RESUMEN
Recently, the development of several strategies based on immunotherapy has raised hopes for a more promising way to treat cancer patients. Here, we describe how interleukin (IL)-10, a seemingly unlikely candidate, stimulates the immune system in a particularly efficacious way. IL-10, an omnipotent anti-inflammatory cytokine, delivers an equally potent immune stimulation in the context of CD8(+) T cells and tumor immunity. By activation of tumor-resident, tumor-specific CD8(+) T cells, pegylated IL-10 can induce rejection of large and metastasizing tumors in mice. Here, we summarize the mechanisms of action of IL-10, the reasons why the mechanisms may be crucial for the treatment of cancer patients, and the rationale for applying pegylated IL-10 in the clinic.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Interferón gamma/metabolismo , Interleucina-10/farmacología , Polietilenglicoles/farmacología , Animales , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunoterapia/métodos , Interferón gamma/inmunología , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/terapiaRESUMEN
Tumor immune surveillance and CD8+ T cells in particular appear capable of recognizing the antigenic properties of human tumor cells. However, those antigen specific T cells are often excluded from tumor tissue or are functionally limited in their cytotoxic capacity. Instead, the immune response provides proinflammatory cytokines and proteases promoting tumor growth and progression while subverting cytotoxic anti-tumor immunity. The cytokines and the inflammatory mechanisms driving tumor associated inflammation resemble tissue remodeling processes during wound healing and chronic inflammatory diseases. In this chapter, we summarize the current knowledge of how inflammatory cytokines may promote the deviation of anti-tumor immunity toward a tumor promoting, noncytotoxic inflammation.
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Neoplasias/inmunología , Inmunidad Adaptativa , Citocinas/fisiología , Humanos , Vigilancia Inmunológica , Linfocitos T/inmunologíaRESUMEN
IL-33 is constitutively expressed in epithelial barrier tissues, such as skin. Although increased expression of IL-33/IL-33R has been correlated with fibrotic disorders, such as scleroderma and progressive systemic sclerosis, the direct consequences of IL-33 release in skin has not been reported. To determine the effects of dysregulated IL-33 signaling in skin, we administered IL-33 s.c. and monitored its effects at the injection site. Administration of IL-33 resulted in IL-33R-dependent accumulation of eosinophils, CD3(+) lymphocytes, F4/80(+) mononuclear cells, increased expression of IL-13 mRNA, and the development of cutaneous fibrosis. Consistent with extensive cutaneous tissue remodeling, IL-33 resulted in significant modulation of a number of extracellular matrix-associated genes, including collagen VI, collagen III, and tissue inhibitor of metalloproteases-1. We establish that IL-33-induced fibrosis requires IL-13 using IL-13 knockout mice and eosinophils using Delta dblGATA mice. We show that bone marrow-derived eosinophils secrete IL-13 in response to IL-33 stimulation, suggesting that eosinophil-derived IL-13 may promote IL-33-induced cutaneous fibrosis. Collectively, our results identify IL-33 as a previously unrecognized profibrotic mediator in skin and highlight the cellular and molecular pathways by which this pathology develops.
Asunto(s)
Interleucina-13/fisiología , Interleucinas/fisiología , Piel/inmunología , Piel/patología , Animales , Células Cultivadas , Eosinófilos/inmunología , Eosinófilos/metabolismo , Eosinófilos/patología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/fisiología , Fibrosis , Mediadores de Inflamación/administración & dosificación , Mediadores de Inflamación/fisiología , Inyecciones Subcutáneas , Quinasas Asociadas a Receptores de Interleucina-1/deficiencia , Quinasas Asociadas a Receptores de Interleucina-1/fisiología , Interleucina-13/biosíntesis , Interleucina-13/deficiencia , Interleucina-33 , Interleucinas/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Piel/metabolismoRESUMEN
Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.
