IL-12 Acts Selectively on CD8α− Dendritic Cells to Enhance Presentation of a Tumor Peptide In Vivo

Ursula Grohmann, Roberta Bianchi, Maria L. Belladonna, Carmine Vacca, Silvia Silla, Emira Ayroldi, Maria C. Fioretti and Paolo Puccetti
J Immunol September 15, 1999, 163 (6) 3100-3105;

Abstract

Previous work has shown that a significant proportion of murine splenic dendritic cells (DC) express a high affinity receptor for IL-12, thus accounting for the adjuvanticity of the cytokine when DBA/2 mice are transferred with syngeneic DC exposed in vitro to rIL-12 and an otherwise poorly immunogenic tumor peptide. In DBA/2 mice, splenic DC consist of 90–95% CD8− and 5–10% CD8+ cells. To detect any difference in IL-12 responsiveness among phenotypically distinct DC subtypes, enriched CD8− (>99% pure) and CD8+ (∼80% pure) populations of DC from DBA/2 spleens were assayed for APC function in vivo following exposure to rIL-12 and tumor peptide in vitro. Unlike unfractionated DC, the CD8− fraction was capable of effective presentation of the peptide even when the cells had not been pretreated with IL-12 before peptide pulsing. The addition of as few as 3% CD8+ cells during pulsing blocked in vivo priming by the CD8− fraction. However, pretreatment of CD8− DC with IL-12 before cell mixing and peptide pulsing ablated the inhibitory effect of the CD8+ fraction. CD8−, but not CD8+, DC showed significant message expression for the β1 and β2 subunits of the IL-12 receptor. These data suggest that a minority population of CD8+ DC, which appeared to secrete IL-10 in vitro, negatively regulates the induction of T cell reactivity by peptide-loaded CD8− DC in DBA/2 mice. However, the CD8− fraction can be primed by IL-12 to overcome the inhibitory effect of the CD8+ subtype.

Murine dendritic cells (DC),3 which derive from multiple pathways, including the Langerhans cell lineage, a monocyte/macrophage-related lineage, and a lymphoid-related lineage, can be distinguished on the basis of their surface phenotype (1). The two major splenic populations of DC consist of CD8α+ and CD8α− subtypes, which presumably represent lymphoid-related and myeloid-derived DC, respectively (2). Despite common ability to activate T cells, the two DC subtypes differ in their regulatory activity, and the CD8+ DC induce T cell apoptosis and a restricted T cell cytokine production relative to CD8− DC (3, 4). In particular, the CD8+ DC induce only a marginal IL-2 production in CD8+ T cells, compared with a marked cytokine induction by CD8− DC. However, the nature of the signals regulating CD8+ T cell responses to CD8+ or CD8− DC remains unclear (5).

We have previously shown that rIL-12 primes splenic DC in vitro for effective presentation of the class II- and class I-restricted epitopes of a tumor peptide upon transfer of the DC into syngeneic DBA/2 hosts (6, 7, 8). We have also shown that IL-12 acts directly on DC to promote nuclear localization of NF-κB, presumably leading to increased maturation of the DC and enhanced APC function (9). Recent evidence indicates that RelB, a member of the NF-κB family, is essential for the development of CD8− but not of CD8+ DC (10).

In the present study, we investigated whether the CD8− and CD8+ subtypes of splenic DC differ in their ability to present the tumor peptide and in their responsiveness to IL-12. We found that a population of splenic DC virtually devoid of CD8+ DC was capable of presenting the tumor peptide even in the absence of external IL-12. Although the presence of a minority fraction of CD8+ DC would block effective presentation by the CD8− DC, the latter cells could be primed by rIL-12 to overcome the inhibitory effect of the CD8+ DC. We conclude that the CD8− cell fraction of splenic DC is exquisitely sensitive to IL-12 and that the adjuvanticity resulting from IL-12 action on these cells counteracts the suppressive effect of CD8+ DC on presentation and/or recognition of the tumor peptide.

Materials and Methods

Mice

DBA/2J (H-2d) were obtained from Charles River Laboratories (Calco, Milan, Italy). Male mice were used at the age of 2 to 4 mo.

