The Tolerogen DNTB Into an Immunogen

IL-12 Breaks Dinitrothiocyanobenzene (DNTB)-Mediated Tolerance and Converts the Tolerogen DNTB into an Immunogen

This information is current as Helge Riemann, Karin Loser, Stefan Beissert, Mayumi of October 1, 2021. Fujita, Agatha Schwarz, Thomas Schwarz and Stephan Grabbe J Immunol 2005; 175:5866-5874; ; doi: 10.4049/jimmunol.175.9.5866 http://www.jimmunol.org/content/175/9/5866 Downloaded from

References This article cites 36 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/175/9/5866.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-12 Breaks Dinitrothiocyanobenzene (DNTB)-Mediated Tolerance and Converts the Tolerogen DNTB into an Immunogen1

Helge Riemann,2* Karin Loser,2† Stefan Beissert,† Mayumi Fujita,* Agatha Schwarz,‡ Thomas Schwarz,‡ and Stephan Grabbe3†§

Epicutaneous application of dinitrothiocyanobenzene (DNTB) induces tolerance against its related compound dinitrofluorobenzene (DNFB), because DNTB-pretreated mice cannot be sensitized against the potent hapten DNFB. This tolerance is hapten- specific and transferable. In this study, we demonstrate that IL-12 can break DNTB-mediated tolerance. Furthermore, naive mice treated with IL-12 before DNTB application responded to DNFB challenge with a pronounced ear swelling response without previous sensitization to DNFB, showing that IL-12 can convert the tolerogen DNTB into an immunogen. No differences in ,numbers or regulatory activity were observed between CD4؉CD25؉ regulatory T cells isolated from mice treated with DNFB Downloaded from DNTB, or IL-12 followed by DNTB. However, the number of CD207؉ Langerhans cells in regional lymph nodes of DNTB-treated (mice was significantly lower than in animals treated with DNFB or IL-12 plus DNTB. Additionally, CD11c؉ dendritic cells (DC isolated from regional lymph nodes of DNTB-treated mice had a significantly lower ability to stimulate T cell proliferation and produced reduced amounts of inflammatory cytokines. Application of both DNFB and DNTB induced apoptotic cell death of DC in the epidermis and the regional lymph nodes. However, the number of apoptotic DC in regional lymph nodes was significantly

higher in DNTB-treated animals compared with mice treated with DNFB or IL-12 plus DNTB. Therefore, we conclude that http://www.jimmunol.org/ DNTB-mediated tolerance is secondary to inefﬁcient Ag presentation as a result of apoptotic cell death of DC and that IL-12 converts the tolerogen DNTB into an immunogen by preventing DNTB-induced apoptosis of DC. The Journal of Immunology, 2005, 175: 5866–5874.

he heterodimeric cytokine IL-12 is composed of co- that have been exposed to UV radiation does not result in sensi- valently linked 35- and 40-kDa subunits (1). Besides tization but induces tolerance (11). This tolerance is hapten-spe- T stimulatory effects on both, NK cells and CD8ϩ cytotoxic cific and can be adoptively transferred by injecting bulk T cells T lymphocytes, IL-12 has costimulatory and regulatory effects on obtained from animals tolerized in this manner (12). We and others ϩ CD4 Th cells and favors the differentiation of Th1 cells (2–4). have reported that IL-12 is able to prevent UV-induced immuno- by guest on October 1, 2021 Although contact hypersensitivity (CHS)4 (4) is unaltered in IL- suppression when injected i.p. into mice between UV exposure and 12-deficient mice, exogenous IL-12 can enhance the CHS response hapten sensitization (7, 9, 10). In addition, tolerance did not de- ϩ by amplifying the development of hapten-specific CD8 T cells velop in these animals (9). Even more importantly, mice tolerized ϩ and by inhibiting the induction of Ag-specific CD4 regulatory by hapten application on UV-irradiated skin can be fully sensitized cells (5, 6). Furthermore, neutralization of IL-12 by injection of with the same hapten when IL-12 is injected before re-sensitiza- blocking Abs inhibits the induction of CHS in wild-type mice (7) tion, demonstrating that IL-12 cannot only prevent but also break and even induces hapten-specific tolerance (8). In contrast, admin- established UV-mediated tolerance (9, 10). Although IL-12 seems istration of exogenous IL-12 can overcome UV-induced immune to act on regulatory T cells (13), the detailed mechanism by which tolerance (9, 10). Epicutaneous application of haptens onto mice IL-12 breaks established tolerance remains to be determined. Epicutaneous application of dinitrothiocyanobenzene (DNTB) *Department of Dermatology, UCHSC at Fitzsimons, Aurora, CO 80010; †Ludwig has been reported to induce tolerance against its related compound Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of dinitrofluorobenzene (DNFB) because DNTB-pretreated mice can- ‡ Dermatology, University of Mu¨nster, Germany; Department of Dermatology, Uni- not be sensitized against the potent hapten DNFB (14, 15). Specific versity of Kiel, Germany; §Department of Dermatology, University of Essen, Ger- many immune tolerance is induced by topical application of DNTB 7 Received for publication January 1, 2005. Accepted for publication August 18, 2005. days before sensitization to DNFB. Tolerance can be abrogated by The costs of publication of this article were defrayed in part by the payment of page cyclophosphamide, indicating that DNTB-induced suppressor/reg- charges. This article must therefore be hereby marked advertisement in accordance ulatory T cells may be involved, and adoptive transfer studies with 18 U.S.C. Section 1734 solely to indicate this fact. showed that DNTB induces hapten-specific regulatory T cells (15). 1 This work was supported by grants from the German Research Foundation [DFG In addition, lymph node cells from DNTB-treated mice are defec- Schw 1177/1-1 (to A.S.), SFB 415-A16 (to T.S.), SFB 293-B1 and SFB492-B2 (to S.G.), DFG 1580/6-2 and SFB 293-B8 (to S.B.), and IZKF Lo2/065/04 (to K.L. and tive in proliferation and produce significantly lower amounts of S.B.)]. IL-1, IL-2, and IL-4 compared with DNFB-treated animals, sug- 2 H.R. and K.L. contributed equally to this study. gesting that DNTB-induced tolerance results in deficient Th1 as 3 Address correspondence and reprint requests to Dr. Stephan Grabbe, Department of well as deficient Th2 responses to hapten re-exposure (16). Dermatology, University of Essen, Hufelandstrasse 55, D-45122 Essen, Germany. Here, we show that injection of IL-12 into DNTB-treated mice E-mail address: [email protected]. before sensitization with DNFB restores CHS responses and that 4 Abbreviations used in this paper: CHS, contact hypersensitivity; CBA, cytometric bead array; DC, dendritic cell; DNFB, 2,4-dinitrofluorobenzene; DNTB, 2,4,-dinitro- injection of IL-12 before DNTB treatment enables subsequent sen- thiocyanobenzene; LC, Langerhans cell. sitization against DNFB. In addition, when naive mice are injected

