HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grohmann, U. Articles by Puccetti, P. Search for Related Content PubMed PubMed Citation Articles by Grohmann, U. Articles by Puccetti, P. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-TRYPTOPHAN

Functional Plasticity of Dendritic Cell Subsets as Mediated by CD40 Versus B7 Activation ¹

Department of Experimental Medicine, University of Perugia, Perugia, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Murine dendritic cells (DCs) can present Ag in an immunogenic or tolerogenic fashion, the distinction depending on either the occurrence of specialized DC subsets or the maturation or activation state of the DC. Although DC subsets may be programmed to direct either tolerance or immunity, it is not known whether appropriate environmental stimulation can result in complete flexibility of a basic program. Using splenic CD8^- and CD8⁺ DCs that mediate the respective immunogenic and tolerogenic presentation of self peptides, we show that both the in vivo and in vitro activities of either subset can be altered by ligation of specific surface receptors. Otherwise immunogenic CD8^- DCs become tolerogenic upon B7 ligation by soluble CTLA-4, a maneuver that initiates immunosuppressive tryptophan catabolism. In contrast, CD40 ligation on tolerogenic CD8⁺ DCs makes these cells capable of immunogenic presentation. Thus, environmental conditioning by T cell ligands may alter the default function of DC subsets to meet the needs of flexibility and redundancy.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs) ³ are potent APCs that possess the ability to stimulate naive T cells. They exist as distinct subsets that differ in their lineage affiliation, surface molecule expression, and biological function. These factors seem to determine the T cell-polarizing signals and type of T cell response, namely Th1, Th2, or T regulatory, induced by DCs (1, 2, 3, 4). Over the last several years, the model of DCs as natural adjuvants that promote the immune response to foreign Ags has been modified after the realization that the APCs that are involved in immunity must also be involved in tolerance to self Ags. It has become increasingly clear that DCs play an important role in both central and peripheral tolerance (3, 4). Two general mechanisms have been proposed by which DCs might maintain peripheral tolerance. The first is that a subtype of specialized regulatory DCs is involved (5, 6). The second is that all DCs have a capacity for initiating tolerance or immunity, the distinction depending on the maturation and/or activation state of the DCs (7, 8, 9, 10).

Much evidence suggests now that the capacity of DCs to orchestrate the immune response is not, in large part, an intrinsic quality of the cell, but, rather, it is the result of environmental stimulation. Among the factors that contribute to environmental conditioning of DCs are cytokine milieu (11), ligation of pattern recognition receptors for microbial products (12, 13), dose of Ag (14), and state of maturation (4). An additional level of DC conditioning may be represented by the expression of specific ligands by T cells, which signal the DC through cell-to-cell contact and engagement of surface receptors (15). All these environmental stimuli, either singly or in combination, may alter the presentation pattern of a DC in the steady state and after maturation. For example, splenic mature CD8^- DCs mediate host priming to the tumor/self peptide P815AB (11). In contrast, not only do CD8⁺ DCs inhibit the induction of immunity by the former cells, but they also initiate a P815AB-specific tolerant state that may have the characteristics of either anergy or deletional tolerance depending on the activation state of the CD8⁺ DCs (16, 17). However, these cells show no inhibitory or tolerogenic activity after CD40 ligation (17) or exposure to IL-6 (18) or IL-23 (19).

Regulatory T cells are known to express surface CTLA-4, which mediates suppressive effects via a combination of inhibitory T cell signaling and blockade of the CD28/B7 costimulatory pathway (20, 21). We have recently shown that CTLA-4 may function as a ligand for B7 receptor molecules expressed by DCs, resulting in tolerogenic effects that are mediated by the induction of tryptophan catabolism (22). Thus, CD40 ligand and CTLA-4, the expression of which is reciprocally regulated in T cells (23), might both alter the presentation programs of DC subsets. In this study we provide evidence for a complete functional plasticity of tolerogenic and immunogenic DC subsets, as mediated by the opposing effects of CD40 and B7 engagement on their surface.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and reagents

Female DBA/2J (H-2^d) mice were obtained from Charles River Breeding Laboratories (Calco, Milan, Italy). The source and characteristics of the hamster anti-murine CD40 (HM40-3) mAb used in combination with goat anti-hamster IgG were previously described (17, 18, 24). Neutralizing, affinity-purified sheep anti-mouse IL-12 p70 polyclonal Ab was provided by Genetics Institute (Cambridge, MA). Rat mAb 6B4 (anti-mouse IL-6) and 15A7 (anti-mouse IL-6R) were previously described (18). CTLA-4-Ig was a fusion protein generated from the extracellular domain of murine CTLA-4 and the Fc portion of a murine IgG3, with native IgG3 representing the control treatment (22). The enzyme inhibitor 1-methyl-D,L-tryptophan (1-MT) was purchased from Sigma-Aldrich (Milan, Italy). The P815AB (amino acid sequence LPYLGWLVF) and NRP-A7 (KYNKANAFL) peptides were synthesized and purified as previously described (17, 18). All in vivo studies were performed in compliance with National and Perugia University animal care and use committee guidelines.

