IL-12p40 is a natural antagonist which inhibits IL-12- and IL-23-mediated biological activity by blocking the binding of IL-12/23 to their receptors. Recently, IL-12p40 was also shown to have immune-enhancing activity through the activation of macrophages or dendritic cells. In this study, we investigated the effects of IL-12p40 as a genetic adjuvant on immune modulation using recombinant adenoviruses expressing IL-12p40 (rAd/IL-12p40) and OVA (rAd/OVA). Coimmunization of rAd/IL-12p40 at a low dose (1 × 104 PFU) with rAd/OVA resulted in OVA-specific immune enhancement, while a high dose of rAd/IL-12p40 (1 × 108 PFU) caused significant suppression of CD8+ T cell responses. In addition, the enhancement and suppression of OVA-specific CD8+ T cell responses correlated with antitumor activity against E.G7-OVA tumor challenge, which subsequently affected the survival rate. Moreover, the differential CD8+ T cell response by IL-12p40 was still observed in IL-12Rβ2 knockout (IL-12Rβ2KO), but not in IL-12Rβ1 knockout (IL-12Rβ1KO) mice, indicating that IL-12p40 is a cytokine which can modulate Ag-specific T cell responses depending on IL-12Rβ1. Our findings provide a novel insight on the physiological role of IL-12p40, which can be informative in the design of vaccine strategies and therapeutic regimens.

Interleukin 12 is a proinflammatory cytokine produced by APCs such as macrophages, dendritic cells (DCs),3 and B cells (1, 2, 3, 4). It consists of p35 and p40 subunits, and the expression of p40 is induced by the activation of APCs while p35 is constitutively expressed (3, 5, 6 ; reviewed in Ref. 7). p40 subunit is also covalently linked to a p19 subunit to form IL-12-related cytokine, IL-23, which is secreted from activated DCs and stimulates memory T cells or Th17 cells (8, 9, 10). The p40 subunit can be secreted as a monomer, or a homodimer, as well as a form of either an IL-12 or IL-23 heterodimer (11). The production of IL-12p40 monomer or homodimer is in large excess over IL-12 in vitro (12, 13, 14) and in vivo (15), and IL-12p40 monomer is produced more abundantly than homodimeric IL-12p40 (p80) by ∼2 to 5-fold (16, 17). However, the monomer binds to IL-12 receptor with only low affinity, whereas the homodimer has higher binding affinity to the receptor (13, 17, 18).

Previous reports have demonstrated that IL-12p40 homodimer inhibits IL-12- or IL-23-mediated biological activity by blocking the binding of IL-12 or IL-23 to their receptors (17, 19, 20), suggesting that IL-12p40 is a natural antagonist of IL-12 and IL-23. Consistent with this hypothesis, IL-12p40 was shown to reduce IL-12-mediated Th1 responses in vivo (21, 22, 23) and antitumor activity by IL-12 gene therapy (24). Moreover, when an IL-12N220L mutant, which selectively inhibits the secretion of IL-12p40, was used as a genetic vaccine adjuvant, the stronger Th1 response and antitumor activity were induced, indicating the inhibitory function of IL-12p40 on IL-12-mediated biological activity (14, 25). Similarly, IL-12p40 impaired the IL-23-mediated immune responses in vivo, leading to the abrogation of antitumor activity by IL-23 (20). IL-12p40 homodimer is at least 20-fold more effective than monomer in this antagonism, and it is known that monomeric IL-12p40 has little effect on immune regulation despite of its abundance in vivo (13, 17, 18).

In contrast, there have been reports showing that IL-12p40 itself could enhance immune responses in several experimental conditions. The administration of IL-12p40 homodimer exacerbated the cardiac allograft rejection by enhancing alloantigen-specific Th1 development (26, 27). Also, it was reported that the expression of IL-12p40 induced macrophage recruitment in a tumor model, resulting in the infiltration of T cells and inhibition of tumor growth in vivo (28). In the murine Mycobacterium tuberculosis (Mtb) model, the delivery of IL-12p40 homodimer restored its protection in IL-12p40 knockout (KO) mice which were highly susceptible to Mtb infection (29), indicating that IL-12p40 could exert a protective function against Mtb. Recently, it was found that IL-12p40 was required for DC migration and T cell priming after Mtb infection (30). Collectively, these findings suggest that IL-12p40 is not only a negative immune modulator inhibiting IL-12 signaling, but also a positive regulator of immune induction depending on experimental conditions. Therefore, the physiological role of IL-12p40 in vivo needs to be clarified more.

In this study, we investigated the effects of IL-12p40 on Ag-specific immune responses by the adenovirus-mediated delivery of IL-12p40 gene. We showed that IL-12p40 could positively or negatively regulate the codelivered Ag-specific CD8+ T cell response in a dose-dependent manner. In addition, this modulation was dependent on IL-12Rβ1, but not on IL-12Rβ2, indicating that IL-12p40 has its own immune-modulating activity independent of IL-12-mediated function.

Recombinant replication-defective adenoviruses (rAd) were generated according to an AdEasy Vector System (Qbiogene) (14). Briefly, the cDNA of OVA was subcloned into a pShuttleCMV vector (Qbiogene) using the BglII site. Mouse cDNAs encoding IL-12p35 (GenBank no. NM_008351) and IL-12p40 (GenBank no. NM_008352) subunits were isolated from RAW264.7 murine macrophage cell line with RT-PCR (25). After RT-PCR, the IL-12 expression cassette in which IL-12p35, internal ribosomal entry site of encephalomyocarditis virus, and IL-12p40 genes were linked in a tandem, unidirectional arrangement was constructed. IL-12p40 gene and IL-12 expression cassette were inserted into pShuttleCMV using XhoI/XbaI sites. Each pShuttleCMV construct was cotransfected with pAdEasy into Escherichia coli BJ5183 by electroporation, and the recombinants were transfected into QBI-293A cells using a calcium phosphate method. The adenovirus carrying the enhanced GFP (EGFP) was similarly produced and the generated recombinant adenoviruses were expanded and purified by cesium gradient ultracentrifugation. A titer of each purified virus was determined by tissue culture infectious dose 50 assay.