Peptides

Peptides were synthesized as described (11, 12), purified by means of reverse-phase HPLC, and characterized by amino acid analysis. The single letter code sequence of the peptides used is as follows: H-2Ld-restricted P815AB.35–43, LPYLGWLVF; and H-Ld-restricted P91A-.15–24, QNHRALDLVA.

Cytokines and Abs

Murine rIL-12 was a generous gift from Dr. B. Hubbard (Genetics Institute, Cambridge, MA). IL-12 was 98.8% pure, as assessed by SDS-PAGE, and endotoxin contamination was <0.9 EU/mg on Limulus amebocyte assay. The specific activity of the purified rIL-12 preparation, measured as ability to stimulate proliferation in human phytohemoagglutinin-activated blasts, was 3.1 × 106 U/mg. Endotoxin was removed from all solutions as described (9). Murine rIL-10 was from PharMingen (San Diego, CA).

PE-conjugated anti-murine CD8α mAb 53-6.7 and anti-mouse IL-10 mAbs SXC-1 and JES5-2A5 were from PharMingen. Affinity purified anti-CD11c (N418, rat IgG2a) was conjugated to FITC using conventional methods (9).

DC preparation

DC were prepared from collagenase-treated spleens (collagenase type IV; Sigma, St. Louis, MO), as described (6, 7, 8, 9). In brief, total spleen cells were suspended in dense BSA (p = 1.080), overlaid with 1 ml of RPMI medium, and centrifuged in a swingout bucket rotor at 7500 rpm for 20 min at 4°C. The low density fraction at the interface was collected and washed several times. The recovered cells were resuspended in RPMI medium supplemented with 10% FCS and allowed to adhere for 2 h, and this was followed by an additional 18-h incubation to allow DC to detach. Contaminating B cells were further removed by one round of panning on polyvalent goat anti-mouse Ig (Sigma). The recovered cells were routinely >96% CD11c+ and appeared to consist of 90–95% CD8α− and 5–10% CD8α+ cells. For preparation of CD8α+ and CD8α− fractions, purified DC were separated using a positive selection column and CD8α MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany). The recovered CD8α− cells typically contained less than 0.8% contaminating CD8α+ DC and were referred to as the CD8− fraction, whereas the CD8+ fraction was made up of ∼80% CD8α+ DC.

Immunization and skin test assay

Cytokine treatments of DC were performed at 37°C by an 18-h incubation with 100 ng/ml rIL-12 or 40 ng/ml rIL-10 before peptide pulsing. Control cultures were incubated with medium alone. For in vivo priming, unfractionated DC, single fractions of DC (CD8− or CD8+), or mixtures thereof were pulsed with 5 μM P815AB peptide at 37°C for 2 h. Cells were then irradiated (3000 rad) and washed, and each mouse received an i.v. injection of 3 × 105 peptide-pulsed DC.

A skin test assay for measuring class I-restricted delayed-type hypersensitivity (DTH) responses was employed in which 50 μg of P815AB peptide in 30 μl of 6% DMSO in saline was inoculated into the left hind footpads of mice transferred with DC 2 wk earlier (6, 7, 11, 12). The right hind footpad received the same volume of vehicle. The DTH reaction was recorded 24 h later, when the animals were killed, their hind feet were cut off at the hair line, and weights were recorded as a measure of swelling, edema, and cellular infiltration. Results were expressed as the increase in footpad weight over that in the vehicle-injected counterpart. Data are the mean ± SD for at least six mice per group.

Cytofluorometric analysis

After blocking the Fcγ receptor with 2.4G2 mAb, phenotypic analysis of DC subpopulations was performed after double staining with FITC-conjugated anti-CD11c and PE-conjugated anti-CD8α mAbs. All the staining steps were performed on ice in PBS supplemented with 1% FCS. Stained cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using LYSYS software (Becton Dickinson).

