondingcontrol DCs twice in a 4-d interval into TG4 mice. Thereafter EAE wasinduced and the disease was assessed according to the internationalEAE score. Mice in groups that received CTRpep-transduced DCsdeveloped severe symptoms of EAE 1 week after induction and 100%died within 3 weeks (Fig. 6a). In contrast, mice that received TCRpep-secreting DCs had substantially less severe EAE symptoms and 70% ofthe animals survived.

We were next interested in investigating whether injections ofTCRpep-transduced DCs may have therapeutic effects on the courseof EAE after the onset of the disease. We first induced EAE in TG4mice, and then we injected TCRpep-transduced DCs on days 5, 7 and9. This treatment resulted in a marked reduction of EAE symptoms ingroups of mice that received TCRpep-expressing DCs (Fig. 6b). Incontrol groups—that is, in groups that received CTRpep-transducedDCs and that received DCs only—EAE symptoms increased over time,causing death of 100% of the animals within 3 weeks after induction.This demonstrated that TCRpep-transduced DCs might be a usefultool to induce antigen-specific immune suppression in a variety of dif-ferent autoimmune conditions in vivo.

DISCUSSIONHere we report on the generation of immunosuppressive DCs bytransduction with a cDNA encoding an 8-amino-acid peptide homol-ogous to the transmembrane domain of the TCR α-chain. ThisTCRpep can interfere with the TCR assembly within the T-cell mem-brane, leading to a block in T-cell activation4. Because of its specificinteraction with the CD3 subunit, the TCRpep does not block prolifer-ation of B-cells and macrophages. It has been shown to successfullysuppress T-cell activation in several autoimmune diseases such asrheumatoid arthritis and diabetes5. However, systemic application ofthis peptide resulted in a generalized abrogation of T-cell function. Inan attempt to circumvent a generalized immunosuppression,researchers topically applied the peptide to inflamed skin and showeda regression of the symptoms in different inflammatory skin diseases7.Moreover, bio-ballistic transduction of epidermal skin cells withcDNA encoding the TCRpep has been observed to result in suppres-sion of CHS reactions7.

Here we describe a method to transduce DCs generated in vitro withthe cDNA encoding a secreted form of TCRpep and inject these cellsinto animals. This resulted in a constant in vivo supply of the peptideand because of its local secretion, no generalized immunosuppressionwas observed. Among antigen-presenting cells, only DCs are capableof migrating to regional lymph nodes in sizeable numbers and are ableto interact with naive T cells as a result of high expression of adhesionmolecules as well as T-cell costimulatory molecules8. We chose DCs ascarriers for the TCRpep to exploit their ability to form intense in vivocontact with T cells, ensuring that enough peptide is transferred intothe T-cell membrane to generate a block of T-cell activation. Mostimportantly, we demonstrated antigen specificity of the suppressiveeffect. According to current models, the DC–T-cell interaction isshort-lived as long as a T cell does not bind its ‘matching’ MHC-peptide complex9,10. By injecting antigen-pulsed DCs, we were able torecruit T cells specific for that respective antigen and thus the secretedpeptide preferentially blocked T cells antigen specifically. This notionis further supported by results showing that injection of TCRpep-transfected fibroblasts (that constantly secrete the TCRpep, but do not

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express T-cell-specific adhesion molecules) failed to produce antigen-specific immunosuppression in vivo (not shown).

By transducing the bone marrow–derived DCs with a TCRpep-expression vector, we converted ‘normal’ DCs into ‘suppressor DCs.’ Acomparable approach to generate so-called ‘killer DCs’ has beenreported11. Here, the expression of CD95L enables DCs to ‘kill’ Fasligand–bearing T cells, resulting in suppression of CHS and host-versus-graft reactions in animals. However, in our approach the T cellswere not ‘killed’ after contact with the TCRpep, because we were ableto overcome our TCRpep-induced T-cell unresponsiveness by addi-tion of αCD3 antibodies. T-cell responsiveness was re-established ∼ 7 dafter the final injection of the ‘suppressor’ DCs. Thus these data sug-gest that the T-cell population affected by the TCRpep is still viableand injection of TCRpep-secreting DCs does not induce deletionaltolerance12 towards a specific antigen.

