Two subunits of the IL-12 receptor (IL-12R), IL-12Rβ1 and IL-12Rβ2, have been identified and cloned. Previous studies demonstrated that the IL-12Rβ1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12Rβ2 in IL-12 signaling, we have generated IL-12Rβ2-deficient (IL-12Rβ2−/−) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12Rβ2−/− mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12Rβ2−/− mice failed to produce IFN-γ or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12Rβ2−/− splenocytes was not induced when incubated with IL-12. IL-12Rβ2−/− splenocytes were deficient in IFN-γ secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12Rβ2−/− mice were deficient in vivo in their ability to produce IFN-γ following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12Rβ1 is the subunit primarily responsible for binding IL-12, IL-12Rβ2 plays an essential role in mediating the biological functions of IL-12 in mice.

Interleukin-12 is a heterodimeric cytokine that is composed of a disulfide-bonded 40-kDa subunit and a 35-kDa subunit. It is predominantly produced by APCs, such as stimulated macrophages, monocytes, dendritic cells, neutrophils, and some B cells. IL-12 is an important immunologic regulator of T and NK cell functions. Its biological activities include promotion of cell-mediated immunity by inducing Th1 responses, induction of IFN-γ production by both T and NK cells, stimulation of the proliferation of activated T and NK cells, and enhancement of T and NK cell-mediated cytolytic lymphocyte responses (reviewed in Refs. 1, 2).

The biological functions of IL-12 are mediated through specific receptors on T and NK cells. To date, two IL-12 receptor subunits have been identified in mouse and humans (3, 4, 5). These receptor subunits are members of the cytokine receptor superfamily. Their cytoplasmic regions contain the box1 and box2 motifs that exist in other cytokine receptors, and they are related closely over their entire length to the “β type” cytokine receptor glycoprotein 130 and the receptors for leukemia-inhibitory factor and G-CSF. Thus, the two IL-12 receptor subunits were designated IL-12Rβ1 and IL-12Rβ2. Analyses of IL-12 binding to these receptors demonstrated that, when expressed in COS-7 cells individually, human IL-12Rβ1 and IL-12Rβ2 bind IL-12 with low affinity; however, when the two subunits are coexpressed, they confer both high and low affinity binding of IL-12 and IL-12 responsiveness (5). In contrast to the human IL-12 receptors, IL-12Rβ1 is the primary binding component in the mouse conferring both high and low affinity binding sites, whereas IL-12Rβ2 only binds weakly to IL-12 (4, 6).

IL-12Rβ1-deficient mice have been generated previously (7). IL-12Rβ1 has been found to be required for high affinity binding of IL-12 and for mouse T and NK cells to respond to IL-12 in vitro and in vivo. To investigate the role of IL-12Rβ2 in mediating IL-12 biological functions, mice deficient in IL-12Rβ2 were generated using homologous recombination in embryonic stem (ES)5 cells. IL-12Rβ2-deficient (IL-12Rβ2−/−) mice were visually indistinguishable from their wild-type littermates. Development of T and B cells in IL-12Rβ2−/− mice was not affected as compared with wild-type littermates. Con A-activated splenocytes from IL-12Rβ2−/− mice still bind IL-12 with both high and low affinity. Nevertheless, no IL-12-mediated biological functions can be detected in the IL-12Rβ2−/− mice. The data presented in this paper demonstrate that although mouse IL-12Rβ1 binds IL-12 with both high and low affinity, IL-12Rβ2 plays an essential role in mediating the biological functions of IL-12 in mice.

C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the animal facilities at Hoffmann-LaRoche (Nutley, NJ). YAC-1 lymphoma cells were maintained in culture as previously described (8).

Tissue culture medium (TCM) was a 1:1 mixture of RPMI 1640 and DMEM (Life Technologies, Grand Island, NY) supplemented as previously described (9) with 5% heat-inactivated FBS (Life Technologies). Complete culture medium used in the experiments consisted of RPMI 1640 (Life Technologies) supplemented as previously described (10). Ab to mouse CD3 was obtained from PharMingen (San Diego, CA). Purified mouse rIL-12 was prepared as previously reported (9). Purified human rIL-2 was supplied by Dr. F. Khan (Hoffmann-LaRoche). mAbs R4–6A2 and XMG1.2 to mouse IFN-γ were obtained from PharMingen. Purified mouse rIFN-γ was provided by Dr. G. Alber (Hoffmann-LaRoche, Basel, Switzerland). Con A and Salmonella enteritidis LPS were obtained from Sigma (St. Louis, MO).

Five mouse IL-12Rβ2 genomic clones were isolated from a 129/Sv library. The inserts were digested with NotI and EcoRI, and several digested fragments were subcloned into pBluescript KS(+) (Stratagene, La Jolla, CA). Restriction mapping, sequencing, and PCR were used to determine the genomic structure. Analysis of the subclones to determine the genomic structure and generation of the targeting vector were performed using standard protocols (11). The targeting vector contains four DNA fragments: 5′-flanking sequence, 3′-flanking sequence, the PGK-1 neo gene (12), and the pMC1-tk gene (13). A 3.2-kb EcoRI-EcoRI fragment located upstream of the 4.2-kb EcoRI-EcoRI fragment containing exons 2 and 3 from the IL-12Rβ2 gene was isolated from the genomic subclone in pBluescipt KS(+) and inserted into the NotI site of pPGKneotk (14). The resulting plasmid, pGKneotk/3.2, was then digested by HindIII, filled in with T4 DNA polymerase (New England Biolabs, Beverly, MA) in the presence of all four deoxyribonucleotides, and then treated with calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN). Finally, a 6.6-kb EcoRI-EcoRI fragment containing exons 4–6 was inserted into the HindIII-digested pPGKneotk/3.2 to generate the targeting vector. The orientation of the 4.2- and 6.6-kb fragments in the resulting plasmid was checked by further restriction digestion. The targeting vector was digested with SalI to linearize the plasmid before electroporation into ES cells.

