CD28 induces immunostimulatory signals in dendritic cells ... - Nature

© 2004 Nature Publishing Group http://www.nature.com/natureimmunology

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CD28 induces immunostimulatory signals in dendritic cells via CD80 and CD86 Ciriana Orabona1, Ursula Grohmann1, Maria Laura Belladonna1, Francesca Fallarino1, Carmine Vacca1, Roberta Bianchi1, Silvia Bozza1, Claudia Volpi1, Benoıˆt L Salomon2, Maria Cristina Fioretti1, Luigina Romani1 & Paolo Puccetti1 Bidirectional signaling along the B7–CTLA-4 coreceptor pathway enables reciprocal conditioning of T cells and dendritic cells. Although T cells can instruct dendritic cells to manifest tolerogenic properties after CTLA-4 engagement of B7, such a B7-mediated signaling is not known to occur in response to CD28. Here we show that mouse dendritic cells were induced by soluble CD28 to express interleukin 6 and interferon-c. Production of interleukin 6 required B7-1 (CD80), B7-2 (CD86) and p38 mitogen-activated protein kinase and prevented interferon-c-driven expression of immunosuppressive tryptophan catabolism. In vivo, an adjuvant activity of soluble CD28 was demonstrated as enhanced T cell-mediated immunity to tumor and self peptides and protection against microbial and tumor challenge. Thus, different ligands of B7 can signal dendritic cells to express functionally distinct effector responses.

The ability of naive T cells to clonally expand and acquire effector functions depends on the strength of signals received by the T cell receptor (TCR) and by a growing set of costimulatory receptors, the most important of which remains CD28 (refs. 1–3). The biological consequences of CD28 costimulation are numerous and include control of the T cell cycle, survival and differentiation, as well as amplification of the membrane-proximal signaling generated by TCR ligation4. As a result, CD28-deficient mice have reduced responses to infectious pathogens as well as allograft and contact hypersensitivity antigens5–7. B7-1 and B7-2 represent the two structurally homologous ligands of CD28, which are expressed by antigen-presenting cells (APCs), including dendritic cells (DCs). B7-1 is nearly absent from immature DCs, whereas B7-2 is expressed in low amounts. Expression of each is upregulated by infection, tissue injury or the production of inflammatory cytokines or after the contact of APCs with activated T cells8. There is evidence that constitutively expressed B7 can suppress T cell activation and maintain self-tolerance by sustaining a population of regulatory T (TR) cells9. In contrast to CD28, which is a positive regulator of T cell function, the inhibitory receptor cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) mediates downregulation of the T cell response through interference with TCR signals10 and favors the onset of antigenspecific tolerance7,11,12. In addition, CTLA-4, whose expression is enhanced by CD28, acts by competing with the latter for B7 molecules on APCs, which are bound by CTLA-4 with a much higher affinity13. A third level of CTLA-4 interference with an ongoing immune

response is represented by its ability to act as a ligand for B7 coreceptor molecules on DCs, resulting in signal transduction (‘reverse signaling’) and the onset of suppressive activity14. Both a soluble form of CTLA-4 (ref. 15) and membrane-anchored CTLA-4 on the surface of TR cells16 will activate the immunosuppressive pathway of tryptophan catabolism in DCs, leading to the induction of specific tolerance to alloantigens and self-antigens17. It is, however, unknown whether CD28 might also act as an agonist ligand of B7 receptor molecules and initiate signal transduction and downstream effector responses. Several forms of soluble CD28 have been generated and used mostly in studies of binding affinity and cellular interactions in vitro18. In one study, the binding of a fusion protein of CD28 and immunoglobulin (CD28-Ig) to B7-1 and its binding to B7-2 transfectants were essentially indistinguishable. Binding of a CTLA4–Ig fusion protein to both transfectants was also similar but was of higher affinity than CD28-Ig binding19. Here we made use of a soluble fusion protein of mouse CD28 and IgG3 Fc (CD28-Ig) to explore the effects of CD28 as a potential agonist for receptors expressed on DCs both in vitro and in vivo. We found B7-dependent as well as p38 MAPK–dependent production of interleukin 6 (IL-6) in vitro after exposure of DCs to CD28-Ig. Such rapid and sustained production of IL-6 dominated over interferon-g (IFN-g)–regulated expression of immunosuppressive tryptophan catabolism. We found IL-6-dependent effects in vivo, all consistent with an unexpected and unusual pattern of adjuvant activity of the fusion protein in three different experimental settings.

1Department of Experimental Medicine, University of Perugia, 06126 Perugia, Italy. 2Centre National de la Recherche Scientifique UMR 7087, Ho ˆ pital Pitie´-Salpeˆtrie`re, 75013 Paris, France. Correspondence should be addressed to P.P. ([email protected]).

Published online 3 October 2004; doi:10.1038/ni1124

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activation20,21 (Fig. 2b). The production of IFN-g was approximately one half the amount induced by CTLA-4–Ig (Fig. 2b). Both IL-6 and IFN-g were produced early in response to CD28-Ig, being detectable at about 8 h, and their production was unaffected by the presence of rat antibody to CD16/32 (anti-CD16/32) as a means of blocking Fc receptors on DCs (data not shown). As with CD28-Ig, human Jurkat T cells expressing mouse CD28 were capable of inducing DC production of IL-6 (Fig. 2c). We also noted this effect when the Jurkat transfectants had been fixed with glutaraldehyde. We found no effect with mock-transfected cells (transfected with empty vector), ruling out the possibility of a contribution of B7 engagement by human CD28 on Jurkat cells. Thus, both soluble and membrane-anchored CD28 induce a cytokine response in DCs that is characterized by preponderant production of IL-6.

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RESULTS CD28-Ig induces IL-6 and IFN-c in DCs To investigate whether CD28-Ig activates a functional response in mouse DCs, we first tested the fusion protein for its binding ability to splenic DCs in comparison with B7-1 (HTR.B7-1) and B7-2 (HTR.B7-2) transfectants. CD28-Ig seemed to bind all cell types except for control HTR.C cells (Fig. 1). No substantial changes were induced by CD28-Ig in the surface expression of major histocompatibility complex (MHC) class II, CD40 or B7 molecules by the DCs, as determined by flow cytometry (data not shown). Because B7 engagement by CTLA-4–Ig in vitro is known to induce a cytokine response dominated by IFN-g15, we assessed the production of IL-1a, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, tumor necrosis factor and IFN-g by DCs treated for 24 h with various concentrations of CD28-Ig, control IgG3 or a control construct containing only the IgG3 heavy chain tail (Ig-Cg3; Fig. 2a). We found a notable dosedependent effect only with IL-6 and IFN-g production in response to CD28-Ig, although we could also detect IL-23. We focused our attention on IL-6 and IFN-g production. The amount of IL-6 induced by CD28-Ig was much greater than that induced by equal concentrations of CTLA-4–Ig and was similar to that resulting from CD40