‘Suppressive’ DCs have also been established without geneticmanipulation. For example, IL-10 treatment of DCs leads to inductionof ‘tolerogenic’ DCs13,14 and immature DCs induce regulatory T cells(Treg)15–17. However, studies with immature DCs injected in vivoalways bear the risk that DCs mature in vivo. If this happens, sensitiza-tion, but not induction of tolerance, will ensue. According to previousstudies3–5, the TCRpep acts directly on the TCR of effector T cells andmost likely does not induce Treg3–5. Our results showing the absenceof CD25 expression on T cells support this notion. CD25 expression isnot sufficient to characterize Treg, but additional results corroborateour conclusion. For example, crosslinking via anti-CD3 antibodiesleads to proliferation of TCRpep-treated T cells. Furthermore, 7 d afterthe initial treatment with TCRpep, T cells proliferate normally uponrestimulation. Neither feature applies to genuine Treg, and thereforewe conclude that the TCRpep acts directly on the TCR of effector Tcells. In addition, once activated, Treg suppress T-cell activation anti-gen nonspecifically18 (unlike the TCRpep), which may cause a rathergeneralized immunosuppression.

Compared to other immunosuppressive drugs such as corticos-teroids, the advantages of the therapeutic use of the TCRpep lies inthe antigen specificity conveyed by the DCs and its short half-lifethat prevents a long-lasting generalized immunosuppression. Thepeptide integrates into CD4+ as well as into CD8+ T cells5, thus

providing a potent block of different T-cell-mediated diseases. Oursystem limits its immunosuppressive action to the local site of in vivoproduction; thus secretion of the TCRpep by DCs demonstrated astrategy to produce ‘designer DCs’19 that might be a useful tool tosuppress T-cell activation in allergic and autoimmune diseases.Because the sequence of the TCRpep is conserved in various speciesranging from mouse to man5, suppression of human T cells in vivo ismost likely possible and further investigations will reveal its impacton human autoimmune diseases.

METHODSAnimal experiments. All experiments were approved by the state of RheinlandPfalz, Germany, permission number 177-07/031-2.

Generation of DCs. Bone marrow–derived DCs were prepared according tostandard methods20 using bone marrow cultures with GM-CSF-supplementedRPMI medium (PAA Biochemicals). For transduction, DCs were incubatedwith the respective adenovirus for 4 h; thereafter DCs were washed and cul-tured in GM-CSF containing medium for an additional 48 h.

Generation of adenovirus. Recombinant adenovirus was generated accordingto a method described by Hardy et al.21 Briefly, the cDNA encoding the TCR-peptide (TCRpep; sequence: ELRILLLKV) or the control peptide (CTRpep,sequence: LGILLLGV) was cloned behind a B cell–derived leader sequence intothe pAdlox vector kindly provided by T. Tueting, University of Bonn, Germany.For recombination of adenovirus, CIN cells were transfected with this pAdloxconstruct harboring the cDNAs using the CaPO4 method. Virus productionwas observed in the form of by lytic plaques and virus was further amplified,harvested and purified using standard CsCl ultracentrifugation.

T-cell proliferation assays and FACS. MLR-graded doses of DCs were dis-pensed into 96-well plates (Falcon) and 2 × 105 magnetobead (Milteni,Germany) purified allogeneic CD4+ T cells were added to a total volume of200 µl/well. After 3 d, T-cell proliferation was assayed by [3H]thymidine(Amersham) uptake followed by scintillation counting (Wallac).

For ovalbumin-specific in vitro restimulation of T cells, CD4+ T cells wereprepared from lymph nodes using magnetobeads. 105 T cells were then cocul-tured with DCs pulsed with ovalbumin (Sigma) and proliferation was assayedby [3H]thymidine uptake.