The W9.5 ES cell line was maintained on irradiated primary murine embryonic fibroblasts as previously described (15) in medium containing 15% FBS and 100 U/ml leukemia-inhibitory factor (Life Technologies). ES cells from a confluent dish were harvested and electroporated with 25 μg of linearized targeting vector using a Gene Pulser (Bio-Rad, Richmond, CA). The cells were then cultured in medium containing G418 (350 μg/ml; Life Technologies) for 1 day after electroporation. Ganciclovir (2 mM; a gift from Syntex, Palo Alto, CA) was added 2 days after electroporation, and selection was conducted for 7 days. Colonies were then picked and expanded.

Southern blot analysis of ES cell DNA was used to determine which of the colonies contained a correctly targeted event. ES cell DNA was extracted as previously described (16). DNA was digested with HindIII and StuI, fractionated by agarose gel electrophoresis, transferred to Hybond-N membrane (Amersham, Arlington Heights, IL) by a electroblot apparatus (Hoefer Scientific Instrument, Canberra Packard, Vancouver, Canada), and UV cross-linked using a Stratalinker (Stratagene). A 1.2-kb EcoRI-BglII probe 3′ to the targeted region was labeled using Prime-It II Random Primer Labeling Kit (Stratagene) as per the manufacturer’s protocol. The membrane was hybridized with the probe for 1 h in Rapid-Hyb buffer (Amersham) at 65°C. The membrane was then washed in 2× SSC/0.1% SDS at room temperature followed by 0.1× SSC/0.1% SDS at 65°C. The membrane was analyzed by a Molecular Dynamics phosphorImager (Sunnyvale, CA). Additional Southern blot analyses using probes from the neomycin gene and the 5′ end of the IL-12Rβ1 gene were performed to confirm the structure of the targeted allele.

Four correctly targeted ES cell clones, G4, J1, J3, and I1, were used to generate chimeric mice. ES cells were injected into host C57BL/6J blastocysts, and embryos were transplanted into the uterine horns of pseudopregnant C57BL/6J × CBA/J F1 females. Highly chimeric agouti males, as judged by agouti coat color, were bred to C57BL/6J and BALB/cByJ females. Progeny carrying one copy of the targeted allele were identified by PCR. Heterozygotes were intercrossed to obtain mice homozygous for the mutation.

PCR analysis of tail DNA was used to identify mice carrying the mutant allele. Tail DNA was isolated as described (16). Two sets of primers were used for the analysis: 5′-GAAGCGGGAAGGGACTGGCTGCTA-3′ (PGK-1 neo); 5′-CGGGAGCGGCGATACCGTAAAGC-3′ (PGK-1 neo); 5′-GTGTGCAAGCTTGGCACTGTGACCGTCCAG-3′ (exon 3); and 5′-GTTTAGCTTGCAGACAAACAAGGTCATACC-3′ (exon 3). An Invitrogen (Carlsbad, CA) Optimized Buffer J Kit was used as per the manufacturer’s protocol. The amplification parameters used in these experiments were: incubation at 94°C for 7 min (1 cycle); denaturation at 94°C for 1 min, annealing at 64°C for 1 min, and extension at 72°C for 1 min (35 cycles); and incubation at 72°C for 6 min (1 cycle). Amplicons were fractionated in gels containing 0.8% NuSieve (FMC BioProducts, Rockland, ME) and 0.8% agarose (Life Technologies) and visualized by ethidium bromide staining.

Recombinant mouse IL-12 was expressed, purified, and labeled with 125I as previously described (17). Activation of splenocytes with Con A (18) and mouse 125I-IL-12 binding assays were performed as previously described (17, 19). All binding assays were performed in triplicate. Receptor binding data were analyzed by using the nonlinear regression program RADLIG 4.0 (20) and plotted by the method of Scatchard (21).

Con A-activated splenocytes were incubated in 96-well plates (Costar, Cambridge, MA) at a final density of 5 × 105 cells/ml in TCM in the presence or absence of the indicated concentrations of cytokines. After incubation at 37°C for 48 h, cells were pulsed with 0.5 μCi/well [3H]TdR (New England Nuclear, Boston, MA) overnight at 37°C. The incorporation of [3H]TdR into cellular DNA was measured by harvesting the contents of each well onto glass fiber filters using a Tomtec cell harvester (Wallac, Gaithersburg, MD). All samples were assayed in triplicate.

Cytolytic assays using 51Cr-labeled YAC-1 target cells were performed as previously described (9). Briefly, splenocytes were cultured at 4 × 106 cells/ml in the presence of indicated concentrations of IL-12 or IL-2 for 4 days at 37°C. Control cultures contained splenocytes without added cytokines. The cells were then harvested and tested for lytic activity. Lytic assays were performed in quadruplicate, and spontaneous 51Cr release ranged from 2.1 to 13.1%.

LPS-induced IFN-γ production in vivo was performed as previously described (10). For IL-12-induced IFN-γ production in vivo, mice were injected with 1 μg/mouse IL-12 i.p. once daily for 5 days and bled 6 h after the final dose was given (9). For the induction of IFN-γ production in vitro, Con A-activated splenocytes were incubated at a density of 3 × 106 cells/ml in TCM in increasing doses of IL-12. Cultures were conducted in 48-well plates in duplicate wells. After incubation at 37°C for 48 h, cell-free culture fluids were harvested. IFN-γ in sera or culture fluids was assayed using an ELISA as previously described (10).

For induction of Th1 responses in vivo, mice were immunized with keyhole limpet hemocyanin (KLH; Calbiochem, La Jolla, CA), followed by culture of the immune lymph node cells (LNCs) with KLH to elicit cytokine production in vitro. Mice were immunized s.c. at the base of the tail with 100 μg alum-precipitated KLH together with 100 μg heat-killed Propionibacterium acnes (Elkins-Sinn, Cherry Hill, NJ). On day 5, the subinguinal, axillary, and para-aortic lymph nodes were removed aseptically, passed through a wire mesh, washed, and cultured in TCM supplemented with 10% FBS (Life Technologies) and 0 or 100 μg/ml of KLH. For measurement of IFN-γ production, LNCs were incubated in 1-ml cultures in 24-well plates (Costar) at 6 × 106 cells/well. Cell-free culture fluids were harvested by centrifugation after 48 h for IFN-γ ELISA and stored at −20°C until assayed. IFN-γ production by LNCs cultured without KLH was below the level of detection (data not shown).