IL-6 induction requires B7 and p38 MAPK To ascertain the DC receptors for CD28-Ig, we used mice genetically deficient in B7-1 or B7-2 or both. We measured IL-6 and IFN-g production in DCs from B7-deficient and wild-type mice after exposure in vitro to CD28-Ig (Fig. 3a). The absence of B7-1 or B7-2 greatly impaired IL-6 and IFN-g production, and we found no response in mice lacking both B7 molecules. To assess any possible contribution of Toll-like receptor (TLR) signaling by endotoxin contamination, we used mice genetically deficient in either TLR4 or the intracellular adapter protein myeloid differentiation factor 88 (MyD88; Fig. 3a). Neither TLR4 nor the MyD88-dependent pathway seemed to be required for DC production of IL-6 and IFN-g in response to CD28-Ig. The promoter of the gene encoding IL-6 contains an NF-kB site that is made accessible to the transcription factor by covalent modification

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Figure 1 Flow cytometry assessing the binding of CD28-Ig to cell transfectants and splenic DCs. HTR.B7-1 and HTR.B7-2 cells expressing surface B7-1 and B7-2, respectively, and splenic DCs from DBA/2 mice were treated for 30 min on ice with 1 mg/ml of CD28-Ig and then with phycoerythrin-conjugated anti-mouse IgG3 (shaded histograms). HTR.C indicates mock-transfected control cells. The control treatment consisted of IgG3 in place of CD28-Ig (dotted lines). The background staining is that of cells treated with anti-mouse IgG3 alone. Blocking of Fc receptors was ensured by the use of rat anti-CD16/32. No specific binding occurred with DCs from mice with genetic deficiency of B7-1 and B7-2 (data not shown). Data are representative of three independent experiments.

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Figure 2 Cytokine production by DCs in vitro in response to CD28-Ig, CTLA-4–Ig or membrane-anchored CD28. (a) Splenic DCs from DBA/2 mice were exposed for 24 h to 10, 20, or 40 mg/ml of CD28-Ig (thick lines), IgG3 (thin lines) or Ig-Cg3 (dotted lines) and cytokines in the supernatants were measured by ELISA. Data are means of replicate samples from one representative experiment out of three performed; s.d., usually less than 5% of the mean, has been omitted. TNF, tumor necrosis factor. (b) Comparison of the production of IL-6 and IFN-g in response to CD28-Ig or CTLA-4–Ig in the conditions as described in a. Production of IL-6 by DCs was also assayed after CD40 ligation by agonist antibody (Anti-CD40). Data are mean 7 s.d. of triplicate samples in one assay representative of four experiments with different ranges of fusion protein concentrations. (c) IL-6 in the supernatants of DCs (1 106 cells/ml) cultured with 40 mg/ml of CD28-Ig or cocultured with Jurkat cells (2 105 cells/ml) transfected with empty vector (J-pEF-BOS) or mouse CD28 (J-CD28). Transfected Jurkat cells were also assayed after fixation with 0.05% glutaraldehyde (Fixed). Data are mean 7 s.d. of triplicate samples. Graph is representative of three independent experiments.

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of the N-terminal tail of histone H3 (ref. 22). Phosphorylation of histone H3 dependent on p38 MAPK is necessary for the production of IL-6 by DCs in response to inflammatory stimuli23. We examined the production of IL-6 in DCs treated with CD28-Ig in the presence or absence of SB202190, an inhibitor of p38 MAPK. Blockade of p38 activity resulted in complete suppression of IL-6 release (Fig. 3b). To confirm that p38 and NF-kB were among the transcriptional mediators of CD28-Ig activity and to compare the early signaling by CD28-Ig and CTLA-4–Ig, we used promoter-driven expression of luciferase activity in DCs transfected with reporter plasmids containing the luciferase gene in combination with appropriate promoter sequences. To investigate MAPK activation, we electroporated DCs from wild-type or B7-deficient mice with the pSRE-Luc reporter

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Figure 3 Cytokine induction by CD28-Ig requires B7 expression and p38 signaling. (a) Production of IL-6 and IFN-g in DCs from wild-type (WT) mice or mice genetically deficient in B7-1 (Cd80 / ), B7-2 (Cd86 / ) or both. Mice deficient in TLR4 (Tlr4 / ) or MyD88 (Myd88 / ) were also assayed. DCs were treated for 24 h with 40 mg/ml of CD28-Ig or IgG3 and cytokines were measured in supernatants. Data are mean 7 s.d. from one of two experiments. (b) Obligatory function of p38 MAPK in IL-6 induction by CD28-Ig. DCs from DBA/2 mice were treated for 1 h with SB202190 at a concentration of 10 mM followed by the addition of CD28-Ig or IgG3 (control). IL-6 production was measured at 24 h. SB202474 is an inactive analog of SB202190. Data are mean 7 s.d. from one of three experiments. *, P o 0.001, presence versus absence of inhibitor.

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plasmid regulated by the serum-responsive element from the Fos promoter. To detect NF-kB activation, we transfected cells with pGL3-TK-kBPD. We incubated DCs with Ig-Cg3, CD28-Ig or CTLA-4–Ig for 4 h (Fig. 4a) or 8 h (Fig. 4b). For comparison, we treated groups of DCs with 1 mg/ml of lipopolysaccharide (LPS) or with anti-CD40 in place of the fusion proteins. CD28-Ig but not CTLA-4–Ig induced an increase of approximately threefold in the expression of luciferase activity controlled by the Fos promoter element at 4 h (Fig. 4a). This expression was abolished by preincubation with the p38 MAPK inhibitor SB202190 (data not shown). Activation of MAPK-dependent transcription by CD28-Ig and CTLA-4–Ig was comparable at 8 h (Fig. 4b). For NF-kB, the effects of CD28-Ig and CTLA-4–Ig were similar at 4 h, although the efficacy of

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Figure 4 CD28-Ig, CTLA-4–Ig and CD28 transfectants activate NF-kB- and MAPK-dependent transcription in B7-expressing DCs. DCs purified from wild-type C57BL/6 mice or mice genetically deficient in B7-1 and B7-2 were transfected with pRL-TK in combination with pSRE-Luc or pGL3-TK-kBPD. After 24 h, cells were incubated with Ig-Cg3, CD28-Ig, CTLA-4–Ig, LPS or anti-CD40. Luciferase activity was assayed at 4 and 8 h. (a) MAPK-dependent transcription at 4 h, assessed as pSRE-Luc activity; NF-kB activity, as pGL3-TK-kBPD activity. (b) The assay in a, repeated at 8 h. Results are expressed as fold induction (mean 7 s.d. of three independent experiments) of the sample incubated with Ig-Cg3, CD28-Ig, CTLA-4–Ig, LPS or anti-CD40 versus the corresponding untreated sample, with the control value being 1. (c) Luciferase activity in CD11c+ DCs after culture for 4 h with Jurkat cells as in Figure 2c. Controls or transfected Jurkat cells were also assayed after fixation with glutaraldehyde. Results are mean 7 s.d. of three independent experiments.