DC preparations and treatments and immunization

DCs were prepared and fractionated according to CD11c/CD8 expression using positive selection columns in combination with CD11c and CD8 MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany) and in the presence of EDTA to disrupt DC-T cell complexes, as described previously (18). The recovered cells were >98% CD11c⁺, >99% MHC I-A⁺, >98% B7-2⁺, <0.1% CD3⁺, <0.5% B220⁺, and appeared to consist of 90–95% CD8^- and 5–10% CD8⁺ cells. After cell fractionation, the recovered CD8^- cells were 45% CD4⁺ and typically contained <0.5% contaminating CD8⁺ DCs, whereas the CD8⁺ fraction was made up of >95% CD8⁺ DCs. In all CD40 stimulations (17, 24), DCs were incubated on ice for 10 min in PBS plus 10% mouse serum, for 20 min with hamster anti-mouse CD40 mAb (5 µg/ml) and then overnight at 37°C with goat anti-hamster Ab (5 µg/ml) in Iscove’s medium plus 10% FCS. To check for nonspecific effects of anti-CD40 ligation, appropriate controls included incubation of DCs in the presence of the second Ab alone, which appeared to be devoid of any functional effect. For cytokine neutralization, DCs were subjected to CD40 activation in vitro in the presence of 6B4 and 15A7 mAbs (for IL-6 neutralization; each at 10 µg/ml) or anti-mouse IL-12p70 polyclonal Ab (10 µg/ml) as previously described (18, 24). CD8^- DCs were exposed to 40 µg/ml CTLA-4-Ig or IgG3 for 24 h at 37°C in the presence or the absence of 2 µM 1-MT. For immunization, cells were washed between and after incubations before peptide loading (5 µM, 2 h at 37°C), irradiation, and i.v. injection into recipient hosts. CD8^- (3 x 10⁵) or, where indicated, CD40-modulated CD8⁺ DCs were injected either alone or in combination with 5% CD8⁺ (or CTLA-4-Ig-treated CD8^-) DCs.

Skin test assay

A skin test assay was used for measuring class I-restricted, delayed-type hypersensitivity responses to synthetic peptides as previously described (17, 18). Results were expressed as the increase in footpad weight of peptide-injected footpads over that of vehicle-injected counterparts. Data are the mean ± SD for at least six mice per group. The statistical analysis was performed using Student’s paired t test by comparing the mean weight of experimental footpads with that of control counterparts. The data reported are representative of at least three independent experiments.

Kynurenine assay

Indoleamine 2,3-dioxygenase (IDO) functional activity was measured in vitro in terms of the ability of DCs to metabolize tryptophan to kynurenine, whose concentrations were measured by HPLC as previously described (22).

Th clones and in vitro assays

The P815AB-specific Th1 cell clone F76 and the NRP-A7-specific Th1 cell clone FF3 were derived by limiting dilution of cultured lines generated from the popliteal lymph nodes of DBA/2 mice immunized with P815AB- or NRP-A7-pulsed DCs, respectively, as described previously (23) and were maintained by weekly restimulation of 1 x 10⁵ cells with 5 µM peptide and 6 x 10⁶ irradiated spleen cells in complete medium containing 40 U/ml human rIL-2. Proliferation assays were performed in triplicate in flat-bottom, 96-well microtiter plates in a total volume of 200 µl. Cultures containing T cell clones (5 x 10⁵ cells/well), purified DCs (10⁴ cells/well), and 5 µM P815AB or NRP-A7 peptide were incubated for 48 h at 37°C. The proliferation of T cells was determined as previously described (23). For cytokine determinations, cultures were established using 5 x 10⁴ T cells and 5 x 10³ DCs in a 200-µl volume in the presence of 5 µM P815AB or NRP-A7 peptide, and supernatants were harvested at 24 h for evaluation of IL-2 contents (23). IL-2 titers (mean ± SD of replicate samples) were expressed as units per milliliter, calculated by reference to standard curves.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD40 activation and cytokines either enforce or suppress the presentation programs of DC subsets

The spleens of DBA/2 mice contain a minority fraction (10%) of mature CD8⁺ DCs that mediate the tolerogenic presentation of the synthetic tumor/self nonapeptide P815AB, such that peptide-loaded CD8⁺ DCs initiate durable Ag-specific anergy upon transfer into recipient hosts (25, 26). The addition of as few as 3–5% CD8⁺ DC inhibits the induction of immunity to P815AB by purified CD8^- DCs in the same model system in vivo, when Ag-specific skin test reactivity is measured 2 wk after cell transfer. A series of cytokines, including IL-12 (11), IFN- (16), IL-6 (18), and IL-23 (19), either reinforce or ablate the activities of the two subsets. CD40 activation on CD8⁺ DCs abolishes their tolerogenic potential, and the same maneuver enables CD8^- DCs to overcome inhibition by unconditioned cells of the other subset (17). In line with previous data (24, 27), Fig. 1 shows that both effects are triggered by cytokines acting in an autocrine fashion, with IL-6 mediating the effect of CD40 activation on CD8⁺ DCs, and IL-12 mediating the corresponding effect on CD8^- DCs. These data are consistent with the inflammatory, Th1-promoting, or adjuvant properties of CD40 activation and the associated cytokine response. However, they do not clarify to which extent a default program can be varied besides being blocked or implemented in its expression. This particularly applies to the possible acquisition of tolerogenic properties by CD8^- DCs.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. CD40 activation affects both the priming ability of CD8- DCs and the negative regulatory function of CD8+ DCs in the induction of immunity to P815AB. DCs fractionated according to CD8 expression and pulsed with P815AB in vitro were transferred into recipient mice to be assayed for skin test reactivity to the eliciting peptide. The DC fractions were used either as such (CD8-, CD8+) or after treatment with anti-CD40 mAb for receptor cross-linking (CD8+/CD40:CL, CD8-/CD40:CL). Different combinations of DC fractions were injected as indicated. CD40 cross-linking occurred in the presence or the absence of cytokine-neutralizing Abs (indicated). The skin test assay was performed at 2 wk. *, p < 0.001, experimental vs control footpads.

