Dysregulation of lymphocyte interleukin-12 receptor expression in Sézary syndrome.

Initial phase I and II clinical trials with recombinant human interleukin-12 have demonstrated the therapeutic efficacy of this cytokine in early stage cutaneous T cell lymphoma as compared with more advanced stages such as the leukemic Sézary syndrome. In an effort to optimize the use of recombinant human interleukin-12, using flow cytometry we studied the regulation of the interleukin-12 receptor beta1 (high affinity chain) and beta2 (chain necessary for interleukin-12 signal transduction) on normal volunteer CD4+ and CD8+ T cells and CD4+ and CD8+ cells from eight patients with different degrees of leukemic involvement with Sézary syndrome. The beta1 chain was not readily detectable on resting normal and T cells from Sézary patients, but expression was induced following T cell activation with phytohemagglutinin. Similarly, the beta2 chain was not detectable on resting normal volunteer T cells, but could be induced following phytohemagglutinin stimulation. Moreover, the beta2 chain on normal volunteer T cells was markedly upregulated following short-term culture with interferon-gamma or recombinant human interleukin-12. CD8+ T cells routinely exhibited a greater expression of beta2 than did CD4+ T cells. In marked contrast, both CD4+ and CD8+ T cells from patients with Sézary syndrome and a high tumor cell burden (> 50% circulating atypical Sézary T cells) failed to express the beta2 chain under any culture conditions. Although, culture with anti-interleukin-10 also markedly increased beta2 expression on normal volunteer T cells, this failed to induce expression on either CD4+ or CD8+ T cells from Sézary patients and a high tumor burden. Investigation of patients with Sézary syndrome and a low tumor cell burden (< 15% circulating Sézary T cells) revealed a pattern of beta2 expression that was intermediate between advanced Sézary syndrome and normal volunteers. Both CD4+ and CD8+ peripheral blood T cells from these earlier stage patients were induced to express the beta2 chain, although at a lower frequency of positivity than T cells from normals, following culture with phytohemagglutinin, interferon-gamma, recombinant human interleukin-12, or anti-interleukin-10. These results indicate that short-term culture with interferon-gamma and recombinant human interleukin-12 potently upregulates beta2 chain expression on T cells from normal volunteers, whereas a similar, but less marked effect occurs on T cells from Sézary syndrome patients and a low circulating tumor cell burden. In contrast, the beta2 chain appears to be suppressed on both CD4+ and CD8+ T cells from Sézary patients with a heavy circulating tumor cell burden and it is not induced by interferon-gamma or recombinant human interleukin-12. Therefore, recombinant human interleukin-12 is likely to be most effective for early stage cutaneous T cell lymphoma due to a greater display of beta2 receptors on responding CD8+ anti-tumor cytotoxic T cells.

IL-12 receptor beta 2 (IL-12R beta 2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding sites.

Two subunits of the IL-12 receptor (IL-12R), IL-12R beta 1 and IL-12R beta 2, have been identified and cloned. Previous studies demonstrated that the IL-12R beta 1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12R beta 2 in IL-12 signaling, we have generated IL-12R beta 2-deficient (IL-12R beta 2(-/-)) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12R beta 2(-/-) 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-12R beta 2(-/-) mice failed to produce IFN-gamma or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12R beta 2(-/-) splenocytes was not induced when incubated with IL-12. IL-12R beta 2(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12R beta 2(-/-) mice were deficient in vivo in their ability to produce IFN-gamma 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-12R beta 1 is the subunit primarily responsible for binding IL-12, IL-12R beta 2 plays an essential role in mediating the biological functions of IL-12 in mice.

Efficient cotransduction of tumors by multiple herpes simplex vectors: implications for tumor vaccine production.