Asunto(s)
Interferón-alfa/fisiología , Interferón beta/fisiología , Interleucinas/metabolismo , Melanoma/metabolismo , Melanoma/patología , Transducción de Señal/fisiología , Muerte Celular/fisiología , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Humanos , Interferón-alfa/biosíntesis , Interferón beta/biosíntesis , Interleucinas/aislamiento & purificación , Interleucinas/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Tumor-reactive T cell exhaustion prevents the success of immune therapies. Pegilodecakin activates intratumoral CD8+ T cells in mice and induces objective tumor responses in patients. Here we report that pegilodecakin induces hallmarks of CD8+ T cell immunity in cancer patients, including elevation of interferon-γ and GranzymeB, expansion and activation of intratumoral CD8+ T cells, and proliferation and expansion of LAG-3+ PD-1+ CD8+ T cells. On pegilodecakin, newly expanded T cell clones, undetectable at baseline, become 1%-10% of the total T cell repertoire in the blood. Elevation of interleukin-18, expansion of LAG-3+ PD-1+ T cells and novel T cell clones each correlated with objective tumor responses. Combined pegilodecakin with anti-PD-1 increased the expansion of LAG-3+ PD-1+ CD8+ T cells.
Asunto(s)
Antineoplásicos/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Inmunoterapia/métodos , Interleucina-10/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias/inmunología , Polietilenglicoles/uso terapéutico , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Granzimas/sangre , Humanos , Interferón gamma/sangre , Interleucina-10/química , Interleucina-10/uso terapéutico , Interleucina-18/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidoresRESUMEN
Interleukin-24 (IL-24), also known as melanoma differentiation-associated gene-7 (mda-7), is a member of the IL-10 family that exhibits both tumor suppressor and proinflammatory properties. We describe the purification of this novel dual-function tumor suppressor/cytokine from the supernatant of IL-24 gene-transfected HEK 293 cells and define the biochemical and functional properties of the soluble human IL-24 protein. Size exclusion chromatography demonstrates that an IL-24 macromolecular complex fractionates in a broad peak with a median of 110 kDa and comprises several IL-24 isoforms, identified by immunoblotting with anti-IL-24 polyclonal antibody after reducing SDS-PAGE analysis. IL-24 was found to associate with two serum components, albumin and C1q. Cation exchange purification results in the isolation of at least two N-linked glycosylated IL-24 dimers covalently associated via intermolecular disulfide bonds. These molecularly defined N-glycosylated IL-24 dimers elicited dose-dependent secretion of tumor necrosis factor-alpha (TNF-alpha) and IL-6 from human monocytes, as well as cytotoxicity to human melanoma cell lines. Thus, we demonstrated that the secreted, glycosylated, dimeric, human IL-24 is immunomodulatory to monocytes and exhibits tumor cell growth inhibition.
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Adyuvantes Inmunológicos/farmacología , Proliferación Celular/efectos de los fármacos , Interleucinas/farmacología , Melanoma/inmunología , Monocitos/efectos de los fármacos , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/aislamiento & purificación , Albúminas/aislamiento & purificación , Apoptosis , Línea Celular Tumoral , Cromatografía por Intercambio Iónico , Dimerización , Glicosilación , Humanos , Interleucina-6/metabolismo , Interleucinas/biosíntesis , Interleucinas/aislamiento & purificación , Monocitos/inmunología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The melanoma differentiation-associated gene 7 (mda-7), also called interleukin (IL)-24, suppresses the growth of some cancers in vitro and in vivo as a result of the ectopic expression of its protein. However, the function of the secreted form of the protein in cancer has not been previously studied. The purpose of this study was to determine the antiangiogenic function of a secreted form of the MDA-7/IL-24 protein (sMDA-7/IL-24). In vitro, sMDA-7/IL-24 inhibited both endothelial cell differentiation and migration of endothelial cells induced by vascular endothelial growth factor and basic fibroblast growth factor. The sMDA-7/IL-24-mediated inhibitory effect was 10-50 times more potent than endostatin, IFN-gamma, and IFN-inducible protein 10 in vitro. Furthermore, the inhibitory effect was not mediated by IFN or IFN-inducible protein 10. IL-22 receptor mediated the antiangiogenic activity of sMDA-7/IL-24. Administration of a blocking antibody to IL-22 receptor in conjunction with sMDA-7/IL-24 led to abrogation of inhibition of endothelial differentiation. sMDA-7/IL-24 inhibited vascular endothelial growth factor-induced angiogenesis as evidenced by reduced vascularization and hemoglobin content in in vivo Matrigel plug assays. In vivo, the growth of human lung tumor cells was significantly inhibited, and vascularization was reduced when the cells were mixed with 293 cells stably expressing sMDA-7/IL-24. Systemic administration of sMDA-7/IL-24 inhibited lung tumor growth in a mouse xenograft model. Associated with tumor growth inhibition was decreased tumor microvessel density and hemoglobin content, indicating the presence of antiangiogenic activity. These data demonstrate that sMDA-7/IL-24 is a novel and potent antiangiogenic effector and support the development of MDA-7/IL-24-based therapeutics.