EL4 and E.G7-OVA were purchased from American Type Culture Collection and maintained in DMEM and RPMI 1640 (Cambrex) medium supplemented with 10% FBS (HyClone) and 100 U/ml penicillin-100 μg/ml streptomycin (Invitrogen).

Six- to 8-wk-old female C57BL/6 mice were purchased from Charles River Breeding Laboratories (Shizuoka). IL-12Rβ1KO, IL-12Rβ2KO, and Thy1.1-congenic mice were purchased from The Jackson Laboratory. The OT-I TCR-transgenic mice were provided by W. Heath (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). To obtain Thy1.1-positive OT-I cells, Thy1.1 mice were crossed to OT-I mice. All mice were kept under the animal care facility in POSTECH.

OT-I cells were purified by negative selection using a CD8a+ T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. The purified cells were adoptively transferred into normal C57BL/6 mice (1 × 106 cells/mouse in 200 μl of RPMI 1640 medium) into the tail vein. One day after transfer, the mice were immunized i.m. with adenoviruses. Four weeks after immunization, the mice were sacrificed and the spleen cells were prepared for subsequent immunological assays.

A dose of 5 × 105 E.G7-OVA cells in 100 μl of PBS was s.c. injected into the right hind flank of C57BL/6 syngenic mice. The tumor size was measured two or three times per week with a digital caliper for the two-dimensional longest axis (L in mm) and shortest axis (W in mm). The tumor volume was calculated according to the formula: volume in mm3 = (L*W2)/2.

The sera were collected from the orbital vein of rAd-immunized mice one day after i.m. injection. IL-12 and IL-12p40 levels were determined by the IL-12 and IL-12p40 ELISA kit (R&D Systems) according to the manufacturer’s instructions. For the IFN-γ measurement, sera were collected 5 days after injection and assayed using the IFN-γ capture and the biotinylated rat anti-mouse IFN-γ Ab (BD Biosciences).

Nitrite was measured using a Griess Reagent System (Promega) according to the manufacturer’s instructions. Briefly, 5 days after immunization serum samples were collected and mixed with a sulfanilamide solution, followed by incubation with an N-1-napthylethylenediamine dihydrochloride solution. OD was measured at 520 nm using an ELISA reader (Bio-Tek Instruments).

Four weeks after immunization, OVA-specific IFN-γ-producing cells were quantified as described before (27). Briefly, splenocytes were incubated with OVA257–264 (SIINFEKL) peptide on an IFN-γ capture Ab (5 μg/ml; BD Biosciences)-coated 96-well ELISPOT plate (Millipore). After a 24-h incubation, the plates were washed and 50 μl of 2.5 μg/ml biotinylated rat anti-mouse IFN-γ Ab (BD Biosciences) was added and incubated for 4 h at room temperature. After washing, streptavidin-alkaline phosphatase (BD Biosciences) was added and the mixture was incubated for an additional 30 min. Spots were visualized by adding a 5-bromo-4-chloro-3-indolyl phosphate/tetra-NBT substrate solution (Promega). The reaction was stopped by washing with tap water, and the numbers of spots were counted using an AID ELISPOT Reader System (Autoimmune Diagnostika).

Four weeks after immunization, the mice were sacrificed and the spleen cells were resuspended in FACS buffer (1% FCS and 0.02% sodium azide in PBS) at a concentration of 1 × 107 cells/ml. A total of 100 μl of these cells (1 × 106 cells) were stained for CD8 or Thy1.1 and fixed in PBS containing 2% formaldehyde and samples were acquired on a FACSCalibur (BD Biosciences). For intracellular IFN-γ staining, cells were stained for surface markers, washed, and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences). Gates were set on lymphocytes by forward and side scatter profiles, and the data were analyzed using CellQuest Pro (BD Biosciences).

CTL activity was measured using a conventional 51Cr release assay 4 wk after immunization. A total of 3 × 107 splenocytes were expanded in vitro for 5 days with mitomycin C-treated 1 × 106 E.G7-OVA cells in complete RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 20 μM 2-ME, and mouse IL-2 (10 U/ml) and used as effector cells. Cytotoxicity was examined against CD8 epitope peptide (OVA257–264, SIINFEKL)-loaded 104 EL4 target cells labeled with 51Cr. The maximum or spontaneous releases of 51Cr were determined from the cells treated with either 2% Triton X-100 or the medium alone, respectively. The percentage of specific lysis was calculated from the formula: [(cpm experimental release − cpm spontaneous release)/(cpm maximum release − cpm spontaneous release)] × 100.

Serum of each mouse was collected 4 wk after immunization and the relative levels of anti-OVA Abs in mice were determined by ELISA as described previously (31).

The statistical difference between groups was assessed using a two-tailed Student’s t test. For all cases, differences were considered significant when the p values were <0.05.

Although IL-12p40 was shown to inhibit the immune response as an antagonist to IL-12 (21, 22, 23, 24), it also has been reported that IL-12p40 could enhance the immunity to infectious disease, tumor challenge, or graft rejection (26, 27, 28, 29). To investigate the effects of IL-12p40 on Ag-specific immune responses in vivo, we used an adenoviral vector-based gene delivery system. OVA-specific CD8+ T cells were adoptively transferred to C57BL/6 mice. The next day, rAd/OVA (106 PFU) was coimmunized with different doses of rAd/IL-12p40 or rAd/IL-12 (102–108 PFU) to the mice. rAd/EGFP was used to complete the total virus up to 1.01 × 108 PFU. Four weeks after immunization, the IFN-γ ELISPOT assay and CTL assay were performed to asses the OVA-specific T cell responses as described in Materials and Methods. As expected, the addition of rAd/IL-12 at low or intermediate doses (1 × 102–1 × 106 PFU) enhanced OVA-specific T cell responses in terms of IFN-γ production and cytotoxicity (p < 0.01; Fig. 1, A and B) (32). Interestingly, coinjection of rAd/IL-12p40 at a low dose (1 × 102–1 × 104 PFU) also increased the OVA-specific T cell responses (p < 0.01; Fig. 1, C and D), which is contradictory to previous reports that IL-12p40 has an inhibitory effect on T cell response (21, 22, 23). The high dose (1 × 108 PFU) of rAd/IL-12, however, markedly abolished the Ag specific IFN-γ production and cytotoxicity which is consistent with previous report (p < 0.001) (32). Also, IL-12p40 at a high dose (1 × 108 PFU) exhibited substantial suppressive effects on T cell responses (p < 0.005), showing a similar pattern of immune modulation as IL-12. These findings indicate that like IL-12, IL-12p40 has differential immunoregulatory effects on Ag-specific T cell responses depending on the dose.