After resting overnight in RPMI 1640 with 1% BSA, 2.5 × 107 Con A-activated splenocytes were plated in 1 ml TCM per well and stimulated with the indicated cytokines for 15 min. Following stimulation, lysis, immunoprecipitation, PAGE, and immunoblotting were performed as previously described (22).

To introduce a null mutation in the IL12Rβ2 gene, a replacement targeting vector was constructed as shown in Fig. 1,A. Upon homologous recombination, exons 2 and 3 were replaced with a neo cassette. This deletion removes the ATG translation initiation codon and deletes both the signal peptide and the N-terminal immunoglobulin domain of the receptor and thus would be expected to result in a null allele. The targeting vector was electroporated into W9.5 ES cells (23), and colonies that were both G418- and ganciclovir-resistant were isolated. Southern blot analysis using a probe outside the targeting vector was used to identify correctly targeted homologous recombination events by detecting a restriction fragment length polymorphism between wild-type and mutated alleles (data not shown). Approximately 25% of the isolated ES cell clones contained a correctly targeted mutation. Four of these clones were used to generate chimeric animals by injection into C57BL/6J blastocysts. All clones gave rise to highly chimeric animals that were bred to wild-type mice to generate mice heterozygous for the mutation. These heterozygous mice were then intercrossed, and the resulting progeny were screened by PCR using the primers shown in Fig. 1,A. A 500-bp fragment amplified from the neo gene is diagnostic of the mutant allele. A 265-bp fragment amplified from exon 3 of the IL-12Rβ2 gene is diagnostic for the wild-type allele. Thus, the presence of only the 500-bp PCR fragment indicates that the mouse is homozygous for the mutant allele (IL-12Rβ2−/−); both PCR fragments are detected in DNA from IL-12Rβ2+/− heterozygous mice, and wild-type mice (IL-12Rβ2+/+) are characterized by the presence of only the 265-bp PCR fragment. IL-12Rβ2−/− mice were generated from each of the four ES cell clones in the expected Mendelian frequency (Fig. 1 B and data not shown). Experiments using mice generated from each of the four different ES cell clones gave similar results and therefore will not be described separately.

FIGURE 1.

Disruption of the IL-12Rβ2 gene. A, IL-12Rβ2 targeting strategy. IL-12Rβ2 genomic structure is represented; black boxes indicate coding exons, open boxes indicate noncoding exon. The MC1-tk gene is indicated by the gray box; the PGK-neo gene is indicated by the hatched box. EcoRI, HindIII (H3), and StuI restriction endonuclease sites are indicated. The probe used for screening by genomic Southern blot analysis of ES cell clones is indicated by the closed bar. Primers used for PCR analysis are indicated by the arrows. B, Identification of IL-12Rβ2−/− mice. PCR analysis was conducted on genomic DNA isolated from tails of intercross progeny using the primers indicated in A. +/+, wild-type; +/−, heterozygous; −/−, homozygous IL-12Rβ2 mutant mice.

FIGURE 1.

Disruption of the IL-12Rβ2 gene. A, IL-12Rβ2 targeting strategy. IL-12Rβ2 genomic structure is represented; black boxes indicate coding exons, open boxes indicate noncoding exon. The MC1-tk gene is indicated by the gray box; the PGK-neo gene is indicated by the hatched box. EcoRI, HindIII (H3), and StuI restriction endonuclease sites are indicated. The probe used for screening by genomic Southern blot analysis of ES cell clones is indicated by the closed bar. Primers used for PCR analysis are indicated by the arrows. B, Identification of IL-12Rβ2−/− mice. PCR analysis was conducted on genomic DNA isolated from tails of intercross progeny using the primers indicated in A. +/+, wild-type; +/−, heterozygous; −/−, homozygous IL-12Rβ2 mutant mice.

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Northern blot analysis was used to study IL-12Rβ2 mRNA expression by IL-12Rβ2−/− and wild-type mouse splenocytes. A shortened 3.5-kb transcript was detected at significantly reduced levels in Con A-activated splenocytes from IL-12Rβ2−/− mice compared with wild-type mice (data not shown). The targeted allele results in a 0.5-kb deletion consistent with the decrease in mRNA length observed in the mutant mouse. Because exon 2 contains the ATG translation initiation codon and exons 2 and 3 encode both the signal peptide and the N-terminal immunoglobulin domain of the receptor, a truncated transcript would be expected to result in a null allele. Lack of cell surface expression of IL-12Rβ2 was substantiated by flow cytometric analysis of activated splenocytes from wild-type and mutant mice stained with a mAb that recognizes the mouse IL-12Rβ2 subunit (data not shown).

IL-12Rβ2−/− mice were visually indistinguishable from their IL-12Rβ2+/+ littermates. Quantitative analysis of splenic CD3+ T cells, CD4+ T cells, CD8+ T cells, B220+ B cells, and 2B4+ NK cells demonstrated that there are no obvious differences between IL-12Rβ2−/− mice and wild-type controls (data not shown). These data are in agreement with previously reported results from characterization of IL-12p40- (10) and IL-12Rβ1-deficient mice (7), suggesting that IL-12 signaling is not required for development of T and B cells in vivo.

Splenocytes from IL-12Rβ2−/− mice were activated with Con A in the presence of IL-2. After 3 days of stimulation, activated splenocytes were tested for 125I-IL-12 binding. Results are shown in Fig. 2. Mouse IL-12 binds to Con A blasts from IL-12Rβ2−/− mice with a high affinity (Kd) of 12.8 pM, 27 sites/cell, and a low affinity (Kd) of 8.45 nM, 1553 sites/cell, which is very similar to the results obtained from Con A-activated splenocytes from wild-type mice (7). These results demonstrate that deletion of IL-12Rβ2 does not diminish the number of high or low affinity binding sites for IL-12 on the Con A-activated splenocytes. This is in agreement with previously reported results from studies of IL-12 binding to Ba/F3 cells expressing IL-12Rβ1 that mouse IL-12Rβ1 appears to be predominantly responsible for mediating both high and low affinity binding of IL-12 (6). Therefore, IL-12Rβ2 does not contribute significantly to mouse 125I-IL-12 binding.