RNA preparation and PCR

These procedures were previously described in detail (9). β-actin primers were purchased from Clontech Laboratories (Palo Alto, CA), and IL-12R primers were synthesized according to published sequences, as described (9). The sequences of 5′ sense primer and 3′ antisense primer of IL-12Rβ1 and IL-12Rβ2 were as follows: 5′ IL-12Rβ1, TAT-GAG-TGC-TCC-TGG-CAG-TAT; 3′ IL-12Rβ1, GGC-ATG-CTC-CAA-TCA-CTC-CAG; 5′ IL-12Rβ2, ACA-TTA-CTG-CCA-TCA-CAG-AG; and 3′ IL-12Rβ2, AGG-AGA-TTA-TCC-GTA-GGT-AG. PCR products were separated in a 1% agarose gel, blotted to a ζ-probe membrane, and hybridized with one of the following oligo probes: IL-12Rβ1, 5′-CTT-GGG-AAC-CGA-ACC-ATG-AA-3′; IL-12Rβ2, 5′-GTC-CTA-TGG-ATG-ACA-GCT-GTG-3′; β-actin, 5′- GCA-TTG-TGA-TGG-ACT-CCG-GT-3′. The intensity of each PCR product band was measured using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Spot enzyme-linked immunosorbent (ELISPOT) assay

IL-10-producing DC were enumerated by ELISPOT assay, as described (6). Briefly, unfractionated, CD8−, or CD8+ DC were incubated overnight with RPMI medium containing 10% FCS in 96-well flat-bottom plates precoated with anti-murine IL-10 mAb JES5-2A5. The secondary Ab was biotinylated SXC-1, the enzyme was avidin-alkaline phosphatase conjugate (Vector Laboratories, Burlingame, CA), and the substrate was 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt (Life Technologies, Gaithersburg, MD). Results were expressed as the mean number of IL-10-producing cells (± SD) per 104 cells, calculated using replicates of serial twofold dilutions of DC.

Results

IL-12 in vitro confers priming ability on DC pulsed with a tumor peptide before transfer into recipient hosts

Our previous studies have shown that a synthetic peptide related to a murine self-protein and tumor rejection Ag, P815AB, can result in a reversible state of Ag-specific T cell anergy when hosts are transferred with peptide-pulsed DC without added adjuvanticity. The anergic state involves unresponsiveness in CD8+ T cells, as detected by skin test assay in vivo and IFN-γ production in vitro, and suppression of IL-2 production by CD4+ T cells. In contrast, transfer of DC exposed sequentially to IL-12 and P815AB in vitro confers CD4+ T cell-dependent skin test (i.e., DTH) reactivity mediated by CD8+ T cells on prospective recipients of an intrafootpad challenge with the tumor peptide. This is accompanied by increased resistance to challenge with tumor cells expressing the otherwise poorly immunogenic rejection Ag P815AB (6, 7, 8). Fig. 1⇓ shows the effect of sensitization with P815AB using splenic DC exposed to IL-12 before pulsing with P815AB and transfer into hosts to be assayed for DTH at 2 wk. Consistent with our previous results, P815AB-specific footpad reactivity was observed only in mice receiving DC exposed sequentially to rIL-12 and P815AB.

FIGURE 1.
FIGURE 1.

Induction of skin test reactivity to P815AB by host transfer with P815AB-pulsed DC. Mice received cells exposed to the tumor peptide and treated or not with rIL-12. Class I-restricted DTH was measured after 2 wk, when mice received an intrafootpad challenge with the tumor peptide. Values are expressed as the mean footpad weight increase ± SD. Specificity controls involved the use of the antigenically unrelated P91A for footpad challenge in IL-12 + P815AB-primed mice. ∗, Indicates p < 0.001 (experimental vs control footpads). One experiment representative of six.