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ARTICLES Figure 5 IL-6 has a dominant function in the effects of CD28-Ig in vitro. (a) DCs from DBA/2 mice were treated with 40 mg/ml of CD28-Ig in the presence (+) or absence ( ) of antibodies to IL-6 and IL-6 receptor (collectively indicated as anti-IL-6). Control treatments included the use of IgG3 (in place of CD28-Ig) and rat IgG (in place of anti-IL-6). IDO activity was measured in terms of tryptophan conversion to kynurenine. Data are mean 7 s.d. of triplicate samples and one of 100 101 102 103 104 100 101 102 103 104 three representative experiments is presented. Fluorescence intensity *, P o 0.001, anti-IL-6 versus rat IgG treatment. (b) Cells were treated with 40 mg/ml of CD28-Ig or IgG3 for 48 h before flow cytometry. Control cultures were treated with isotype-matched antibody (open histograms). Mean channel fluorescence intensity is indicated for one experiment representative of four, in which the flow cytometry parameters were kept constant so that data could be compared from one experiment to the next. The mean channel fluorescence intensity values 7 s.d. for the four experiments were 11.6 7 1.7 and 7.3 7 2.0 for IgG3 and CD28-Ig treatments, respectively (P o 0.01).

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CD28-Ig was higher at 8 h, with an induction value of almost 10-fold. This suggested that the early differences in MAPK activity induced by CD28-Ig and CTLA-4–Ig might be reflected by differential expression of NF-kB-dependent transcription by the two fusion proteins at 8 h. LPS seemed to affect mostly early transcription regulated by NF-kB, and anti-CD40 had a greater effect on MAPK-dependent transcription. For both NF-kB and MAPK, the effects of CD28-Ig were similar to or greater than those of LPS and anti-CD40. Lack of B7 expression ablated DC responsiveness to CD28-Ig but not LPS or anti-CD40, as assessed by the induction of luciferase activity controlled by either promoter at 4 h (Fig. 4a). CD28 Jurkat transfectants but not mock-transfected cells showed a pattern of p38 and NF-kB activation similar to that of CD28-Ig, and we also noted the effect after glutaraldehyde fixation of the transfectants (Fig. 4c). Thus, induction of IL-6 by soluble or cell-bound CD28 requires both B7-1 and B7-2 and is associated with early p38 MAPK activation and later high NF-kB activity. IL-6 effects are dominant in CD28-Ig–treated DCs In DCs treated with CTLA-4–Ig, the presence of autocrine IFN-g correlates with induction of the enzyme indoleamine 2,3-dioxygenase (IDO), responsible for tryptophan conversion to kynurenine in vitro and for the appearance of immunosuppressive effects in vivo15. CD40 activation24,25 and IL-6 (ref. 20) are negative regulators of tryptophan catabolism in mouse DCs. Because of the presence of IL-6 and IFN-g in supernatants of DCs exposed to CD28-Ig (Fig. 2), we measured kynurenine concentrations in cultures treated with CD28-Ig in the presence or absence of IL-6-specific and IL-6 receptor–specific monoclonal antibodies (Fig. 5a). We found negligible kynurenine production in DCs exposed to CD28-Ig, yet blocking the effects of IL-6 resulted in enhanced tryptophan catabolism. Induction of Indo, which encodes IDO, in DCs requires IFN-gmediated activation of the transcriptional factor STAT1 (ref. 15). A series of studies with various experimental approaches demonstrated that IL-6-induced STAT3 activation is notably enhanced in cells lacking suppressor of cytokine signaling 3 (SOCS3)26–28. In particular, in the absence of SOCS3, IL-6 becomes immunosuppressive and activates genes typically induced by interferons. Therefore, SOCS3 is involved in the prevention of IFN-g-like responses in cells stimulated by IL-6 and acts as an essential and specific regulator of the IFN-g pathway29. Using RT-PCR, we found that treatment with CD28-Ig for 24 h enhanced the transcriptional expression of SOCS3 in DCs (data not shown). Modulation of IFN-g-inducible genes by IL-6 via SOCS3 would itself explain the ability of IL-6 neutralization to rescue tryptophan

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catabolism in DCs treated with CD28-Ig. IL-6 can also act to downregulate IFN-g receptor expression in DCs, which correlates with a reduced ability of the cells to metabolize tryptophan to kynurenine20. We measured IFN-g receptor expression in DCs exposed in vitro to CD28-Ig (Fig. 5b). Cytofluorometry showed that IFN-g receptor expression was downregulated in DCs treated with CD28-Ig. This effect, however, was negated by antibody-mediated blockade of IL-6 activity concomitant with CD28-Ig treatment (data not shown). Therefore, the overall effects of IL-6 are dominant in vitro and prevent the IFN-g-regulated expression of tryptophan catabolism. CD28-Ig favors DC induction of cell-mediated immunity Mouse splenic DCs can present peptide antigens in an immunogenic or tolerogenic way, with the distinction depending on either the occurrence of specialized DC subsets or the maturation or activation state of the DC30. Environmental factors are crucial in conditioning the outcome of DC presentation of the synthetic nonapeptides P815AB24,25 and NRP-A7 (ref. 16). The former is related to a poorly immunogenic antigen of mouse mastocytoma P1.HTR31; the latter acts as a peptide mimotope of a potential antigen in autoimmune diabetes in mice32. After transfer of peptide-pulsed DCs into recipient mice, the induction of immunity versus tolerance can be monitored by skin test assay, through intrafootpad challenge with the eliciting peptide16,20,25. We pulsed DCs in vitro with P815AB or NRP-A7 and injected them into recipient hosts that we assayed at 2 weeks for the development of MHC class I–restricted skin test reactivity. We either left the DCs untreated or treated them with CD28-Ig in the presence or absence of anti-IL-6 and anti–IL-6 receptor. We also used mice deficient in IL-6 expression as a source of DCs (Fig. 6a). In line with previous results with unfractionated DCs presenting poorly immunogenic peptides16,20,25, we found no reactivity after the transfer of unconditioned DCs. DC conditioning by CD28-Ig elicited strong footpad reactivity, and this effect was negated by the neutralization of IL-6 or by the use of IL-6-deficient mice as donors of DCs. Relative to results with mice receiving IgG3-treated DCs pulsed with P815AB, the use of DCs exposed to CD28-Ig resulted in a fourfold increase in the frequency of antigen-specific IL-2-producing CD4+ T cells (from 20 to 80 cells per 104 cells) and in a fivefold increase in the frequency of IFN-g-producing CD8+ T cells (from 30 to 150 cells per 104 cells) in the spleens of recipient mice at 2 weeks after cell transfer. We next examined the effect of B7-1 or B7-2 deficiency on CD28-Ig adjuvant activity in the model of DC induction of skin test reactivity to P815AB or NRP-A7 (Fig. 6b). We also used DCs lacking B7-1 or B7-2 to immunize mice against a fully immunogenic synthetic peptide, the influenza virus nucleoprotein peptide. The absence of