FIGURE 1.

IL-12p40 modulates Ag-specific CD8+ T cell response in a dose-dependent manner. Four weeks after immunization of 106 PFU of rAd/OVA with different doses of rAd/IL-12 or rAd/IL-12p40, the mice (n ≥ 5/group) were sacrificed and the splenocytes were prepared. A and C, The frequency of OVA-specific IFN-γ-producing cells was determined by an IFN-γ ELISPOT assay. Data are presented as mean ± SEM of five to six mice in triplicate cultures. B and D, For measuring cytotoxic activity against OVA-expressing target cells, splenocytes were cocultured with mitomycin C-treated E.G7-OVA cells for 5 days. The resulting splenocytes were used as effector cells and the 51Cr-labeled, SIINFEKL peptide-pulsed EL4 cells were used as target cells in a 51Cr-release assay. Data are presented as the mean percentage of specific lysis for the indicated E:T ratio and SEM in triplicate cultures of five to six mice. A and B, These are representative of three independent experiments with similar results.

FIGURE 1.

IL-12p40 modulates Ag-specific CD8+ T cell response in a dose-dependent manner. Four weeks after immunization of 106 PFU of rAd/OVA with different doses of rAd/IL-12 or rAd/IL-12p40, the mice (n ≥ 5/group) were sacrificed and the splenocytes were prepared. A and C, The frequency of OVA-specific IFN-γ-producing cells was determined by an IFN-γ ELISPOT assay. Data are presented as mean ± SEM of five to six mice in triplicate cultures. B and D, For measuring cytotoxic activity against OVA-expressing target cells, splenocytes were cocultured with mitomycin C-treated E.G7-OVA cells for 5 days. The resulting splenocytes were used as effector cells and the 51Cr-labeled, SIINFEKL peptide-pulsed EL4 cells were used as target cells in a 51Cr-release assay. Data are presented as the mean percentage of specific lysis for the indicated E:T ratio and SEM in triplicate cultures of five to six mice. A and B, These are representative of three independent experiments with similar results.

Close modal

To quantify in vivo expression of the cytokines, serum IL-12 and IL-12p40 were measured by ELISA on days 1–3 after immunization of adenoviruses. As shown in Fig. 2,A, IL-12 was detected at high level (42.3 ± 16.7 ng/ml on day 1) only in the mice immunized with 108 PFU of rAd/IL-12, whereas it was undetectable in other groups that received rAd/OVA alone or 102–106 PFU of rAd/IL-12 (Fig. 2,A). The expression reached its peak level on day 1 and rapidly decreased thereafter as previously reported (14). The levels of circulating IL-12p40 showed a similar pattern to that of IL-12, with a much higher concentration (167.1 ± 15.5 ng/ml on day 1) in the mice injected with 108 PFU of rAd/IL-12p40 (Fig. 2 A). Because the exogenous IL-12p40 can bind with endogenous IL-12p35 to increase IL-12 expression in vivo, we determined the level of IL-12 in the mice injected with rAd/IL-12p40. However, there was no significant increase in IL-12 level (data not shown).

FIGURE 2.

Neither IFN-γ nor NO is related with IL-12p40-mediated immune modulation. The 106 PFU of rAd/OVA and different doses of rAd/IL-12 or IL-12p40 (102–108 PFU) were injected into the mice (n ≥ 5/group) i.m. rAd/EGFP was injected to complete the total virus up to 1.01 × 108 PFU. A, One day after immunization, sera were collected and the levels of IL-12 and IL-12p40 were measured by ELISA. B, On day 5, the levels of IFN-γ and NO were measured as described in Materials and Methods. Data are presented as mean ± SEM of five to six mice and representative of three independent experiments.

FIGURE 2.

Neither IFN-γ nor NO is related with IL-12p40-mediated immune modulation. The 106 PFU of rAd/OVA and different doses of rAd/IL-12 or IL-12p40 (102–108 PFU) were injected into the mice (n ≥ 5/group) i.m. rAd/EGFP was injected to complete the total virus up to 1.01 × 108 PFU. A, One day after immunization, sera were collected and the levels of IL-12 and IL-12p40 were measured by ELISA. B, On day 5, the levels of IFN-γ and NO were measured as described in Materials and Methods. Data are presented as mean ± SEM of five to six mice and representative of three independent experiments.

Close modal

Since the immune suppression with a high dose of IL-12 was caused by the elevation of IFN-γ and NO in serum (32, 33), we measured the level of IFN-γ and NO to address whether these factors were related with the immunomodulatory effect of IL-12p40. As a result, the levels of IFN-γ and NO were dramatically increased in serum by a high dose of rAd/IL-12 (1 × 108 PFU; Fig. 2,B), which eventually might suppress the OVA-specific CD8+ T cell response (Fig. 1, A and B) as previously reported (32). However, the coimmunization of the high dose of rAd/IL-12p40 (1 × 108 PFU) failed to induce IFN-γ or NO in serum (Fig. 2 B), indicating that IFN-γ and NO are not involved in the immune modulation by IL-12p40.

