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IL-6 Inhibits the Tolerogenic Function of CD8⁺ Dendritic Cells Expressing Indoleamine 2,3-Dioxygenase¹

Ursula Grohmann^*, Francesca Fallarino^*, Roberta Bianchi^*, Maria Laura Belladonna^*, Carmine Vacca^*, Ciriana Orabona^*, Catherine Uyttenhove, Maria Cristina Fioretti^* and Paolo Puccetti^2,*

^* Department of Experimental Medicine, University of Perugia, Perugia, Italy; and Ludwig Institute for Cancer Research, Brussels, Belgium

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The outcome of dendritic cell (DC) presentation of tumor and/or self peptides, including P815AB (a tumor peptide of murine mastocytoma cells) and NRP-A7 (a synthetic peptide mimotope recognized by diabetogenic T cells), may depend on a balance between the activities of immunogenic (CD8^-) and tolerogenic (CD8⁺) DC. By virtue of their respective actions on CD8^- and CD8⁺ DC, IL-12 and IFN- have functionally opposing effects on peptide presentation by the CD8^- DC subset, and IFN--activated CD8⁺ DC mediate tolerogenic effects that prevail over the adjuvant activity of IL-12 on CD8^- DC. We have previously shown that CD40 ligation abrogates the tolerogenic potential of CD8⁺ DC, an effect associated with an impaired capacity of the CD40-modulated and IFN--treated DC to degrade tryptophan and initiate T cell apoptosis in vitro. We report here that IL-6 may both replace (upon administration of the recombinant cytokine) and mediate (as assessed by the use of neutralizing Abs) the effect of CD40 ligation in ablating the tolerogenic activity of CD8⁺ DC. The activity of IL-6 includes down-regulation of IFN-R expression in the CD8⁺ DC subset and correlates to a reduced ability of these cells to metabolize tryptophan and initiate T cell apoptosis in vitro.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Using an in vivo model of tumor/self peptide presentation for induction of class I-restricted skin test reactivity (1, 2), we have previously shown that CD8⁺ dendritic cells (DC)³ negatively regulate the induction of T cell reactivity by CD8^- DC presenting P815AB, a nonameric peptide related to a major rejection Ag of murine mastocytoma cells (3), or NRP-A7, a synthetic peptide that acts as a mimotope for autoimmune diabetes in nonobese diabetic (NOD) mice (4, 5). However, CD8^- DC can be primed by IL-12 to overcome inhibition by the CD8⁺ subset and initiate immunogenic presentation in vivo when the two types of peptide-pulsed DC are cotransferred into recipient hosts (6, 7, 8). Although IFN- enhances the tolerogenic activity of CD8⁺ DC on Ag presentation by the other subset, CD40 activation on the former cells will abolish their tolerogenic capacity or even trigger the potential for immunogenic presentation of P815AB (9, 10). This effect is accompanied in vitro by an impaired ability of the CD40-modulated and IFN--treated CD8⁺ DC to produce indoleamine 2,3-dioxygenase (IDO) and to initiate apoptosis of Ag-specific CD4⁺ T cells (10).

Much evidence indicates that DC can present Ag in an immunogenic or tolerogenic fashion and that autoimmunity can be primed by DC (11, 12, 13). Many autoimmune states involve an imbalance of cytokines, and it has been proposed that DC may induce autoimmunity because of intrinsic defects in genes controlling DC function or following their differentiation by certain cytokines (14). In several experimental models, IL-6 appears to be required for the development of Ag- or collagen-induced arthritis (15, 16), myelin oligodendrocyte protein-induced experimental autoimmune encephalomyelitis (17, 18, 19, 20), and autoantibodies against DNA in pristane-induced systemic lupus erythematosus (21). However, how IL-6 acts to induce disease whereas TNF- (22) and IFN- (23, 24, 25) can, in at least some cases, protect against autoimmunity is not clear (14). Regarding the possible role of IFN-, we have previously proposed that the cytokine acting on CD8⁺ DC might contribute to the maintenance of T cell tolerance to self Ags via tryptophan degradation affecting T cell responses (8, 10).