FIGURE 2.

Scatchard analysis of 125I-IL-12 binding on Con A-stimulated splenocytes from IL-12Rβ2−/− mice. Splenocytes were stimulated at 1 × 106/ml in TCM in the presence of 2 mg/ml Con A and 20 U/ml IL-2 for 3 days. Cells were harvested, washed, and examined for 125I-IL-12 binding. Con A blasts were incubated with increasing concentrations of mouse 125I-IL-12 as indicated. Specific binding of 125I-labeled IL-12 was obtained by subtracting nonspecific binding from total binding (inset). Binding data were analyzed by nonlinear regression analysis and plotted by the method of Scatchard. Similar results were obtained in one additional experiment.

FIGURE 2.

Scatchard analysis of 125I-IL-12 binding on Con A-stimulated splenocytes from IL-12Rβ2−/− mice. Splenocytes were stimulated at 1 × 106/ml in TCM in the presence of 2 mg/ml Con A and 20 U/ml IL-2 for 3 days. Cells were harvested, washed, and examined for 125I-IL-12 binding. Con A blasts were incubated with increasing concentrations of mouse 125I-IL-12 as indicated. Specific binding of 125I-labeled IL-12 was obtained by subtracting nonspecific binding from total binding (inset). Binding data were analyzed by nonlinear regression analysis and plotted by the method of Scatchard. Similar results were obtained in one additional experiment.

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Because both high and low affinity IL-12 binding sites are detected on splenocytes from IL-12Rβ2−/− mice, we investigated whether IL-12 binding could mediate IL-12-induced biological activity. First, we checked whether IL-12 was able to induce IFN-γ production by Con A-activated splenocytes obtained from IL-12Rβ2−/− mice. Con A-activated splenocytes from IL-12Rβ2+/+ mice were used as a positive control. Cells were cultured with increasing doses of IL-12 at 37°C for 48 h, and the levels of IFN-γ in the cell-free culture fluids were measured by ELISA. Results are shown in Fig. 3. IL-12 induced the production of high levels of IFN-γ by Con A-activated splenocytes from IL-12Rβ2+/+ mice; however, no IFN-γ could be detected in culture fluids from Con A-activated splenocytes of IL-12Rβ2−/− mice, even when the cells were treated with very high concentrations of IL-12 (up to 5000 ng/ml). In vivo induction of IFN-γ production by IL-12 was also deficient in IL-12Rβ2−/− mice. Administration of 1 μg/mouse of mouse rIL-12 for 5 days resulted in high levels of IFN-γ production detected in the sera of IL-12Rβ2+/+ mice but not in sera of IL-12Rβ2−/− mice (Fig. 4).

FIGURE 3.

IL-12-induced IFN-γ production by Con A-activated splenocytes from IL-12Rβ2+/+ and IL-12Rβ2−/− mice. Con A-activated splenocytes (1 × 106 cells/ml) were incubated in the presence or absence of various concentrations of IL-12 at 37°C for 24 h. Cell-free culture fluids were collected and assayed for IFN-γ by ELISA. Data are mean ± SEM (some error bars fall within the symbols) and are representative of two experiments.

FIGURE 3.

IL-12-induced IFN-γ production by Con A-activated splenocytes from IL-12Rβ2+/+ and IL-12Rβ2−/− mice. Con A-activated splenocytes (1 × 106 cells/ml) were incubated in the presence or absence of various concentrations of IL-12 at 37°C for 24 h. Cell-free culture fluids were collected and assayed for IFN-γ by ELISA. Data are mean ± SEM (some error bars fall within the symbols) and are representative of two experiments.

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FIGURE 4.

IFN-γ production induced by in vivo administration of rIL-12. IL-12Rβ2+/+ and IL-12Rβ2−/− mice (five mice per treatment group) were injected with 1 μg/mouse IL-12 once daily for 5 days and bled 6 h after the final dose was given. Levels of IFN-γ in sera were measured by ELISA. The mean and SE are shown. The results observed in this experiment were representative of two separate experiments.

FIGURE 4.

IFN-γ production induced by in vivo administration of rIL-12. IL-12Rβ2+/+ and IL-12Rβ2−/− mice (five mice per treatment group) were injected with 1 μg/mouse IL-12 once daily for 5 days and bled 6 h after the final dose was given. Levels of IFN-γ in sera were measured by ELISA. The mean and SE are shown. The results observed in this experiment were representative of two separate experiments.

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Con A-activated splenocytes from IL-12Rβ2+/+ mice proliferated in response to IL-12 in a dose-dependent manner (Fig. 5,A). In contrast, no proliferation was detected in Con A-activated splenocytes from IL-12Rβ2−/− mice even at concentrations of IL-12 as high as 67 nM (Fig. 5,A). In contrast, Con A-activated splenocytes from both IL-12Rβ2+/+ and IL-12Rβ2−/− mice proliferated equally well when stimulated with IL-2 (Fig. 5 B). These results indicate that IL-12Rβ2−/− T cells have a defective proliferative response to IL-12 but not to IL-2.

FIGURE 5.

Proliferation of Con A-activated splenocytes from IL-12Rβ2+/+ and IL-12Rβ2−/− mice in response to IL-12 (A) or IL-2 (B). Con A-activated splenocytes were incubated in 96-well plates at a final density of 5 × 105 cells/ml in the presence or absence of the indicated concentrations of cytokines at 37°C for 48 h. Cells were then pulsed with 0.5 μCi/well [3H]TdR overnight at 37°C. Data are the mean ± SEM (some error bars fall within the symbols). Similar results were observed in two additional experiments.

FIGURE 5.

Proliferation of Con A-activated splenocytes from IL-12Rβ2+/+ and IL-12Rβ2−/− mice in response to IL-12 (A) or IL-2 (B). Con A-activated splenocytes were incubated in 96-well plates at a final density of 5 × 105 cells/ml in the presence or absence of the indicated concentrations of cytokines at 37°C for 48 h. Cells were then pulsed with 0.5 μCi/well [3H]TdR overnight at 37°C. Data are the mean ± SEM (some error bars fall within the symbols). Similar results were observed in two additional experiments.