with IL-12 followed by DNTB treatment, primary contact with PC61), anti-CTLA-4 (clone UC10-4F10-11), anti-CD86 (clone GL1), peri- DNFB results in a specific ear swelling response, indicating that dinin chlorophyll protein-conjugated anti-CD3 (clone 145-2C11), anti- IL-12 converts the tolerogen DNTB into an immunogen. Further- CD4 (clone RM4-5), allophycocyanin-conjugated anti-CD4 (clone RM4- 5), anti-CD25 (clone PC61), anti-CD11c (clone HL3), Cy5-conjugated more, we show that treatment with DNTB leads to increased num- anti-CD207 (Langerin, clone 929F3; kindly provided by Dr. S. Saeland, bers of apoptotic dendritic cells (DC) both in epidermis and re- Schering-Plough, Dardilly, France). CD207 staining was performed after gional lymph nodes and that this effect can be mitigated by IL-12. cell permeabilization. Isotype-matched control Abs were included in each staining. All Abs as well as isotype-matched controls were obtained from BD Pharmingen unless otherwise noted. Apoptotic and necrotic cells were Materials and Methods identified using the Annexin V apoptosis detection kit (BD Pharmingen) Mice according to the manufacturer’s instructions. BALB/c mice between 8 and 12 wk of age were purchased from Charles Proliferation assays River Laboratories. Animals were housed under specific pathogen-free conditions and treated according to institutional guidelines. Naive CD4ϩCD25Ϫ and CD4ϩCD25ϩ cells were sorted by MACS as described (18). Proliferation assays were performed in triplicate, and T cell Contact hypersensitivity proliferation was assessed by [3H]thymidine incorporation. CD4ϩCD25Ϫ ϩ ϩ ϫ 6 Mice were sensitized by painting 25 ␮l of 2,4-dinitrofluorobenzene solu- and CD4 CD25 T cells (1 10 /ml alone or mixed at indicated ratios) tion (Sigma-Aldrich; 0.5% in acetone/olive oil 4:1) on the shaved back on were cultured in 96-well round-bottom plates, and cells were stimulated ␮ with 1 ␮g/ml anti-CD3 (clone 145-2C11) and 1 ␮g/ml anti-CD28 (clone day 0. On day 5, the left ear was challenged by applying 20 l of 0.3% ␮ DNFB, and the right ear was treated with acetone/olive oil alone. Ear 37.51). Proliferation assays were done for 96 h in a final volume of 200 l; ␮ 3 swelling was measured in a blinded fashion with a spring-loaded micro- 1 Ci/well [ H]thymidine was added for the last 12 h of the experiment, meter (Mitutoyo) 24 h after challenge. Contact hypersensitivity was deter- and thymidine incorporation was measured by liquid scintillation counting. mined as the amount of swelling of the hapten-challenged ear compared Mixed lymphocyte reactions Downloaded from with the thickness of the vehicle-treated ear and was expressed in centimeters ϫ 10Ϫ3 (mean Ϯ SD). Mice that were ear challenged without pre- Proliferation of CD4ϩCD25Ϫ T cells isolated from lymph nodes of naive vious sensitization served as negative controls. Sensitization against ox- BALB/c mice was assessed by [3H]thymidine incorporation. Cells (1 ϫ azolone was performed by applying 100 ␮l of a 2% oxazolone solution on 106/ml) were cultured in triplicate in 96-well round-bottom plates in the day 0, and ears were challenged using 20 ␮l of 0.5% oxazolone on day 5. presence of 1 ϫ 104 DC isolated from lymph nodes of naive BALB/c mice Each group consisted of at least 5 mice, experiments were performed at or mice treated with DNFB, DNTB, or IL-12 followed by DNTB. Mixed ␮

least three times. lymphocyte reactions were done for 72 h in a ﬁnal volume of 200 l. T cell http://www.jimmunol.org/ proliferation was evaluated by adding 1 ␮Ci/well [3H]thymidine for the DNTB treatment last 12 h of the experiment, and thymidine incorporation was measured by Mice were painted with 100 ␮l of DNTB (Lancaster Synthesis) solution liquid scintillation counting. (1% in acetone/olive oil 4:1) on the shaved abdomen. On day 7, mice were Cytometric bead array (CBA) sensitized with 0.5% DNFB, and DNFB challenge was performed 5 days later. The cytokine activity in culture supernatants of CD11cϩ cells from skin draining lymph nodes of DNFB-sensitized, DNTB-sensitized, and IL-12- Injection of rIL-12 treated, and DNTB-sensitized mice was assayed by CBA (BD Pharmingen) ϫ 6 For in vivo injection, 250 ng of recombinant murine IL-12 (Genetics In- according to the manufacturer’s instructions. Cells (2 10 /ml) were incubated for 3 days without any further stimulation at 37°C and 5% CO in