Many gene therapy strategies would be enhanced by efficient transfer of multiple genes into the same cell. Herpes simplex viral amplicon (HSV) vectors are good vehicles for gene transfer because they accommodate large pieces of foreign DNA and transfer genes rapidly and efficiently. The current studies examine whether efficient cotransduction of tumor cells can be accomplished using multiple HSV vectors in a manner useful for clinical gene therapy. Interleukin-12 (IL-12) exists as a heterodimer, with components (m35 and m40) coded for by genes on two separate chromosomes. We constructed HSV vectors carrying either IL12m35 (HSVm35) or IL12m40 (HSVm40) or both genes (HSVm75) separated by an internal ribosome entry site to assess whether gene transfer using a single HSV vector constructed to carry multiple genes has any advantage over gene transfer using multiple vectors that are each carrying single genes. Because IL-12 and IL-2 have been found to have synergistic antitumoral activity, we further analyzed the biologic activity of tumor cells cotransduced by separate HSV vectors carrying genes coding for these two cytokines. The results demonstrate that multiple genes can be inserted into the same cell efficiently using multiple HSV vectors, and that these vectors allow rapid production of tumor vaccines expressing multiple cytokine genes. Thus, gene transfer using HSV may not be limited by the size of the DNA that each vector can accommodate. Immunizations with tumors cotransduced with HSVm35 and HSVm40 were equally effective in eliciting a cytolytic T-lymphocyte response and in protecting against tumor growth in vivo as immunization with tumors treated with HSVm75. Immunization with tumors cotransduced with HSVm75 and HSVil2 was superior to immunization with tumors transduced with either alone. The combination of IL-2- and IL-12-secreting tumor cells may be used as an effective immunization strategy against solid tumors.

Down-regulation of the pharmacokinetic-pharmacodynamic response to interleukin-12 during long-term administration to patients with renal cell carcinoma and evaluation of the mechanism of this "adaptive response" in mice.

BACKGROUND: Interleukin-12 (IL-12) is a cytokine that promotes type-1 helper T-cell responses and may have therapeutic utility in the treatment of cancer, asthma, and a variety of infectious diseases. METHODS: In a phase I trial, recombinant human IL-12 (rHuIL-12) was administered subcutaneously once a week at a fixed dose of 0.1 to 1.0 microg/kg to 24 patients with renal cell carcinoma. A similar study was later performed in mice to evaluate the mechanism of down-regulation of pharmacokinetic-pharmacodynamic response observed in patients with cancer. RESULTS: Adverse events, serum IL-12 levels, and serum levels of interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) produced in response to IL- 12 were all maximum in the week after the first dose of rHuIL-12 and decreased after long-term administration. Similar to these results, repetitive subcutaneous administration of recombinant mouse IL-12 (rMoIL-12) to normal mice led to down-regulation of serum levels of IL-12 and IFN-gamma measured 5 hours after rMoIL-12 injection. Down-regulation of IL-12 serum levels was inversely correlated with the up-regulation of IL-12 receptor expression and may be the result of increased clearance of rMoIL-12 from serum by binding to lymphoid cells expressing increased amounts of IL-12 receptor. The down-regulation of serum IFN-gamma levels correlated with decreased IFN-gamma messenger ribonucleic acid expression and may result from feedback inhibition of IL-12 signaling or from a more specific inhibition of IFN-gamma synthesis. CONCLUSION: Administration of rHuIL-12 in fixed weekly doses resulted in decreased serum levels of IL-12 and of IFN-gamma, a secondary cytokine believed to be critical to response of IL-12. A better understanding of the complex regulation of the pharmacokinetic-pharmacodynamic response to IL-12 should facilitate the development of more effective dosing regimens for its use in the clinic.

Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-bonded subunits, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL-12, a homodimer of the p40 subunit, designated (p40)2, has been shown to be a potent IL-12 antagonist. To study the interaction between (p40)2 and the known IL-12 receptor (IL-12R) subunits, IL-12Rbeta1 and IL-12Rbeta2, we directly measured the binding activity of mouse (p40)2 to ConA-activated lymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These results demonstrated the presence of both high (Kd about 5 pM) and low affinity (Kd about 15 nM) binding sites for mouse 125I-labeled (p40)2. To elucidate which of the IL-12R subunits binds mouse (p40)2, binding studies of mouse 125I-labeled (p40)2 to Ba/F3 cells expressing recombinant mouse IL-12Rbeta1 and/or mouse IL-12Rbeta2 were carried out. Mouse IL-12Rbeta1 bound mouse 125I-labeled (p40)2 with high and low affinities, comparable to that observed on Con A blasts and B cells. In contrast, mouse IL-12Rbeta2 bound mouse 125I-labeled (p40)2 very poorly. These data demonstrate that similar to IL-12, mouse (p40)2 binds with both high and low affinity to Con A blasts and B cells, and that IL-12Rbeta1 is responsible for mediating the specific binding of mouse (p40)2.

Antibodies to the IL-12 receptor beta 2 chain mark human Th1 but not Th2 cells in vitro and in vivo.