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Interleucinas/farmacología , Receptores de Interleucina/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL10 , Quimiocinas CXC/fisiología , Colágeno/farmacología , Proteínas de Unión al ADN/metabolismo , Endostatinas , Endotelio Vascular/citología , Femenino , Genes Supresores de Tumor , Humanos , Interferón gamma/fisiología , Interleucinas/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Interleukin-10 (IL-10) is a multifunctional cytokine that exerts potent context specific immunostimulatory and immunosuppressive effects. We have investigated the mechanism by which PEGylated rIL-10 regulates plasma cholesterol in mice and humans. In agreement with previous work on rIL-10, we report that PEGylated rIL-10 harnesses the myeloid immune system to control total plasma cholesterol levels. We have discovered that PEG-rMuIL-10's dramatic lowering of plasma cholesterol is dependent on phagocytotic cells. In particular, PEG-rHuIL-10 enhances cholesterol uptake by Kupffer cells. In addition, removal of phagocytotic cells dramatically increases plasma cholesterol levels, suggesting for the first time that immunological cells are implicitly involved in regulating total cholesterol levels. These data suggest that treatment with PEG-rIL-10 potentiates endogenous cholesterol regulating cell populations not currently targeted by standard of care therapeutics. Furthermore, we show that IL-10's increase of Kupffer cell cholesterol phagocytosis is concomitant with decreases in liver cholesterol and triglycerides. This leads to the reversal of early periportal liver fibrosis and facilitates the restoration of liver health. These data recommend PEG-rIL-10 for evaluation in the treatment of fatty liver disease and preventing its progression to non-alcoholic steatohepatitis. In direct confirmation of our in vivo findings in the treatment of hypercholesterolemic mice with PEG-rMuIL-10, we report that treatment of hypercholesterolemic cancer patients with PEG-rHuIL-10 lowers total plasma cholesterol by up to 50%. Taken together these data suggest that PEG-rIL-10's cholesterol regulating biology is consistent between mice and humans.
Asunto(s)
Colesterol/sangre , Hipercolesterolemia/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Interleucina-10/uso terapéutico , Macrófagos del Hígado/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Colesterol/inmunología , Femenino , Humanos , Hipercolesterolemia/sangre , Hipercolesterolemia/inmunología , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Interleucina-10/química , Interleucina-10/farmacología , Macrófagos del Hígado/inmunología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Polietilenglicoles/química , Polietilenglicoles/farmacología , Polietilenglicoles/uso terapéutico , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Adulto JovenRESUMEN
IL-10 has been classically defined as a broad-spectrum immunosuppressant and is thought to facilitate the development of regulatory CD4(+) T cells. IL-10 is believed to represent one of the major suppressive factors secreted by IDO(+)FoxP3(+)CD4(+) Tregs. Contrary to this view, we have previously reported that PEGylated recombinant IL-10 (PEG-rIL-10) treatment of mice induces potent IFNγ and CD8(+) T-cell-dependent antitumor immunity. This hypothesis is currently being tested in clinical trials and we have reported that treatment of cancer patients with PEG-rHuIL-10 results in inhibition and regression of tumor growth as well as increased serum IFNγ. We have continued to assess PEG-rIL-10's pleiotropic effects and report that treatment of tumor-bearing mice and humans with PEG-rIL-10 increases intratumoral indoleamine 2, 3-dioxygenase (IDO) in an IFNγ-dependent manner. This should result in an increase in Tregs, but paradoxically our data illustrate that PEG-rIL-10 treatment of mice reduces intratumoral FoxP3(+)CD4(+) T cells in an IDO-independent manner. Additional investigation indicates that PEG-rIL-10 inhibits TGFß/IL-2-dependent in vitro polarization of FoxP3(+)CD4(+) Tregs and potentiates IFNγ(+)T-bet(+)CD4(+) T cells. These data suggest that rather than acting as an immunosuppressant, PEG-rIL-10 may counteract the FoxP3(+)CD4(+) Treg suppressive milieu in tumor-bearing mice and humans, thereby further facilitating PEG-rIL-10's potent antitumor immunity.