We have recently proposed a novel model of tolerance induction by CTLA-4, that occurs through B7-dependent signaling in DCs. Using an experimental system of allogeneic islet transplant tolerance, we have shown that B7 engagement by CTLA-4-Ig conditions the DC to produce IFN-. The cytokine acts in an autocrine or paracrine manner to promote induction of the enzyme IDO, which initiates tolerogenic mechanisms dependent on tryptophan catabolism (22, 28). We therefore assayed CTLA-4-Ig for possible effects on the presentation of P815AB by CD8^- DCs. Fig. 2A shows that exposure of these cells to CTLA-4-Ig before peptide loading and transfer into recipient hosts abolished the induction of skin test reactivity. The effect was associated with high level production of IFN- in culture supernatants (i.e., >500 pg/ml at 24 h) in the absence of detectable IL-12 production, which is consistent with previous data of CTLA-4-Ig treatment of unfractionated DCs (22). Also, the effect was due to active suppression involving IDO, as it could be reversed by the addition of the enzyme inhibitor 1-MT during cell exposure to CTLA-4-Ig. The latter treatment and 1-MT also had opposing effects on IDO activity in vitro, as measured by the conversion of tryptophan to kynurenines (Fig. 2B).

View larger version (14K): [in this window] [in a new window]   FIGURE 2. B7 activation confers suppressive properties on CD8- DCs through mechanisms associated with tryptophan catabolism. A, DCs fractionated according to CD8 expression and pulsed with P815AB were transferred into recipient mice to be assayed for skin test reactivity. The CD8- DC fraction was used as such or after treatment with CTLA-4-Ig or control IgG3. Experimental groups included the use of CD8- DCs treated with 2 µM 1-MT during exposure to CTLA-4-Ig. An additional group consisted of the combination of unconditioned CD8- DCs and 5% CD8- DCs treated with CTLA-4-Ig. *, p < 0.001, experimental vs control footpads. B, The functional activity of IDO produced by CD8- DCs in response to CTLA-4-Ig treatment was measured in terms of tryptophan degradation to kynurenine, the levels of which were measured by HPLC. Results are the mean ± SD of triplicate samples.

CTLA-4 ligation of B7 molecules on CD8^- DCs induces specific tolerance

To further explore the effect induced by CD8^- DCs treated with CTLA-4-Ig, we used an experimental design previously adopted to ascertain the nature of the suppressive properties imparted by IFN- to CD8⁺ DCs (17). We studied the impact of a previous exposure to CTLA-4-Ig-treated CD8^- DCs on the priming ability of subsequent, otherwise immunogenic, vaccination to P815AB. Groups of mice were first injected with either CTLA-4-Ig-treated CD8^- DCs or a mixture of unconditioned CD8^- DCs plus 5% CTLA-4-Ig-treated cells, as described for the experiment in Fig. 2A. On day 15 animals received a second cell transfer using peptide-pulsed, unconditioned CD8^- DCs. Mice were finally assayed for skin test reactivity to P815AB after an additional 2 wk. Fig. 3 shows that exposure of mice to CTLA-4-Ig-treated CD8^- DCs resulted in a tolerant state that could not be reversed by the use of unconditioned CD8^- DCs. As unresponsiveness persisted when the second cell transfer was delayed up to 90 days after the tolerogenic priming (data not shown), these findings suggested the occurrence of deletional tolerance initiated by the action of CTLA-4-Ig on CD8^- DCs. This condition appeared to be similar to the state of prolonged unresponsiveness (i.e., deletional tolerance) induced by host transfer with P815AB-pulsed DCs pre-exposed to IFN- (16, 17). Thus, not only will CTLA-4-Ig-treated CD8^- DCs inhibit priming by unconditioned cells of the same subset, but they also initiate a state of durable Ag-specific unresponsiveness.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Peptide-pulsed CD8- DCs treated with CTLA-4-Ig induce Ag-specific tolerance. DCs fractionated according to CD8 expression and pulsed with P815AB were transferred into recipient mice. The CD8- DC fraction was used as such or after treatment with CTLA-4-Ig, as indicated in Fig. 2. On day 15 after primary cell transfer, mice were treated with an otherwise effective priming consisting of P815AB-pulsed CD8- DCs. Mice were assayed for skin test reactivity after an additional 2 wk. Controls included the use of mice injected on day 15 with CD8- DCs pulsed with the antigenically unrelated P91A peptide (26 ) as a second cell transfer, and then assayed after 2 wk for skin test reactivity to the same peptide. *, p < 0.001, experimental vs control footpads.