The augmentation or depression of T cell responses can result from the differential modulation of either T cell proliferation in the early stage or the maintenance after peak response. To determine when IL-12p40 influence Ag-specific CD8+ T cell response, we observed the T cell response from day 6, the early activation phase, to day 40, the memory phase. As a result, the percentages of OVA-specific IFN-γ-producing T cells from PBMCs were found to be increased by low doses of rAd/IL-12p40 (104 PFU), but decreased much more with a high dose of rAd/IL-12p40 (108 PFU) on days 6 and 12 (p < 0.05 on day 12; Fig. 3, A and B). Also, the tendency of the T cell response was maintained up to day 40 (p < 0.05 for enhancement and p < 0.01 for suppression; Fig. 3, A and B).

FIGURE 3.

IL-12p40 modulates the CD8+ T cell response by affecting the population of Ag-specific T cells from the early phase. Six or 12 days after injection, PBMCs were prepared from the blood and the percentages of OVA-specific IFN-γ-producing cells were determined by an intracellular IFN-γ staining and FACS analysis. On day 40, mice were sacrificed and the intracellular IFN-γ staining assay was performed with the splenocytes. A, The percentages of OVA-specific IFN-γ-producing CD8+ T cells from PBMCs or splenocytes were analyzed by flow cytometer (day 6, ▨; day 12, ▦; and day 40, ▪). Data are presented as mean ± SEM of four mice. B, Numbers in quadrants indicate the percentages of IFN-γ-positive cells in CD8+ T cells. Results are representative of four mice. C, On day 40, the percentages of adoptively transferred Thy1.1+ CD8+ T cells were analyzed and presented as mean ± SEM (upper panel) and as representative density plots of four mice (lower panel). These data are representative of three independent experiments with similar results.

FIGURE 3.

IL-12p40 modulates the CD8+ T cell response by affecting the population of Ag-specific T cells from the early phase. Six or 12 days after injection, PBMCs were prepared from the blood and the percentages of OVA-specific IFN-γ-producing cells were determined by an intracellular IFN-γ staining and FACS analysis. On day 40, mice were sacrificed and the intracellular IFN-γ staining assay was performed with the splenocytes. A, The percentages of OVA-specific IFN-γ-producing CD8+ T cells from PBMCs or splenocytes were analyzed by flow cytometer (day 6, ▨; day 12, ▦; and day 40, ▪). Data are presented as mean ± SEM of four mice. B, Numbers in quadrants indicate the percentages of IFN-γ-positive cells in CD8+ T cells. Results are representative of four mice. C, On day 40, the percentages of adoptively transferred Thy1.1+ CD8+ T cells were analyzed and presented as mean ± SEM (upper panel) and as representative density plots of four mice (lower panel). These data are representative of three independent experiments with similar results.

Close modal

Next, we investigated whether the enhancement or suppression is due to the changes in Ag-specific CD8+ T cell populations. As shown in Fig. 3 C, the percentages of the transferred CD8+Thy1.1+ T cells were increased by the codelivery of 104 PFU of rAd/IL-12p40, while only the basal level of CD8+Thy1.1+ T cells was detected in the mice coimmunized with 108 PFU of rAd/IL-12p40. However, the apoptotic populations of Thy1.1+ cells determined by propodium iodide/annexin V staining were similar regardless of the dose of rAd/IL-12p40 (data not shown). Thus, these data suggest that IL-12p40 modulates Ag-specific T cell responses by enhancing or suppressing the proliferation of CD8+ T cells in the early stage without affecting T cell apoptosis.

It was previously reported that the humoral responses to the adenoviral vector Ag were increased by the addition of rAd/IL-12 at a high dose (32). To compare the effects of IL-12p40 on the Ab responses with that of IL-12, we analyzed the level of OVA-specific serum IgG at 4 wk after immunization. Although the OVA-specific B cell response was enhanced by IL-12 in terms of total serum IgG, it was not affected by IL-12p40 (p < 0.05 for rAd/IL-12 and p = 0.60 for IL-12p40; Fig. 4,A). Also, the IgG1:IgG2a ratio, the indicator of Th1 response, was not significantly altered along with the doses of rAd/IL-12p40 (from 1.66 for rAd/OVA alone to 1.47 for rAd/OVA plus 108 PFU of rAd/IL-12p40), while the addition of rAd/IL-12 gave rise to the reduction of the IgG1:IgG2a ratio (from 1.62 for rAd/OVA alone to 0.93 for rAd/OVA plus 108 PFU of rAd/IL-12; Fig. 4 B). Together, these results indicate that IL-12p40 has little effect on the B cell response.

FIGURE 4.

IL-12p40 has no effect on humoral response to codelivered Ag. rAd/OVA with rAd/IL-12p40 or rAd/IL-12 were injected into B6 mice (n ≥ 5/group). Four weeks after immunization, sera were collected and the levels of OVA-specific serum IgGs were determined by Ab ELISA. Sera were diluted by 50-fold and anti-OVA total IgG (A), IgG1, and IgG2a (B) isotypes were measured. The numbers on each bar represent the IgG1:IgG2a ratio. These results are representative of three independent experiments.

FIGURE 4.

IL-12p40 has no effect on humoral response to codelivered Ag. rAd/OVA with rAd/IL-12p40 or rAd/IL-12 were injected into B6 mice (n ≥ 5/group). Four weeks after immunization, sera were collected and the levels of OVA-specific serum IgGs were determined by Ab ELISA. Sera were diluted by 50-fold and anti-OVA total IgG (A), IgG1, and IgG2a (B) isotypes were measured. The numbers on each bar represent the IgG1:IgG2a ratio. These results are representative of three independent experiments.

Close modal

It is known that the IL-12p40 homodimer inhibits IL-12 function by interfering with the binding of IL-12 to its receptor (17, 19) and that IL-12Rβ2 subunit is required for IL-12 function (34, 35). To define the action mechanism of IL-12p40-mediated immune modulation, we compared the OVA-specific CD8+ T cell responses in B6, IL-12Rβ1KO, and IL-12Rβ2KO mice. As expected, the number of OVA-specific IFN-γ-secreting cells were increased by a low dose of rAd/IL-12p40 (p < 0.05) and dramatically reduced by a high dose of rAd/IL-12p40 (p < 0.05) in B6 mice. A similar pattern of T cell responses was observed in IL-12Rβ2KO. Interestingly, this differential immune modulation mediated by IL-12p40 disappeared in IL-12Rβ1KO mice regardless of the rAd/IL-12p40 doses (p = 0.80 and p = 0.34, respectively), showing that the IL-12p40-mediated immune regulation is IL-12Rβ1-dependent (Fig. 5). Administration of rAd/IL-12 failed to bring the enhancing or suppressive effect in both β1 and β2 KO mice as previously reported (data not shown) (36, 37).