Ability of CD8− but not CD8+ DC to prime the host to P815AB

In the spleen, two DC populations can be distinguished, the CD8α+ DEC-205+ CD11b− and CD8α− DEC-205− CD11b+, representing putative lymphoid-related and putative myeloid-derived DC, respectively (1, 2). Both of these DC show a capacity to activate T cells, but they differ in their ability to limit or regulate the response of the T cells they activate (3, 4, 5). To determine whether the CD8− and CD8+ components of the DC cultures in our model system might contribute differentially to the induction of P815AB-specific T cell reactivity, >96% CD11c+ splenic DC were fractionated to yield a population of >99% CD8− cells and a fraction highly enriched in CD8+ cells (Fig. 2⇓). After pulsing with P815AB, cells were injected into recipient hosts to be assayed for footpad reactivity to P815AB. A group of mice received CD8− cells admixed with 3% CD8+ cells at the time of peptide pulsing (Fig. 3⇓). Somewhat unexpectedly, we found that the CD8− fraction alone was highly effective in priming hosts to P815AB, consistently yielding footpad responses greater than those of unfractionated DC preexposed to rIL-12 (Fig. 1⇑, and data not shown). In contrast, mice transferred with the CD8+ cell-enriched population did not show significant DTH. Of interest, the presence of 3% CD8+ cells in the CD8− fraction completely blocked the ability of the latter cells to prime the host to P815AB. Similar to our previous results with unfractionated DC in the absence of adjuvants (7), we found that both the CD8+ cells alone and the combination of 97% CD8− and 3% CD8+ cells would result in a long-lasting state of P815AB-specific anergy (data not shown).

FIGURE 2.
FIGURE 2.

Coexpression of CD11c and CD8α in DC cultures either unfractionated or subjected to cell fractionation according to CD8α distribution. Numbers within boxes indicate percentage of double-positive cells.

FIGURE 3.
FIGURE 3.

Induction of skin test reactivity to P815AB by host transfer with fractionated DC. DC, either unfractionated or fractionated according to CD8 expression, were pulsed with P815AB and injected into recipient mice to be assayed for DTH. A group of mice received a mixture of both types of DC. Specificity controls included the use of P91A for footpad challenge. ∗, Indicates p < 0.001 (experimental vs control footpads). One of four experiments.

Ability of IL-12 to counteract suppression by CD8+ DC

Because our previous studies indicated that rIL-12 can make DC capable of effective presentation of P815AB (6, 7), we analyzed the effect of the cytokine on the different DC fractions used in the experiments above. CD8− cells were used either singly or in combination with 3% CD8+ cells, and either fraction was used either as such or after exposure to IL-12. Cell mixing occurred at the time of DC loading with the peptide. At 2 wk after cell transfer, the animals were assayed for DTH to P815AB. Fig. 4⇓ shows that the blockade of DTH induction resulting from the addition of CD8+ cells to the CD8− DC was reversed only by preexposure of the latter cells to IL-12. In contrast, no effect was apparently exerted by the cytokine on CD8+ cells. Interestingly, concomitant treatment of CD8− and CD8+ cells with IL-12 did not improve the effect of CD8− cell treatment alone.

FIGURE 4.
FIGURE 4.

Induction of skin test reactivity to P815AB by host transfer with DC subtypes treated with IL-12. DC fractionated according to CD8 expression and treated (CD8−/IL-12, CD8+/IL-12) or not (CD8−, CD8+) with IL-12 were mixed before peptide pulsing and transfer into recipient hosts. P815AB-specific DTH was assessed at 2 wk. ∗, Indicates p < 0.001 (experimental vs control footpads). One experiment is reported of three performed.

Expression of IL-12 receptor mRNA in CD8− DC

We have previously shown that murine DC from DBA/2 mice express transcripts for the β1 and β2 subunits of the IL-12 receptor, although RT-PCR analysis revealed that DC present a different β1 isoform relative to T cells (9). Using RT-PCR and the same β1 and β2 primers employed in our previous studies, we comparatively analyzed the expression of β1 and β2 messages in CD8− and CD8+ DC (Fig. 5⇓). Consistent with the finding of biological activity of rIL-12 in CD8− DC, we found that these cells clearly expressed β1 and β2 transcripts. Low-intensity bands of β1 and β2 PCR products were found in CD8+ DC. This might reflect a reduced but still definite expression of the receptor by the latter cells or merely the incomplete purification of the CD8+ fraction. When the intensity of the β1 and β2 bands was measured, the levels of their respective expressions in CD8− DC were more than 5-fold and 2.5-fold higher than those in CD8+ cells.

FIGURE 5.
FIGURE 5.