Great attention has been placed on the possibility of distinguishing Th1 from Th2 cells on the basis of differential expression of surface receptors. We have recently shown that the differential expression of the IL-12R beta 2 chain in Th1 and Th2 cells, as measured at the mRNA level, accounts for an important regulatory mechanism in the differentiation of the two cell subsets. In this study, we identify IL-12R expression at the protein level. We have generated an anti-IL-12R beta 2-specific mAb and analyzed IL-12R beta 2 expression on polarized Th cell populations generated in vitro and on T cells derived from patients with Th1- or Th2-mediated inflammatory conditions. Although IL-12R beta 2 was absent in freshly isolated PBMC and in cord blood cells, we were able to detect IL-12R beta 2 expression selectively in differentiated Th1 and T cytotoxic 1, but not Th2 or T cytotoxic 2 cells. In the presence of IL-12, cell surface expression of the IL-12R beta 2 subunit was readily detected on T cells after 24 h, reached the maximum at day 5, and declined thereafter. Most importantly, the anti-IL-12R beta 2 mAb recognizes lung T cells from patients with sarcoidosis, a disease characterized by a typical cell-mediated, Th1-type inflammatory response. In contrast, IL-12R beta 2 was absent in lung T cells from patients with allergic asthma, a disease characterized by a Th2-type inflammatory response. The mAb reported in this study should represent a powerful tool to investigate the role of Th1 and Th2 cells in inflammatory conditions and to monitor therapies aimed at altering the balance of Th cell subsets.

Expression of the IL-12 receptor beta 1 and beta 2 subunits in human tuberculosis.

To determine whether the Th1 response in tuberculosis correlated with IL-12R expression, we measured expression of the IL-12R beta 1 and IL-12R beta 2 subunits, as well as IL-12R beta 2 mRNA expression in tuberculosis patients and healthy tuberculin reactors. In tuberculosis patients, IFN-gamma production by Mycobacterium tuberculosis-stimulated PBMC was reduced, the percentages of T cells expressing IL-12R beta 1 and IL-12R beta 2 were significantly decreased, and IL-12R beta 2 mRNA expression was also markedly reduced. In contrast, in pleural fluid and lymph nodes at the site of disease in tuberculosis patients, in which IFN-gamma production is enhanced, IL-12R beta 2 mRNA expression was also increased. In M. tuberculosis-stimulated peripheral blood T cells from tuberculosis patients, anti-IL-10 and anti-TGF-beta enhanced IL-12R beta 1 and IL-12R beta 2 expression, and IFN-gamma production. In M. tuberculosis-stimulated peripheral blood T cells from healthy tuberculin reactors, recombinant IL-10 and TGF-beta reduced IL-12R beta 1 and IL-12R beta 2 expression, as well as IFN-gamma production. In combination with prior studies showing increased production of TGF-beta by blood monocytes from tuberculosis patients, this suggests that increased TGF-beta production is the underlying abnormality that reduces IL-12R beta 1 and IL-12R beta 2 expression in tuberculosis. Our findings provide evidence that IL-12R expression correlates well with IFN-gamma production in human tuberculosis, and that expression of IL-12R beta 1 and IL-12R beta 2 may play a central role in mediating a protective Th1 response.

The role of Stat4 in species-specific regulation of Th cell development by type I IFNs.

Type I IFNs (IFN-alpha/beta), in addition to IL-12, have been shown to play an important role in the differentiation of human, but not mouse, Th cells. We show here that IFN-alpha/beta act directly on human T cells to drive Th1 development, bypassing the need for IL-12-induced signaling, whereas IFN-alpha cannot substitute IL-12 for mouse Th1 development. The molecular basis for this species specificity is that IFN-alpha/beta activate Stat4 in differentiating human, but not mouse, Th cells. Unlike IL-12, which acts only on Th1 cells, IFN-alpha/beta can activate Stat4 not only in human Th1, but also in Th2 cells. However, restimulation of human Th2 lines and clones in the presence of IFN-alpha does not induce the production of IFN-gamma. These results suggest that activation of Stat4, which is necessary for the differentiation of naive T cells into polarized Th1 cells, is not sufficient to induce phenotype reversal of human Th2 cells.

The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses.

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays a central role in promoting type 1 T helper cell (Th1) responses and, hence, cell-mediated immunity. Its activities are mediated through a high-affinity receptor composed of two subunits, designated beta 1 and beta 2. Of these two subunits, beta 2 is more restricted in its distribution, and regulation of its expression is likely a central mechanism by which IL-12 responsiveness is controlled. Studies with neutralizing anti-IL-12 antibodies and IL-12-deficient mice have suggested that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens. However, IL-12 appears also to play a central role in the genesis of some forms of immunopathology. Inhibition of IL-12 synthesis or activity may be beneficial in diseases associated with pathologic Th1 responses, such as multiple sclerosis or Crohn's disease. On the other hand, administration of recombinant IL-12 may have utility in the treatment of diseases associated with pathologic Th2 responses such as allergic disorders and asthma.