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Figure 6 CD28-Ig triggers IL-6-dependent adjuvant properties in B7-expressing DCs. (a) The peptides P815AB and NRP-A7 were used to pulse wild-type or Il6 / DCs in vitro. Cells were pulsed with either peptide in the presence of IgG3 or CD28-Ig, with (+) or without ( ) anti-IL-6 treatment. The priming ability of the conditioned, peptide-pulsed DCs was examined by transfer into wild-type recipient mice, which were assayed at 2 weeks for skin test reactivity to the eliciting peptide. Reactivity was measured as the increase in footpad weight of peptide-injected footpads over that of vehicle-injected counterparts. (b) DCs were purified from mice lacking B7-1 or B7-2 and were pulsed with P815AB, NRP-A7 or the immunogenic nucleoprotein peptide (NP) in the presence or absence of CD28-Ig. Cells were injected into wild-type recipients. For the sets of data shown in a and b, the results (mean 7 s.d.) are from one experiment representative of three, with at least six mice per group per experiment. *, P o 0.001, experimental versus control footpads.

B7-1 or B7-2 negated the induction of immunity to P815AB or NRPA7 after transfer of CD28-Ig–treated DCs, as measured by footpad reactivity. In contrast, lack of B7-1 or B7-2 did not affect the onset of footpad reactivity to the nucleoprotein peptide, and this reactivity developed independently of DC exposure to CD28-Ig. Thus, in this model system with nonimmunogenic peptides, CD28-Ig exerted an adjuvant effect on DCs that was dependent on the presence of B7-1 and B7-2 as well as on the autocrine effects of IL-6. CD28-Ig enhances antifungal resistance by DC vaccination In addition to the choice between tolerance and immunity, environmental factors affect the T cell–polarizing signals and type of T cell response (T helper type 1 (TH1), TH2 or TR) induced by DCs33. Mouse infection with the fungus Candida albicans has become a prototypic model of the mutual conditioning of T cells and DCs, leading to protective TH1 or nonprotective TH2 responses34–36. DCs pulsed with viable C. albicans yeasts but not those pulsed with hyphae are capable of inducing protective immunity when adoptively transferred in vivo37. To comparatively assess the effect of DC modulation with CD28-Ig and CTLA-4–Ig on vaccine-induced antifungal resistance, we used either type of fusion protein to condition DCs before pulsing with C. albicans yeasts (CTLA-4–Ig) or hyphae (CD28-Ig). In parallel experimental groups, anti-IFN-g was present during DC

conditioning with CTLA-4–Ig, whereas anti-IL-6 and anti–IL-6 receptor were present in DCs treated with CD28-Ig. Mice received two injections of conditioned candida-pulsed DCs, then were infected with virulent fungus 1 week after the last immunization37. We measured susceptibility to infection in terms of fungal clearance from the organs and patterns of cytokine production by splenic CD4+ T cells. Protective vaccination with yeast-pulsed DCs was rendered nonprotective by CTLA-4–Ig, which required IFN-g (Fig. 7a) and resulted in a TH2-dominated overwhelming infection (data not shown). In contrast, nonprotective DCs pulsed with hyphae were made protective by exposure to CD28-Ig, through mechanisms contingent on autocrine IL-6 (Fig. 7b). Compared with that of mice receiving DCs pulsed with hyphae in the absence of CD28-Ig, the frequency of IFN-g-producing T cells was increased and that of IL-4-producing cells was decreased, as the mice developed durable anticandidal protection (data not shown). Therefore, in this infectious model, different ligands of B7, through different cytokine responses in target DCs, induced qualitatively different TH cell responses. CD28-Ig given in vivo eradicates a growing tumor Based on the finding that CD28-Ig will trigger MHC class I–restricted immunity to P815AB (Fig. 6a), we assayed administration of the fusion protein in vivo for effects on host resistance to challenge with

Figure 7 CD28-Ig and CTLA-4–Ig have disparate effects on DC ability to initiate immunity to C. albicans in vivo. (a) Assay of CTLA-4–Ig for possible interference with the development of protective immunity to the fungus as induced by host vaccination with DCs pulsed with yeasts. DCs were coexposed to C. albicans yeasts and CTLA-4–Ig, with or without anti-IFN-g (or control rat IgG2a), before transfer into BALB/c recipient hosts. Controls include the combination of antiIFN-g with Ig-Cg3. Breaks in bars and vertical axis indicate scale interruption. (b) Assay of the ability of CD28-Ig to interfere with the development of nonprotective immunity to fungus after vaccination with DCs pulsed with hyphae. DCs were coexposed to C. albicans hyphae and Ig-Cg3 or CD28-Ig, with or without anti-IL-6 (or control rat IgG) treatment, before transfer into BALB/c mice. In both a and b, mice were then challenged with virulent Candida and were assayed for fungal growth in various organs. Data are mean colony-forming units (CFU) 7 s.d. per organ and are from one experiment representative of three with at least eight mice per group per experiment. *, P o 0.005–0.0001, CTLA-4–Ig (with or without IgG2a) or CD28-Ig (with or without rat IgG) treatment versus Ig-Cg3 treatment of DCs.

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Figure 8 CD28-Ig interferes with CD4+CD25+ TR cell suppression in tumorbearing mice. CD4+CD25+ cells were obtained from tumor-draining lymph nodes of mice challenged with P1.HTR cells. Cocultures of CD4+CD25 cells (1 105) from naive mice were established with various numbers of CD4+CD25+ cells (horizontal axis) from the tumor-bearing hosts in the presence of soluble anti-CD3 (1 mg/ml) and APCs. The tumor-bearing mice were treated either with CD28-Ig or control IgG3. CD4+CD25+ cells from naive mice were used to determine baseline values of TR cell activity. Data are mean 7 s.d. of quadruplicate samples and are from one experiment representative of three. *, P o 0.01, CD28-Ig versus IgG3 treatment.