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IL-12 was initially identified on the basis of its ability to enhance the lytic activity of NK cells (24). Therefore, NK lytic activity of IL-12Rβ2−/− splenocytes was examined. Cytolytic assays using 51Cr-labeled YAC-1 target cells were performed. Splenocytes from both IL-12Rβ2−/− and IL-12Rβ2+/+ mice were cultured in the presence of the indicated concentrations of IL-12 or IL-2 for 4 days at 37°C. Cells were then harvested and tested for lytic activity. NK lytic activity of IL-12Rβ2−/− splenocytes was enhanced when incubated with IL-2 (Fig. 6,B) but not when incubated with IL-12 (Fig. 6,A). In contrast, NK lytic activity of IL-12Rβ2+/+ splenocytes was enhanced in response to both IL-12 (Fig. 6,A) and IL-2 (Fig. 6 B).

FIGURE 6.

Cytokine-induced NK lytic activity of splenocytes from IL-12Rβ2+/+ and IL-12Rβ2−/− mice. Splenocytes were incubated for 4 days with various concentrations of IL-12 (A) or IL-2 (B). The cells were then harvested and tested for their lytic ability to 51Cr-labeled YAC-1 cells. Data are the mean ± SEM observed at an E:T ratio of 40:1 and are representative of two experiments.

FIGURE 6.

Cytokine-induced NK lytic activity of splenocytes from IL-12Rβ2+/+ and IL-12Rβ2−/− mice. Splenocytes were incubated for 4 days with various concentrations of IL-12 (A) or IL-2 (B). The cells were then harvested and tested for their lytic ability to 51Cr-labeled YAC-1 cells. Data are the mean ± SEM observed at an E:T ratio of 40:1 and are representative of two experiments.

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IL-12 has been found to play an important role in promoting Th1 responses both in vitro and in vivo (reviewed in Ref. 2). Upon subsequent activation with mitogens or specific Ags, Th1 cells can produce a large amount of IFN-γ (25, 26, 27). Therefore, Ag-induced IFN-γ production in IL-12Rβ2−/− and IL-12Rβ2+/+ mice treated with or without IL-12 was compared. For this purpose, mice were immunized with alum-precipitated KLH combined with heat-killed P. acnes as adjuvant. Lymph nodes were harvested after 4 days, and IFN-γ production by LNCs cultured with KLH was measured by ELISA. Results are shown in Fig. 7. LNCs from KLH-immunized IL-12Rβ2−/− mice were unable to produce IFN-γ when cultured with KLH, whereas IL-12Rβ2+/+ LNCs secreted large amounts of IFN-γ.

FIGURE 7.

Effects of in vivo administration of rIL-12 on IFN-γ production by Ag-stimulated immune LNCs in vitro. IL-12Rβ2+/+ and IL-12Rβ2−/− mice (four mice per treatment group) were immunized with KLH once and injected with 100 ng/mouse rIL-12 i.p. or a comparable volume of vehicle daily for 4 days. Lymph nodes were harvested 4 days after KLH immunization, and LNCs were cultured in the presence or absence of KLH. Cell-free culture fluids were collected and assayed for IFN-γ levels. The mean and SEM are shown. Similar results were observed in one additional experiment.

FIGURE 7.

Effects of in vivo administration of rIL-12 on IFN-γ production by Ag-stimulated immune LNCs in vitro. IL-12Rβ2+/+ and IL-12Rβ2−/− mice (four mice per treatment group) were immunized with KLH once and injected with 100 ng/mouse rIL-12 i.p. or a comparable volume of vehicle daily for 4 days. Lymph nodes were harvested 4 days after KLH immunization, and LNCs were cultured in the presence or absence of KLH. Cell-free culture fluids were collected and assayed for IFN-γ levels. The mean and SEM are shown. Similar results were observed in one additional experiment.

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It has been found that mitogens like Con A and anti-CD3 mAb are able to induce IFN-γ production by mouse splenocytes. To investigate whether IL-12Rβ2 plays a role in this process, splenocytes from both IL-12Rβ2+/+ and IL-12Rβ2−/− mice were stimulated with an anti-CD3 mAb and Con A for 48 h, cell-free culture fluids were collected, and levels of IFN-γ were measured by ELISA (Fig. 8). Splenocytes from IL-12Rβ2−/− mice are defective in secreting IFN-γ in response to either anti-CD3 mAb or Con A stimulation. These results are similar to those observed using IL-12Rβ1−/− mice (7).

FIGURE 8.

IFN-γ production by splenocytes from IL-12Rβ2+/+ and IL-12Rβ2−/− mice activated by polyclonal stimulation in vitro. Splenocytes were cultured in 48-well plates at a density of 2 × 106/ml in TCM containing 5% FBS in the presence or absence of Con A (2 μg/ml) or anti-CD3 (1 μg/ml). After 48 h of incubation, cell-free culture fluids were collected and levels of IFN-γ were measured by ELISA. The results are the mean ± SEM. Similar results were observed in one additional experiment.

FIGURE 8.

IFN-γ production by splenocytes from IL-12Rβ2+/+ and IL-12Rβ2−/− mice activated by polyclonal stimulation in vitro. Splenocytes were cultured in 48-well plates at a density of 2 × 106/ml in TCM containing 5% FBS in the presence or absence of Con A (2 μg/ml) or anti-CD3 (1 μg/ml). After 48 h of incubation, cell-free culture fluids were collected and levels of IFN-γ were measured by ELISA. The results are the mean ± SEM. Similar results were observed in one additional experiment.

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IL-12Rβ1 is involved in elevating IFN-γ production when mice are treated with endotoxin LPS in vivo (7). To investigate whether IL-12Rβ2 plays a role in stimulating IFN-γ production in vivo, IL-12Rβ2+/+ and IL-12Rβ2−/− mice were injected with LPS i.p., serum samples were collected 6 h later, and levels of IFN-γ were assayed by ELISA. Similar to previous results with IL-12Rβ1−/− mice (7), IFN-γ production in IL-12Rβ2−/− mice was markedly reduced (0.27 ± 0.07 ng/ml) as compared with IL-12Rβ2+/+ mice (1.40 ± 0.32 ng/ml) (Fig. 9).