We have adopted an experimental design relying on the in vitro treatment of DC with cytokines before their transfer into recipient hosts to confine the analysis of cytokine effects to DC in the face of the high pleiotropy of these molecules in vivo. Our earlier studies were largely based on the use of P815AB, which was shown by us to possess both class I- and class II-restricted epitopes (3), such that transfer of peptide-pulsed DC would result in an IL-12-dependent response in the host requiring the presence of CD4⁺ T cells (1, 2). However, because of the low intrinsic immunogenicity of the peptide, effective priming in vivo called for the use of added adjuvanticity in vitro (2, 6, 7) or ablation of the tolerogenic effect of a specific subset of splenic DC (8, 10). In the present study we have further explored the issue of cytokine regulation of Ag presentation in an immunogenic vs tolerogenic fashion as resulting from the combined actions of different DC subsets. We demonstrate that, similar to CD40 activation, and largely mediating the effect of this maneuver, IL-6 will abolish the susceptibility of CD8⁺ DC to the tolerogenic activity of IFN- in the induction of T cell reactivity to P815AB or NRP-A7. These data strengthen the hypothesis that the DC system can present tumor/self Ags in an immunogenic or tolerogenic fashion depending on the cytokine balance and that IL-6 may be a major cytokine involved in the induction of autoimmunity by DC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice, cytokines, and reagents

DBA/2J (H-2^d) mice were obtained from Charles River Laboratories (Calco, Milan, Italy). Male mice were used at the age of 2–4 mo. The source and characteristics of murine rIL-12 (6), murine rIFN- (8), hamster anti-murine CD40 (HM40-3) mAb used in combination with goat anti-hamster IgG (9, 10), and biotinylated rat IgG to murine IFN-R -chain (10) were previously described. IL-12 was 98.8% pure, as assessed by SDS-PAGE, and endotoxin contamination was <0.9 endotoxin U/mg on Limulus amebocyte assay. Murine rIL-6 (10⁹ U/mg) was produced in the baculovirus system, as previously described (26, 27). Endotoxin was removed from all solutions containing rIL-12, anti-CD40 Abs, or rIL-6 with Detoxi-gel (Pierce, Rockford, IL), resulting in endotoxin contamination below the detection limit (0.05 endotoxin U/ml) of the assay (Coatest Endotoxin, Chromogenix AB, Molndal, Sweden) (9). Rat monoclonal 6B4 (anti-mouse IL-6) and 15A7 (anti-mouse IL-6R) Abs were previously described (28, 29). The enzyme inhibitor 1-methyl-DL-tryptophan (1-MT) waspurchased from Aldrich (Milan, Italy).

Peptides

P815AB (amino acid sequence LPYLGWLVF) and NRP-A7 (KYNKANAFL) were synthesized on solid phase using F-moc for transient N-terminal protection, purified by means of reverse phase HPLC, and characterized by amino acid analysis. NRP-A7 is an alanine mutant analog, with superior agonistic properties, of NRP, a synthetic peptide mimotope recognized in the context of H-2K^d class I molecules by a prevailing, pancreatic -cell-specific T lymphocyte population in NOD mice (4, 5).

DC purification

DC were prepared and fractionated according to CD11c/CD8 expression using positive selection columns in combination with CD11c and CD8 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described (6, 7, 8). Briefly, DC were obtained from collagenase-treated spleens (collagenase type IV, Sigma, St. Louis, MO). Total spleen cells were treated with EDTA to disrupt DC-T cell complexes according to a previous described procedure (30), and EDTA was also present in subsequent steps involving the use of positive selection columns. Cells were resuspended in a 1.080 g/cm³ isoosmotic Nycodenz medium (Sigma, St. Louis, MO), and centrifuged at 3000 rpm for 15 min at 4°C. The low density fraction at the interface was collected and washed several times. The recovered cells were incubated with CD11c microbeads and separated using a positive selection column. Cells were then resuspended in RPMI medium supplemented with 10% FCS and allowed to adhere for 2 h; this was followed by an additional 18-h incubation to allow DC to detach. The recovered cells were routinely 96–98% CD11c⁺, >99% I-A⁺, >98% B7-2⁺, and <0.1% CD3⁺ and appeared to consist of 90–95% CD8^- and 5–10% CD8⁺ cells. For preparation of CD8⁺ and CD8^- fractions, the purified DC were separated using a positive selection column and CD8 microbeads. After cell fractionation the recovered CD8^- cells typically contained <0.5% contaminating CD8⁺ DC, whereas the CD8⁺ fraction was made up of >90% CD8⁺ DC.