P1.HTR. This tumor line exploits immunosuppressive tryptophan catabolism to downregulate the host response to a tumor-specific antigen related to the P815AB peptide used in the skin test assay31,38. We also treated a group of mice on CD28-Ig immunotherapy with anti-IL-6 and anti–IL-6 receptor administered at the time of challenge. Treatment with CD28-Ig resulted in IL-6-dependent regression of the transplanted tumor, according to a growth and rejection pattern typical of immune-mediated protection38 (Supplementary Fig. 1 online). The mice that had been cured were later found to resist rechallenge in the absence of immunotherapy (data not shown). We assayed splenocytes from tumor-bearing mice treated with IgG3 or CD28-Ig for cytotoxicity to P815AB-positive (P1.HTR) or P815ABnegative (P1.204) cells (Supplementary Fig. 1 online). A potent, antigen-specific cytotoxic T lymphocyte response was induced to P1.HTR cells by CD28-Ig but not IgG3 immunotherapy. Thus, CD28-Ig is capable of IL-6-dependent immunoregulatory effects in vivo, detectable as anti-tumor protection and induction of a cytotoxic T lymphocyte response in vitro. IL-6 induced by TLRs after recognition of microbial products can block TR cell activity39. In addition, persistent TLR signals are required for reversal of TR cell–mediated CD8 tolerance40. We therefore investigated whether the protective response induced by CD28-Ig in the tumor-bearing mice involved interference with TR activity. To examine whether CD28-Ig treatment affected TR cell suppression of autologous T cells in P1.HTR tumor–bearing mice, we collected draining lymph node cells from these mice 2 weeks after tumor implantation and purified CD4+CD25+ cells. We cocultured CD4+CD25 cells from naive mice with irradiated T cell–depleted splenocyte samples as APCs, soluble anti-CD3 and various numbers of CD4+CD25+ cells from the tumor-challenged mice treated with IgG3 or CD28-Ig. We measured the proliferation of the CD4+CD25 cells at the end of the coculture (Fig. 8). CD28-Ig treatment in vivo significantly reduced the suppressive activity of CD4+CD25+ TR cells from the tumor-bearing hosts. Thus, the therapeutic efficacy of CD28-Ig in the in vivo tumor growth inhibition assay might involve combined effects on the effector function of tolerogenic DCs as well as on the directive function of TR cells in orchestrating peripheral tolerance.

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DISCUSSION Although they are known to mediate positive and inhibitory costimulation through their respective interactions with CD28 and CTLA4, B7 molecules could have a complex function in immunoregulation, contributing to self-tolerance by sustaining a population of TR cells9. Among the functions of B7 is its ability to signal DCs and activate IDO expression after engagement by CTLA-4 (refs. 15,16). This raises the question of how general the mechanism of B7 signaling could be in the interaction of T and dendritic cells8,14,41. In this study we used CD28-Ig, which represents a soluble monovalent agent, to mimic membrane-anchored homodimeric CD28 (refs. 18,19). Although CD28 and CTLA-4 have distinct association and dissociation rates and form different types of dimers with B7 (ref. 41), we used CD28-Ig in conditions comparable to those of the soluble bivalent agent CTLA4–Ig15 to gain comparative insight into the possible physiological functions of CTLA-4 and CD28 as ligands of B7 receptor molecules. In vitro studies of DC conditioning by CD28-Ig or CD28 transfectants showed that CD28 engagement of B7 induced a cytokine response dominated by IL-6, whose release required early activation of p38 MAPK. The dominant production of IL-6 induced by CD28-Ig prevented the expression of the IFN-g-inducible Indo gene. Maximum production of IL-6 by DCs required coexpression of B7-1 and B7-2, and the absence of either molecule negated the potentiating effect of DC conditioning with CD28-Ig in an in vivo assay with synthetic peptides. In contrast, B7-1 but not B7-2 was absolutely required for the effects of CTLA-4–Ig in vitro and in vivo (data not shown). CD28 and CTLA-4 share two ligands, B7-1 and B7-2, but B7 molecules bind to CTLA-4 with substantially higher affinity than they bind CD28. Although both CD28 and CTLA-4 interact with B7 by virtue of the MYPPPY sequence, each CTLA-4 dimer can bind two different B7 homodimers, thereby forming a stable zipper-like complex not seen with CD28, which can bind only one B7 homodimer at a time. There is evidence in different in vitro systems for a multistate model of receptor activation in which ligand-specific conformations are capable of differentially activating distinct signaling partners42,43. The molecular mechanisms of B7 signaling are obscure at present. However, assuming that B7 receptor molecules exist in multiple conformational states conditioned by dimerization, and allowing for agonist-specific conformation, a pattern of pathway-dependent agonist efficacy and potency can be predicted44. CD28-Ig and CTLA-4–Ig could thus stabilize distinct receptor conformations that allow qualitatively and/or quantitatively different signaling events. As a result, CD28-Ig and CTLA-4–Ig would be characterized by distinct potency and efficacy in the induction of specific cellular responses. This might explain the early, selective induction of MAPK in DCs by CD28-Ig, the differential cytokine response and the disparate ‘downstream’ effects induced by the fusion proteins on DC function. Metabotropic receptors and activation of MAPK as a signaling system have been used to show that a ligand can have opposing efficacies on two effector systems engaged by the same receptor42. Interferons, through STAT1 activation, have crucial actions in host defense, serving as positive regulators of immune responses. But interferons have many important tolerogenic actions as well. As a result, genetic deficiency in STAT1 results in increased susceptibility to autoimmune disease45. IL-6 is a multifunctional cytokine that regulates inflammatory responses, and overproduction of IL-6 is associated with autoimmunity and chronic inflammatory diseases29. IL-6 induced during the innate response to microbial pathogens can block the suppressor activity of TR cells39. However, in the absence of SOCS3, IL-6 becomes immunosuppressive and activates genes typically induced by interferons26–28. This indicates that through SOCS3,