IL-12 receptor β1 and β2 mRNA expression in different DC fractions. cDNA prepared from unfractionated or fractionated DC populations was PCR amplified using β1 and β2 primers specific for the respective subunits of the IL-12R in DC. PCR products were separated on a 1% agarose gel, transferred to a ζ-probe membrane, and hybridized with specific oligo probes for β1, β2, or β-actin. The intensity of each PCR product band was measured as described in Materials and Methods. The figure shows an ethidium bromide-stained agarose gel with the PCR products. The ratios of intensities of the β1 and β2 product bands to the β-actin band are also indicated.

Production of IL-10 by CD8+ DC and effect of rIL-10 on CD8− cell function

One possible explanation for the suppressive activity of CD8+ DC on the in vivo priming to P815AB mediated by CD8− DC could be differences in regulatory functions affecting Ag presentation and/or recognition by specific T cells. Because IL-10 is known to be released by DC (13) and to affect different components of an ongoing immune response (14, 15, 16), we became interested in analyzing the frequencies of IL-10-producing cells in the different DC fractions. Unfractionated, CD8−, and CD8+ DC were incubated overnight in FCS-enriched medium and then assayed for IL-10 production in an ELISPOT assay (Fig. 6⇓). We found that the frequency of IL-10-producing cells was much higher in the CD8+ cell-enriched fraction than in the unfractionated or CD8− populations.

FIGURE 6.
FIGURE 6.

Frequency of IL-10-producing cells in different DC populations. Numbers of cells secreting IL-10 were evaluated by ELISPOT assay in unfractionated DC, CD8− DC, and CD8+ DC following overnight incubation in the absence of deliberate Ag stimulation. Analogous results (not reported in the figure) were obtained when 5 μM P815AB was present in the cultures during the overnight incubation.

We also assayed rIL-10 for possible effects on the ability of CD8− DC to prime the host in vivo to P815AB. CD8− cells were incubated overnight with control medium or 40 ng/ml rIL-10 before P815AB pulsing and transfer into recipient hosts. The DTH reactivity to the tumor peptide was tested at 2 wk. Fig. 7⇓ shows that exposure of CD8− DC to IL-10 before peptide pulsing abolished their ability to confer P815AB-specific reactivity on prospective recipients of an intrafootpad challenge with the tumor peptide. Although these data might suggest a direct effect of CD8+ cell-derived IL-10 on CD8− DC during coculture in vitro, we found that the presence of anti-IL-10 mAb in the mixtures of CD8+ and CD8− DC during peptide pulsing did not restore the ability of the latter cells to prime the host to P815AB in vivo (data not shown). On the other hand, neither supernatants from relevant numbers of CD8+ DC cultured with P815AB for 2 h nor rIL-10 concentrations (up to 100 pg/ml) in the range of those released by CD8+ DC during coculture could block the ability of CD8− DC to prime the host to P815AB in vivo when either type of material was added to the CD8− DC during peptide pulsing (data not shown). This suggested that most of the inhibition exerted by CD8+ DC could take place in vivo.

FIGURE 7.
FIGURE 7.

Effect of IL-10 on the induction of footpad reactivity to P815AB by CD8− DC. DC exposed overnight to rIL-10 or control cultures were injected into recipient mice that were assayed at 2 wk for DTH reactivity. Values are the mean difference ± SD in weight between experimental and control footpads. ∗, Indicates p < 0.001 (experimental vs control footpads). One of three experiments.

Inhibitory activity of CD8+ DC admixed with CD8− DC at the time of transfer

Experiments were thus performed to compare the coculture of CD8− and CD8+ DC before transfer vs the injection of CD8− and CD8+ DC mixed at the time of transfer without prior in vitro coculture. CD8− DC were admixed with 3% CD8+ DC at the time of peptide pulsing or after incubation with P815AB immediately before injection into recipient hosts. Fig. 8⇓ shows that the two procedures were equally effective in preventing the induction of P815AB-specific footpad reactivity by the peptide-loaded CD8− DC.

FIGURE 8.
FIGURE 8.