DC treatments and immunization

Cytokine treatments of DC were performed at 37°C by incubation with 100 ng/ml rIL-12, 200 U/ml rIFN-, or 10 ng/ml rIL-6 for 18 h before peptide pulsing, unless otherwise stated. Control cultures were incubated with medium alone. In all CD40 stimulations (9, 10), DC 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. CD40 ligation on DC routinely involved the use of the second cross-linking Ab, as the latter appears to be necessary for effective DC activation. To check for possible nonspecific effects of anti-CD40 ligation, appropriate controls included incubation of the CD8⁺ DC in the presence of the second Ab alone, which treatment appeared to be devoid of any functional effect. As an additional control, isotype-matched anti-mouse H-2K^d mAb 31-3-4S, capable of binding to DC, was also used in place of the primary anti-CD40 reagent (9). For IL-6 neutralization, DC were subjected to CD40 activation in vitro in the presence of 6B4 (anti-IL-6) and 15A7 (anti-IL-6R) mAbs, each at 10 µg/ml. In all experiments, appropriate controls for anti-IL-6 treatments included the use of isotype-matched mAbs in the place of 6B4 and 15A7 mAbs. In general, preincubation of the DC with cytokines or anti-CD40 Abs significantly reduced their viability, with the CD8⁺ subset being apparently more affected than the CD8^- subset. For most experiments, the viability of the CD8^- DC that were used for in vivo immunization would exceed 90% and that of CD8⁺ DC would exceed 50%. For some experiments, nonviable cells in the CD8⁺ DC fraction were removed by selecting low density viable cells using a Ficoll centrifugation procedure. Cells were washed between and after the incubations before peptide loading (5 µM, 2 h at 37°C), irradiation, and i.v. injection into recipient hosts of 3 x 10⁵ CD8^- DC, either alone or in combination with 3% CD8⁺ DC. Appropriate controls included the use of rTNF- (8) and baculovirus-derived IL-9 (31) to check for possible nonspecific effects of cytokine treatment, and none was found.

Skin test assay

A skin test assay was employed for measuring class I-restricted delayed-type hypersensitivity responses to P815AB and NRP-A7, as previously described (1, 2, 3). Measurements were made in a blind fashion, and 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 (1, 2, 3). The data reported are from representative experiments, and experiments with similar results were performed three to six times.

Cytofluorometric analysis

Surface expression of the IFN-R -chain was performed using biotinylated rat IgG to murine CD119 (clone GR20; PharMingen, San Diego, CA) in conjunction with avidin-FITC. Biotinylated rat IgG2a (PharMingen) was used as an isotype-matched control mAb.

Kynurenine assay

The kynurenine assay was performed as previously described (10). Briefly, CD8⁺ DC, either untreated or treated with different agents, were washed and resuspended in HBSS containing 100 µM tryptophan (Sigma, St. Louis, MO). Cells were incubated for an additional 4 h at 37°C, followed by harvest of supernatant that was stored at -80°C before quantitation of kynurenine by HPLC. IDO activity was expressed as the concentration (micromoles per liter kynurenine) in the sample.

Induction and assay of apoptosis

DC (2.5 x 10⁵) treated overnight with IL-6 and/or IFN- were washed and cultured for 3 days with 5 x 10⁵ F76 cells, a Th1-type P815AB-specific CD4⁺ T cell clone (10), in the presence of 5 µM P815AB peptide. At the end of the coculture the CD11c⁺ cells were removed by the use of CD11c microbeads (Miltenyi Biotec), and the remainder of the population was surface stained with anti-CD4-PE and FITC-labeled annexin V and propidium iodide (PI; PharMingen). For measurement of apoptosis, a gate was set on CD4⁺ T cells, and the percentage of cells in the very early stages of apoptosis was determined by annexin V staining excluding PI⁺ cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD40 ligation inhibits IFN- effects on CD8⁺ DC expressing IDO activity