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ARTICLES IL-6 inhibits the STAT-dependent expression of interferon-inducible genes. In keeping with this hypothesis, we found that inhibition of autocrine IL-6 enabled induction of tryptophan catabolism in DCs treated with CD28-Ig, thus resembling the effects of CTLA-4–Ig. This indicates that the relative magnitude of IL-6 and IFN-g responses is one crucial factor conditioning the in vitro effects of CD28-Ig versus CTLA-4–Ig signaling through B7. IL-6 induced by CD28-Ig also produced autocrine effects in vitro that might be independent of interference with IFN-g signaling but traceable to direct immunoregulatory actions20,36. Otherwise tolerogenic synthetic peptides were presented in an immunogenic way after DC conditioning with CD28-Ig. In a second model system, the focus of CD28-Ig effects in vitro centered on the qualitative development of TH cell responses to C. albicans34,35. Nonprotective DC vaccination with fungal hyphae37 was made highly protective by DC conditioning with CD28-Ig, which required IL-6 and involved redirecting the TH response. In the same conditions, CTLA-4–Ig would instead turn a protective response37 into a nonprotective one, through IFN-g-dependent mechanisms. CD28 has been widely recognized as the main costimulation pathway for naive T cell activation. However, CD28 is also prominent in the regulation of immune responses and the maintenance of peripheral tolerance46. Administration of CD28-Ig in vivo could thus affect costimulatory and regulatory signals with disparate effects on immune reactivity. In the tumor model with P1.HTR, CD28-Ig exerted potent immunostimulatory effects that were dependent on endogenous IL-6 production. Besides autocrine regulation of DC function, a multiplicity of effects might contribute to IL-6-dependent immunopotentiation by CD28-Ig given in vivo. The finding that TR cell activity is reduced in tumor-bearing mice on CD28-Ig therapy suggests that direct modulation of TR function can be involved in the antitumor effects of the fusion protein. Additional IL-6-dependent mechanisms involving TR activity might include the release of tumor-reactive T lymphocytes from the control of TR cells, as well as enhanced DC maturation, which would in turn facilitate overcoming TR cell– mediated suppression on DCs39,40,47. Natural TR cells constitutively express CTLA-4. One chief function of CD28 as an agonist ligand of B7 could be the release of DCs from the homeostatic control of TR cells. CD4+CD25 T cells from naive mice induced IL-6 production when cocultured with DCs for 48 h. This induction of IL-6 persisted when the T cells were fixed with glutaraldehyde, but could no longer be found when these cells were from Cd28 / mice (data not shown). Bidirectional signaling through the B7–CTLA-4 coreceptor pair will act to downregulate immune responses and favor tolerance to alloantigens as well as tumor and self peptides, providing a mechanistic clue to the action of CTLA-4-expressing TR cells15,16,25. Our data here suggest that both CD28 forward signaling and B7 reverse signaling positively affect the induction of immune responses after B7 engagement by CD28. This adds complexity to the intricacies of T cell costimulation involving CD28, B7-1, B7-2 and CTLA-4, yet the new data point to a unitary basis for the action of the four coreceptors, both in health and disease. Deciphering the bidirectional pathways emanating from a B7-coreceptor complex not only will help in the understanding of basic biological processes but also could provide new opportunities for the development of immunotherapies targeting costimulatory pathways.

targeted mutations (Cd80 / Cd86 / ) were as described15. Singly deficient Cd80 / or Cd86 / mice on a BALB/c background were also used48. Mice homozygous for the TLR4 (Tlr4 / ) or MyD88 (Myd88 / ) targeted mutation, raised on the C57BL/6 background, were as described49. The generation of BALB/c IL-6–deficient (Il6 / ) mice has also been described36. All in vivo studies were in compliance with National and Perugia University Animal Care and Use Committee guidelines. Fusion proteins. For the construction and expression of the fusion protein of CD28 and IgG3 Fc, cDNA encoding the extracellular domain of mouse CD28 was generated from the 3DO T cell hybridoma. The cDNA was amplified with primers (sense, 5¢-TGCAGCACTAGTCCTCATCAGAACA-3¢; antisense, 5¢-GTGCCCTGATCAACTTAGGAGATGAC-3¢) containing SpeI (sense) and BclI (antisense) restriction enzyme site sequences. PCR products were digested with the appropriate restriction enzymes and were cloned into a modified pEFBOS plasmid upstream of a region comprising the hinge, CH2 and CH3 domains of mouse IgG3 Fc50. Clones with the correct inserts were stably transfected by electroporation into P815 (clone P511) mastocytoma cells. Similar to the natural monovalent dimer, CD28-Ig seemed to be a dimeric protein about 110 kDa in molecular size, as judged by immunoblot analysis in reducing versus nonreducing conditions (Supplementary Fig. 2 online). CTLA-4–Ig consisted of a fusion protein of the extracellular domain of CTLA-4 and the same Fc portion as CD28-Ig15. Fusion proteins were routinely used in vitro at a concentration of 40 mg/ml. For all experiments involving fusion proteins, control treatments consisted of either or both of two different controls: native IgG3, which was produced by the anti-trinitrophenyl (TNP) hybridoma C3110 used for amplification of the mouse IgG3 heavy chain tail51; and Ig-Cg3, a construct that was produced by the same cell line as CD28-Ig and CTLA-4–Ig, and consisted of the hinge, CH2 and CH3 regions of mouse IgG3 Fc inserted downstream of the IgG3 signal peptide in the absence of CD28 or CTLA-4 domains. For all affinity-purified fusion proteins, endotoxin contamination was below the detection limit (0.05 endotoxin units/ml) of the specific assay (Coatest endotoxin; Chromogenix AB).

METHODS

Cells and reagents. Splenic DCs expressing CD11c were obtained as described, with a positive selection column in combination with CD11c MicroBeads (Miltenyi Biotec) in the presence of EDTA to disrupt DC–T cell complexes15,16. The recovered cells were more than 99% CD11c+, more than 99% MHC I-A+, more than 98% B7-2+, less than 0.1% CD3+ and 1–5% B220+. HTR transfectants expressing B7-1 (HTR.B7-1) or B7-2 (HTR.B7-2) were obtained as described10. CD28 transfectants were generated by amplification of cDNA encoding mouse CD28 from 3DO cells with primers (sense, 5¢-TGCAGCACTAGTCCTCATCAGAACA-3¢; antisense, 5¢-TACCAGGCGGCCGCCTTCTGGATAGGG-3¢) containing SpeI (sense) and NotI (antisense) restriction enzyme site sequences, followed by cloning into pEF-BOS. The CD28-encoding plasmid was introduced into human Jurkat T cells by electroporation and transfectants were selected in puromycin. The transfectants selectively expressed surface CD28 recognized by hamster anti–mouse CD28 (Supplementary Fig. 3 online). CD40 ligation on DCs was accomplished with a combination of antiCD40 and a second crosslinking reagent24. Antibody reagents for enzymelinked immunosorbent assay (ELISA) of IL-1a, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, tumor necrosis factor and IFN-g were from commercially available kits or have been described16,24,50. MP5-20F3 and biotinylated MP5-32C11 were used for IL-6 ELISA, whereas R4-6A2 and biotinylated XMG1.2 were used for IFN-g (all from PharMingen). The XMG1.2 antibody was also used for neutralization of IFN-g in vitro16. Rat monoclonal IgG1 6B4 (anti–mouse IL-6) and IgG2b 15A7 (anti–mouse IL-6 receptor) were used to block IL-6 activity20. The pyridinyl imidazole SB202190, a potent and specific inhibitor of p38 MAPK, and SB202474, an inactive analog of SB202190, were purchased from Calbiochem. Peptide P815AB (amino acid sequence, LPYLGWLVF), nucleoprotein peptide (TYQRTRALV) and peptide NRP-A7 (KYNKANAFL) were synthesized and purified as described16,20,52. These peptides are recognized in the context of the MHC class I H-2Ld (P815AB) or H-2Kd (NRP-A7 and nucleoprotein peptide) molecules.

