NMR structure of the human oncofoetal fibronectin ED-B domain, a specific marker for angiogenesis

BACKGROUND: The process of angiogenesis (i.e. the formation of new blood vessels from pre-existing ones) is fundamental to physiological processes such as reproduction, development and repair, as well as to pathological conditions such as tumor progression, rheumathoid arthritis and ocular disorders. The oncofoetal ED-B domain, a specific marker of angiogenesis, consists of 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule. RESULTS: The NMR structure of the ED-B domain is reported and reveals important differences from other FN type III domains. A comparison of the ED-B domain with the crystal structure of a four-domain FN fragment shows the novel features of ED-B to be located in loop regions that are buried at interdomain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potential specific recognition site. Epitope mapping with 82 decapeptides that span the ED-B sequence revealed that three ED-B-specific monoclonal antibodies, which selectively target newly forming blood vessels in tumor-bearing mice, bind to adjacent regions on the ED-B surface. CONCLUSIONS: The NMR structure enables the identification of a large surface area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED-B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the formation of new blood vessels.     

huBC1-IL12, an immunocytokine which targets EDB-containing oncofetal fibronectin in tumors and tumor vasculature,shows potent anti-tumor activity in human tumor models

IL-12 is a cytokine which showed anti-tumor effects in clinical trials, but also produced serious toxicity. We describe a fusion protein, huBC1-IL12, designed to achieve an improved therapeutic index by specifically targeting IL-12 to tumor and tumor vasculature. huBC-1 is a humanized antibody that targets a cryptic sequence of the human ED-B-containing fibronectin isoform, B-FN, present in the subendothelial extracellular matrix of most aggressive tumors. B-FN is oncofetal and angiogenesis-associated, and is undetectable in most normal adult tissues. The original murine BC-1 antibody has been used successfully for immunoscintigraphy to image brain tumor mass in glioblastoma patients. In huBC1-IL12, each of the IgG heavy chains is genetically fused to the N-terminus of the IL-12 p35 subunit, which in turn is disulfide-bonded to the p40 subunit, resulting in a hexameric molecule of MW of ∼300 kDa. Since human IL-12 has no biological activity in mice, we produced huBC1-muIL12 as a surrogate molecule for animal tumor models. Despite the relatively poor PK profile of this molecule in mice and the apparent drawbacks of xenogeneic models in SCID mice, which lack T and B cells, one cycle of treatment with huBC1-muIL12 was efficacious in the PC3mm2, A431, and HT29 subcutaneous tumor models and PC3mm2 lung metastasis model. This molecule also was found to have surprisingly low toxicity in immunocompetent mice. A fusion protein that contains human IL-12 (huBC1-huIL12), which is a suitable molecule for investigation as a therapeutic, has also been produced. This protein has been shown to have a longer serum half-life than huBC1-muIL12 in mice, and retains both antigen binding and IL-12 activity in in vitro assays.     

Enhanced sensitivity to IL-2 signaling regulates the clinical responsiveness of IL-12-primed CD8(+) T cells in a melanoma model

The optimal expansion, trafficking, and function of adoptively transferred CD8(+) T cells are parameters that currently limit the effectiveness of antitumor immunity to established tumors. In this study, we addressed the mechanisms by which priming of self tumor-associated Ag-specific CD8(+) T cells influenced antitumor functionality in the presence of the inflammatory cytokine IL-12. In vitro priming of mouse tumor-specific CD8(+) T cells in the presence of IL-12 induced a diverse and rapid antitumor effector activity while still promoting the generation of memory cells. Importantly, IL-12-primed effector T cells dramatically reduced the growth of well-established s.c. tumors and significantly increased survival to highly immune resistant, established intracranial tumors. Control of tumor growth by CD8(+) T cells was dependent on IL-12-mediated upregulation of the high-affinity IL-2R (CD25) and a subsequent increase in the sensitivity to IL-2 stimulation. Finally, IL-12-primed human PBMCs generated tumor-specific T cells both phenotypically and functionally similar to IL-12-primed mouse tumor-specific T cells. These results highlight the ability of IL-12 to obviate the strict requirement for administering high levels of IL-2 during adoptive cell transfer-mediated antitumor responses. Furthermore, acquisition of a potent effector phenotype independent of cytokine support suggests that IL-12 could be added to adoptive cell transfer clinical strategies in cancer patients.     