FIGURE 9.

LPS-induced IFN-γ production in vivo in IL-12Rβ2+/+ and IL-12Rβ2−/− mice. Mice (five mice per treatment group) were injected with LPS i.p. and bled 6 h later. IFN-γ in the sera was determined by ELISA. Data are the mean ± SEM. Similar results were obtained in one additional experiment.

FIGURE 9.

LPS-induced IFN-γ production in vivo in IL-12Rβ2+/+ and IL-12Rβ2−/− mice. Mice (five mice per treatment group) were injected with LPS i.p. and bled 6 h later. IFN-γ in the sera was determined by ELISA. Data are the mean ± SEM. Similar results were obtained in one additional experiment.

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IL-12-induced phosphorylation of STAT-4 is absent in Con A-activated splenocytes from IL-12Rβ2−/− mice

To address whether the signal transduction pathways mediated by IL-12 were also impaired, Con A-activated splenocytes were analyzed for their ability to induce STAT-4 phosphorylation in response to IL-12. In contrast to cells obtained from IL-12Rβ2+/+ mice, phosphorylation of STAT-4 was not observed in response to IL-12 in splenocytes obtained from IL-12Rβ2−/− mice (Fig. 10). However, cells obtained from IL-12Rβ2+/+ and IL-12Rβ2−/− mice were equally responsive to IL-2-mediated signal transduction as measured by phosphorylation of STAT-5 following IL-2 treatment (Fig. 10).

FIGURE 10.

STAT4 phosphorylation in Con A-activated splenocytes stimulated with IL-12. Con A-activated splenocytes from IL-12Rβ2+/+ (lanes 1, 2, 5, and 6) or IL-12Rβ2−/− (lanes 3, 4, 7, and 8) mice were untreated (lanes 1, 3, 5, and 7) or treated with 10 ng/ml IL-12 (lanes 2 and 4) or 1000 IU/ml IL-2 (lanes 6 and 8) for 15 min, lysed, and immunoprecipitated with anti-STAT4 (lanes 1–4) or anti-STAT5 (lanes 5–8), then subjected to immunoblotting with antiphosphotyrosine (top) or indicated STAT antisera (bottom).

FIGURE 10.

STAT4 phosphorylation in Con A-activated splenocytes stimulated with IL-12. Con A-activated splenocytes from IL-12Rβ2+/+ (lanes 1, 2, 5, and 6) or IL-12Rβ2−/− (lanes 3, 4, 7, and 8) mice were untreated (lanes 1, 3, 5, and 7) or treated with 10 ng/ml IL-12 (lanes 2 and 4) or 1000 IU/ml IL-2 (lanes 6 and 8) for 15 min, lysed, and immunoprecipitated with anti-STAT4 (lanes 1–4) or anti-STAT5 (lanes 5–8), then subjected to immunoblotting with antiphosphotyrosine (top) or indicated STAT antisera (bottom).

Close modal

Two IL-12 receptor subunits have been cloned and characterized in both mouse and humans to date. These have been designated IL-12Rβ1 and IL-12Rβ2 due to their sequence homology to other β type cytokine receptors like gp130 and leukemia-inhibitory factor receptor. Previous studies have demonstrated the important role of IL-12Rβ1 in IL-12 responsiveness and the generation of Th1 cells (7). To determine the role of IL-12Rβ2 and elucidate its function in IL-12-mediated events, we generated and characterized IL-12Rβ2-deficient mice. These mice are viable, fertile, of normal size and weight, have no gross abnormalities, and appear to have normal B and T cell development, indicating that IL-12Rβ2 is not involved in normal mouse development, including the development of immune cells. However, consistent with the deficiency in IL-12p40−/− and IL-12Rβ1−/− mice (10, 7), defects in several immunological responses were observed in IL-12Rβ2−/− mice. These include unresponsiveness to IL-12 as measured by multiple parameters as well as severely impaired Th1 responses in vitro and in vivo.

Several lines of evidence suggest that responsiveness to IL-12 requires a functional receptor consisting of both IL-12Rβ1 and IL-12Rβ2 subunits. Blocking of IL-12 binding to IL-12Rβ1 by using an anti-IL-12Rβ1 Ab (28) or IL-12p40 homodimer, a known IL-12 receptor antagonist, abrogates the biological activities induced by IL-12 (29). Likewise, Con A-activated splenocytes from IL-12Rβ1−/− mice fail to proliferate, produce IFN-γ, or generate Th1 cells in response to IL-12 (7). It has been recently reported that PBMCs from IL-12Rβ1-deficient patients produce much lower amounts of IFN-γ than PBMCs from normal donors when cells are stimulated with Ags and mitogens (30, 31). IL-12 could not induce proliferation or IFN-γ production by PBMCs from these patients. Importantly, patients with IL-12Rβ1 deficiency were extremely susceptible to mycobacterial and salmonella infections, suggesting that IL-12-induced IFN-γ production and Th1 generation are essential for controlling resistance to these pathogens in humans (30, 31). Data presented in this paper demonstrates the additional requirement for IL-12Rβ2 in the mouse system.