Mice. Male 8- to 10-week-old DBA/2 (H-2d), BALB/c (H-2d) and C57BL/6 (H-2b) mice were purchased from Charles River Laboratories. B7-deficient C57BL/6 mice homozygous for both the CD80 (B7-1) and CD86 (B7-2)

Luciferase assay. DCs (6 106) were electroporated (230 V, 75 Ohm and 1,500 microfarads) with 40 mg of pGL3-TK-kBPD53 or pSRE-Luc (Stratagene)

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plasmid. The first construct contained two palindromic NF-kB binding sites and the thymidine kinase promoter upstream of the firefly luciferase coding sequence, and the second contained repeats of the serum responsive element of the Fos promoter. Another reporter plasmid, pRL-TK (1 mg; Promega) encoding Renilla luciferase, was coelectroporated as an internal control of the transfection process. Cells were seeded in 48-well plates at a density of 1 106 cells/ml. The next day, cells were stimulated for 4 or 8 h with 40 mg/ml of CD28-Ig or CTLA-4–Ig before lysis. Luciferase assays used the dual luciferase reporter assay kit (Promega). Flow cytometry and kynurenine determination. For all flow cytometry, cells were treated for 30 min at 41C with rat anti-CD16/32 (2.4G2) for blockade of Fc receptors. Phycoerythrin-conjugated goat anti-mouse IgG3 was from Southern Biotechnology. Surface expression of the IFN-g receptor a-chain was measured with biotinylated rat IgG to mouse CD119 (clone GR20; PharMingen), with biotinylated rat IgG2a being the isotype-matched control20. IDO functional activity was measured in vitro in terms of ability to metabolize tryptophan to kynurenine, whose concentrations were measured by HPLC15. In vivo assays. A skin test assay was used for measurement of MHC class I– restricted delayed-type hypersensitivity responses to synthetic peptides16,20,25. For P815AB, the afferent induction of reactivity by peptide-loaded DCs requires CD4+ T cells, whereas the effector phase of the response is mediated by CD8+ T cells54. We transferred peptide-loaded DCs (3 105) intravenously into recipients that we assayed at 2 weeks for the development of peptidespecific delayed-type hypersensitivity in response to intrafootpad challenge with the peptide. Results were expressed as the increase in footpad weight of peptide-injected footpads over that of vehicle-injected counterparts. The conditions of in vivo challenge of mice with C. albicans after protective or nonprotective vaccination with DCs pulsed with yeasts (strain PCA-2) or hyphae (strain CA-6), respectively, have been described in detail37. DCs pulsed with either fungal form (5 105) were injected twice, 1 week apart, into recipient hosts that were later infected intravenously with virulent CA-6 cells (6 105). Fungal growth was evaluated at 2–4 d of infection by quantification of the number of colony-forming units in various organs36,37. For the in vivo tumor growth inhibition assay38, living P1.HTR (H-2d) cells (2 105) were injected in a volume of 100 ml through a 27-gauge needle into the left flanks of DBA/2 mice. Tumor size was assessed twice per week with calipers; the longest and the shortest diameters were measured and an average value was calculated. Measurements were continued for 4 weeks; that is, up to the time when tumor-bearing mice would succumb to challenge and mice that had been cured were free of visible tumor. Cytotoxic T lymphocyte generation. The cytolytic assay was done as described38. Splenocytes (5 106) were collected 2 weeks after tumor challenge and were stimulated with irradiated P1.HTR cells (2.5 105). After 5 d, responder cells were recovered and tested for lytic activity against 2 103 51Cr-labeled P1.HTR or P1.204 target cells, according to standard conditions. Results were expressed as the mean 7 s.d. of quadruplicate samples at different effector/target cell ratios. TR cell purification and suppression assay. CD4+CD25+ and CD4+CD25 T cells were isolated from lymph nodes by magnetic-activated cell sorting as described16. The purity of either T cell fraction was more than 95%. For polyclonal TR cell suppression assay, CD4+CD25 cells were cocultured with irradiated T cell–depleted splenocyte samples and CD4+CD25+ cells for 3 d in the presence of soluble anti-CD3. Proliferation was measured by incorporation of [3H]thymidine according to standard procedures. Statistical analysis. Student’s t-test was used for analysis of in vitro and in vivo data. Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS We thank V. Poli (University of Turin, Turin, Italy) for the gift of IL-6-deficient mice and G. Andrielli for technical assistance. Supported by the Italian Association for Cancer Research.

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COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 13 April; accepted 2 September 2004 Published online at http://www.nature.com/natureimmunology/