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.     

High fat diet induces formation of spontaneous liposarcoma in mouse adipose tissue with overexpression of interleukin 22

Interleukin 22 (IL-22) is a T-cell secreted cytokine that modulates inflammatory response in nonhematopoietic tissues such as epithelium and liver. The function of IL-22 in adipose tissue is currently unknown. We generated a transgenic mouse model with overexpression of IL-22 specifically in adipose tissue. The IL-22 transgenic mice had no apparent changes in obesity and insulin resistance after feeding with high fat diet (HFD). Unexpectedly, all the IL-22 transgenic mice fed with HFD for four months developed spontaneous tumors in epididymal adipose tissue. Histological analysis indicated that the tumors were well-differentiated liposarcomas with infiltration of inflammatory cells. IL-22 overexpression promotes production of inflammatory cytokines such as IL-1β and IL-10 and stimulates ERK phosphorylation in adipose tissue. Furthermore, IL-22 treatment in differentiated 3T3-L1 adipocytes could induce IL-1β and IL-10 expression, together with stimulation of ERK phosphorylation. Taken together, our study not only established a novel mouse model with spontaneous liposarcoma, but also revealed that IL-22 overexpression may collaborate with diet-induced obesity to impact on tumor development in mouse.     

C-reactive protein mediates protection from lipopolysaccharide through interactions with Fc gamma R

C-reactive protein (CRP) is a component of the acute phase response to infection, inflammation, and trauma. A major activity of acute phase proteins is to limit the inflammatory response. It has been demonstrated that CRP protects mice from lethal doses of LPS. In the mouse, CRP binds to the regulatory receptor, FcgammaRIIb, and to the gamma-chain-associated receptor, FcgammaRI. The goal ofthis study was to determine whether FcgammaRs are necessary for the protective effect of CRP. The ability of CRP to protect mice from a lethal dose of LPS was confirmed using injections of 500 and 250 micro g of CRP at 0 and 12 h. CRP treatment of FcgammaRIIb-deficient mice increased mortality after LPS challenge and increased serum levels of TNF and IL-12 in response to LPS. CRP did not protect FcR gamma-chain-deficient mice from LPS-induced mortality. Treatment of normal mice, but not gamma-chain-deficient mice, with CRP increased IL-10 levels following LPS injection. In vitro, in the presence of LPS, CRP enhanced IL-10 synthesis and inhibited IL-12 synthesis by bone marrow macrophages from normal, but not gamma-chain-deficient mice. The protective effect of CRP appears to be mediated by binding to FcgammaRI and FcgammaRII resulting in enhanced secretion of the anti-inflammatory cytokine IL-10 and the down-regulation of IL-12. These results suggest that CRP can alter the cytokine profile of mouse macrophages by acting through FcgammaR leading to a down-regulation of the inflammatory response.     

Polymorphisms in Toll-Like Receptor 4 Underlie Susceptibility to Tumor Induction by the Mouse Polyomavirus