To confirm and expand our previous observations with P815AB and NRP-A7 (10), we conducted parallel experiments aimed at ascertaining any regulatory function of CD40 activation on CD8⁺ DC treated with IFN- and assayed for suppressive activity. The effect of CD40 activation on IDO induction by IFN- was also investigated. Recipient mice were injected with CD8^- DC or a combination of CD8^- and 3% CD8⁺ DC pulsed with P815AB or NRP-A7. Each DC fraction was used either as such or after cytokine (IL-12 or IFN-) treatment in vitro. Groups of CD8⁺ DC were exposed to CD40 activation as described in Materials and Methods before IFN- treatment (200 U/ml for 6 h). After 2 wk recipients of DC transfer were challenged intrafootpad with the appropriate peptide. Fig. 1A shows that CD40 activation of CD8⁺ DC blocked the IFN--induced inhibition of CD8^- DC presentation of P815AB or NRP-A7. Thus, similar to the pattern of P815AB, negative modulation of CD8^- DC presentation of NRP-A7 is induced by IFN- acting on CD8⁺ DC, an effect that can be prevented by CD40 activation in the latter cells. In addition, Fig. 1B shows that CD40 activation blocked IDO induction by IFN- in CD8⁺ DC. The effect was quantitatively similar to that observed with the use of the enzyme inhibitor 1-MT.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. CD40 activation affects the negative regulatory role of CD8+ DC in vivo and tryptophan metabolism in vitro as induced by IFN-. A, DC fractionated according to CD8 expression and pulsed with P815AB or NRP-A7 were transferred into recipient mice to be assayed for skin test reactivity to the eliciting peptide. The DC fractions were used as such or after treatment with IL-12, IFN-, anti-CD40 mAb, or a combination of anti-CD40 mAb and IFN-. Different combinations of DC fractions were injected as indicated. The skin test assay was performed at 2 wk. *, p < 0.001, experimental vs control footpads. B, The functional activity of IDO produced by activated CD8+ DC was measured in terms of tryptophan degradation to kynurenine. CD8+ DC were exposed sequentially to anti-CD40 mAb and IFN-. Kynurenine levels were measured by HPLC. Results are the mean ± SD of triplicate samples. Controls included the use of CD8+ DC treated with 2 µM 1-MT during exposure to IFN-. Data shown are from one of three experiments with similar results.

IL-6 blocks the effect of IFN- on CD8⁺ DC

In several experimental models it has been shown that IL-6 acts to induce autoimmune disease and that the cytokine may exert effects on DC that could contribute to the priming of autoimmunity (14). Therefore, we wanted to investigate whether exposure of CD8⁺ DC to IL-6 might affect their negative regulatory function in the priming to P815AB or NRP-A7. Recipient mice were injected with a combination of peptide-pulsed CD8^- and 3% CD8⁺ DC. Each DC fraction was used either as such or after cytokine treatment in vitro. Fig. 2 shows that IL-6 treatment of CD8⁺ DC blocked their inhibitory function on peptide presentation by the CD8^- subset. In addition, this maneuver abolished the effect of IFN- on CD8⁺ DC when these cells were exposed sequentially to IL-6 (18 h) and IFN- (6 h) before peptide pulsing. Fig. 2 also shows that combined treatment of mice with IL-12-treated CD8^- DC and IL-6-treated CD8⁺ DC would not result in increased reactivity in vivo to P815AB or NRP-A7, according to a pattern previously observed on testing the effect of combined treatment of DC with IL-12 and anti-CD40 agonistic mAb (9). In experiments not reported here we also found that the modulatory effect of IL-6 was lost when the CD8⁺ DC were exposed concurrently, rather than sequentially, to IL-6 and IFN- for 18 h.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. IL-6 abrogates the negative regulatory role of CD8+ DC either as such or activated by IFN-. DC fractionated according to CD8 expression were transferred into recipient mice to be assayed for reactivity to P815AB or NRP-A7. The DC fractions were used as such or after treatment with IL-12, IFN-, IL-6, or a combination of IL-6 and IFN-. After pulsing with P815AB and NRP-A7, the different fractions were injected in different combinations. *, p < 0.001, experimental vs control footpads.