2 by guest on October 1, 2021 stitute) diluted in sterile endotoxin-free saline (PAA) were used. The cy- ␮ tokine was injected i.p. 24 and 3 h before treatment with DNTB or DNFB. 96-well round-bottom plates (BD Falcon) in a volume of 200 l of RPMI Control mice were treated i.p. with equal volumes of saline, which had no 1640 containing 10% FCS. Supernatants were collected and subjected to effect on the outcome of the sensitization procedure or on the suppressive cytokine quantification using CBA kits. effect of DNTB. Statistical analysis Adoptive transfer of immune response Data were analyzed by Student’s t test and differences were considered Ͻ Donor mice were treated with DNTB, draining lymph nodes and spleens significant at p 0.05. Each experiment was performed at least three were removed 7 days later, and single cell suspensions were prepared. Cell times. number was adjusted to 5 ϫ 108 cells/ml, and 200 ␮l(1ϫ 108 cells) were injected i.v. into naive recipients. Recipients were sensitized 24 h after Results injection by painting 0.5% DNFB on the shaved back. Recipient mice were Induction of hapten-specific tolerance against DNFB by challenged on the left ear 5 days later, and ear swelling was evaluated. epicutaneous application of DNTB Immunofluorescence and in situ TUNEL analysis DNTB (1%) was painted on the shaved abdomen of BALB/c mice. Immunofluorescence stainings were performed on cryostat sections of A sensitizing dose of DNFB was applied 7 days later epicutane- mouse ears according to standard methods (17). Epidermal Langerhans ously on the shaved back and challenge with DNFB was per- cells (LC) were identified using anti-CD207 (clone 929F3, diluted 1/50 in formed 5 days thereafter. Although animals sensitized with DNFB PBS; kindly provided by Dr. S. Saeland, Schering-Plough, Dardilly, showed a significant ear swelling response upon challenge, ani- France) and OregonGreen-coupled secondary Ab (Molecular Probes). Ap- optosis of Langerhans cells was detected using TUNEL staining and the mals pretreated with DNTB could not be sensitized with DNFB, as Texas Red in situ cell death detection kit (Roche) according to the man- indicated by the absent ear swelling response. Likewise, animals ufacturer’s instructions. Slides were examined using an Olympus BX61 challenged with DNFB without prior sensitization showed no ear microscope and the MetaMorph software (Visitron Systems). swelling response (Fig. 1A). The tolerizing effect of DNTB ap- Cell preparation and flow cytometry peared to be dose dependent, because both lower and higher concentrations of DNTB failed to induce tolerance (data not shown). Single cell suspensions of regional lymph nodes were prepared as described before (18). DC were isolated from lymph node cell suspensions by DNTB-mediated tolerance against DNFB is transferable MACS (Miltenyi Biotec) using anti-CD11c-coupled microbeads according to the manufacturer’s instructions. Expression of cell surface and intracel- Next we analyzed whether DNTB-mediated tolerance was trans- lular markers was analyzed by standard four-color flow cytometry with a ferable. Therefore, 1% DNTB was painted on the shaved abdomen FACSCalibur flow cytometer and CellQuest software (BD Pharmingen). of BALB/c mice. Animals were sacrificed 7 days later, and re- Cells were stained for FACS analysis in PBS containing 1% FCS with the following mAbs: anti-neuropilin-1 (clone H-286; Santa Cruz Biotechnol- gional lymph nodes and spleens were removed. Single cell sus- ogy), FITC-conjugated anti-CD103 (clone 2E7), anti-CD80 (clone 16- pensions were prepared as described and injected i.v. into naive 10A1), goat anti-rabbit Ig (Dianova), PE-conjugated anti-CD25 (clone recipient BALB/c. Recipients were sensitized with DNFB 1 day 5868 Conversion of a Tolerogen into an Immunogen by IL-12

challenge with DNFB, no ear swelling response was observed in recipients of cells from DNTB-treated donors, indicating that these animals could not be sensitized against DNFB (Fig. 1B).

DNTB-mediated tolerance is hapten-speciﬁc To exclude that DNTB induces tolerance against an unrelated hapten, mice were treated by epicutaneous application of DNTB onto the shaved abdomen. Sensitization with oxazolone was performed on the shaved back 7 days later, and ear challenge with oxazolone was performed 5 days later. Upon challenge, mice pretreated with DNTB and sensitized with oxazolone had ear swelling reponses similar to positive control animals sensitized with oxazolone alone, showing that DNTB-mediated tolerance is speciﬁc for DNFB (Fig. 1C).

IL-12 prevents DNTB-mediated tolerance To evaluate whether IL-12 can prevent DNTB-mediated tolerance, IL-12 was injected 24 and 3 h before DNTB application. Seven

days later, sensitization with DNFB was performed, and ears were Downloaded from challenged with DNFB 5 days thereafter. CHS response in DNTB- pretreated mice was signiﬁcantly suppressed compared with positive control animals. In contrast, when IL-12 was injected 24 and 3 h before DNTB application, the tolerizing effect of DNTB was not observed because animals responded with a marked ear swell- A ing response to sensitization with DNFB (Fig. 2 ). http://www.jimmunol.org/

IL-12 breaks DNTB-mediated tolerance To study whether IL-12 can break established DNTB-mediated tolerance, BALB/c mice were treated by epicutaneous application of DNTB. Animals were sensitized with 0.5% DNFB 7 days after application of DNTB, and challenge with DNFB was performed 5 days later. One group of BALB/c mice received i.p. injections of IL-12, 24 and 3 h before sensitization with DNFB. Control animals sensitized with DNFB showed a signiﬁcant ear swelling response by guest on October 1, 2021 upon challenge, whereas animals pretreated with DNTB could not be sensitized against DNFB. However, when IL-12 was injected into DNTB-pretreated mice before application of DNFB, a significant CHS response was observed (Fig. 2B).

IL-12 converts the tolerogen DNTB into an immunogen To study whether injection of IL-12 before application of DNTB enables a CHS response to DNFB challenge without prior DNFB FIGURE 1. A, DNTB induces tolerance against DNFB. Mice were sensitization, mice received IL-12 i.p. 24 and 3 h before DNTB tolerized by epicutaneous application of 100 ␮l of 1% DNTB on the ab- application. Challenge with DNFB was performed 7 days later. ␮ domen, followed by sensitization against DNFB (25 l of 0.5% painted on Although animals treated with DNTB alone showed no ear swell- ␮ the back) 7 days later. Ears were challenged with DNFB (20 l 0.3%) 5 ing response upon challenge with DNFB, mice treated with the days after sensitization with DNFB. B, DNTB-mediated tolerance is trans- combination of IL-12 injection followed by DNTB application ferable. Donor mice were painted with DNTB or left untreated, and single cell suspensions were prepared from draining lymph nodes and spleens 7 showed a significant CHS response upon challenge with DNFB days later. Cells (1 ϫ 108) were injected i.v. into naive recipients followed (Fig. 3). This ear swelling response appeared hapten-specific, be- by DNFB sensitization 1 day after transfer. Recipient mice were ear chal- cause the combination of IL-12 injection followed by DNTB ap- lenged with DNFB 5 days after sensitization. C, DNTB-induced tolerance plication did not allow for a CHS response upon challenge with the is hapten-specific; IL-12 followed by DNTB does not allow for an un- unrelated hapten oxazolone (Fig. 1C). specifc challenge. Mice were treated with DNTB, and a sensitizing dose of oxazolone was applied 7 days later. One group of mice was injected with IL-12 Concomitant application of DNTB and an irrelevant hapten followed by DNTB application but not sensitized against oxazolone. All mice does not reconstitute DNFB sensitization were challenged with oxazolone 5 days after sensitization with oxazolone. Ear swelling response is expressed as the difference (centimeters ϫ 10Ϫ3, mean Ϯ Because DNFB sensitizes whereas DNTB induces tolerance against the same hapten, we reasoned that DNTB might fail to ,ء .SD) between the thickness of the challenged ear and the vehicle-treated ear p Ͻ 0.001 versus positive control; n.s., nonsignificant. generate the immunostimulatory signal(s) necessary for induction of CHS. To substitute for these signal(s), animals were sensitized by epicutaneous application of DNTB in conjunction with the non- after transfer, and challenge was performed with DNFB 5 days related hapten oxazolone in the conventional sensitizing dose onto after sensitization. Whereas control animals that had received cells the same area. Ears were challenged with DNFB 5 days later. from untreated donors showed a significant CHS response upon Upon challenge with DNFB, no CHS response was observed in The Journal of Immunology 5869