Ability of CD8+ DC to inhibit induction of skin test reactivity when admixed with CD8− DC at the time of transfer. Purified CD8− DC were pulsed with P815AB and injected into recipient mice to be assayed for DTH. Groups of mice received mixtures of CD8− and CD8+ DC that had been either cocultured during peptide pulsing or mixed immediately before injection. P815AB-specific DTH was assessed at 2 wk. ∗, Indicates p < 0.001 (experimental vs control footpads). One of three experiments.

Discussion

At least two different types of DC can be found in adult mouse spleens, and they are characterized by distinct surface phenotypes (2, 17, 18, 19, 20, 21), ontogeny (18, 19, 22), the cytokines required for development (22, 23, 24), and regulatory features (3, 4). In the inbred mouse strains so far examined, the CD8− and CD8+ subtypes each account for ∼50% of the whole splenic DC population (3, 5, 21). We were therefore surprised to observe disparate percentages of the two subtypes (90–95% CD8− and 5–10% CD8+) in splenic DC from DBA/2 mice (Fig. 2⇑). Yet, under the same conditions of testing, we were able to detect equal proportions of CD8− and CD8+ DC in C57BL/6 mice (data not shown).

In DBA/2 mice, nonameric P815AB, a synthetic peptide related to a tumor rejection and self Ag encoded by gene P1A (8, 25), is not sufficiently immunogenic on its own when presented by peptide-loaded DC (12), presumably as a result of poor ability to recruit CD4+ T cells to the early response (6). However, transfer of DC exposed to IL-12 before pulsing leads to the induction of P815AB-specific class II- and class I-restricted reactivities (6, 7). The latter reactivity can be evidenced at 2 wk as a DTH-like cutaneous response to intrafootpad challenge with the peptide in saline (11, 12). By means of this skin test assay, we have previously shown that P815AB-pulsed DC can present the peptide not only in an immunogenic but also in a tolerogenic fashion, depending on whether or not the DC have been pretreated with IL-12 (7).

We have also shown that the adjuvanticity of IL-12 in this model system results from direct effects of the recombinant cytokine on the DC, via interaction with a specific high affinity receptor that comprises a β1 isoform different from that in T cells (9). Signaling through this receptor initiates nuclear localization of members of the NF-κB family, leading to increased maturation and APC function of the DC. One major member of this family that appears to be activated by IL-12 is RelB (9). Besides being involved in the development/differentiation of DC (26) and effective APC function (27), this transcription factor has been recently shown to selectively regulate the myeloid-related CD8− lineage of splenic DC (10). The present study demonstrates that IL-12 acts selectively on CD8− DC, which in fact express transcripts for the β1 and β2 subunits of the IL-12 receptor (Fig. 5⇑).

By using a highly purified CD8− DC population (Fig. 2⇑), we were able to demonstrate that removal of CD8+ cells confers increased ability on the former cells to prime the host for skin test reactivity to P815AB (Fig. 3⇑). This effect is at variance with that of transfer of whole DC populations and is similar to the transfer of IL-12-treated, peptide-pulsed DC (Fig. 1⇑). However, the presence of a minority fraction of CD8+ DC in the transferred population blocked its priming ability (Fig. 3⇑), an effect that could be reversed by exposure of CD8−, but not of CD8+, DC to IL-12 (Fig. 4⇑). Of interest, mice transferred with P815AB-pulsed CD8+ DC or with a combination of CD8+ and CD8− DC would develop Ag-specific anergy (data not shown), similar to animals treated with whole DC populations in the absence of IL-12 adjuvanticity (7, 8).