FIGURE 5.

IL-12Rβ1 is required for the immune modulation by IL-12p40. Two weeks after immunization, PBMCs were prepared from B6 (▪), IL-12Rβ1KO (▦), and IL-12Rβ2KO (▧) mice (n ≥ 3/group). A, The percentages of OVA-specific IFN-γ-producing CD8+ T cells from PBMCs were analyzed by flow cytometer. Data are presented as mean ± SEM of three to five mice. B, Numbers in quadrants indicate the percentages of IFN-γ-positive cells in CD8+ T cells. Results are representative of three to five mice. A and B, These data are representative of two independent experiments.

FIGURE 5.

IL-12Rβ1 is required for the immune modulation by IL-12p40. Two weeks after immunization, PBMCs were prepared from B6 (▪), IL-12Rβ1KO (▦), and IL-12Rβ2KO (▧) mice (n ≥ 3/group). A, The percentages of OVA-specific IFN-γ-producing CD8+ T cells from PBMCs were analyzed by flow cytometer. Data are presented as mean ± SEM of three to five mice. B, Numbers in quadrants indicate the percentages of IFN-γ-positive cells in CD8+ T cells. Results are representative of three to five mice. A and B, These data are representative of two independent experiments.

Close modal

CD8+ T cells are known to play a pivotal role in antitumor activity in various tumor models (38, 39, 40). To investigate whether the differential effects of IL-12p40 on induction of CD8+ T cell responses correlate with antitumor activity, the immunized mice were challenged with E.G7-OVA, an OVA-expressing syngenic tumor cell line. When the tumor growth and survival rate were examined up to day 42, immunized mice with rAd/OVA exhibited a retardation of tumor growth compared with the PBS-injected group (p ≪ 0.001, day 18; Fig. 6 A). However, the coinjection of rAd/IL-12p40 at a high dose (108 PFU) with rAd/OVA markedly accelerated the tumor growth and reduced survival rate compared with the mice immunized with rAd/OVA alone (p ≪ 0.001, day 18). Interestingly, the immunization of rAd/OVA with rAd/IL-12p40 at a low dose (104 PFU) rather increased antitumor activity, resulting in delayed tumor growth and reduced lethality compared with the rAd/OVA-alone group (p < 0.05, day 30). These results indicate that Ag-specific CD8+ T cell responses also correlate with antitumor activity in our prophylactic vaccine model.

FIGURE 6.

IL-12p40 modulates the antitumor activity in a dose-dependent manner. Five weeks after immunization, mice (n = 10/group) were s.c. injected with 5 × 105 of E.G7-OVA cells. Individual evolution of the tumor mass (A) and survival rate (B) were monitored two to three times per week. Data are presented as the mean ± SEM of the tumor volume (A). These are representative of two independent experiments.

FIGURE 6.

IL-12p40 modulates the antitumor activity in a dose-dependent manner. Five weeks after immunization, mice (n = 10/group) were s.c. injected with 5 × 105 of E.G7-OVA cells. Individual evolution of the tumor mass (A) and survival rate (B) were monitored two to three times per week. Data are presented as the mean ± SEM of the tumor volume (A). These are representative of two independent experiments.

Close modal

In this study, we demonstrated the effects of IL-12p40 on the generation of the Ag-specific CD8+ T cell response in a recombinant adenovirus-based vaccine model. rAd/IL-12p40 as a genetic adjuvant showed an immune-enhancing effect on Ag-specific CD8+ T cell responses at a low dose, while it negatively modulated the responses at a high dose. We also observed the similar effect of IL-12p40 in hepatitis C virus E2-specific CD8+ T cell responses using DNA vaccine and the adenovirus vaccine in the naive BALB/c mice model (data not shown), implying independency of the Ag-specific CD8+ T cell precursor frequency as well as antigenic nature and vaccine regimen.

It was reported that IL-12p40 has a positive regulatory effect on the immune response in several experimental models (16, 26, 27, 28, 30). In the Mtb infection model, the exogenous IL-12p40 homodimer partially restored the protection in IL-12p40-deficient mice (29), and IL-12p40 was known to be required for the migration of pulmonary DCs to lymph nodes (30), which is an essential event for T cell priming and activation. Since we observed that IL-12p40-induced immune modulation occurred at the early stages of T cell activation (day 6) without apparent differences in T cell apoptosis, it is possible to speculate that a low dose of rAd/IL-12p40 increased migration of APCs, which eventually enhanced T cell responses. It is worth noting that unlike T cells, DCs and macrophages constitutively express IL-12R subunits (41, 42, 43), which further supports our suggestion that IL-12p40 can modulate immune responses via APCs at an initial phase.

Interestingly, a similar pattern of CD8+ T cell responses was also shown in IL-12Rβ2KO mice as well as in wild-type B6 mice by the coadministration of rAd/IL-12p40, whereas the effect of IL-12p40 disappeared in IL-12Rβ1KO mice. These results indicate that IL-12p40 has an IL-12Rβ1-mediated immunomodulatory function. Previously, it was reported that the IL-12p40 homodimer positively or negatively modulated macrophage chemotaxis in a dose-dependent manner and that IL-12Rβ1, but not β2, was required and sufficient for the migration of macrophages (44). Also, IL-12Rβ1 was found to form oligomeric structures on the cell surface and to bind to IL-12p40 at a high affinity (45). Thus, it is likely that IL-12p40 enhanced or suppressed CD8+ T cell responses by the modulation of APC migration via IL-12Rβ1. Besides, it is possible that IL-12p40 binds to B cells which constitutively express only IL-12Rβ1 (36, 46, 47, 48). However, it is still unclear whether IL-12p40 can modulate the CD8+ T cell responses via B cells.