Because the effect of rIL-6 on the basal or IFN--induced suppressive activity of CD8⁺ DC appeared to be similar to that of CD40 activation, we examined whether induction of IL-6 might be involved in the modulatory properties of CD40 activation. In preliminary experiments we found that triggering of CD40 would result in the production of high levels of IL-6 (up to 5 ng/ml) by both CD8⁺ and CD8^- DC. We thus examined the effect of IL-6-neutralizing Abs added to cultures of CD8⁺ DC treated with CD40-stimulating Abs. Mixtures of CD8^- and CD8⁺ DC pulsed with P815AB or NRP-A7, as illustrated above, were injected into recipient mice. Each DC fraction was used either as such or after cytokine (IL-12 or IFN-) treatment. Groups of CD8⁺ DC were also exposed to CD40 agonistic mAb either alone or in combination with anti-IL-6 mAbs. Fig. 3 shows that the presence of IL-6-neutralizing Abs would completely block the effect of CD40 activation in triggering reactivity to P815AB or NRP-A7. An absolute requirement for IL-6 induced by CD40 activation appeared to be evident both in the modulation of the basal inhibitory activity of CD8⁺ DC and in the reversal of the effect mediated by IFN-. It is interesting that the ability of IL-6 neutralization to ablate the effect of CD40 activation argues against the possibility that selective death or paralysis of CD8⁺ DC in culture is a major mechanism through which CD40 ligation exerts its effect. In addition, we have previously demonstrated that CD40 ligation renders CD8⁺ DC capable of presenting P815AB in an immunogenic fashion (10). Furthermore, in experiments not reported here we have obtained evidence that both CD40 ligation and IL-6 treatment up-regulate the expression of B7-1 in CD8⁺ DC. Therefore, differential cell death in culture and the mere selection of nonsuppressive cells are unlikely to underlie the apparent loss of suppressive function resulting from CD40 ligation or treatment with IL-6 in vitro.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. IL-6 mediates the effect of CD40 activation on CD8+ DC either as such or activated by IFN-. CD8- and CD8+ DC were transferred into recipient mice to be assayed for skin test reactivity to P815AB or NRP-A7. The different fractions were used as such or after treatment with IL-12, IFN-, anti-CD40 mAb, anti-CD40 mAb plus IFN-, or a combination of anti-CD40, anti-IL-6, and IFN-. The skin test was performed at 2 wk. *, p < 0.001, experimental vs control footpads.

IL-6 mediates the antagonistic effect of CD40 activation on IDO induction by IFN-

We have previously shown that the effect triggered by IFN- in CD8⁺ DC involves interference with tryptophan metabolism in vivo upon transfer of P815AB-pulsed DC (8). Furthermore, CD40 activation prevents the effect of IFN- on IDO expression by CD8⁺ DC (10). Therefore, we sought to determine whether IL-6 might interfere with IDO activity as induced by IFN- and whether the former cytokine might be involved in the effect of CD40 activation. The functional activity of IDO produced by activated CD8⁺ DC was measured in terms of its ability to metabolize tryptophan to kynurenine. CD8⁺ DC were treated with IFN- or exposed sequentially to IL-6 (18 h) and IFN- (6 h). Alternatively, the CD8⁺ DC were exposed sequentially to a combination of anti-CD40 mAb plus anti-IL-6 for 18 h, followed by IFN- treatment. Fig. 4 shows that IL-6 would greatly impair the effect of IFN- on IDO production. Furthermore, IL-6 neutralization ablated the effect of CD40 modulation on IDO induction by IFN-.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Effect of IL-6 on functional activity of IDO produced by CD8+ DC in response to IFN-. CD8+ DC were exposed sequentially to IL-6 and IFN-. The effect of IL-6 neutralization was also examined upon CD40 ligation followed by IFN- treatment in CD8+ DC. Kynurenine levels were measured by HPLC. Results are the mean ± SD of replicate samples in one of five experiments.

Down-regulation of IFN-R expression by IL-6

We have previously shown that CD40 activation on CD8⁺ DC negatively regulates the expression of the IFN-R. We therefore examined any possible effect of IL-6 on this expression. We assayed the surface expression of the IFN-R -chain by flow cytometry using biotinylated rat IgG to murine CD119. CD8^- DC were included as a control because CD40 activation on these cells has previously been shown to lack any effect on IFN-R expression. Fig. 5 shows that IL-6 resulted in a marked decrease in the -chain of the IFN-R. This effect was in contrast with that on CD8^- DC, where no significant changes were observed.