RESUMEN
Purpose Interleukin-10 (IL-10) stimulates the expansion and cytotoxicity of tumor-infiltrating CD8+ T cells and inhibits inflammatory CD4+ T cells. Pegylation prolongs the serum concentration of IL-10 without changing the immunologic profile. This phase I study sought to determine the safety and antitumor activity of AM0010. Patients and Methods Patients with selected advanced solid tumors were treated with AM0010 in a dose-escalation study, which was followed by a renal cell cancer (RCC) dose-expansion cohort. AM0010 was self-administered subcutaneously at doses of 1 to 40 µg/kg once per day. Primary end points were safety and tolerability; clinical activity and immune activation were secondary end points. Results In the dose-escalation and -expansion cohorts, 33 and 18 patients, respectively, were treated with daily subcutaneous injection of AM0010. AM0010 was tolerated in a heavily pretreated patient population. Treatment-related adverse events (AEs) included anemia, fatigue, thrombocytopenia, fever, and injection site reactions. Grade 3 to 4 nonhematopoietic treatment-related AEs, including rash (n = 2) and transaminitis (n = 1), were observed in five of 33 patients. Grade 3 to 4 anemia or thrombocytopenia was observed in five patients. Most treatment-related AEs were transient or reversible. AM0010 led to systemic immune activation with elevated immune-stimulatory cytokines and reduced transforming growth factor beta in the serum. Partial responses were observed in one patient with uveal melanoma and four of 15 evaluable patients with RCC treated at 20 µg/kg (overall response rate, 27%). Prolonged stable disease of at least 4 months was observed in four patients, including one with colorectal cancer with disease stabilization for 20 months. Conclusion AM0010 has an acceptable toxicity profile with early evidence of antitumor activity, particularly in RCC. These data support the further evaluation of AM0010 both alone and in combination with other immune therapies and chemotherapies.
Asunto(s)
Citocinas/sangre , Interleucina-10/efectos adversos , Neoplasias/tratamiento farmacológico , Polietilenglicoles/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Anemia/inducido químicamente , Carcinoma de Células Renales/tratamiento farmacológico , Erupciones por Medicamentos/etiología , Exantema/inducido químicamente , Fatiga/inducido químicamente , Femenino , Fiebre/inducido químicamente , Humanos , Inyecciones Subcutáneas/efectos adversos , Interferón gamma/sangre , Interleucina-10/inmunología , Interleucina-10/farmacocinética , Interleucina-10/uso terapéutico , Interleucina-4/sangre , Interleucina-8/sangre , Neoplasias Renales/tratamiento farmacológico , Masculino , Melanoma/tratamiento farmacológico , Persona de Mediana Edad , Neoplasias/patología , Polietilenglicoles/farmacocinética , Polietilenglicoles/uso terapéutico , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Trombocitopenia/inducido químicamente , Factor de Crecimiento Transformador beta/sangre , Neoplasias de la Úvea/tratamiento farmacológico , Adulto JovenRESUMEN
The melanoma differentiation association gene-7 (mda-7) is a novel tumor suppressor gene of which the protein expression decreases to nearly undetectable levels in metastatic melanoma. In contrast, expression of inducible nitric oxide synthase (iNOS) is increased in advanced stages of melanoma, and iNOS expression has been proposed as a potential prognostic marker in this disease. Thus, expression of these molecules in the same tumor appears to exhibit reciprocal characteristics. We hypothesize that the relative ratios of these melanoma progression molecules may define either tumor progression or tumor suppression in human melanoma. The first goal of this study was to determine whether MDA-7 expression in melanoma negatively correlates with iNOS expression. The second goal was to determine whether iNOS expression could be regulated by MDA-7 expression in melanoma cells. Expression of MDA-7 and iNOS proteins were evaluated by immunohistochemistry in a total of 81 tumor samples: 38 primary melanomas and 43 metastatic melanomas. Evaluation of these melanoma patient samples showed that expression of MDA-7 and iNOS exhibits a significant negative correlation (P = 0.03). In vitro studies revealed that iNOS expression in melanoma cell lines is lost in a dose-dependent fashion after treatment with an adenoviral vector encoding the mda-7 gene (Ad-mda7) or with rhMDA-7 protein, demonstrating that MDA-7 down-regulates iNOS expression. Furthermore, we demonstrate that the STAT-3-modulated expression of IFN regulatory factors 1 and 2 is regulated by MDA-7, which may alter iNOS gene expression. These studies demonstrate that expression of the MDA-7 tumor suppressor can negatively regulate iNOS expression in malignant melanoma cell lines.