Although it is well known that IL-12Rβ2 is the main signal transducing subunit by which the Jak-STAT pathway is activated (34, 35, 49), the downstream molecules involved in IL-12Rβ1 signaling have not been well investigated except for the p38 MAPK and NF-κB activation in microglial cells or macrophages (50, 51). Further study for understanding of IL-12p40-mediated signaling is needed.

IL-12p40 could directly exert its suppressive activity on T cells in addition to action on APCs at the initial phase. When CD8+ T cells are activated, they express a higher level of IL-12R and become responsive to IL-12, in which IL-12p40 can exert its antagonist activity on T cells. It is worth noting that the codelivery of rAd/IL-12p40 at a high dose (108 PFU) resulted in a significant level of serum IL-12p40 (167.1 ± 15.5 ng/ml on day1 and 30.1 ± 10.9 ng/ml on day 3), which might be sufficient for the inhibition of IL-12-mediated function. However, the level of IL-12p40 expressed from a low dose of rAd/IL-12p40 was undetectable in blood. Thus, it is possible that the high concentration of IL-12p40 suppressed both APC migration and T cell activation in vivo, while low levels of IL-12p40 could exist only at the initial phase, resulting in increased APC migration.

IL-23 was known to act on T cells to enhance the Th1 (9, 52) or Th17 responses (53), and IL-12p40 also can antagonize its function by inhibiting the binding of IL-23 to its receptor (20). It is possible that high expression of IL-12p40 leads to the enhancement or suppression of T cell responses by increasing IL-23 production or blocking IL-23-mediated function. However, we observed that IL-23 was not detected in the serum of the immunized mice, indicating no effect of IL-12p40 gene transfer on IL-23 expression (data not shown). Moreover, it is unlikely that IL-12p40 significantly influences CD8+ T cell responses via antagonizing the function of IL-23, since the expression of IL-23R subunit is restricted on the late or activated/memory phase of T cells (54) and IL-12p40 was shown to exhibit its differential effect from the early phase of T cell response in this study.

In conclusion, we demonstrated that IL-12p40 can modulate Ag-specific CD8+ T cell responses in a dose-dependent manner using genetic vaccination. These effects of IL-12p40 were IL-12Rβ1 dependent and IL-12Rβ2 independent, suggesting that IL-12p40 is not only a passive antagonist to IL-12 but also an independent cytokine which can affect Ag-specific adaptive immune responses. Also, these data could widen our understanding of IL-12p40, which can be informative to design vaccine strategies and therapeutic regimen.

We thank Sang-Chun Lee, Kwan Seok Lee, and Bok Chae Cho for devoted animal care and So Young Choi for the technical assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A020598), supported by a grant from Science Research Center fund to Immunomodulation Research Center at University of Ulsan from the Korea Science and Engineering Foundation and the Korea Ministry of Science and Technology, supported by Growth Engine Technology Development Program by the Ministry of Commerce, Industry and Energy (10028480-2006-11), and supported by Generic Technology Development Program, Minister of Commerce, Industry and Energy (10020817).

3

Abbreviations used in this paper: DC, dendritic cell; Mtb, Mycobacterium tuberculosis; rAd, recombinant adenovirus; EGFP, enhanced GFP; KO, knockout.