View larger version (72K): [in this window] [in a new window]   FIGURE 5. IL-6 down-modulates IFN-R expression in CD8+ DC. CD8+ and CD8- DC were treated with IL-6 for 48 h before Cytofluorometric analysis. Control cultures (C) consisted of freshly harvested cells unexposed to IL-6. The filled histograms indicate cultures treated with isotype-matched Ab. Additional controls (data not shown) included cultures assayed over time in the absence of IL-6 treatment, which showed no detectable changes in CD119 expression. One experiment representative of three is shown.

Antagonistic effects of IL-6 and IFN- on induction of apoptosis by CD8⁺ DC

We have previously suggested that IDO might contribute to the negative regulatory and tolerogenic properties of IFN- via induction of T cell apoptosis in our model system with P815AB. Using a P815AB-specific CD4⁺ T cell clone cultured with DC treated with IFN-, we have found that CD40 activation may block the induction of T cell apoptosis by CD8⁺ DC (10). We therefore became interested in ascertaining any possible regulatory activity of IL-6 on T cell apoptosis mediated by IFN--activated CD8⁺ DC. The P815AB-specific CD4⁺ T cell clone F76 was used for measurement of apoptosis upon 24- or 72-h coculture of the latter cells with CD8⁺ DC exposed to IFN- either alone or in combination with IL-6. No significant apoptosis was observed in the 24-h cocultures (data not shown). In contrast, Fig. 6 shows that the coculture of clone F76 cells and CD8⁺ DC for 72 h in the presence of P815AB resulted in apoptosis of approximately 10% CD4⁺ cells. This effect could be explained at least in part by the production of IFN- by the Th1 clone cells, and in fact, the proportion of apoptotic T cells in the absence of DC would not exceed 5% (data not shown). Upon exposure of the CD8⁺ DC to rIFN- (6 h), the proportion of apoptotic cells rose to >30%. However, sequential exposure of the CD8⁺ DC to IL-6 (18 h) and IFN- (6 h) ablated the modulatory role of externally added IFN-. The effect of IL-6 was quantitatively similar to that observed upon addition of the competitive inhibitor of IDO, 1-MT, to the cocultures of DC and CD4⁺ T cells. These data suggest that IL-6 may act through modulation of IDO induction by IFN- in CD8⁺ DC to regulate T cell apoptosis. It is interesting to note that although annexin V staining measures the very early stages of apoptosis, the generation of effective proapoptotic signals in the cocultures required prolonged exposure of the CD4⁺ T cells to CD8⁺ DC in the presence of cognate Ag, as no apoptosis was observed at 24 h. This suggests that temporally distinct events contribute to activation of the apoptotic response in T cells.

View larger version (41K): [in this window] [in a new window]   FIGURE 6. Effect of IL-6 on IDO-dependent apoptosis mediated by CD8+ DC. Apoptosis was measured in Ag-specific CD4+ T cells cultured with CD8+ DC preexposed to IFN- or a combination of IL-6 and IFN-. Controls included cocultures established in the presence of 2 µM 1-MT. Representative dot blots are shown of annexin V staining of gated CD4+ PI- cells. Numbers within boxes indicate percentages of CD4+ apoptotic cells in one of four experiments with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

DC could be regarded as a multilineage system of leukocytes with variable function rather than as a homogenous cell type with predetermined functional properties (35, 36). Because the DC system as a whole can present Ags in an immunogenic or tolerogenic fashion, it is possible that the outcome of an immune response initiated by DC depends on a combination of several factors, including Ag presentation by a specific type of DC, the nature of the DC-activating signal, and the stage of DC maturation (37). IL-6 could be acting at several levels to regulate DC maturation (38) and function (39). Although CD8⁺ DC function has been associated with the development of Th1 responses to foreign Ags (40, 41), the protection against potential autoreactivity within inflammatory contexts dominated by IFN- is also believed to involve specific actions of the cytokine on DC (14, 42), which might be able to discriminate self from nonself (8).