PERA/Ei (PE) mice are susceptible to tumor induction by polyomavirus (Py), while C57BR/cdJ (BR) mice are resistant. Antigen-presenting cells from BR mice respond to the virus with interleukin-12 (IL-12) and those from PE mice with IL-10. These polarized cytokine responses underlie the development of effective antitumor immunity in BR mice and the lack thereof in PE mice. An ex vivo cytokine production assay using spleen cells from infected [PE × BR] F2 mice together with a genome-wide SNP (single-nucleotide polymorphism)-based QTL (quantitative trait locus) analysis was used to map the determinant of cytokine production to a region of chromosome 4 carrying the Toll-like receptor 4 (TLR4) gene. Genotyping of infected F2 mice showed concordance of TLR4 allele-specific DNA sequences with the cytokine profile. Cytokine responses elicited by Py are MyD88 dependent. Bacterial lipopolysaccharide (LPS), a known TLR4 ligand, induced the same polarized responses as the virus in these host strains. Spleen cells from C3H/HeJ and C57BL/10ScNJ LPS-nonresponsive mice challenged in vitro with Py showed an impaired IL-12 response but were unaffected in IL-10 production. TLR4s of strains PE and BR differ by 3 amino acid substitutions, 2 in the extracellular domain and 1 in the intracellular domain. cDNAs encoding the TLR4s signaled equally to an NF-κB reporter in 293 cells in a ligand-independent manner. When introduced into TLR2/TLR4 double-knockout macrophages, the TLR4 cDNA from BR mice conferred a robust IL-12 response to Py and no IL-10 response. The TLR4 cDNA from PE mice failed to confer a response with either cytokine. These results establish TLR4 as a key mediator of the cytokine response governing susceptibility to tumor induction by Py.     

Virulizin® induces production of IL-17E to enhance antitumor activity by recruitment of eosinophils into tumors

Virulizin has demonstrated strong antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. Our previous studies have demonstrated that macrophages, NK cells, and cytokines are important in the antitumor mechanism of Virulizin. Virulizin treatment of tumor bearing mice results in the expansion as well as increased activity of monocytes/macrophages and production of cytokines IL-12 and TNFalpha and activation of NK cells. In this study we show that the inflammatory cytokine IL-17E (IL-25) is induced by Virulizin treatment and is part of its antitumor mechanism. IL-17E is a proinflammatory cytokine, which induces a T(H)2 type immune response, associated with eosinophil expansion and infiltration into mucosal tissues. IL-17E was increased in sera of Virulizin-treated mice bearing human melanoma xenografts, compared to saline-treated controls, as shown by 2D gel electrophoresis and ELISA. Treatment of splenocytes in vitro with Virulizin resulted in increased IL-17E mRNA expression, which peaked between 24 and 32 h post-stimulation. Both in vitro and in vivo experiments demonstrated that B cells produced IL-17E in response to Virulizin treatment. Furthermore, Virulizin treatment in vivo resulted in increased blood eosinophilia and eosinophil infiltration into tumors. Finally, injection of recombinant IL-17E showed antitumor activity towards xenografted tumors, which correlated with increased eosinophilia in blood and tumors. Taken together, these results support another antitumor mechanism mediated by Virulizin, through induction of IL-17E by B cells, leading to recruitment of eosinophils into tumors, which may function in parallel with macrophages and NK cells in mediating tumor destruction.     

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.     

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.     

Direct determination of the interleukin-6 binding epitope of the interleukin-6 receptor by NMR spectroscopy

All cytokines belonging to the interleukin-6 (IL-6)-type family of cytokines utilize receptors that have a modular build of several immunoglobulin-like and fibronectin type III-like domains. Characteristic of these receptors is a cytokine receptor homology region consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 first binds to its specific receptor and subsequently to a homodimer of the signal transducer protein gp130. The interleukin-6 receptor consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane proximal fibronectin-like domain accounts for more than 90% of the binding energy to IL-6. Here, the key residues of this fibronectin-like domain involved in the interaction with IL-6 are described. Chemical shift mapping data with 15N-labeled IL-6R-D3 and unlabeled IL-6 coupled with recent structural data clearly reveal the epitope within the IL-6R-D3 responsible for mediating the high affinity interaction with its cognate cytokine.     