We have previously shown that CD40 activation in CD8⁺ DCs inhibits the tolerogenic potential of these cells (17). This occurs through induction of autocrine IL-6, which blocks IFN--induced activation of IDO by down-regulating the expression of IFN- receptors on the cell surface (18). We therefore wanted to investigate whether B7 engagement, which results in the release of IFN- (22), and CD40 engagement, which ultimately prevents intracellular signaling of the cytokine (17, 18), would exert reciprocal influence on the suppressive capacity of CD8⁺ DCs. Experiments were conducted using a combination of P815AB-pulsed CD8^- and 5% CD8⁺ DCs in the experimental model illustrated above. The CD8⁺ DCs were either untreated or subjected to B7 and/or CD40 activation. As expected, Fig. 4 shows that CD40 activation blocked the baseline suppressive effect of the CD8⁺ subset. However, the copresence of CTLA-4-Ig during CD40 activation fully restored this activity. Thus, the two maneuvers, CD40 activation and B7 activation, have opposing effects on CD8⁺ DC function, and the impact of B7 activation appears to be dominant when CD8⁺ DCs are coexposed to the cross-linkers in vitro. This could be due to a greater latency in the induction of downstream effects by CD40 activation. In experiments not reported here, we found that sequential, rather than concurrent, exposure to the CD40 cross-linker and CTLA-4-Ig would result in no suppression by the CD8⁺ subset. Of interest, Fig. 4 also shows that when CD8⁺ DCs were coexposed to the CD40 cross-linker and CTLA-4-Ig in the presence of 1-MT, the suppressive activity of these cells was again lost. Therefore, once IDO activity is blocked, the combined effects of CD40 and B7 activation do not result in an effective inhibitory action by the CD8⁺ DC subset.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. CD40 engagement on CD8+ DCs blocks their suppressive activity in the priming to P815AB and NRP-A7, which is abolished by B7 activation. DCs fractionated according to CD8 expression were pulsed with a peptide and transferred into recipient mice to be assayed for reactivity to the eliciting peptide. The CD8+ fraction was used as such or after treatment with the CD40 cross-linker or a combination of CD40 cross-linker and CTLA-4-Ig. Exposure of CD8+ DCs to the CD40 cross-linker and CTLA-4-Ig was also performed in the presence of 2 µM 1-MT. After pulsing with P815AB or NRP-A7, the different fractions were injected in different combinations. *, p < 0.001, experimental vs control footpads.

CD40 engagement on CD8⁺ DCs and B7 engagement on CD8^- DCs alter the immune function of these cells in vitro

The skin test response to P815AB that is triggered by transfer of peptide-pulsed DCs is a class I-restricted response that requires class II-restricted CD4⁺ T cells for afferent induction in vivo (25, 26). Using P815AB-specific CD4⁺ T cell clones, we have recently shown that CD8^- and CD8⁺ DCs manifest differential ability to sustain Th1 cell proliferation and cytokine production in vitro (23). We therefore extended the exam of DC conditioning to this model of secondary response in vitro. We measured the proliferation and IL-2 production of a Th1 clone cultured with either type of DC subset in the presence of P815AB. Fig. 5 shows that the poor response sustained by unconditioned CD8⁺ DCs was converted into a strong response by CD40 activation in the latter cells. In contrast, activation of B7 by CTLA-4-Ig in CD8^- DCs dramatically reduced their ability to stimulate Th1 cell proliferation and IL-2 production. A similar pattern of baseline reactivity and the induction of similar changes by CD40 or B7 activation were observed on assaying the proliferative response and IL-2 production of a Th1 clone specific for NRP-A7 (Fig. 5). Thus, CD40 and B7 activation will produce changes in DC subsets that may alter the immune function of these cells in a primary as well as in a secondary response.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Presentation of peptide Ag by CD8- and CD8+ DCs to the Th1 clones F76 (for P815AB) and FF3 (for NRP-A7). The proliferative response of T cells exposed to peptide-loaded DCs was measured in terms of radiolabel uptake at 48 h. Counts are expressed as the mean counts per minute of replicate samples ± SE. IL-2 production was measured in parallel culture supernatants harvested at 24 h. CD8+ and CD8- DC fractions were used either as such or after CD40 activation (CD8+/CD40:CL) or B7 activation (CD8-/CTLA-4-Ig).

Complete flexibility of DC programs would require that each subset be able to substitute for the other upon appropriate conditioning in vitro. We therefore investigated the combined effects of CD40-activated CD8⁺ DCs and B7-activated CD8^- DCs on the induction of skin test reactivity to P815AB. Fig. 6 shows that CD40-activated CD8⁺ DCs would present P815AB in an immunogenic fashion when transferred into recipient hosts in sufficient amounts (e.g., with an inoculum size similar to that of untreated CD8^- DCs in the induction of immunity, i.e., of at least 10⁵ cells). However, the addition of 5% CD8^- DCs treated with CTLA-4-Ig completely blocked the induction of reactivity by CD40-activated CD8⁺ DCs. Similar to the results in Fig. 3, the coinjection of CD40-activated CD8⁺ DCs and B7-activated CD8^- DCs resulted in a state of specific unresponsiveness that could not be reversed by the injection of unconditioned CD8^- DCs or CD40-activated CD8⁺ DCs for at least 90 days after the first cell transfer (data not shown). This demonstrates that environmental stimulation can condition each subset to mimic the default function of the other subset.