1. Schwartz, R.H. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71, 1065–1068 (1992). 2. Lenschow, D.J., Walunas, T.L. & Bluestone, J.A. CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14, 233–258 (1996). 3. Egen, J.G., Kuhns, M.S. & Allison, J.P. CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat. Immunol. 3, 611–618 (2002). 4. Acuto, O. & Michel, F. CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat. Rev. Immunol. 3, 939–951 (2003). 5. Shahinian, A. et al. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261, 609–612 (1993). 6. Kondo, S., Kooshesh, F., Wang, B., Fujisawa, H. & Sauder, D.N. Contribution of the CD28 molecule to allergic and irritant-induced skin reactions in CD28 / mice. J. Immunol. 157, 4822–4829 (1996). 7. Salomon, B. & Bluestone, J.A. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu. Rev. Immunol. 19, 225–252 (2001). 8. Sansom, D.M., Manzotti, C.N. & Zheng, Y. What’s the difference between CD80 and CD86? Trends Immunol. 24, 314–319 (2003). 9. Lohr, J., Knoechel, B., Jiang, S., Sharpe, A.H. & Abbas, A.K. The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens. Nat. Immunol. 4, 664–669 (2003). 10. Fallarino, F., Fields, P.E. & Gajewski, T.F. B7-1 engagement of cytotoxic T lymphocyte antigen 4 inhibits T cell activation in the absence of CD28. J. Exp. Med. 188, 205–210 (1998). 11. Thompson, C.B. & Allison, J.P. The emerging role of CTLA-4 as an immune attenuator. Immunity 7, 445–450 (1997). 12. Oosterwegel, M.A., Greenwald, R.J., Mandelbrot, D.A., Lorsbach, R.B. & Sharpe, A.H. CTLA-4 and T cell activation. Curr. Opin. Immunol. 11, 294–300 (1999). 13. Alegre, M.L., Frauwirth, K.A. & Thompson, C.B. T-cell regulation by CD28 and CTLA-4. Nat. Rev. Immunol. 1, 220–228 (2001). 14. Finger, E.B. & Bluestone, J.A. When ligand becomes receptor—tolerance via B7 signaling on DCs. Nat. Immunol. 3, 1056–1057 (2002). 15. Grohmann, U. et al. CTLA-4–Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3, 1097–1101 (2002). 16. Fallarino, F. et al. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4, 1206–1212 (2003). 17. Grohmann, U., Fallarino, F. & Puccetti, P. Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol. 24, 242–248 (2003). 18. Linsley, P.S. et al. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 173, 721–730 (1991). 19. Lanier, L.L. et al. CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL. J. Immunol. 154, 97–105 (1995). 20. Grohmann, U. et al. IL-6 inhibits the tolerogenic function of CD8a+ dendritic cells expressing indoleamine 2,3-dioxygenase. J. Immunol. 167, 708–714 (2001). 21. Mann, J., Oakley, F., Johnson, P.W. & Mann, D.A. CD40 induces interleukin-6 gene transcription in dendritic cells: regulation by TRAF2, AP-1, NF-kB, and CBF1. J. Biol. Chem. 277, 17125–17138 (2002). 22. Saccani, S., Pantano, S. & Natoli, G. Two waves of nuclear factor kB recruitment to target promoters. J. Exp. Med. 193, 1351–1359 (2001). 23. Saccani, S., Pantano, S. & Natoli, G. p38-Dependent marking of inflammatory genes for increased NF-kB recruitment. Nat. Immunol. 3, 69–75 (2002). 24. Grohmann, U. et al. CD40 ligation ablates the tolerogenic potential of lymphoid dendritic cells. J. Immunol. 166, 277–283 (2001). 25. Grohmann, U. et al. Functional plasticity of dendritic cell subsets as mediated by CD40 versus B7 activation. J. Immunol. 171, 2581–2587 (2003). 26. Croker, B.A. et al. SOCS3 negatively regulates IL-6 signaling in vivo. Nat. Immunol. 4, 540–545 (2003). 27. Lang, R. et al. SOCS3 regulates the plasticity of gp130 signaling. Nat. Immunol. 4, 546–550 (2003). 28. Yasukawa, H. et al. IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunol. 4, 551–556 (2003). 29. Johnston, J.A. & O’Shea, J.J. Matching SOCS with function. Nat. Immunol. 4, 507– 509 (2003). 30. Shortman, K. & Heath, W.R. Immunity or tolerance? That is the question for dendritic cells. Nat. Immunol. 2, 988–989 (2001). 31. Uyttenhove, C. et al. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat. Med. 9, 1269–1274 (2003). 32. Amrani, A. et al. Progression of autoimmune diabetes driven by avidity maturation of a T-cell population. Nature 406, 739–742 (2000). 33. Medzhitov, R. & Janeway, C. Jr. Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173, 89–97 (2000).

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ARTICLES 34. Puccetti, P., Romani, L. & Bistoni, F. A TH1-TH2-like switch in candidiasis: new perspectives for therapy. Trends Microbiol. 3, 237–240 (1995). 35. Romani, L. Immunity to fungal infections. Nat. Rev. Immunol. 4, 1–23 (2004). 36. Romani, L. et al. Impaired neutrophil response and CD4+ T helper cell 1 development in interleukin 6-deficient mice infected with Candida albicans. J. Exp. Med. 183, 1345–1355 (1996). 37. d’Ostiani, C.F. et al. Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans. Implications for initiation of T helper cell immunity in vitro and in vivo. J. Exp. Med. 191, 1661–1674 (2000). 38. Fallarino, F. et al. Th1 and Th2 cell clones to a poorly immunogenic tumor antigen initiate CD8+ T cell-dependent tumor eradication in vivo. J. Immunol. 165, 5495– 5501 (2000). 39. Pasare, C. & Medzhitov, R. Toll pathway-dependent blockade of CD4+CD25+ T cellmediated suppression by dendritic cells. Science 299, 1033–1036 (2003). 40. Yang, Y., Huang, C.-T., Huang, X. & Pardoll, D.M. Persistent Toll-like receptor signals are required for reversal of regulatory T cell–mediated CD8 tolerance. Nat. Immunol. 5, 508–515 (2004). 41. Rothstein, D.M. & Sayegh, M.H. T-cell costimulatory pathways in allograft rejection and tolerance. Immunol. Rev. 196, 85–108 (2003). 42. Azzi, M. b-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc. Natl. Acad. Sci. USA 100, 11406–11411 (2003). 43. Andres, P.G. et al. Distinct regions in the CD28 cytoplasmic domain are required for T helper type 2 differentiation. Nat. Immunol. 5, 435–442 (2004). 44. Chen, C.Y., Cordeaux, Y., Hill, S.J. & King, J.R. Modelling of signalling via G-protein coupled receptors: pathway-dependent agonist potency and efficacy. Bull. Math. Biol. 65, 933–958 (2003).

45. Nishibori, T., Tanabe, Y., Su, L. & David, M. Impaired development of CD4+ CD25+ regulatory T cells in the absence of STAT1: increased susceptibility to autoimmune disease. J. Exp. Med. 199, 25–34 (2004). 46. Bour-Jordan, H. & Bluestone, J.A. CD28 function: a balance of costimulatory and regulatory signals. J. Clin. Immunol. 22, 1–7 (2002). 47. Serra, P. et al. CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells. Immunity 19, 877–889 (2003). 48. Montagnoli, C. et al. B7/CD28-dependent CD4+CD25+ regulatory T cells are essential components of the memory-protective immunity to Candida albicans. J. Immunol. 169, 6298–6308 (2002). 49. Bellocchio, S. et al. The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo. J. Immunol. 172, 3059–3069 (2004). 50. Belladonna, M.L. et al. IL-23 and IL-12 have overlapping, but distinct, effects on murine dendritic cells. J. Immunol. 168, 5448–5454 (2002). 51. Gajewski, T.F., Fallarino, F., Uyttenhove, C. & Boon, T. Tumor rejection requires a CTLA-4 ligand provided by the host or expressed on the tumor: superiority of B7-1 over B7-2 for active tumor immunization. J. Immunol. 156, 2909–2917 (1996). 52. Grohmann, U. et al. IFN-g inhibits presentation of a tumor/self peptide by CD8a dendritic cells via potentiation of the CD8a+ subset. J. Immunol. 165, 1357–1363 (2000). 53. Richard, M., Louahed, J., Demoulin, J.B. & Renauld, J.C. Interleukin-9 regulates NF-kB activity through BCL3 gene induction. Blood 93, 4318–4327 (1999). 54. Grohmann, U. et al. CD8+ cell activation to a major mastocytoma rejection antigen, P815AB: requirement for tum- or helper peptides in priming for skin test reactivity to a P815AB-related peptide. Eur. J. Immunol. 25, 2797–2802 (1995).

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