Asunto(s)
Interleucinas/genética , Melanoma/enzimología , Melanoma/genética , Óxido Nítrico Sintasa/genética , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Inmunohistoquímica , Factor 1 Regulador del Interferón , Interleucinas/análisis , Melanoma/patología , Metástasis de la Neoplasia , Óxido Nítrico Sintasa de Tipo II , Fosfoproteínas/análisis , Fosfoproteínas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción/análisis , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Recent advances in immunoncology have dramatically changed the treatment options available to cancer patients. However, the fundamental challenges with this therapeutic modality are not new and still persist with the current wave of immunoncology compounds. These challenges are centered on the activation and expansion, induction of intratumoral infiltration and persistence of highly activated, cytotoxic, tumor antigen specific CD8+ T cells. We have investigated the anti-tumor mechanism of action of pegylated recombinant interleukin-10, (PEG-rIL-10) both pre-clinically with murine (PEG-rMuIL-10) and now clinically (AM0010) with human pegylated interleukin-10. The preponderance of data suggest that IL-10's engagement of its receptor on CD8+ T cells enhances their activation status leading to antigen specific expansion. Quantitation of CD8+ T cell tumor infiltration reveals that treatment of both humans and mice with pegylated rIL-10 results in 3-4 fold increases of intratumoral, cytotoxic, CD8+ T cells. In addition, mice cured of their tumors with PEG-rMuIL-10 exhibit long term immunological protection from tumor re-challenge and long term treatment of cancer patients with AM0010 results in the persistence of highly activated CD8+ T cells. Cumulatively, these data suggest the IL-10 represents an emerging therapeutic that specifically addresses the fundamental challenges of the current wave of immunoncology assets.
RESUMEN
The melanoma differentiation associated gene-7 (mda-7) cDNA was isolated by virtue of being induced during melanoma differentiation. Initial gene transfer studies convincingly demonstrated potent antitumor effects of mda-7. Further studies showed that the mechanism of antitumor activity was due to induction of apoptosis. Most striking was the tumor-selective killing by mda-7 gene transfer--normal cells were unaffected by Adenoviral delivery of mda-7 (Ad-mda7). A variety of molecules implicated in apoptosis and intracellular signaling are regulated by Ad-mda7 transduction. Different apoptosis effector proteins are regulated in different tumor types, suggesting that Ad-mda7 may regulate various signaling pathways. mda-7 encodes a secreted protein, MDA-7, which has now been designated as IL-24, and is a novel member of the IL-10 cytokine family. MDA-7/IL-24 protein is actively secreted from cells after mda-7 gene transfer. In human peripheral blood mononuclear cells (PBMC), STAT3 activation by MDA-7/IL-24 is followed by elaboration of secondary Th1 cytokines, demonstrating that MDA-7/IL-24 is a pro-Th1 cytokine. Furthermore, MDA-7/IL-24 is antagonized by the prototypic Th2 cytokine IL-10. MDA-7/IL-24 protein is endogenously expressed in cultured NK and B-cells and is also expressed in dendritic cells in tissues. MDA-7/IL-24 protein is expressed in nevi and melanoma primary tumors, to varying degrees, but is rarely expressed in malignant melanoma or other human tumors evaluated. Indeed, loss of MDA-7/IL-24 protein expression correlates strongly with melanoma tumor invasion and disease progression. The "bystander" effects proposed for MDA-7/IL-24 protein include immune stimulation, antiangiogenesis and receptor-mediated cytotoxicity. Thus, mda-7 is a unique multifunctional cytokine in the IL-10 family and may have potent antitumor utility in a clinical setting.
Asunto(s)
Citocinas/farmacología , Genes Supresores de Tumor/efectos de los fármacos , Interleucina-10/clasificación , Interleucinas/clasificación , Interleucinas/farmacología , Citocinas/clasificación , Citocinas/genética , Humanos , Interleucina-10/genética , Interleucina-10/farmacología , Interleucinas/genéticaRESUMEN
Interleukin-10 (IL-10) is considered to be an immunosuppressive cytokine. However, the continuous administration of pegylated IL-10 (PEG-IL10) leads to the rejection of large, firmly established and metastatic syngeneic tumors. PEG-IL10 therapy induces the expansion and activation of intratumoral, tumor antigen-specific CD8(+) T cells, leading to interferon γ (IFNγ)-mediated Th1 like immunity and tumor rejection.