View larger version (11K): [in this window] [in a new window]   FIGURE 6. Cross-regulation of DC subsets after conditioning via CD40 or B7 activation. Otherwise suppressive CD8+ DCs were assayed for priming ability to P815AB following CD40 activation according to the experimental conditions illustrated in Fig. 1. The priming ability of CD40-activated CD8+ DCs (3 x 105) was also assayed in the presence of a minority fraction (5%) of CD8- DCs rendered suppressive by treatment with CTLA-4-Ig, IgG3 representing the control treatment. *, p < 0.001, experimental vs control footpads.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Using self peptides that express class I- and class II-restricted epitopes, we show that appropriate stimuli can alter the default function of DC subsets, resulting in immunity or tolerance according to the type of prevailing environmental conditioning. Not only does CD40 activation enhance priming by CD8^- DCs and ablate suppression by the other subset, as previously reported (17, 18), but it also makes CD8⁺ DCs capable of immunogenic presentation of self peptides. In contrast, CTLA-4-Ig engagement of B7 confers suppressive properties on CD8^- DCs, mimicking the qualitative and quantitative expression of the inhibitory activity of unconditioned CD8⁺ DCs. A significant portion of these regulatory effects is probably mediated by the release of specific cytokines, most notably IL-12 and IFN- from CD8^- DCs (in response to CD40 and B7 activation, respectively) and IL-6 from CD8⁺ DCs (in response to CD40 ligation). The suppressive activity induced by CTLA-4-Ig in CD8^- DCs is dependent upon effective tryptophan catabolism, thus suggesting that the occurrence of IDO-mediated regulatory effects involving the DC is the principal mediator of tolerance (4, 7), as previously described (22).

Although the exact mechanisms by which DC exposure to 1-MT in vitro can affect their activity in vivo once the cells have been separated from the enzyme inhibitor are unclear, we have consistently observed prolonged IDO inhibition in this type of experimental setting. This suggests that the inhibition of IDO by 1-MT is both a time-dependent and slowly reversible phenomenon (17).

One important feature of the biological activity of CD8^- DCs in our model systems with P815AB and NRPA-7 is that the action of this subset, upon conditioning by CTLA-4-Ig treatment, is not limited to impaired priming by unconditioned CD8^- DCs. Similar to the effect of IFN--treated CD8⁺ DCs (17), host transfer with CD8^- DCs exposed to CTLA-4-Ig, either alone or in combination with untreated cells, will result in specific unresponsiveness that is not reversed by a subsequent (i.e., at 90 days), otherwise effective priming with the peptide. Also, the cotransfer of CD40-activated CD8⁺ DCs and B7-activated CD8^- DCs resulted in a state of unresponsiveness that could not be overcome by later transfer of unconditioned CD8^- DCs or CD40-activated CD8⁺ DCs. This indicates that either subset can, under specific conditions, initiate a state of durable tolerance to the peptide.

Several considerations can be made in this regard. First, analogous to the condition of IFN--treated CD8⁺ DCs (17), the effect of CD8^- DCs treated with CTLA-4-Ig appears to be different from the anergic state induced by host transfer with unfractionated DCs pulsed with P815AB, because the latter represents a reversible phenomenon that is no longer observable 40–60 days after tolerogenic priming (26). Second, the recent observation that selected tryptophan catabolites, namely kynurenine derivatives, are strong inducers of apoptosis in T cells is compatible with an important role for DCs as mediators of deletional tolerance occurring by IDO-dependent effects (28, 33). Finally, and perhaps more importantly in the present context, the demonstration of specific tolerance in mice receiving CD8^- DCs exposed to CTLA-4-Ig indicates that B7 activation in these cells is an effective means of inducing a switch between immunity and tolerance. Although switching between different types of immunity has been reported as a result of DC flexibility in directing Th cell development (14), this effect is unlikely to contribute to the tolerant state observed in mice receiving CTLA-4-Ig-treated DCs.

Another interesting observation in our current data may be represented by the opposing effects of CD40 activation and B7 activation on CD8⁺ DC function, with the impact of B7 activation being dominant when CD8⁺ DCs were coexposed to the cross-linkers in vitro. Under such exposure conditions, the cells did produce IL-6 in response to CD40 activation (>1 ng/ml) as well as IFN- in response to CTLA-4-Ig (>600 pg/ml). However, it is possible that a greater latency in the onset of downstream effects by IL-6 would enable the IFN- response driven by B7 activation to prevail in terms of inhibitory activity by the conditioned CD8⁺ subset. This is consistent with the data that sequential, rather than concurrent, exposure to the CD40 cross-linker and CTLA-4-Ig would result in no suppression by the CD8⁺ subset. On the other hand, the observation that CD8⁺ DCs subjected to simultaneous CD40 and B7 activation in the presence of 1-MT will lose their suppressive activity can be taken to indicate that upon blockade of IDO activity, the combined effects of CD40 and B7 activation do not result in suppression by whatever additional/alternative mechanism. Thus, IFN--induced activation of the IDO mechanism appears to be the major effector mode of suppression in this model system. Interestingly, in a series of parallel experiments not reported here, we also found that the concurrent exposure of CD8^- DCs to the CD40 cross-linker and CTLA-4-Ig would lead to tolerance induction to P815AB, with high levels of both IL-12 and IFN- in culture supernatants.