While studying the induction of immunity vs tolerance to tumor and/or self peptides, we have previously found that the outcome of DC presentation of P815AB (a tumor/self peptide) and NRP-A7 (a peptide mimotope for autoimmune diabetes) depends on a balance between the respective immunogenic and tolerogenic properties of CD8^- (myeloid) and CD8⁺ (lymphoid) DC (7, 8). IL-12 (6, 7) and IFN- (8) have functionally opposing effects on peptide presentation by CD8^- DC, and IFN--activated CD8⁺ DC mediate tolerogenic activity that can be prevented by CD40 activation on these cells (10). The effect of CD40 activation is accompanied by an impaired capacity of the CD8⁺ DC to initiate T cell apoptosis in vitro, presumably through modulation of tryptophan degradation affecting lymphocyte function (10, 43). We demonstrate here that IL-6 has profound effects on the presentation of tumor or self peptides by CD8^- DC via modulation of the regulatory activity of CD8⁺ DC. In addition, we provide evidence that autocrine IL-6 is involved in the regulatory effect of CD40 activation in CD8⁺ DC. Blockade of the tolerogenic activity of CD8⁺ DC is observed both under basal conditions and when the activity of the latter cells is potentiated by IFN- treatment.

Nonameric P815AB is a synthetic peptide that is related to a major tumor rejection Ag encoded by the P1A gene in murine mastocytoma cells (44). Although an efficient target for rejection responses in immunized mice, this Ag is not sufficiently immunogenic per se, and induction of protective immunity by P815AB-pulsed DC requires the use of rIL-12 (3). Effective priming to P815AB and the detection of class I-restricted responses in vivo require the presence of an intact CD4⁺ T cell compartment in the host (3). Of interest, adoptive transfer of class II-restricted CD4⁺ T cell clones with specificity for P815AB also results in the induction of protective antitumor immunity (45). Spontaneous autoimmune diabetes in NOD mice is the result of a CD4⁺ and CD8⁺ T cell-dependent autoimmune process directed against the pancreatic -cells. NRP-A7 represents a peptide ligand with strong agonistic activity for CD8⁺ T cells in autoimmune diabetes and also induces the deletion of specific CD8⁺ T cells under selected conditions of immunization (4, 5). In both model systems with synthetic peptides, we found that the pattern of immune response induced by transfer of peptide-pulsed DC was strongly influenced by IL-6 acting on CD8⁺ DC. These findings may be relevant to an improved understanding of the possible role of IL-6 in avoiding tolerance induction in cancer immunotherapy, particularly with regard to the idea that Ag-specific T cell tolerance is known to limit the efficacy of therapeutic cancer vaccines. Given the exceptional capacity of DC to induce immunity in vivo, recent reports of the first successful clinical trials based on vaccination of tumor patients with autologous blood DC pulsed in vitro with tumor Ags come as no surprise. However, to maximize antitumor immunity and avoid tolerance induction, a number of technical questions still need to be addressed, including the frequency and route of administration, the subset and number of DC to be used, and the concentration and duration of cytokine treatment. On the other hand, our findings with the NRP-A7 peptide may help to explain how DC can cause autoimmune disease and the possible role of IL-6 in this process.

Cytokines are known to regulate the progression and maintenance of autoimmunity. Although the roles of most cytokines are generally controversial (14), the role of IL-6, on the one hand, and that of TNF-/IFN-, on the other, appear to be more clearly defined. For example, experiments using knockout mice have demonstrated an absolute requirement for IL-6 in several models of autoimmunity (15, 16, 17, 18, 19, 20, 21). The ability of IL-6 to down-modulate the function of tolerogenic CD8⁺ DC could provide a model for explaining at least a portion of the disease-promoting effects of IL-6 in autoimmunity. In contrast, in systems of experimental autoimmune encephalomyelitis (23, 24) and diabetes (25), IFN- exerts a protective role. Although IFN- may have an essential function in stimulating APC to produce nitric oxide (an inducer of T cell apoptosis) (46), we have suggested that IFN--dependent production of IDO by DC may also result in apoptosis of T lymphocytes (10). We demonstrate here that IL-6 blocks the stimulatory activity of IFN- on IDO production by CD8⁺ DC, an effect that can at least in part be explained by down-modulation of IFN-R expression. Interestingly, we have previously observed that CD40 activation also results in reduced IFN-R expression by CD8⁺ DC (10), thus supporting the hypothesis that autocrine IL-6 mediates most of the effects of CD40 activation in CD8⁺ DC.