Recent studies suggest that IL-12Rβ2 expression in both humans and mice may be confined to Th1 cells and suppressed on Th2 cells. Following differentiation of CD4+ T cells in vitro, Th2 cells expressing IL-12Rβ1, but not IL-12Rβ2, fail to respond to IL-12. Expression of IL-12Rβ2 appears to be regulated by cytokines. IFN-α in humans, IFN-γ in mice, and IL-12 itself enhanced, whereas IL-4 inhibited IL-12Rβ2 expression (32, 33, 34). It was shown in this study that Con A-activated splenocytes from IL-12Rβ2−/− mice, which displayed both high and low affinity binding sites for IL-12, were still deficient in the IL-12-induced biological activities tested. These included IL-12-induced proliferation, IFN-γ secretion in vitro and in vivo, and enhancement of NK lytic activity. In agreement with previous findings in IL-12p40- and p35-deficient mice and in IL-12Rβ1-deficient mice, IL-12Rβ2−/− mice had a severe defect in their ability to generate Th1 responses, as measured by production of IFN-γ. The lack of IL-12 responsiveness was further substantiated by the observation that in contrast to the wild-type control cells, STAT-4 phosphorylation is not observed after treatment of IL-12Rβ2-deficient splenocytes with IL-12. STAT-4 phosphorylation has been previously shown to be an important downstream event in IL-12-mediated signal transduction (reviewed in Ref. 35). In contrast, treatment of splenocytes from both wild-type and IL-1212Rβ2-deficient mice with IL-2 resulted in phosphorylation of STAT-5 as expected (36, 37). Taken together, the control of IL-12Rβ2 expression may constitute an important mechanism for regulating IL-12 responsiveness.

Previous studies have identified two IL-12 receptor subunits to date in both mice and humans (3, 4, 5) that have differing affinities for IL-12. The relative contribution of the IL-12 receptor subunits to IL-12 binding differs significantly between the human and mouse systems. This was demonstrated using mouse pro-B cells (Ba/F3). Ba/F3 cells transfected with only human IL-12Rβ1 or IL-12Rβ2 bind human IL-12 with only low affinity. Coexpression of both human subunits resulted in the appearance of both high (Kd = 50 pM) and low affinity (Kd = 5 nM) binding sites, which correspond to the high and low affinity binding sites detected on human PHA lymphoblast cells (17). In contrast to human IL-12 receptor subunits, Ba/F3 cell lines expressing mouse IL-12Rβ1 alone displayed both high (Kd = 50–100 pM) and low (Kd = 0.5–2 nM) affinity IL-12 binding sites (6), whereas Ba/F3 cells expressing mouse IL-12Rβ2 alone bound IL-12 very poorly. Thus, in the mouse system, IL-12Rβ1 is the primary binding component, and IL-12Rβ2 adds very little additional binding capacity.

IL-12 appears to interact with IL-12Rβ1 via domains on the IL-12p40 subunit and with IL-12Rβ2 via a heterodimer-specific region of IL-12 to which the IL-12p40 and p35 subunits may both contribute (38). In vitro studies using blocking Abs or purified mouse IL-12p40 homodimer indicate that IL-12p40 interacts primarily with IL-12Rβ1 because Ba/F3 cells expressing mouse IL-12Rβ1 alone bound mouse IL-12p40 homodimer with both high and low affinity binding sites (39). Multiple interactions between IL-12 heterodimer and IL-12R complex can also be observed with human receptor subunits transfected into COS-7 cells. Two classes of IL-12 inhibitors were identified based on their ability to interfere with the binding of 125I-IL-12 to these receptor subunits expressed on COS-7 cells. Anti-IL-12Rβ1 Ab (2B10) and mouse Il-12p40 homodimer blocked the binding of IL-12 to IL-12Rβ1 but not to IL-12Rβ2 (38). In contrast, anti-human IL-12 heterodimer-specific mAb (20C2) selectively inhibits the binding of IL-12 to IL-12Rβ2-transfected COS-7 cells. These two classes of IL-12 inhibitors have a synergistic effect on blocking IL-12-mediated proliferation and IFN-γ production (38).

Activated splenocytes from IL-12Rβ2−/− mice bind IL-12 with both high and low affinity, corresponding to that observed in lymphoblasts from wild-type mice. This study as well as our previous results suggest that IL-12Rβ1 is primarily responsible for binding IL-12 in the mouse system, and IL-12Rβ2 is further required for IL-12 responsiveness. Disruption of either IL-12Rβ1 or IL-12Rβ2 activity abrogates IL-12-mediated biological functions.

5

Abbreviations used in this paper: ES, embryonic stem; TCM, tissue culture medium; LNCs, lymph node cells; KLH, keyhole limpet hemocyanin;