Several considerations can be made from these experiments. First, in DBA/2 mice, a minority population of CD8+ DC counteracts the ability of the CD8− fraction to prime the host in vivo to the P815AB peptide. Both the cellular targets and the signaling mechanisms responsible for the inhibitory activity of CD8+ DC remain unclear. Although the negative regulatory function of CD8+ DC has been demonstrated in several experimental models and a role has been established for Fas/Fas-ligand-induced apoptosis in CD4+ T cells (3), the nature of the signals regulating CD8+ T cell responses to CD8+ DC is poorly understood (5). Our present data may suggest a role for CD8+ DC-derived IL-10 (Fig. 6⇑), perhaps via regulation of both the APC function of CD8− DC (Fig. 7⇑) and recognition of P815AB epitopes at the site of T cell priming (16). Considering the ability of IL-10 to suppress DC function (28) and of IL-10-treated DC to induce tolerance (29), a direct effect of IL-10 on CD8− DC would be an intriguing possibility. Yet, we were unable to demonstrate any effect of anti-IL-10 mAb added to mixtures of CD8− and CD8+ DC during peptide priming (data not shown). Furthermore, in experiments not reported here, we have examined whether the effect of CD8+ DC on the CD8− population could be transferred with culture supernatants of the former cells added to CD8− DC during peptide pulsing. Under these conditions, no inhibition of CD8− DC activity was observed. Therefore, while the inhibitory effect of CD8+ DC on the activity of the CD8− fraction could still be mediated by IL-10, it is likely that most of the inhibition occurs in vivo once the DC have been transferred into recipient hosts. In line with this hypothesis was the observation that the CD8+ DC retain their inhibitory activity when admixed with CD8− DC at the time of cell transfer, without prior in vitro coculture (Fig. 8⇑).

Second, when subtracted to the inhibitory function of CD8+ DC, the CD8− subtype can present P815AB in an immunogenic fashion, a condition similar to that of whole DC preexposed to IL-12. Since the nature of the inhibitory signals of CD8+ DC is unclear, the mechanisms allowing CD8− DC to prime the host to P815AB in the absence of CD8+ DC are not easily explained. Because one major function of DC is the production of IL-12 (30), one possibility is that the transferred CD8− DC initiate IL-12 production in vivo, which, unopposed by IL-10 (31, 32), may result in optimal development of cell-mediated reactivity (33, 34).

Third, externally added IL-12 conditions the CD8− DC to overcome the suppressive effect of the CD8+ fraction. This observation does not necessarily imply that both IL-12 and the negative regulatory signals of CD8+ DC act primarily via modulation of the APC function of the CD8− fraction. However, it is interesting to note that IL-12 and IL-10 have opposite effects on the accessory function of APC, including DC (9, 28, 31), and that IL-12 induces increased expression of fully mature class II molecules on DC4 whereas IL-10 decreases class II Ag expression (29). Persistent expression of class II/peptide complexes is a likely mechanism through which rIL-12 exerts adjuvant effects in the priming to P815AB (7, 9).

In conclusion, our results indicate that a minority population of CD8+ DC appears to regulate the induction of T cell reactivity by splenic, peptide-loaded CD8− DC transferred into recipient DBA/2 mice. Relative to CD8− DC, the CD8+ cell-enriched fraction contains a higher proportion of IL-10-secreting cells, and CD8− DC lose their ability to prime the host to the tumor peptide when exposed to rIL-10 in vitro. In contrast, treatment of CD8− DC with rIL-12 makes the cells capable of an effective priming to the peptide even when the copresence of CD8+ DC would negate the induction of peptide-specific T cell reactivity. Although the cellular targets and the mechanisms of the inhibitory activity of the CD8+ DC in our model are unclear, the present data may be of importance for designing therapeutic approaches with DC and synthetic peptides in antitumor immunotherapy.

Acknowledgments

We thank Prof. Thierry Boon for continued support in our studies with tumor-specific peptides and Genetics Institute (Cambridge, MA) for the generous gift of rIL-12.

Footnotes

  • ↵1 This work was supported by the Italian Association for Cancer Research (AIRC).

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

  • ↵3 Abbreviations used in this paper: DC, dendritic cells; DTH, delayed-type hypersensitivity.

  • ↵4 U. Grohmann, C. Orabona, R. Bianchi, M. L. Belladonna, M. C. Fioretti, and P. Puccetti. IL-12 induces SDS-stable class II αβ dimers in murine dendritic cells. Submitted for publication.

  • Received April 2, 1999.
  • Accepted July 6, 1999.

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The Journal of Immunology: 163 (6)
The Journal of Immunology
Vol. 163, Issue 6
15 Sep 1999
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