FIGURE 3. IL-12 converts the tolerogen DNTB into an immunogen. Mice were treated with DNTB, and ear challenge with DNFB was performed 5 days later. One group received IL-12, 24 and 3 h before DNTB application. As a positive control, mice were sensitized against DNFB. All groups were ear challenged with DNFB. Ear swelling response is expressed as the difference (centimeters ϫ 10Ϫ3, mean Ϯ SD) between the thickness p Ͻ 0.005. Downloaded from ,ء .of the challenged ear and the vehicle-treated ear

sitized mice were ear challenged with either DNTB alone or with DNTB in combination with a conventional challenging dose of the irrelevant hapten oxazolone. Although DNFB-sensitized animals challenged with DNTB alone failed to develop a CHS response, a

signiﬁcant ear swelling could be observed in animals simulta- http://www.jimmunol.org/ neously challenged with DNTB plus oxazolone. Moreover, the ear swelling response was clearly dependent on the dose of DNTB FIGURE 2. A, IL-12 prevents DNTB-mediated tolerance toward applied (Fig. 4, lines 11–13).

DNFB. Mice were treated with DNTB on the shaved abdomen and sensi- ϩ ϩ tized against DNFB on the back 7 days later. All groups were ear chal- IL-12 does not affect the induction of CD25 CD4 regulatory T lenged with DNFB 5 days thereafter. One group received IL-12 injected cells i.p. 24 and 3 h before DNTB application. B, IL-12 breaks DNTB-mediated We next investigated the mechanism by which DNTB might exert tolerance. Mice were treated with DNTB on the shaved abdomen and sen- its tolerogenic effects during primary hapten exposure. As shown sitized against DNFB on the back 7 days later. All groups were ear chal- ϩ ϩ by guest on October 1, 2021 previously, CD4 CD25 regulatory T cells can control CHS re- lenged with DNFB 5 days later. One group received IL-12 injected i.p. 24 ϩ ϩ and 3 h before DNFB sensitization. Ear swelling response is expressed as sponses (20). To study the role of CD4 CD25 regulatory T cells the difference (centimeters ϫ 10Ϫ3, mean Ϯ SD) between the thickness of in DNTB-induced tolerance, mice were left untreated or were p Ͻ 0.05, treated with DNFB, DNTB, or IL-12 plus DNTB. Lymph nodes ,ءء ;p Ͻ 0.01 ,ء .the challenged ear and the vehicle-treated ear n.s., nonsignificant. and spleens were removed 7 days later, and single cell suspensions were prepared. CD4ϩCD25Ϫ and CD4ϩCD25ϩ cells were separated. CD4ϩCD25Ϫ T cells from naive mice were stimulated with animals sensitized with DNTB alone or in combination with ox- anti-CD3 and anti-CD28 Ab in the absence or presence of azolone (Fig. 4, lines 3 and 4). Thus, unlike IL-12 treatment, con- CD4ϩCD25ϩ T cells from animals treated with DNFB, DNTB, or comitant application of an immunologically unrelated hapten IL-12 plus DNTB. Proliferation was measured by the amount of could not convert the tolerogen DNTB into an immunogen, sug- [3H]thymidine incorporation (Fig. 5A). Whereas CD4ϩCD25Ϫ gesting that the tolerogenic effects of DNTB are not due to the lack cells from naive mice alone showed vigorous proliferation, prolif- of an immunostimulatory effect. eration was significantly inhibited by addition of CD4ϩCD25ϩ cells from mice treated with DNFB, DNTB, or IL-12 plus DNTB. DNTB fails to elicit a CHS response in DNFB-sensitized CD4ϩCD25ϩ cells from different groups exhibited similar inhib- animals, but CHS reactivity is restored by simultaneous itory capacities. Flow cytometric analyses of CD4ϩ T cells iso- application of an irrelevant hapten lated from lymph nodes of naive, DNFB-sensitized, DNTB- It has been shown earlier that an inherent feature of contact aller- treated, and IL-12 plus DNTB-treated mice showed no significant ϩ ϩ gens is their capacity to induce a nonspecific inflammatory re- differences in the numbers of CD4 CD25 T cells (Fig. 5B). sponse necessary for elicitation of CHS (19). Thus, to investigate Moreover, the expression of surface markers typically associated whether DNTB, in addition to its tolerogenic effects during hapten with regulatory T cells like CTLA-4, CD103, or neuropilin-1 was sensitization, might also have an altered capacity to elicit CHS not changed (data not shown). These findings suggest that the gen- ϩ ϩ responses in DNFB-sensitized mice, animals were DNFB sensi- eration of CD4 CD25 cells is not the primary mechanism of ϩ ϩ tized and subsequently challenged with DNTB. Indeed, application tolerance induction by DNTB and that generation of CD4 CD25 of DNTB also failed to elicit a CHS response in DNFB-sensitized T cells is not affected by IL-12. mice, suggesting that it not only lacks sensitizing capacity but also signal(s) necessary for elicitation of a CHS response (Fig. 4, lines DC from DNTB-treated animals fail to prime naive T cells 8–10). Mice were treated with DNFB, DNTB, or IL-12 plus DNTB, and We were interested whether concomitant application of a non- DC were isolated from regional lymph nodes. DC were coincu- related hapten could substitute for these signals. Thus, DNFB-sen- bated with CD4ϩCD25Ϫ T cells from naive BALB/c mice for 3 5870 Conversion of a Tolerogen into an Immunogen by IL-12 Downloaded from