Blockade of IL-12 during the induction of collagen-induced arthritis (CIA) markedly attenuates the severity of the arthritis.

The effect of blocking IL-12, a potent inducer of interferon-gamma (IFN-gamma) and promoter of Th1 cell responses, during the induction phase of CIA was investigated. Arthritis was elicited in male DBA/1 mice by immunizing with type II collagen (CII) in Freund's complete adjuvant. Neutralizing anti-IL-12 antibodies were administered twice weekly from CII immunization. It was found that administration of anti-IL-12 from immunization until the onset of clinical arthritis did not lower the incidence of arthritis, but dramatically attenuated the severity of the disease, both clinically and histopathologically. This regime was associated with reduced IFN-gamma levels produced by ex vivo CII-stimulated draining lymph node cells, and with diminished spontaneous ex vivo production of tumour necrosis factor (TNF), IL-6 and IL-10 by freshly isolated synovial cells. Total anti-CII antibody serum levels in these mice were lower than in the controls, but there was no change in the IgG2a/IgG1 ratio. These findings confirm that IL-12 has a major role in the induction of murine CIA and suggests that this disease is propagated, in part, by cells of the Th1 phenotype.

Analysis of the multiple interactions between IL-12 and the high affinity IL-12 receptor complex.

IL-12 is a heterodimeric cytokine, composed of a p40 and a p35 subunit, that exerts its biological effects by binding to specific cell surface receptors. Two IL-12R proteins, designated human IL-12 (huIL-12) receptor beta1 (huIL-12Rbeta1) and huIL-12Rbeta2, have been previously identified. These IL-12R individually bind huIL-12 with low affinity and in combination bind huIL-12 with high affinity and confer IL-12 responsiveness. In this study the interactions of hulL-12 with these two identified human IL-12R protein subunits are examined. The heterodimer-specific anti-huIL-12 mAb 20C2, which neutralizes huIL-12 bioactivity but does not block 125I-huIL-12 binding to huIL-12Rbeta1, blocked binding of huIL-12 to huIL-12Rbeta2. In contrast, anti-huIL-12Rbeta1 mAb 2B10 and mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to huIL-12Rbeta1, but not to huIL-12Rbeta2. Therefore, two classes of IL-12 inhibitors can be identified that differ in their ability to block huIL-12 interaction with either huIL-12Rbeta1 or huIL-12Rbeta2. Both mo(p40)2 and 20C2 blocked high affinity binding to huIL-12Rbeta1/beta2-cotransfected COS-7 cells, although, as previously reported, mo(p40)2 does not block high affinity binding to IL-12R on PHA-activated human lymphoblasts. Furthermore, these two classes of IL-12 inhibitors synergistically decreased hulL-12-stimulated proliferation and IFN-gamma production. Therefore, IL-12, in binding to the high affinity IL-12R, interacts with IL-12Rbeta1 primarily via regions on the IL-12 p40 subunit and with IL-12Rbeta2 via 20C2-reactive, heterodimer-specific regions of IL-12 to which the p35 and p40 subunits both contribute.

Treatment with homodimeric interleukin-12 (IL-12) p40 protects mice from IL-12-dependent shock but not from tumor necrosis factor alpha-dependent shock.

The role of interleukin-12 (IL-12) was investigated in different shock models using anti-IL-12 reagents. IL-12 is composed of two disulfide-bonded subunits, p35 and p40. The IL-12 p40 homodimer (p40)2 has been shown to be a potent IL-12 antagonist in vitro. We investigated its in vivo inhibitory capacity in different shock models of mice. We could demonstrate that (p40)2 is able to protect mice from septic shock in primarily IL-12-dependent models such as the Shwartzman reaction and lipopolysaccharide (LPS)-induced shock, whereas (p40)2 has no effect in the tumor necrosis factor alpha-dependent LPS/D-GalN shock model. In IL-12-dependent shock models, (p40)2 inhibits IL-12-induced gamma interferon production and thereby interferes with the cascade of cytokine release, finally leading to death.

Selective expression of an interleukin-12 receptor component by human T helper 1 cells.