1
Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri.
1989
. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes.
J. Exp. Med.
170
:
827
-845.
2
Macatonia, S. E., N. A. Hosken, M. Litton, P. Vieira, C. S. Hsieh, J. A. Culpepper, M. Wysocka, G. Trinchieri, K. M. Murphy, A. O'Garra.
1995
. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells.
J. Immunol.
154
:
5071
-5079.
3
Stern, A. S., F. J. Podlaski, J. D. Hulmes, Y. C. Pan, P. M. Quinn, A. G. Wolitzky, P. C. Familletti, D. L. Stremlo, T. Truitt, R. Chizzonite, et al
1990
. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells.
Proc. Natl. Acad. Sci. USA
87
:
6808
-6812.
4
Murtaugh, M. P., D. L. Foss.
2002
. Inflammatory cytokines and antigen presenting cell activation.
Vet. Immunol. Immunopathol.
87
:
109
-121.
5
Wolf, S. F., P. A. Temple, M. Kobayashi, D. Young, M. Dicig, L. Lowe, R. Dzialo, L. Fitz, C. Ferenz, R. M. Hewick, et al
1991
. Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.
J. Immunol.
146
:
3074
-3081.
6
Schoenhaut, D. S., A. O. Chua, A. G. Wolitzky, P. M. Quinn, C. M. Dwyer, W. McComas, P. C. Familletti, M. K. Gately, U. Gubler.
1992
. Cloning and expression of murine IL-12.
J. Immunol.
148
:
3433
-3440.
7
Ma, X., M. Aste-Amezaga, G. Gri, F. Gerosa, G. Trinchieri.
1997
. Immunomodulatory functions and molecular regulation of IL-12.
Chem. Immunol.
68
:
1
-22.
8
Aggarwal, S., N. Ghilardi, M. H. Xie, F. J. de Sauvage, A. L. Gurney.
2003
. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17.
J. Biol. Chem.
278
:
1910
-1914.
9
Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al
2000
. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.
Immunity
13
:
715
-725.
10
Vanden Eijnden, S., S. Goriely, D. De Wit, F. Willems, M. Goldman.
2005
. IL-23 up-regulates IL-10 and induces IL-17 synthesis by polyclonally activated naive T cells in human.
Eur. J. Immunol.
35
:
469
-475.
11
D'Andrea, A., M. Rengaraju, N. M. Valiante, J. Chehimi, M. Kubin, M. Aste, S. H. Chan, M. Kobayashi, D. Young, E. Nickbarg, et al
1992
. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells.
J. Exp. Med.
176
:
1387
-1398.
12
Podlaski, F. J., V. B. Nanduri, J. D. Hulmes, Y. C. Pan, W. Levin, W. Danho, R. Chizzonite, M. K. Gately, A. S. Stern.
1992
. Molecular characterization of interleukin 12.
Arch. Biochem. Biophys.
294
:
230
-237.
13
Gillessen, S., D. Carvajal, P. Ling, F. J. Podlaski, D. L. Stremlo, P. C. Familletti, U. Gubler, D. H. Presky, A. S. Stern, M. K. Gately.
1995
. Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist.
Eur. J. Immunol.
25
:
200
-206.
14
Jin, H. T., J. I. Youn, H. J. Kim, J. B. Lee, S. J. Ha, J. S. Koh, Y. C. Sung.
2005
. Enhancement of interleukin-12 gene-based tumor immunotherapy by the reduced secretion of p40 subunit and the combination with farnesyltransferase inhibitor.
Hum. Gene Ther.
16
:
328
-338.
15
Trinchieri, G..
1998
. Interleukin-12: a cytokine at the interface of inflammation and immunity.
Adv. Immunol.
70
:
83
-243.
16
Heinzel, F. P., A. M. Hujer, F. N. Ahmed, R. M. Rerko.
1997
. In vivo production and function of IL-12 p40 homodimers.
J. Immunol.
158
:
4381
-4388.
17
Ling, P., M. K. Gately, U. Gubler, A. S. Stern, P. Lin, K. Hollfelder, C. Su, Y. C. Pan, J. Hakimi.
1995
. Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity.
J. Immunol.
154
:
116
-127.
18
Mattner, F., S. Fischer, S. Guckes, S. Jin, H. Kaulen, E. Schmitt, E. Rude, T. Germann.
1993
. The interleukin-12 subunit p40 specifically inhibits effects of the interleukin-12 heterodimer.
Eur. J. Immunol.
23
:
2202
-2208.
19
Gately, M. K., D. M. Carvajal, S. E. Connaughton, S. Gillessen, R. R. Warrier, K. D. Kolinsky, V. L. Wilkinson, C. M. Dwyer, G. F. Higgins, Jr, F. J. Podlaski, et al
1996
. Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo.
Ann. NY Acad. Sci.
795
:
1
-12.
20
Shimozato, O., S. Ugai, M. Chiyo, H. Takenobu, H. Nagakawa, A. Wada, K. Kawamura, H. Yamamoto, M. Tagawa.
2006
. The secreted form of the p40 subunit of interleukin (IL)-12 inhibits IL-23 functions and abrogates IL-23-mediated antitumour effects.
Immunology
117
:
22
-28.
21
Kato, K., O. Shimozato, K. Hoshi, H. Wakimoto, H. Hamada, H. Yagita, K. Okumura.
1996
. Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection.
Proc. Natl. Acad. Sci. USA
93
:
9085
-9089.
22
Abdi, K., S. H. Herrmann.
1997
. CTL generation in the presence of IL-4 is inhibited by free p40: evidence for early and late IL-12 function.
J. Immunol.
159
:
3148
-3155.
23
Yoshimoto, T., C. R. Wang, T. Yoneto, S. Waki, S. Sunaga, Y. Komagata, M. Mitsuyama, J. Miyazaki, H. Nariuchi.
1998
. Reduced T helper 1 responses in IL-12 p40 transgenic mice.
J. Immunol.
160
:
588
-594.
24
Chen, L., D. Chen, E. Block, M. O'Donnell, D. W. Kufe, S. K. Clinton.
1997
. Eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cDNAs for the IL-12 heterodimer and its inhibition by the IL-12 p40 subunit homodimer.
J. Immunol.
159
:
351
-359.
25
Ha, S. J., J. Chang, M. K. Song, Y. S. Suh, H. T. Jin, C. H. Lee, G. H. Nam, G. Choi, K. Y. Choi, S. H. Lee, et al
2002
. Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization.
Nat. Biotechnol.
20
:
381
-386.
26
Piccotti, J. R., S. Y. Chan, R. E. Goodman, J. Magram, E. J. Eichwald, D. K. Bishop.
1996
. IL-12 antagonism induces T helper 2 responses, yet exacerbates cardiac allograft rejection: evidence against a dominant protective role for T helper 2 cytokines in alloimmunity.
J. Immunol.
157
:
1951
-1957.
27
Piccotti, J. R., S. Y. Chan, K. Li, E. J. Eichwald, D. K. Bishop.
1997
. Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-γ-producing cells.
J. Immunol.
158
:
643
-648.
28
Ha, S. J., C. H. Lee, S. B. Lee, C. M. Kim, K. L. Jang, H. S. Shin, Y. C. Sung.
1999