FIGURE 4. The combination of DNTB and oxazolone fails to induce

CHS against DNFB but induces ear challenge in DNFB-sensitized mice. http://www.jimmunol.org/ Mice were sensitized by epicutaneous application of DNFB, or by application of DNTB in combination with oxazolone (2%). Ear challenge was performed 5 days later by applying either DNFB (0.3%), DNTB (0.5, 1, or FIGURE 5. A, CD4ϩCD25ϩ T cells are suppressive in vitro. 2%), or DNTB (0.5, 1, or 2%) in combination with oxazolone (0.5%). Ear CD4ϩCD25Ϫ and CD4ϩCD25ϩ T cells were separated by MACS, and swelling response was measured 24 h later and is expressed as the differ- proliferation assays were perfomed by stimulating CD4ϩCD25Ϫ T cells Ϫ3 ence (centimeters ϫ 10 , mean Ϯ SD) between the thickness of the chal- from naive BALB/c mice (1 ϫ 105) with anti-CD3 and anti-CD28 Ab in -p Ͻ 0.005 versus line 1, n.s., the absence or presence of 7.5 ϫ 104 CD4ϩCD25ϩ T cells from DNFB ,ء .lenged ear and the vehicle-treated ear nonsignificant versus line 1; #, nonsignificant versus line 5. sensitized, DNTB-sensitized, or IL-12-treated and DNTB-sensitized mice. Ͻ ϩ Ϫ ϩ ء , p 0.05 versus CD4 CD25 . B, Flow cytometric analyses of CD4 by guest on October 1, 2021 lymph node T cells from naive BALB/c, DNFB-sensitized, DNTB-sensi- days, and T cell proliferation was measured. Whereas T cells in- tized, and IL-12-treated plus DNTB-sensitized mice. cubated with DC from DNFB-treated animals proliferated vigor- ously, T cells incubated with DC from DNTB-treated mice showed only a weak proliferative response, suggesting that DNTB fails to Staining with propidium iodide and annexin V showed that the activate DC for efficient priming of naive T cells. This effect could number of apoptotic CD11cϩ cells was increased in lymph nodes partially be reversed by treatment with IL-12 (Fig. 6A). from DNTB-sensitized mice compared with control, DNFB, and IL-12 plus DNTB-treated animals (Fig. 7A). To evaluate whether IL-12 prevents DNTB-induced apoptosis of DC in epidermis and DNTB induces apoptosis of DC not only in regional lymph nodes regional lymph nodes but also in the skin, mice were treated with DNFB, DNTB, or Mice were treated with DNFB, DNTB, or IL-12 plus DNTB, and IL-12 plus DNTB. One day later, skin samples were taken from the single cell suspensions were prepared from regional lymph nodes area of DNTB, DNFB, or vehicle application. Biopsies were 3 days later. Lymph node cells were stained for CD11c and the stained for Langerin and for apoptotic cells using the TUNEL as- Langerhans cell-specific intracellular marker Langerin (CD207) say. Although application of DNFB slightly increased the number and analyzed by flow cytometry. Although the number of CD11cϩ of apoptotic cells compared with vehicle-treated skin, the total cells was slightly increased, the number of Langerinϩ cells was number of apoptotic cells and especially of apoptotic Langerhans considerably reduced in the skin draining lymph nodes of DNTB- cells was significantly higher after DNTB treatment. However, in- treated compared with DNFB-treated mice (Fig. 6B). Injection of jection of IL-12 before DNTB application reduced the number of IL-12 into mice before application of DNTB raised the number of apoptotic cells to background levels (Fig. 7B). Therefore, induc- ϩ ϩ Langerinϩ lymph node cells to an amount similar to that observed tion of apoptosis in Langerin CD11c DC correlates with toler- in DNFB-treated animals (Fig. 6B). Interestingly, CD11cϩ cells ance induction in the DNFB/DNTB model, and IL-12 can prevent isolated from skin draining lymph nodes of DNTB-sensitized mice DNTB-induced apoptosis. were less activated than DC isolated from the lymph nodes of DNFB-sensitized or IL-12 plus DNTB-treated mice as demon- Discussion strated by the reduced expression of CD80 and CD86 (Fig. 6C). DNTB has initially been described to induce tolerance to subse- Moreover, lymph node DC from DNTB-sensitized mice produced quent treatment with DNFB (14). Because later reports could not reduced amounts of inflammatory cytokines like TNF-␣ and IFN-␥ confirm the tolerizing capacity of DNTB (21, 22), we first deter- compared with CD11cϩ cells isolated from skin draining lymph mined whether DNTB in our hands acts as a tolerogen. DNTB at nodes of DNFB-sensitized or IL-12 plus DNTB-treated mice a concentration of 1% applied to the abdomen clearly suppressed (Fig. 6D). subsequent sensitization with DNFB, whereas lower and higher The Journal of Immunology 5871 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. Functional and flow cytometric analysis of regional lymph node cells. A,DC(5ϫ 104) were isolated from skin draining lymph nodes of DNFB-sensitized, DNTB-sensitized, or IL-12-treated plus DNTB-sensitized mice. DC were incubated for 3 days with 1 ϫ 105 CD4ϩ T cells from naive ,p Ͻ 0.05 versus DNTB treatment. B, Lymph node cells from mice treated with DNFB, DNTB ,ء .BALB/c mice, and proliferation assays were performed and IL-12 plus DNTB were stained for CD11c or Langerin (CD207) and analyzed by flow cytometry. CD207 staining was performed after cell perme- .p Ͻ 0.05 versus DNFB and IL-12 plus DNTB ,ء .abilization. Percentage of positive cells in total lymph nodes from five independent experiments are shown C, Lymph node cells from DNFB-sensitized, DNTB-sensitized mice, or IL-12 plus DNTB-treated mice were stained for CD80 and CD86 and analyzed by p Ͻ ,ء .flow cytometry. Cells were gated for CD11cϩ and percentage of CD80ϩCD86ϩ cells in lymph nodes from five independent experiments are shown 0.05 versus DNFB and IL-12 plus DNTB. D, Cytokine production of CD11cϩ DC isolated from skin draining lymph nodes of DNFB-sensitized, DNTB-sensitized, and IL-12 plus DNTB-treated mice was measured by CBA. Data are shown as one of three different experiments with similar results. concentrations failed to exert this effect (data not shown). There- because animals that received IL-12 before DNTB application fore, we speculate that the different properties attributed to DNTB could be sensitized to DNFB. Furthermore, although animals are due to different concentrations used and different sites of ap- treated with DNTB alone did not react with ear swelling to DNFB plication (ear vs abdomen). We were able to further consolidate challenge, mice which received IL-12 before DNTB application this tolerance model by confirming previous reports that DNTB- showed a significant ear swelling response following DNFB ap- mediated tolerance is transferable and that this is due to the de- plication on the ear. Because in these mice, ear challenge with velopment of hapten-specific regulatory/suppressor cells (15). DNFB represents the primary contact with DNFB in a concentra- Once we had established the model of DNTB-mediated toler- tion that does not cause ear swelling by itself as shown by the ance we evaluated the effects of IL-12 at different time points, i.e., negative controls, the combination of IL-12 plus DNTB obviously after and before tolerance had developed. IL-12 injected after sensitizes the animals against DNFB. DNTB treatment and just before DNFB application clearly enabled Our data suggest that IL-12 specifically prevents immune toler- DNFB sensitization, indicating that IL-12 can break DNTB-medi- ance, but does not enhance immunity in a general fashion, because ated tolerance. It is important to mention that IL-12 was injected at injection of IL-12 before DNTB application induced CHS against a time point when regulatory T cells had already developed as DNFB, whereas IL-12 in the amounts used throughout this study demonstrated by adoptive transfer of suppression. These findings did not further enhance the induction of CHS if injected before are similar to those observed in UV-mediated tolerance (9, 13). We sensitization with DNFB (data not shown). Although this is similar also found that IL-12 is able to prevent DNTB-mediated tolerance, to results we previously obtained in a different mouse strain (9), 5872 Conversion of a Tolerogen into an Immunogen by IL-12 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 7. Detection of apoptotic CD11cϩ and Langerhans cells in skin draining lymph nodes and in the skin of sensitized mice. A, Mice were treated with DNFB, DNTB, or IL-12 plus DNTB. Lymph node cells were prepared 3 days later, stained for annexin V and propidium iodide (PI), and subjected .to flow cytometry analysis. Cells were gated for CD11c؉. Statistical evaluation of apoptotic lymph node DC from five independent experiments is shown pϽ0.05. B, Immunofluorescence analysis of (i) BALB/c mice as well as (ii) DNFB sensitized, (iii) DNTB-sensitized, or (iv) IL-12 plus DNTB-treated ,ء mice. One day after sensitization cryosections of treated skin areas were stained for CD207 (green) and for apoptotic cells (TUNEL assay, red). Apoptotic Langerhans cells are marked with white arrows. Original magnification, ϫ400. Statistical evaluation of apoptotic Langerhans cells from five independent experiments is shown. In each experiment, numbers of apoptotic LC were determined by two independent investigators in a blinded fashion from 15 .pϽ0.05 ,ء .randomly selected slide sections