One final observation that may be noteworthy in our study is that opposing effects of CD40 and B7 activation on functional DC subset activity were observed not only with the tumor/self peptide P815AB, but also with a peptide mimotope (NRPA-7) that is recognized by diabetogenic T cells in NOD mice, a prototypic model of autoimmune disease (34, 35, 36, 37). In addition, CD40 and B7 activation were found to produce changes in DC subsets that would alter the immune function of these cells in a primary as well as a secondary response to either P815AB or NRPA-7. This indicates that a similar pattern of functional plasticity of DC subsets may operate in the response to tumor/self Ags as well as in the mechanisms of tolerance in autoimmunity and in the regulation of autoreactive T cells in the periphery. This is consistent with recent data in our laboratory that a defect in tryptophan catabolism impairs tolerance in nonobese diabetic mice (38).

Our current understanding of DC regulation of Th cell differentiation favors the idea that lineage determination of Th cells follows an instructive model, in which the net effect of the dose of Ag, the state of maturation of the DC, and environmental signals will determine whether naive T cells develop into Th1 or Th2 cells (8, 14). Our data indicate that a similar instructive model may dictate whether DCs ultimately present Ags in either an immunogenic or tolerogenic manner, according to the combined effects of maturation and activation state of the DC and environmental signals. Thus, besides default programming, which is necessary to face the challenges of their usual setting, each DC subset can acquire disparate abilities in an instructive mode. This might provide DCs with enough flexibility and sufficient redundancy to ensure that an essential function of the immune system, i.e., eradicating pathogens and preserving tolerance to self (7), occurs under optimal conditions. Among the environmental signals that confer flexibility on DCs, specific ligands expressed by T cells, including CD40 ligand and CTLA-4, may be of critical importance. It is of interest that the DCs capable of cross-priming are CD8⁺ cells (30), and that cross-priming requires CD40 (31, 32).

Th1 and Th2 cells do not seem, in general, to manifest a differential pattern of expression of CD40 ligand vs CTLA-4. However, the recent observation in our laboratory that CD40 ligand and CTLA-4 are reciprocally regulated in their expression by a Th1 clone (23) raises the issue of the effects of T cells, including regulatory T cells, on unconditioned CD8^- and CD8⁺ DCs. Although such possible bidirectional influences are currently being examined in our model, the present data can improve our understanding of the functional plasticity, cooperation, and cross-regulation of DC subsets in light of the cross-talk between these cells and T lymphocytes.

	Footnotes

 
¹ This work was supported by grants from the Juvenile Diabetes Research Foundation International (to U.G.) and the Italian Association for Cancer Research (to P.P.).

² Address correspondence and reprint requests to Dr. Paolo Puccetti, Section of Pharmacology, Department of Experimental Medicine, University of Perugia, 06126 Perugia, Italy. E-mail address: plopcc{at}tin.it

³ Abbreviations used in this paper: DC, dendritic cell; IDO, indoleamine 2,3-dioxygenase; 1-MT, 1-methyl-D,L-tryptophan; NOD, nonobese diabetic.