Interleukin-12 (IL-12), a heterodimeric cytokine produced by activated monocytes and dendritic cells, plays a crucial role in regulating interferon (IFN)-gamma production and in the generation of IFN-gamma-producing T helper 1 (Th1) cells. Here we show that the IL-12 receptor (IL-12R) beta 2 subunit, a recently cloned binding and signal transducing component of the IL-12R, is expressed on human Th1 but not Th2 clones and is induced during differentiation of human naive cells along the Th1 but not the Th2 pathway. IL-12 and type I but not type II interferons induce expression of the IL-12R beta 2 chain during in vitro T cell differentiation after antigen receptor triggering. The selective expression and regulation of the IL-12R beta 2 subunit may help to understand the basis of Th1/Th2 differentiation and may provide therapeutic options for altering the Th1/Th2 balance in several immuno-pathological conditions such as autoimmune diseases and allergies.

Differential associations between the cytoplasmic regions of the interleukin-12 receptor subunits beta1 and beta2 and JAK kinases.

The role of the cytoplasmic regions of interleukin-12 receptors (IL-12R) beta1 and beta2 in stimulating proliferation was examined. The transmembrane and cytoplasmic regions of IL-12Rbeta1 or IL-12Rbeta2 were fused to the extracellular domain of the epidermal growth factor (EGF) receptor, yielding chimeric receptors E12R1 and E12R2, respectively. These chimeras were stably transfected into BaF3 cells, a factor-dependent murine pro-B cell line. Only E12R2 or E12R1+E12R2 transfectants were capable of EGF-dependent proliferation. EGF-dependent phosphorylation of E12R2, JAK2, Tyk2, and STAT3 was observed. JAK2 was phosphorylated in E12R1-, E12R2-, and E12R1+E12R2-expressing cells. However, direct associations were detectable only between E12R2 and JAK2. Tyk2 phosphorylation was observed only in cells expressing E12R1 or E12R1+E12R2. In parallel with this activation pattern, direct interactions only between Tyk2 and E12R1 were demonstrable. Phosphorylation of STAT3 was observed in cells expressing E12R1, E12R2, and E12R1+E12R2. The expression levels of STAT4 protein in BaF3 cells are undetectable by the methods employed here; therefore, STAT4 phosphorylation was not observed. Taken together, the data indicate that differential interactions take place between the cytoplasmic regions of the two IL-12R subunits and JAK2/Tyk2 and that the cytoplasmic region of IL-12Rbeta2 alone is capable of delivering a proliferative signal.

Regulation of interleukin-12 receptor beta1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells.

The interleukin-12 receptor (IL-12R)beta1 chain is an essential component of the functional IL-12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti-CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up-regulation of IL-12Rbeta1 expression and IL-12 binding. Kinetic studies revealed that maximum expression of IL-12Rbeta1 and IL-12 binding occurred on days 3-4. Anti-CD3-induced expression of IL-12Rbeta1 chain and IL-12 binding by PBMC was augmented by anti-CD28 mAb, indicating that the potentiating effect of anti-CD28 on T cell responses to IL-12 could be mediated, at least in part, by the enhancement of IL-12R expression. Among 16 cytokines tested, IL-2, IL-7 and IL-15 markedly induced IL-12Rbeta1 expression and IL-12 binding on resting PBMC, whereas IL-1alpha and tumor necrosis factor-alpha had a minimal enhancing effect. In contrast, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-alpha, IFN-gamma, granulocyte/macrophage colony-stimulating factor and transforming growth factor (TGF)-beta2 had no detectable enhancing effect. Anti-CD3-induced expression of IL-12Rbeta1 and of low-affinity IL-12 binding sites was partially inhibited by TGF-beta2, IL-10 and IL-4; however, TGF-beta2 and IL-10 completely abolished anti-CD3-induced expression of high-affinity IL-12 binding sites. Consistent with the reduction of high affinity IL-12 binding sites, PBMC activated with anti-CD3 mAb in the presence of TGF-beta2 or IL-10 failed to produce IFN-gamma or to proliferate in response to IL-12. These results suggest that Th2 cell-derived cytokines can inhibit IL-12-induced biological functions by inhibiting IL-12R expression and that expression of a second subunit of the IL-12R (IL-12Rbeta2), required for the formation of high-affinity IL-12 binding sites, may be more highly regulated by TGF-beta2 and IL-10 than is expression of IL-12Rbeta1.

A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits.