. A novel function of IL-12p40 as a chemotactic molecule for macrophages.
J. Immunol.
163
:
2902
-2908.
29
Holscher, C., R. A. Atkinson, B. Arendse, N. Brown, E. Myburgh, G. Alber, F. Brombacher.
2001
. A protective and agonistic function of IL-12p40 in mycobacterial infection.
J. Immunol.
167
:
6957
-6966.
30
Khader, S. A., S. Partida-Sanchez, G. Bell, D. M. Jelley-Gibbs, S. Swain, J. E. Pearl, N. Ghilardi, F. J. Desauvage, F. E. Lund, A. M. Cooper.
2006
. Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection.
J. Exp. Med.
203
:
1805
-1815.
31
Lee, C. G., S. Y. Choi, S. H. Park, K. S. Park, S. H. Ryu, Y. C. Sung.
2005
. The synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met as a novel adjuvant for DNA vaccine.
Vaccine
23
:
4703
-4710.
32
Lasarte, J. J., F. J. Corrales, N. Casares, A. Lopez-Diaz de Cerio, C. Qian, X. Xie, F. Borras-Cuesta, J. Prieto.
1999
. Different doses of adenoviral vector expressing IL-12 enhance or depress the immune response to a coadministered antigen: the role of nitric oxide.
J. Immunol.
162
:
5270
-5277.
33
Koblish, H. K., C. A. Hunter, M. Wysocka, G. Trinchieri, W. M. Lee.
1998
. Immune suppression by recombinant interleukin (rIL)-12 involves interferon γ induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect.
J. Exp. Med.
188
:
1603
-1610.
34
Bacon, C. M., E. F. Petricoin, III, J. R. Ortaldo, R. C. Rees, A. C. Larner, J. A. Johnston, J. J. O'Shea.
1995
. Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes.
Proc. Natl. Acad. Sci. USA
92
:
7307
-7311.
35
Jacobson, N. G., S. J. Szabo, R. M. Weber-Nordt, Z. Zhong, R. D. Schreiber, J. E. Darnell, Jr, K. M. Murphy.
1995
. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat) 3 and Stat4.
J. Exp. Med.
181
:
1755
-1762.
36
Wu, C., J. Ferrante, M. K. Gately, J. Magram.
1997
. Characterization of IL-12 receptor β1 chain (IL-12Rβ1)-deficient mice: IL-12Rβ1 is an essential component of the functional mouse IL-12 receptor.
J. Immunol.
159
:
1658
-1665.
37
Wu, C., X. Wang, M. Gadina, J. J. O'Shea, D. H. Presky, J. Magram.
2000
. IL-12 receptor β 2 (IL-12Rβ2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding sites.
J. Immunol.
165
:
6221
-6228.
38
Dailey, M. O., E. Pillemer, I. L. Weissman.
1982
. Protection against syngeneic lymphoma by a long-lived cytotoxic T-cell clone.
Proc. Natl. Acad. Sci. USA
79
:
5384
-5387.
39
Greenberg, P. D..
1986
. Therapy of murine leukemia with cyclophosphamide and immune Lyt-2+ cells: cytolytic T cells can mediate eradication of disseminated leukemia.
J. Immunol.
136
:
1917
-1922.
40
Kast, W. M., R. Offringa, P. J. Peters, A. C. Voordouw, R. H. Meloen, A. J. van der Eb, C. J. Melief.
1989
. Eradication of adenovirus E1-induced tumors by E1A-specific cytotoxic T lymphocytes.
Cell
59
:
603
-614.
41
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-κB and primes DC for IL-12 production.
Immunity
9
:
315
-323.
42
Ha, S. J., S. B. Lee, C. M. Kim, H. S. Shin, Y. C. Sung.
1998
. Rapid recruitment of macrophages in interleukin-12-mediated tumour regression.
Immunology
95
:
156
-163.
43
Puddu, P., L. Fantuzzi, P. Borghi, B. Varano, G. Rainaldi, E. Guillemard, W. Malorni, P. Nicaise, S. F. Wolf, F. Belardelli, S. Gessani.
1997
. IL-12 induces IFN-γ expression and secretion in mouse peritoneal macrophages.
J. Immunol.
159
:
3490
-3497.
44
Russell, T. D., Q. Yan, G. Fan, A. P. Khalifah, D. K. Bishop, S. L. Brody, M. J. Walter.
2003
. IL-12 p40 homodimer-dependent macrophage chemotaxis and respiratory viral inflammation are mediated through IL-12 receptor β1.
J. Immunol.
171
:
6866
-6874.
45
Chua, A. O., R. Chizzonite, B. B. Desai, T. P. Truitt, P. Nunes, L. J. Minetti, R. R. Warrier, D. H. Presky, J. F. Levine, M. K. Gately, et al
1994
. Expression cloning of a human IL-12 receptor component: a new member of the cytokine receptor superfamily with strong homology to gp130.
J. Immunol.
153
:
128
-136.
46
Benjamin, D., V. Sharma, M. Kubin, J. L. Klein, A. Sartori, J. Holliday, G. Trinchieri.
1996
. IL-12 expression in AIDS-related lymphoma B cell lines.
J. Immunol.
156
:
1626
-1637.
47
Vogel, L. A., L. C. Showe, T. L. Lester, R. M. McNutt, V. H. Van Cleave, D. W. Metzger.
1996
. Direct binding of IL-12 to human and murine B lymphocytes.
Int. Immunol.
8
:
1955
-1962.
48
Wu, C. Y., R. R. Warrier, D. M. Carvajal, A. O. Chua, L. J. Minetti, R. Chizzonite, P. K. Mongini, A. S. Stern, U. Gubler, D. H. Presky, M. K. Gately.
1996
. Biological function and distribution of human interleukin-12 receptor β chain.
Eur. J. Immunol.
26
:
345
-350.
49
Yu, C. R., J. X. Lin, D. W. Fink, S. Akira, E. T. Bloom, A. Yamauchi.
1996
. Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2, IL-12, and IFN-α.
J. Immunol.
157
:
126
-137.
50
Jana, M., S. Dasgupta, R. N. Saha, X. Liu, K. Pahan.
2003
. Induction of tumor necrosis factor-α (TNF-α) by interleukin-12 p40 monomer and homodimer in microglia and macrophages.
J. Neurochem.
86
:
519
-528.
51
Pahan, K., F. G. Sheikh, X. Liu, S. Hilger, M. McKinney, T. M. Petro.
2001
. Induction of nitric-oxide synthase and activation of NF-κB by interleukin-12 p40 in microglial cells.
J. Biol. Chem.
276
:
7899
-7905.
52
Ha, S. J., D. J. Kim, K. H. Baek, Y. D. Yun, Y. C. Sung.
2004
. IL-23 induces stronger sustained CTL and Th1 immune responses than IL-12 in hepatitis C virus envelope protein 2 DNA immunization.
J. Immunol.
172
:
525
-531.
53
Iwakura, Y., H. Ishigame.
2006
. The IL-23/IL-17 axis in inflammation.
J. Clin. Invest.
116
:
1218
-1222.
54
Parham, C., M. Chirica, J. Timans, E. Vaisberg, M. Travis, J. Cheung, S. Pflanz, R. Zhang, K. P. Singh, F. Vega, et al
2002
. A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rβ1 and a novel cytokine receptor subunit, IL-23R.
J. Immunol.
168
:
5699
-5708.