1
Trinchieri, G..
1995
. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity.
Annu. Rev. Immunol.
13
:
251
2
Gately, M. G., L. M. Renzetti., J. Magram, A. S. Stern, L. Adorini, U. Gubler, D. H. Presky.
1998
. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses.
Annu. Rev. Immunol.
16
:
495
3
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.
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
4
Chua, A. O., V. L. Wilkinson, D. H. Presky, U. Gubler.
1995
. Cloning and characterization of a mouse IL-12 receptor-β component.
J. Immunol.
155
:
4286
5
Presky, D. H., H. Yang, L. J. Minetti, A. O. Chua, N. Nabavi, C.-Y. Wu, M. K. Gately, U. Gubler.
1996
. A functional interleukin-12 receptor complex is composed of two β-type cytokine receptor subunits.
Proc. Natl. Acad. Sci. USA
93
:
14002
6
Wilkinson, V. L., D. Carvajal, L. J. Minetti, A. O. Chua, U. Gubler, M. K. Gately, D. H. Presky.
1996
. Functional characterization of mouse IL-12 receptors.
J. Allergy Clin. Immunol.
99
:
S52
7
Wu, C.-Y., J. Ferrante, M. K. Gately, J. Magram.
1997
. Characterization of IL-12 receptor β1 chain (IL-12Rβ1)-deficient mice.
J. Immunol.
159
:
1658
8
Gately, M. K., T. D. Anderson, T. J. Hayes.
1988
. Role of asialo-GM1-positive lymphoid cells in mediating the toxic effects of recombinant IL-2 in mice.
J. Immunol.
141
:
189
9
Gately, M. K., R. R. Warrier, S. Honasoge, D. M. Carvajal, D. A. Faherty, S. E. Connaughton, T. D. Anderson, U. Sarmiento, B. R. Hubbard, M. Murphy.
1994
. Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-γ in vivo.
Int. Immunol.
6
:
157
10
Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C.-Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately.
1996
. IL-12-deficient mice are defective in IFN-γ production and type 1 cytokine responses.
Immunity
4
:
471
11
Sambrook, J., E. T. Fritsch, T. Maniatis.
1989
.
Molecular Cloning, A Laboratory Manual
2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
12
Soriano, P., C. Montgomery, R. Geske, A. Bradley.
1991
. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice.
Cell
64
:
693
13
Labow, M. A., C. R. Norton, J. M. Rumberger, K. M. Lombard-Gillooly, D. J. Shuster, J. Hubbard, R. Bertko, P. A. Knaack, R. W. Terry, M. L. Harbison, et al
1994
. Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins.
Immunity
1
:
709
14
Kwee, L., H. S. Baldwin, H. M. Shen, C. L. Stewart, C. Buck, C. A. Buck, M. A. Labow.
1995
. Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice.
Development
121
:
489
15
Abbondanzo, S. J., I. Gadi, C. L. Stewart.
1993
. Derivation of embryonic stem cell lines.
Methods Enzymol.
225
:
803
16
Laird, P. W., A. Zijderveld, K. Linders, M. A. Rudnicki, R. Jaenish, A. Berns.
1991
. Simplified mammalian DNA isolation procedure.
Nucleic Acids Res.
19
:
4293
17
Chizzonite, R., T. Truitt, B. B. Desai, P. Nunes, F. J. Podlaski, A. S. Stern, M. K. Gately.
1992
. IL-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts.
J. Immunol.
148
:
3117
18
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
19
Chizzonite, R., T. Truitt, F. J. Podlaski, A. G. Wolitzky, P. M. Quinn, P. Nunes, A. S. Stern, M. K. Gately.
1991
. IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts.
J. Immunol.
147
:
1548
20
McPherson, G. A..
1985
. Analysis of radioligand binding experiments: a collection of computer programs for the IBM PC.
J. Pharmacol. Methods
14
:
213
21
Scatchard, G..
1949
. The attractions of proteins for small molecules and ions.
Ann. NY Acad. Sci.
51
:
660
22
Cho, S. S., C. M. Bacon, C. Sudarshan, R. C. Rees, D. Finbloom, R. Pine, J. J. O’Shea.
1996
. Activation of STAT4 by IL-12 and IFN-α: evidence for involvement of ligand-induced tyrosine and serine phosphorylation.
J. Immunol.
157
:
4781
23
Szabo, P., J. R. Mann.
1994
. Expression and methylation of imprinted genes during in vitro differentiation of mouse parthenogenetic and androgenetic embryonic stem cells.
Development
120
:
1651
24
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 biological effects on human lymphocytes.
J. Exp. Med.
170
:
827
25
Hsieh, C.-S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, K. M. Murphy.
1993
. Development of Th1 CD4+ T cells through IL-12 produced by Lister-induced macrophages.
Science
260
:
547
26
Seder, R. A., R. Gazzinelli, A. Sher, W. E. Paul.
1993
. Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon-γ production and diminishes interleukin 4 inhibition of such priming.
Proc. Natl. Acad. Sci. USA
90
:
10188
27
Schmitt, E., P. Heohn, C. Huels, S. Goedert, N. Palm, E. Rude, T. Germann.
1994
. T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-γ and is inhibited by transforming growth factor-β.
Eur. J. Immunol.
24
:
793
28
Wu, C. Y., R. R. Warrier, D. M. Carvajal, A. O. Chua, L. J. Minetti, R. Chizzonite, P. K. A. 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
29
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
30
Jong, R. D., F. Altare, I. Haagen, D. G. Elferink, T. Boer, P. J. C. Van Breda Vriesman, P. J. Kabel, J. M. T. Draaisma, J. T. van Dissel, F. P. Kroon, et al
1998
. Severe mycobacterial and salmonella infections in interleukin-12 receptor-deficient patients.
Science
280
:
1435
31
Altare, F., A. Durandy, D. Lammas, J.-F. Emile, S. Lamhamedi, F. le Deist, P. Drysdale, E. Jouanguy, R. Doffinger, F. Bernaudin, et al
1998
. Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency.
Science
280
:
1432
32
Szabo, S. J., A. S. Dighe, U. Gubler, K. M. Murphy.
1997
. Regulation of the interleukin (IL)-12Rβ2 subunit expression in developing T helper 1(Th1) and Th2 cells.
J. Exp. Med.
185
:
817
33
Rogge, L., L. Barberis-Maino, M. Biffi, N. Passini, D. H. Presky, U. Gubler, F. Sinigaglia.
1997
. Selective expression of an interleukin-12 receptor component by human T helper 1 cells.
J. Exp. Med.
185
:
825
34
Rogge, L., A. Papi, D. H. Presky, M. Biffi, L. J. Minetti, D. Miotto, C. Agostini, G. Semenzato, L. M. Fabbri, F. Sinigaglia.
1999
. Antibodies to the IL-12 receptorβ2 chain mark human Th1 but not Th2 cells in vitro and in vivo.
J. Immunol.
162
:
3926
35
Wurster, A. L., T. Tanaka, M. J. Grusby.
2000
. The biology of Stat4 and Stat6.
Oncogene
19
:
2577
36
Johnston, J. A., C. M. Bacon, D. S. Finbloom, R. C. Rees, D. Kaplan, K. Shibuya, J. R. Ortaldo, S. Gupta, Y. Q. Chen, J. D. Giri, J. J. O’Shea.
1995
. Tyrosine phosphorylation and activation of STAT5, STAT3, and janus kinases by interleukins 2 and 15.
Proc. Natl. Acad. Sci. USA
92
:
8705
37
Gilmour, K. C., R. Pine, N. C. Reich.
1995
. Interleukin 2 activates STAT5 transcription factor (mammary gland factor) and specific gene transcription in T lymphocytes.
Proc. Natl. Acad. Sci. USA
92
:
10772
38
Presky, D. H., L. J. Minetti, S. Gillessen, V. L. Wilkinson, C. Y. Wu, U. Gubler, R. Chizzonite, M. K. Gately.
1998
. Analysis of the multiple interactions between IL-12 and the high affinity IL-12 receptor complex.
J. Immunol.
160
:
2174
39
Wang. X., V. L., F. J. Wilkinson, C.-Y. Podlaski, A. S. Wu, D. H. Stern, D. H. Presky, J. Magram.
1999
. Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits.
Eur. J. Immunol.
29
:
2007