Received for publication April 11, 2003. Accepted for publication July 8, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
2. Liu, Y. J., H. Kanzler, V. Soumelis, M. Gilliet. 2001. Dendritic cell lineage, plasticity and cross-regulation. Nat. Immunol. 2:585.[Medline]
3. Shortman, K., Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2:151.[Medline]
4. Steinman, R. M., M. C. Nussenzweig. 2002. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc. Natl. Acad. Sci. USA 99:351.[Abstract/Free Full Text]
5. Fazekas de St. Groth, B.. 1998. The evolution of self-tolerance: a new cell arises to meet the challenge of self-reactivity. Immunol. Today 19:448.[Medline]
6. Suss, G., K. Shortman. 1996. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J. Exp. Med. 183:1789.[Abstract/Free Full Text]
7. Shortman, K., W. R. Heath. 2001. Immunity or tolerance? That is the question for dendritic cells. Nat. Immunol. 2:988.[Medline]
8. Dhodapkar, M. V., R. M. Steinman, J. Krasovsky, C. Munz, N. Bhardwaj. 2001. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J. Exp. Med. 193:233.[Abstract/Free Full Text]
9. Roncarolo, M. G., M. K. Levings, C. Traversari. 2001. Differentiation of T regulatory cells by immature dendritic cells. J. Exp. Med. 193:F5.
10. Albert, M. L., M. Jegathesan, R. B. Darnell. 2001. Dendritic cell maturation is required for the cross-tolerization of CD8⁺ T cells. Nat. Immunol. 2:1010.[Medline]
11. Grohmann, U., R. Bianchi, M. L. Belladonna, C. Vacca, S. Silla, E. Ayroldi, M. C. Fioretti, P. Puccetti. 1999. IL-12 acts selectively on CD8^- dendritic cells to enhance presentation of a tumor peptide in vivo. J. Immunol. 163:3100.[Abstract/Free Full Text]
12. Medzhitov, R., C. Janeway, Jr. 2000. Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173:89.[Medline]
13. O’Keeffe, M., H. Hochrein, D. Vremec, I. Caminschi, J. L. Miller, E. M. Anders, L. Wu, M. H. Lahoud, S. Henri, B. Scott, et al 2002. Mouse plasmacytoid cells: long-lived cells, heterogenous in surface phenotype and function, that differentiate into CD8⁺ dendritic cells only after microbial stimulus. J. Exp. Med. 196:1307.[Abstract/Free Full Text]
14. Boonstra, A., C. Asselin-Paturel, M. Gilliet, C. Crain, G. Trinchieri, Y. J. Liu, A. O’Garra. 2003. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential Toll-like receptor ligation. J. Exp. Med. 197:101.[Abstract/Free Full Text]
15. Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4⁺ T-helper and a T-killer cell. Nature 393:474.[Medline]
16. Grohmann, U., R. Bianchi, M. L. Belladonna, S. Silla, F. Fallarino, M. C. Fioretti, P. Puccetti. 2000. IFN- inhibits presentation of a tumor/self peptide by CD8^- dendritic cells via potentiation of the CD8⁺ subset. J. Immunol. 165:1357.[Abstract/Free Full Text]
17. Grohmann, U., F. Fallarino, S. Silla, R. Bianchi, M. L. Belladonna, C. Vacca, A. Micheletti, M. C. Fioretti, P. Puccetti. 2001. CD40 ligation ablates the tolerogenic potential of lymphoid dendritic cells. J. Immunol. 166:277.[Abstract/Free Full Text]
18. Grohmann, U., F. Fallarino, R. Bianchi, M. L. Belladonna, C. Vacca, C. Orabona, C. Uyttenhove, M. C. Fioretti, P. Puccetti. 2001. IL-6 inhibits the tolerogenic function of CD8⁺ dendritic cells expressing indoleamine 2, 3-dioxygenase. J. Immunol. 167:708.[Abstract/Free Full Text]
19. Belladonna, M. L., J. C. Renauld, R. Bianchi, C. Vacca, F. Fallarino, C. Orabona, M. C. Fioretti, U. Grohmann, P. Puccetti. 2002. IL-23 and IL-12 have overlapping, but distinct, effects on murine dendritic cells. J. Immunol. 168:5448.[Abstract/Free Full Text]
20. Bluestone, J. A.. 1997. Is CTLA-4 a master switch for peripheral T cell tolerance?. J. Immunol. 158:1989.[Abstract]
21. Egen, J. G., M. S. Kuhns, J. P. Allison. 2002. CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat. Immunol. 3:611.[Medline]
22. Grohmann, U., C. Orabona, F. Fallarino, C. Vacca, F. Calcinaro, A. Falorni, P. Candeloro, M. L. Belladonna, R. Bianchi, M. C. Fioretti, et al 2002. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3:1097.[Medline]
23. Fallarino, F., U. Grohmann, C. Vacca, R. Bianchi, M. C. Fioretti, P. Puccetti. 2002. CD40 ligand and CTLA-4 are reciprocally regulated in the Th1 cell proliferative response sustained by CD8⁺ dendritic cells. J. Immunol. 169:1182.[Abstract/Free Full Text]
24. Bianchi, R., U. Grohmann, C. Vacca, M. L. Belladonna, M. C. Fioretti, P. Puccetti. 1999. Autocrine IL-12 is involved in dendritic cell modulation via CD40 ligation. J. Immunol. 163:2517.[Abstract/Free Full Text]
25. Bianchi, R., U. Grohmann, M. L. Belladonna, S. Silla, F. Fallarino, E. Ayroldi, M. C. Fioretti, P. Puccetti. 1996. IL-12 is both required and sufficient for initiating T cell reactivity to a class I-restricted tumor peptide (P815AB) following transfer of P815AB-pulsed dendritic cells. J. Immunol. 157:1589.[Abstract]
26. Grohmann, U., R. Bianchi, E. Ayroldi, M. L. Belladonna, D. Surace, M. C. Fioretti, P. Puccetti. 1997. A tumor-associated and self antigen peptide presented by dendritic cells may induce T cell anergy in vivo, but IL-12 can prevent or revert the anergic state. J. Immunol. 158:3593.[Abstract]
27. Grohmann, U., M. L. Belladonna, R. Bianchi, C. Orabona, E. Ayroldi, M. C. Fioretti, P. Puccetti. 1998. IL-12 acts directly on DC to promote nuclear localization of NF-B and primes DC for IL-12 production. Immunity 9:315.[Medline]
28. Fallarino, F., U. Grohmann, C. Vacca, R. Bianchi, C. Orabona, A. Spreca, M. C. Fioretti, P. Puccetti. 2002. T cell apoptosis by tryptophan catabolism. Cell Death Differ. 9:1069.[Medline]
29. Belz, G. T., G. M. Behrens, C. M. Smith, J. F. Miller, C. Jones, K. Lejon, C. G. Fathman, S. N. Mueller, K. Shortman, F. R. Carbone, et al 2002. The CD8⁺ dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens. J. Exp. Med. 196:1099.[Abstract/Free Full Text]
30. den Haan, J. M., S. M. Lehar, M. J. Bevan. 2000. CD8⁺ but not CD8^- dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192:1685.[Abstract/Free Full Text]
31. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478.[Medline]
32. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480.[Medline]
33. Grohmann, U., F. Fallarino, P. Puccetti. 2003. Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol. 24:242.[Medline]
34. Anderson, B., B. J. Park, J. Verdaguer, A. Amrani, P. Santamaria. 1999. Prevalent CD8⁺ T cell response against one peptide/MHC complex in autoimmune diabetes. Proc. Natl. Acad. Sci. USA 96:9311.[Abstract/Free Full Text]
35. Amrani, A., J. Verdaguer, P. Serra, S. Tafuro, R. Tan, P. Santamaria. 2000. Progression of autoimmune diabetes driven by avidity maturation of a T-cell population. Nature 406:739.[Medline]
36. Delovitch, T. L., B. Singh. 1997. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7:727.[Medline]
37. Atkinson, M. A., E. H. Leiter. 1999. The NOD mouse model of type 1 diabetes: as good as it gets?. Nat. Med. 5:601.[Medline]
38. Grohmann, U., F. Fallarino, R. Bianchi, C. Orabona, C. Vacca, M. C. Fioretti, P. Puccetti. 2003. A defect in tryptophan catabolism impairs tolerance in nonobese diabetic mice. J. Exp. Med. 198:153.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. L. Belladonna, U. Grohmann, P. Guidetti, C. Volpi, R. Bianchi, M. C. Fioretti, R. Schwarcz, F. Fallarino, and P. Puccetti Kynurenine Pathway Enzymes in Dendritic Cells Initiate Tolerogenesis in the Absence of Functional IDO J. Immunol., July 1, 2006; 177(1): 130 - 137. [Abstract] [Full Text] [PDF]