We have identified a cDNA from a human phytohemagglutinin-activated lymphoblast library encoding a protein that binds 125I-labeled human interleukin 12 (125I-huIL-12) with a Kd of about 5 nM when expressed in COS-7 cells. When coexpressed in COS-7 cells with the previously identified IL-12 beta receptor (IL-12R beta) protein, two classes of 125I-huIL-12 binding sites were measured with Kds of about 55 pM and 8 nM, corresponding to the high- and low-affinity binding sites seen on phytohemagglutinin-activated lymphoblasts. This newly identified huIL-12R subunit is a member of the cytokine receptor superfamily, with closest resemblance to the beta-type cytokine receptor gp130 and the receptors for leukemia inhibitory factor and granulocyte colony-stimulating factor. Consequently, we have reclassified the previously identified IL-12R beta subunit as huIL-12R beta 1 and designated the newly identified subunit as huIL-12R beta 2. huIL-12R beta 2 is an 862-amino acid type I transmembrane protein with a 595-amino-acid-long extracellular domain and a cytoplasmic tail of 216 amino acids that contains three tyrosine residues. A cDNA encoding the mouse homolog of the huIL12R beta 2 protein has also been isolated. Human and mouse IL-12R beta 2 proteins show a 68% amino acid sequence identity. When expressed in COS-7 cells, huIL-12R beta 2 exists as a disulfide-linked oligomer with an apparent monomeric molecular weight of 130 kDa. Coexpression of the two identified IL-12R subunits in Ba/F3 cells conferred IL-12 responsiveness, and clones of these cotransfected Ba/F3 cells that grew continuously in the presence of IL-12 were isolated and designated LJM-1 cells. LJM-1 cells exhibited dose-dependent proliferation in response to huIL-12, with an ED50 of about 1 pM huIL-12. Interestingly, Ba/F3 cells transfected with IL-12R beta 2 alone proliferated in response to huIL-12 with an ED50 of about 50 pM, although a role for endogenous mouse IL-12R beta 1 in IL-12 signal transduction in these transfectants cannot be ruled out. These results demonstrate that the functional high-affinity IL-12R is composed of at least two beta-type cytokine receptor subunits, each independently exhibiting a low affinity for IL-12.

Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo.

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

Molecular biology of interleukin-12 receptors.

IL-12 receptors are expressed primarily on activated T and NK cells. Using labeled IL-12, three classes of IL-12-binding sites have been identified on human PHA-activated lymphoblasts. Their Kd values are 5-20 pM (high affinity), 50-200 pM (intermediate affinity), and 2-6 nM (low affinity). Using a monoclonal antibody, a cDNA encoding a human IL-12 receptor was isolated by expression cloning. The homologous mouse cDNA was isolated by cross hybridization. These proteins are gp130-like members of the cytokine receptor superfamily. Individually, however they bind IL-12 with low affinity. An expression cloning approach was undertaken to isolate an additional human IL-12 receptor component that would act as a high-affinity converter. A cDNA encoding another IL-12 receptor was isolated. The mouse homologue was obtained by cross hybridization. These IL-12 receptors are also gp130-like cytokine receptor superfamily members. Because these two types of IL-12 receptors have the general makeup of beta-type cytokine receptor subunits, they are designated as IL-12R beta 1 and IL-12R beta 2. Coexpression of IL-12R beta 1 and IL-12R beta 2 leads to the formation of high-affinity IL-12-binding sites and reconstitution of IL-12-dependent signaling.

Evidence for multiple sites of interaction between IL-12 and its receptor.

We have previously described the identification of a protein, now designated IL-12R beta 1, that binds 125I-huIL-12 with a Kd of about 10 nM, corresponding to the low affinity 125I-huIL-12 binding sites seen on PHA-activated human lymphoblasts. Using expression cloning techniques, we have recently identified an additional IL-12-binding protein subunit, IL-12R beta 2, which binds 125I-huIL-12 with a Kd of about 5 nM when expressed alone in COS-7 cells. Coexpression of IL-12R beta 1 and IL-12R beta 2 in COS-7 cells results in formation of two classes of 125 I-huIL-12-binding sites with Kds of about 50 pM and 5 nM. Mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to human IL-12R beta 1, but did not inhibit binding to human IL-12R beta 2. In contrast, anti-huIL-12 monoclonal antibody 20C2, which does not block 125I-huIL-12 binding to human IL-12R beta 1, completely blocked binding to human IL-12R beta 2. These results demonstrate that two classes of IL-12 inhibitors, one that primarily blocks IL-12/IL-12R beta 1 interaction (e.g., mo(p40)2), and one that primarily blocks IL-12/IL-12R beta 2 interaction (e.g., 20C2), can be identified.