It has been shown that human monocyte-derived macrophages suppress T cell proliferation in vitro via IFN--mediated induction of IDO (47). Significant IDO production by human DC has also been shown to occur (43). Because cells synthesizing IDO modulate T cell proliferation by reducing the tryptophan concentration in local tissue microenvironments, tryptophan catabolism may represent a general mechanism in T cell suppression (48). In our experimental model with P815AB-specific CD4⁺ T cells, we have previously found that the blockade of IDO activity by a competitive inhibitor would negate the induction of apoptosis in vitro by IFN--treated DC (8). This suggested that IDO induction is a major mechanism by which IFN- acts on DC to mediate apoptosis of T cells and supported the idea that IDO-dependent modulation of T cell function may involve selective cell death in addition to inhibition of proliferation. In the present experiments we found that IL-6 would ablate most of the proapoptotic effect induced by IFN-, either externally added or putatively produced by the Th1 cells. Although under the adopted experimental conditions (i.e., 18-h incubation) IL-6 was unable to completely block apoptosis, our current finding that IL-6 and IFN- exert opposing effects on IDO induction and T cell apoptosis may be relevant to a better understanding of the immunological mechanisms governed by IDO-dependent tryptophan catabolism. On the other hand, the failure of 1-MT to completely negate the induction of apoptosis may indicate that additional mechanisms, such as production of NO, may contribute to the proapoptotic effect of IFN- in our model system. Finally, we are currently evaluating any possible direct effect of IL-6 on the regulation of IDO expression.

The term lymphoid DC was introduced to describe a mouse DC subset in the thymus that develops from a population of thymic lymphoid progenitor cells, and CD8 was considered to be a characteristic marker for lymphoid DC in mice (49, 50). CD8⁺ DC are also found in peripheral lymphoid organs, and the two types of CD8⁺ DC are thought to play important roles in the establishment and maintenance of central and peripheral tolerance, respectively (11, 51). It is possible that the murine lymphoid CD8⁺ DC lineage corresponds in humans to the progeny of DC precursors with a characteristic surface phenotype and a plasmacytoid appearance (52). It has been shown that human DC expressing significant IDO activity can mediate inhibition of T cell proliferation (43). Our present and previous observations (10) provide the first experimental evidence for the involvement of tryptophan degradation in T cell apoptosis and regulation of anergy of mature T lymphocytes by CD8⁺ DC in the mouse. This may add to our understanding of the complex role of DC in the control of immunity and may provide novel mechanistic insights into how DC tolerize T cells to self Ags and minimize autoimmune reactions (11, 53, 54).

In conclusion, the data reported here reinforce our previous findings that mature, immunologically competent DC may either immunize or tolerize T cells to tumor or self Ags depending on a combination of several factors, including Ag presentation by specific types of DC, the nature of the DC-activating signal, and the balance of cytokine signals (7, 8, 9, 10). In addition, our current data prospect a critical role for IL-6 in mediating the effect of CD40 activation on CD8⁺ DC, in regulating the susceptibility of these cells to the effects of IFN-, and, indirectly, in regulating the tryptophan catabolism pathway that affects T lymphocyte function. These properties of IL-6 may all influence the ability of DC to activate or tolerize autoreactive T cells, prime regulatory T cell subsets, or shift a Th1/Th2 balance, thus confirming the critical role that IL-6 plays in immune reactions to self Ags.

	Acknowledgments

 
We thank Genetics Institute (Cambridge, MA) for the generous gift of rIL-12.

	Footnotes

 
¹ This work was supported by the Italian Association for Cancer Research and a postdoctoral fellowship from Fondazione Italiana per la Ricerca sul Cancro (to F.F.).

² Address correspondence and reprint requests to Prof. Paolo Puccetti, Department of Experimental Medicine, Pharmacology Section, University of Perugia, Via del Giochetto, I-06122 Perugia, Italy. E-mail address: plopcc{at}tin.it

³ Abbreviations used in this paper: DC, dendritic cells; IDO, indoleamine 2,3-dioxygenase; 1-MT, 1-methyl-DL-tryptophan; NOD, nonobese diabetic; PI, propidium iodide.

Received for publication February 2, 2001. Accepted for publication May 7, 2001.

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