For 2,4,6-trinitrobenzene sulfonic acid (TNBS)-specific (Sigma) T-cell pro-liferation, lymph node cells were prepared by passing lymph nodes through amesh. Thereafter cells were incubated with different doses of TNBS (as indi-cated in the figure) for 10 min, washed extensively and cultivated in 96-wellplates (Falcon). Proliferation was determined by [3H]thymidine uptake.

To measure proliferation of OVA-specific DO11.10 T cells in vivo, CD4+ Tcells were prepared from DO11.10 mice and stained with CFSE (MolecularProbes). Next, 5 × 106 labeled cells were injected intravenously into Balb/crecipients and 24 h later OVA pulsed DCs were injected. After 2 d, lymph nodesand spleens were removed and cells were examined in a FACScan by gating onCFSE-positive cells. To analyze CD25 expression, mice were reconstituted withpurified DO11.10 T cells and injected with transduced DCs as described above.Lymph nodes were harvested 5 d later and isolated T cells were stained withFITC-labeled anti-CD4 antibodies, PE labeled anti-CD25 antibodies and corre-sponding isotype controls (all from Caltag).

Contact hypersensitivity. Transduced and control DCs were haptenized by a10-min incubation with 0.1% w/v TNBS. In some experiments DCs were

NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 8 AUGUST 2003 907

Figure 6 Effect of TCRpep-transduced DCs on autoimmune EAE. (a) Micewere injected with MBP-pulsed DCs, TCRpep-transduced DCs or respectivecontrols. Two days after the last injection, EAE was induced and the timecourse of the disease was determined using a score ranging from 1 (mildsymptoms) to 6 (death). (b) EAE was induced in mice. Five days later,TCRpep-transduced DCs or respective control DCs were injected three timesin a 4-d interval and mice were evaluated for EAE symptoms every 3 d. Themean score of groups of four mice is shown in this figure.1

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incubated for 10 min with a solution (0.1% wt/vol) of another contact sensti-tizer 2,4-Dinitrofluorobenzene (Sigma). After extensive washing, DCs wereinjected into the abdomen of Balb/c mice either before or after sensitization.For transcutaneous sensitization, 1% (w/v) TNCB dissolved in a 1:3 mixture ofacetone/olive oil (vol/vol) was applied onto shaved abdominal skin. After 6 d,mice were challenged by application of 0.5% (w/v) TNCB onto the left ear.Application of vehicle only served as control. Ear thickness was measured usinga caliper rule 24 h later. Results are displayed as mean difference of left versusright ear + s.d. In some experiments draining lymph nodes were removed forproliferation assays.

EAE model. For the EAE model, transgenic female TG4 mice (kindly providedby D. Wraith, University of Bristol, UK) were used6. These mice carry trans-genic T cells expressing a TCR that is specific for the Ac1–9 peptide of myelinbasic protein (MBP), an antigen present in myelinated neurons. In these miceEAE can easily be induced by injection of the specific T-cell antigen, MBP orspinal cord homogenate.

According to the experimental settings, TG4-derived DCs that had beentransduced with the TCRpep and respective controls were either injectedbefore or after induction of the EAE. To induce EAE, mice were injectedwith spinal cord homogenate suspended in complete Freund’s adjuvant(CFA; 150 mg/ml; Sigma). On the same day and 2 d later, mice were alsointraperitonally injected with 100 µl of pertussis toxin (Sigma, Germany).Mice were then kept under supervision and EAE symptoms ranging from mild(partial paralysis of the tail) to severe (complete paralysis of all feet and finallydeath) were recorded every 2 d.

ACKNOWLEDGMENTSWe thank Annekatrin Meinl for excellent technical assistance and K. Steinbrink,E. Schmitt and H. Jonuleit for helpful discussions. The work was supported byDeutsche Forschungsgemeinschaft grants EN 209/7-2 and SFB 548-A3 and aMAIFOR grant.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 16 February; accepted 9 May 2003Published online 29 June 2003; doi:10.1038/nbt842

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