IL-12 applied before sensitization did exaggerate CHS responses poorly understood. Enk and Katz (24) showed that tolerizers in in other studies, albeit in significantly higher doses (5, 6). contrast to immunogens do not induce IL-1, MIP-2, IL-10, or class In the model of UV-induced tolerance, the immune suppression II mRNA. Because we demonstrated that IL-12 can prevent seems to be induced by the release of keratinocyte-derived cyto- DNTB-mediated tolerance we hypothesized that DNTB might fail kines, especially IL-10 (23). However, the mechanisms by which to induce IL-12 and thereby induces hapten-specific tolerance. In chemical tolerogens, including DNTB, exert their effects, are the human system, induction of IL-12 has been observed upon The Journal of Immunology 5873 application of haptens (25). However, we were not able to identify by IL-12 (36). Thus, similar mechanisms are responsible for both either p35 or p40 transcripts in murine skin after hapten applica- UV-mediated and hapten-induced tolerance. tion (data not shown). Because IL-12 can prevent DNTB-mediated In addition to its tolerogenic effects during hapten sensitization, tolerance, we studied whether DNTB fails to induce IL-12 in the DNTB also failed to elicit a CHS response even in mice that had regional lymph nodes and thereby induces tolerance. Both p35 and been sensitized to the relevant hapten by using DNFB. This effect p40 transcripts could be detected in regional lymph nodes of both is due to a different mechanism and cannot be explained by in- DNFB- and DNTB-treated animals (data not shown). Thus, there duction of LC apoptosis. We have previously shown that for the is little evidence that a failure to induce IL-12 might account for elicitation of hapten-specific CHS responses, two signals are re- the differences between DNTB and DNFB. quired: 1) the specific Ag, and 2) a nonspecific proinflammatory CD4ϩCD25ϩ regulatory T cells provide an important pathway signal (19). This conclusion was based on the observation that for immune tolerance as shown in different models (20, 26–29). coadministration of an irrelevant hapten with a low dose of the CD4ϩCD25ϩ T cells are classically anergic, unable to proliferate specific hapten in sensitized mice yielded a full CHS response, in vitro in response to mitogenic stimulation or stimulation through whereas application of either substances alone, or the combination the TCR, and inhibit proliferation of CD4ϩCD25Ϫ T cells by in- of haptens applied to nonsensitized animals, failed to elicit a sig- hibiting production of IL-2 (30). It was tempting to hypothesize nificant reaction. Although the nature of the proinflammatory sig- that CD4ϩCD25ϩ T cells have a crucial function in DNTB-me- nal remains to be characterized, there is strong evidence that this diated tolerance and that IL-12 either interferes with their function signal is specifically provided by haptens because it could not be or impairs their development. However, we were not able to detect substituted by application of irritants (19). To determine whether ϩ ϩ DNTB differs from haptens in its ability to provide this second any differences in the suppressive activity of CD4 CD25 regu- Downloaded from latory T cells in mice treated with DNFB, DNTB, or IL-12 plus signal, mice were either treated by epicutaneous application of DNTB. Furthermore, these CD4ϩCD25ϩ regulatory T cells did DNTB alone or in combination with oxazolone in the induction or not differ in their expression of surface markers like CTLA-4, effector phase. While in the effector phase a vigorous ear swelling CD103, or neuropilin-1 (data not shown). It therefore seems un- response could be observed in DNFB-sensitized animals chal- likely that modulation of development or function of CD4ϩCD25ϩ lenged with the combination of DNTB and oxazolone, no sensitization against DNFB was observed in animals treated with the