Tumor necrosis therapy antibody interleukin-2 fusion protein elicits prolonged and targeted antitumor effects in vivo

Interleukin-2 (IL-2) is one of the most successful cytokines applied in tumor immunotherapy because of its ability to stimulate potent cellular immune response. The life-threatening toxicity of vascular leak syndrome (VLS) associated with the high-dose IL-2 treatment regimen has limited its use in tumor immunotherapy. To reverse this situation, a tumor-targeted fusion protein, recombinant human TNT-IL2 (rhTNT-IL2), was generated with both the cytokine activity of IL-2 and the tumor-targeting ability of TNT antibody. TNT is a human tumor necrosis therapy monoclonal antibody capable of binding intracellular antigens which are accessible and abundant in necrotic regions of tumors. The immunotherapeutic potential of this fusion protein was tested in murine melanoma and lung cancer models, and tumor-bearing mice showed satisfied tumor regressions after rhTNT-IL2 immunotherapy. Immunohistochemical study showed a distinct penetration of IL-2 in tumors in mice treated with rhTNT-IL2, indicating its evident tumor-targeting activity. Moreover, the rhTNT-IL2 was well tolerated in cynomolgus monkeys in a 12-week long-term repeated toxicity study. These studies indicate that the targeting of IL-2 to necrotic areas of tumors might be a new approach for the immunotherapy of solid tumors.     

Role of Th1 and Th2 cytokines in BCG-induced IFN-gamma production: cytokine promotion and simulation of BCG effect

Induction of a T-helper-type 1 (Th1) immune response is indispensable for successful treatment of superficial bladder cancer with BCG. In this study possible involvement of various cytokines in BCG action as well as their potential roles in enhancing and mimicking BCG effect were explored. In immunocompetent cell cultures, IFN-gamma, a major Th1 cytokine, appears to be a late responsive cytokine to BCG stimulation. Its induction requires involvement of various endogenously produced Th1 and Th2 cytokines. Functional abolishment of any one of these cytokines (IL-2, IL-6, IL-12, IL-18, GMCSF, TNF-alpha, or IFN-alpha, except IL-10) by neutralizing antibodies leads to reduced IFN-gamma production (19-82% inhibition in mouse and 44-77% inhibition in human systems, respectively). In mice cytokines IL-2, IL-12, IL-18, and GMCSF are observed to synergize with BCG for IFN-gamma production, whereas in human cytokines IL-2, IL-12, TNF-alpha, and IFN-alpha exhibit similar synergistic effects. Rational combinations of these Th1-stimulating cytokines (IL-12 plus IL-18 in mice and IL-2 plus IL-12 in humans, respectively) dramatically up-regulate IFN-gamma production that is incomparably superior to BCG for induction of this cytokine. These results suggest that combined Th1-stimulating cytokines and combinations of BCG plus selected Th1-stimulating cytokines are rational candidates for further study in the treatment of bladder cancer patients.     

Combination of two fiber-mutant adenovirus vectors, one encoding the chemokine FKN and another encoding cytokine interleukin 12, elicits notably enhanced anti-tumor responses

For achieving optimal cancer immunotherapy, it is anticipated that both the activation and infiltration of immune cells into tumor are indispensable. In the present study, fiber-mutant adenovirus vectors (Ad) encoding chemokine FKN, (AdRGD-FKN), and cytokine interleukin 12, (AdRGD-IL-12), were constructed. The in vivo gene expression of AdRGD was confirmed and the combination of both FKN and IL-12 encoding Ad elicited synergistic anti-tumor activity in ovarian carcinoma, which induced tumor regression in all tumor-bearing mice, while using FKN alone did not show notable tumor-suppressive effect. The treatment with both IL-12 and FKN induced long-term specific immunity against OV-HM tumors in tumor-rejected mice. The results of immunohistochemical staining for CD3(+ )and perforin-positive cells suggested that the failure of using FKN alone was because of the inactivation of infiltrated immune cells. In contrast, cotransduction with IL-12 and FKN could induce more activated tumor-infiltrating immune cells than that transducted with FKN or IL-12 alone. The results indicated that using both chemokine and cytokine might be a powerful tool and a promising way for effective cancer immunotherapy.     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor α in mice

 There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. Received: 17 February 1997 / Accepted: 5 August 1997     

Autocrine production of IL-10 mediates defective IL-12 production and NF-kappa B activation in tumor-associated macrophages