				C. Orabona, P. Puccetti, C. Vacca, S. Bicciato, A. Luchini, F. Fallarino, R. Bianchi, E. Velardi, K. Perruccio, A. Velardi, et al. Toward the identification of a tolerogenic signature in IDO-competent dendritic cells Blood, April 1, 2006; 107(7): 2846 - 2854. [Abstract] [Full Text] [PDF]

				M. Delgado, E. Gonzalez-Rey, and D. Ganea The Neuropeptide Vasoactive Intestinal Peptide Generates Tolerogenic Dendritic Cells J. Immunol., December 1, 2005; 175(11): 7311 - 7324. [Abstract] [Full Text] [PDF]

				F. Fallarino, C. Orabona, C. Vacca, R. Bianchi, S. Gizzi, C. Asselin-Paturel, M. C. Fioretti, G. Trinchieri, U. Grohmann, and P. Puccetti Ligand and cytokine dependence of the immunosuppressive pathway of tryptophan catabolism in plasmacytoid dendritic cells Int. Immunol., November 1, 2005; 17(11): 1429 - 1438. [Abstract] [Full Text] [PDF]

				D. Braun, R. S. Longman, and M. L. Albert A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation Blood, October 1, 2005; 106(7): 2375 - 2381. [Abstract] [Full Text] [PDF]

				X. Zhang, H. Huang, J. Yuan, D. Sun, W.-S. Hou, J. Gordon, and J. Xiang CD4-8- Dendritic Cells Prime CD4+ T Regulatory 1 Cells to Suppress Antitumor Immunity J. Immunol., September 1, 2005; 175(5): 2931 - 2937. [Abstract] [Full Text] [PDF]

				J. K. H. Tan and H. C. O'Neill Maturation requirements for dendritic cells in T cell stimulation leading to tolerance versus immunity J. Leukoc. Biol., August 1, 2005; 78(2): 319 - 324. [Abstract] [Full Text] [PDF]

				M. Rossi and J. W. Young Human Dendritic Cells: Potent Antigen-Presenting Cells at the Crossroads of Innate and Adaptive Immunity J. Immunol., August 1, 2005; 175(3): 1373 - 1381. [Abstract] [Full Text] [PDF]

				C. Orabona, M. L. Belladonna, C. Vacca, R. Bianchi, F. Fallarino, C. Volpi, S. Gizzi, M. C. Fioretti, U. Grohmann, and P. Puccetti Cutting Edge: Silencing Suppressor of Cytokine Signaling 3 Expression in Dendritic Cells Turns CD28-Ig from Immune Adjuvant to Suppressant J. Immunol., June 1, 2005; 174(11): 6582 - 6586. [Abstract] [Full Text] [PDF]

				F. Fallarino, R. Bianchi, C. Orabona, C. Vacca, M. L. Belladonna, M. C. Fioretti, D. V. Serreze, U. Grohmann, and P. Puccetti CTLA-4-Ig Activates Forkhead Transcription Factors and Protects Dendritic Cells from Oxidative Stress in Nonobese Diabetic Mice J. Exp. Med., October 18, 2004; 200(8): 1051 - 1062. [Abstract] [Full Text] [PDF]

				C. Haase, K. Skak, B. K. Michelsen, and H. Markholst Local Activation of Dendritic Cells Leads to Insulitis and Development of Insulin-Dependent Diabetes in Transgenic Mice Expressing CD154 on the Pancreatic {beta}-Cells Diabetes, October 1, 2004; 53(10): 2588 - 2595. [Abstract] [Full Text] [PDF]

				F. Fallarino, C. Asselin-Paturel, C. Vacca, R. Bianchi, S. Gizzi, M. C. Fioretti, G. Trinchieri, U. Grohmann, and P. Puccetti Murine Plasmacytoid Dendritic Cells Initiate the Immunosuppressive Pathway of Tryptophan Catabolism in Response to CD200 Receptor Engagement J. Immunol., September 15, 2004; 173(6): 3748 - 3754. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grohmann, U. Articles by Puccetti, P. Search for Related Content PubMed PubMed Citation