T cells is the main mechanism of IL-12 in reversing DNTB-me- http://www.jimmunol.org/ combination of DNTB and oxazolone in the induction phase of diated tolerance. CHS (Fig. 4). These findings suggest that an absent irritative effect Instead, however, a major reduction in the relative numbers of ϩ ϩ of DNTB could be responsible for the inability of DNTB to elicit CD11c Langerin DC in regional lymph nodes was observed a CHS response in DNFB-sensitized animals. In contrast, applica- after application of DNTB when compared with DNFB. Interest- tion of an irrelevant hapten during sensitization did not restore the ingly, injection of IL-12 before DNTB application could raise the ϩ ϩ sensitizing potential of DNTB. number of CD11c Langerin cells to levels in the range observed In summary, the present study shows that DNTB acts as a after DNFB treatment. Initially, we speculated that the reduced tolerogen upon primary hapten exposure and also fails to elicit number of CD11cϩ Langerinϩ cells after DNTB treatment could CHS responses during secondary hapten application. IL-12 can be due to the inability of DNTB to sufficiently activate Langerhans by guest on October 1, 2021 both prevent and overcome DNTB-mediated tolerance and even cells for migration to regional lymph nodes. The hapten then convert the tolerogen DNTB into an immunogen. Our data also would be presented less efficiently, which might lead to tolerance link the tolerogenic effect of DNTB to induction of apoptosis in LC (11, 31). If DNTB failed to activate Langerhans cells to migrate to and demonstrate an anti-apoptotic effect of IL-12 on LC. the lymph nodes, one would expect that simultaneous application of the unrelated hapten oxazolone would substitute for this defi- Acknowledgments ciency as this potent allergen is obviously able to activate DC for The excellent technical assistance of Joachim Windau and Maik Voskort is migration and efficient Ag presentation. However, concomitant ap- gratefully acknowledged. plication of oxazolone and DNTB did not lead to induction of CHS. We then analyzed the possibility that DNTB application in- Disclosures duces LC damage or even death, thereby decreasing the number of The authors have no financial conflict of interest. cells that reached the regional lymph node. Indeed, we found that DNTB consistently induced enhanced apoptotic cell death of DC References both in epidermis and regional lymph nodes. This toxic, apoptosis- 1. Chehimi, J., and G. Trinchieri. 1994. Interleukin-12: a bridge between innate inducing effect of DNTB on APCs is able to explain the dose resistance and adaptive immunity with a role in infection and acquired immuno- dependency we observed for the tolerogenic effects of DNTB. deficiency. J. Clin. Immunol. 14: 149–161. 2. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, Thus, at low concentrations, the damaging effect of DNTB on DC F. Sherman, B. Perussia, and G. Trinchieri. 1989. Identification and purification is absent, but the amount of Ag is too small to induce CHS. At high of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170: 827–845. concentrations, apoptotic death of DC will be so extensive that 3. Stern, A. S., F. J. Podlaski, J. D. Hulmes, Y. C. Pan, P. M. Quinn, A. G. Wolitzky, sufficient numbers of viable DC do not reach the regional lymph P. C. Familletti, D. L. Stremlo, T. Truitt, and R. Chizzonite. 1990. Purification to nodes and thus fail to prime for tolerance. In our study, adminis- homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 87: tration of IL-12 was able to prevent DNTB-induced cell death in 6808–6812. vivo. It has recently been shown that IL-12 has anti-apoptotic 4. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol. 3: 133–146. properties, because it is able to protect keratinocytes from UV- 5. Gorbachev, A. V., N. A. DiIulio, and R. L. Fairchild. 2001. IL-12 augments induced apoptosis by inducing DNA repair (32) and to prevent CD8ϩ T cell development for contact hypersensitivity responses and circumvents anti-CD154 antibody-mediated inhibition. J. Immunol. 167: 156–162. activation-induced cell death in T cells by down-regulating Fas ϩ 6. DiIulio, N. A., H. Xu, and R. L. Fairchild. 1996. Diversion of CD4 T cell ligand and inhibiting caspase activation (33–35). Now, our find- development from regulatory T helper to effector T helper cells alters the contact ings indicate that IL-12 also protects LC against hapten-mediated hypersensitivity response. Eur. J. Immunol. 26: 2606–2612. 7. Muller, G., J. Saloga, T. Germann, G. Schuler, J. Knop, and A. H. Enk. 1995. apoptosis. Likewise, we demonstrated earlier that UV-mediated IL-12 as mediator and adjuvant for the induction of contact sensitivity in vivo. tolerance is also dependent on apoptosis of DC and can be reversed J. Immunol. 155: 4661–4668. 5874 Conversion of a Tolerogen into an Immunogen by IL-12