IL-12 is a central cytokine in the activation of inflammation and immunity and in the generation of Th1-type responses. Tumor-associated macrophages (TAM) from mouse and human tumors showed defective production of IL-12. Defective IL-12 production was associated with lack of p50/p65 NF-kappa B activation. TAM produced increased amounts of the immunosuppressive cytokine IL-10. Abs against IL-10 restored the defective capacity of TAM to produce IL-12. Our data suggest that during tumor growth an IL-10-dependent pathway of diversion of macrophage function can be activated into the tumor microenvironment and results in the promotion of the IL-10+ IL-12- phenotype of TAM. Blocking IL-10, as well as other immunosuppressive cytokines present in the tumor microenvironment, such as TGF-beta, may complement therapeutic strategies aimed at activating type I antitumor immune responses.     

The solution structure of the membrane-proximal cytokine receptor domain of the human interleukin-6 receptor

The members of the interleukin-6-type family of cytokines interact with receptors that have a modular structure and are built of several immunoglobulin-like and fibronectin type III-like domains. These receptors have a characteristic cytokine receptor homology region consisting of two fibronectin type III-like domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 (IL-6) initially binds to its cognate alpha-receptor and subsequently to a homodimer of the signal transducer receptor gp130. The IL-6 receptor (IL-6R) consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane-proximal fibronectin-like domain (IL-6R-D3) accounts for more than 90% of the binding energy to IL-6. Here, we present the solution structure of the IL-6R-D3 domain solved by multidimensional heteronuclear NMR spectroscopy.     

Subtherapeutic doses of interleukin-15 augment the antitumor effect of interleukin-12 in a B16F10 melanoma model in mice.

Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that exhibits antitumor activity in many experimental tumor models. In the present study, we investigated the ability of IL-15, a cytokine sharing many functions of IL-2, to modulate antitumor effectiveness of IL-12 against B16F10 melanoma in mice. In a model of locally growing tumor, intratumoral (i.t.) administration of IL-12, in three cycles of five consecutive daily injections (0.1 mug) followed by 2 days of rest, led to considerable delay of tumor development but no curative response was achieved. When combined with IL-12, subtherapeutic doses of IL-15 (0.4 mug) pontentiated the antitumor effects of IL-12 and induced complete tumor regressions in 50% of mice. Similar results were obtained in a model in which tumor-bearing mice were intravenously co-injected with melanoma cells to induce metastases. Combined administration of IL-12 and IL-15 yielded greater antitumor activity than injections of either cytokine alone and resulted in prolonged survival of mice bearing locally growing tumor and metastases. Studies of immunological parameters in mice treated with both IL-12 and IL-15 have shown enhanced NK activity (against YAC-1 cells) in the spleen and stimulation of both NK activity and specific anti-B16F10 cytotoxic effector cells in tumor-draining lymph nodes (LN). The strong antitumor effect of the IL-12 + IL-15 combination correlated with a high serum level of IFN-gamma in the treated mice. Moreover, increased expression of IL-15Ralpha was demonstrated in LN lymphocytes isolated from mice injected with IL-12. This result together with findings of other authors showing enhanced expression of IL-12 receptor by IL-15 [1] suggests that the augmentation of the antitumor effect during the course of IL-12/IL-15-based therapy could result from reciprocal upregulation of receptors by both cytokines and synergistic effects on IFN-gamma induction.     

Interleukin-12 (IL-12)/STAT4 Axis Is an Important Element for β-Cell Dysfunction Induced by Inflammatory Cytokines

Pathology driving β-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with β-cell dysfunction. Acute in vitro exposure of islets and β-cells to an inflammatory cytokine cocktail (IL-1β/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse β-cell lines and primary mouse islets. Cytokine cooperation is required for β-cell apoptosis with the most potent combinations including IL-1β. Single cytokine exposure did not induce β-cell apoptosis. Expression of endogenous interleukin-12 in β-cells correlated with inflammatory cytokine combinations that induced β-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to β-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced β-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/β-cell viability in established diabetes.