8. Riemann, H., A. Schwarz, S. Grabbe, Y. Aragane, T. A. Luger, M. Wysocka, 22. Dearman, R. J., M. Cumberbatch, J. Hilton, I. Fielding, D. A. Basketter, and M. Kubin, G. Trinchieri, and T. Schwarz. 1996. Neutralization of IL-12 in vivo I. Kimber. 1997. A re-appraisal of the skin-sensitizing activity of 2,4-dinitrothio- prevents induction of contact hypersensitivity and induces hapten-specific toler- cyanobenzene. Food Chem. Toxicol. 35: 261–269. ance. J. Immunol. 156: 1799–1803. 23. Rivas, J. M., and S. E. Ullrich. 1992. Systemic suppression of delayed-type hy- 9. Schwarz, A., S. Grabbe, Y. Aragane, K. Sandkuhl, H. Riemann, T. A. Luger, persensitivity by supernatants from UV-irradiated keratinocytes. An essential role M. Kubin, G. Trinchieri, and T. Schwarz. 1996. Interleukin-12 prevents ultravi- for keratinocyte-derived IL-10. J. Immunol. 149: 3865–3871. olet B-induced local immunosuppression and overcomes UVB-induced tolerance. 24. Enk, A. H., and S. I. Katz. 1992. Early molecular events in the induction phase J. Invest. Dermatol. 106: 1187–1191. of contact sensitivity. Proc. Natl. Acad. Sci. USA 89: 1398–1402. 10. Schmitt, D. A., L. Owen-Schaub, and S. E. Ullrich. 1995. Effect of IL-12 on 25. Muller, G., J. Saloga, T. Germann, I. Bellinghausen, M. Mohamadzadeh, J. Knop, immune suppression and suppressor cell induction by ultraviolet radiation. J. Im- and A. H. Enk. 1994. Identification and induction of human keratinocyte-derived munol. 154: 5114–5120. IL-12. J. Clin. Invest 94: 1799–1805. 11. Toews, G. B., P. R. Bergstresser, and J. W. Streilein. 1980. Epidermal Langer- 26. Jonuleit, H., E. Schmitt, H. Kakirman, M. Stassen, J. Knop, and A. H. Enk. 2002. Infectious tolerance: human CD25ϩ regulatory T cells convey suppressor activity hans cell density determines whether contact hypersensitivity or unresponsive- ϩ ness follows skin painting with DNFB. J. Immunol. 124: 445–453. to conventional CD4 T helper cells. J. Exp. Med. 196: 255–260. 12. Elmets, C. A., P. R. Bergstresser, R. E. Tigelaar, P. J. Wood, and J. W. Streilein. 27. Schwarz, A., A. Maeda, M. K. Wild, K. Kernebeck, N. Gross, Y. Aragane, 1983. Analysis of the mechanism of unresponsiveness produced by haptens S. Beissert, D. Vestweber, and T. Schwarz. 2004. Ultraviolet radiation-induced painted on skin exposed to low dose ultraviolet radiation. J. Exp. Med. 158: regulatory T cells not only inhibit the induction but can suppress the effector 781–794. phase of contact hypersensitivity. J. Immunol. 172: 1036–1043. 28. Skelsey, M. E., E. Mayhew, and J. Y. Niederkorn. 2003. CD25ϩ, interleukin- 13. Schwarz, A., S. Grabbe, K. Mahnke, H. Riemann, T. A. Luger, M. Wysocka, ϩ G. Trinchieri, and T. Schwarz. 1998. Interleukin 12 breaks ultraviolet light in- 10-producing CD4 T cells are required for suppressor cell production and im- duced immunosuppression by affecting CD8ϩ rather than CD4ϩ T cells. J. Invest. mune privilege in the anterior chamber of the eye. Immunology 110: 18–29. Dermatol. 110: 272–276. 29. Joffre, O., N. Gorsse, P. Romagnoli, D. Hudrisier, and J. P. van Meerwijk. 2004. Induction of antigen-specific tolerance to bone marrow allografts with 14. Iijima, M., and S. I. Katz. 1983. Specific immunologic tolerance to dinitroflu- CD4ϩCD25ϩ T lymphocytes. Blood 103: 4216–4221. orobenzene following topical application of dinitrothiocyanobenzene: modulation 30. Thornton, A. M., and E. M. Shevach. 1998. CD4ϩCD25ϩ immunoregulatory T by suppressor T cells. J. Invest. Dermatol. 81: 325–330. cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 15. Sommer, G., D. Parker, and J. L. Turk. 1975. Epicutaneous induction of hypo- Downloaded from production. J. Exp. Med. 188: 287–296. reactivity in contact sensitization. Demonstration of suppressor cells induced by 31. Kurimoto, I., M. Arana, and J. W. Streilein. 1994. Role of dermal cells from contact with 2,4-dinitrothiocyanatebenzene. Immunology 29: 517–525. normal and ultraviolet B-damaged skin in induction of contact hypersensitivity 16. Wei, L., K. M. Muller, J. H. Saurat, and C. Hauser. 1993. Lymphokine profiles and tolerance. J. Immunol. 152: 3317–3323. in contact sensitivity induced by dinitrofluorobenzene and tolerance induced by 32. Schwarz, A., S. Stander, M. Berneburg, M. Bohm, D. Kulms, H. van Steeg, dinitrothiocyanobenzene. Arch. Dermatol. Res. 284: 427–431. K. Grosse-Heitmeyer, J. Krutmann, and T. Schwarz. 2002. Interleukin-12 sup- 17. Scholzen, T. E., S. Stander, H. Riemann, T. Brzoska, and T. A. Luger. 2003. presses ultraviolet radiation-induced apoptosis by inducing DNA repair. Nat. Cell Modulation of cutaneous inflammation by angiotensin-converting enzyme. J. Im- Biol. 4: 26–31. munol. 170: 3866–3873. 33. Lee, S. W., Y. Park, J. K. Yoo, S. Y. Choi, and Y. C. Sung. 2003. Inhibition of http://www.jimmunol.org/ 18. Gunzer, M., S. Janich, G. Varga, and S. Grabbe. 2001. Dendritic cells and tumor TCR-induced CD8 T cell death by IL-12: regulation of Fas ligand and cellular immunity. Semin. Immunol. 13: 291–302. FLIP expression and caspase activation by IL-12. J. Immunol. 170: 2456–2460. 19. Grabbe, S., M. Steinert, K. Mahnke, A. Schwarz, T. A. Luger, and T. Schwarz. 34. Palmer, E. M., L. Farrokh-Siar, v. S. Maguire, and G. A. van Seventer. 2001. 1996. Dissection of antigenic and irritative effects of epicutaneously applied hap- IL-12 decreases activation-induced cell death in human naive Th cells costimu- tens in mice. Evidence that not the antigenic component but nonspecific proin- lated by intercellular adhesion molecule-1. I. IL-12 alters caspase processing and flammatory effects of haptens determine the concentration-dependent elicitation inhibits enzyme function. J. Immunol. 167: 749–758. of allergic contact dermatitis. J. Clin. Invest. 98: 1158–1164. 35. Yoo, J. K., J. H. Cho, S. W. Lee, and Y. C. Sung. 2002. IL-12 provides prolif- 20. Dubois, B., L. Chapat, A. Goubier, M. Papiernik, J. F. Nicolas, and D. Kaiserlian. eration and survival signals to murine CD4ϩ T cells through phosphatidylinositol ϩ ϩ 2003. Innate CD4 CD25 regulatory T cells are required for oral tolerance and 3-kinase/Akt signaling pathway. J. Immunol. 169: 3637–3643. ϩ inhibition of CD8 T cells mediating skin inflammation. Blood 102: 3295–3301. 36. Schwarz, A., S. Grabbe, K. Grosse-Heitmeyer, B. Roters, H. Riemann, 21. Kimber, I., P. A. Botham, N. J. Rattray, and S. T. Walsh. 1986. Contact-sensi- T. A. Luger, G. Trinchieri, and T. Schwarz. 1998. Ultraviolet light-induced im-

tizing and tolerogenic properties of 2,4-dinitrothiocyanobenzene. Int. Arch. Al- mune tolerance is mediated via the Fas/Fas-ligand system. J. Immunol. 160: by guest on October 1, 2021 lergy Appl. Immunol. 81: 258–264. 4262–4270.