RESUMEN
Successful cancer immunotherapy is thought to require de novo priming of tumor specific CD8(+) T cells in lymphatic organs. Contrasting these beliefs, cancer therapy based on interleukin-10 (IL-10) results in tumor rejection without a requirement for T-cell trafficking from lymphatic organs. Rather, IL-10 directly activates autochthonous, tumor-resident CD8(+) T cells.
RESUMEN
The presence of activated intratumoral T cells correlates clinically with better prognosis in patients with cancer. Although tumor vaccines can increase the number of tumor-specific CD8(+) T cells in systemic circulation, they frequently fail to increase the number of active and tumor reactive T cells within the tumor. Here we show that treatment with the pleiotropic cytokine interleukin-10 (IL-10) induces specific activation of tumor-resident CD8(+) T cells as well as their intratumoral expansion in several mouse tumor models. We found that inhibition of T-cell trafficking from lymphoid organs did not impair IL-10-induced tumor rejection or the activation of tumor-resident CD8(+) T cells. Tumor-resident CD8(+) T cells expressed elevated levels of the IL-10 receptor and were directly activated by IL-10, resulting in prominent phosphorylation of STAT3 and STAT1. Although CD4(+) T cells, regulatory T cells, NK cells, and dendritic cells have been reported as prominent targets of IL-10 in the tumor microenvironment, we found that expression of the IL-10R was required only on CD8(+) T cells to facilitate IL-10-induced tumor rejection as well as in situ expansion and proliferation of tumor-resident CD8 T cells. Together, our findings indicate that IL-10 activates CD8(+) T-cell-mediated tumor control and suggest that IL-10 may represent a potential tumor immunotherapy in human patients with cancer.
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Linfocitos T CD8-positivos/inmunología , Interleucina-10/farmacología , Activación de Linfocitos/inmunología , Neoplasias/inmunología , Animales , Femenino , Humanos , Sistema Linfático/inmunología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Interleucina-10/metabolismo , Linfocitos T Reguladores/inmunologíaRESUMEN
Tumor immune surveillance and cancer immunotherapies are thought to depend on the intratumoral infiltration of activated CD8(+) T cells. Intratumoral CD8(+) T cells are rare and lack activity. IL-10 is thought to contribute to the underlying immune suppressive microenvironment. Defying those expectations we demonstrate that IL-10 induces several essential mechanisms for effective antitumor immune surveillance: infiltration and activation of intratumoral tumor-specific cytotoxic CD8(+) T cells, expression of the Th1 cytokine interferon-γ (IFNγ) and granzymes in CD8(+) T cells, and intratumoral antigen presentation molecules. Consequently, tumor immune surveillance is weakened in mice deficient for IL-10 whereas transgenic overexpression of IL-10 protects mice from carcinogenesis. Treatment with pegylated IL-10 restores tumor-specific intratumoral CD8(+) T cell function and controls tumor growth.
Asunto(s)
Interferón gamma/metabolismo , Interleucina-10/metabolismo , Neoplasias Experimentales/inmunología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Granzimas/metabolismo , Humanos , Interferón gamma/genética , Interleucina-10/genética , Interleucina-10/inmunología , Subunidad alfa del Receptor de Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Perforina/metabolismo , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Bazo/metabolismo , Trasplante Heterólogo , Carga Tumoral , Escape del TumorRESUMEN
The melanoma differentiation-associated gene (mda-7; approved gene symbol IL24) is a tumor suppressor gene whose protein expression in normal cells is restricted to the immune system and to melanocytes. Recent studies have shown that mda-7 gene transfer inhibits cell growth and induces apoptosis in melanoma, lung cancer, breast cancer, and other tumor types through activation of various intracellular signaling pathways. In the current study, we demonstrate that Ad-mda7 transduction of human pancreatic cancer cells results in G2/M cell cycle arrest and cell killing. Cytotoxicity is mediated via apoptosis in a time- and dose-dependent manner. Tumor cell killing correlates with regulation of proteins involved in the Wnt and PI3K pathways: beta-catenin, APC, GSK-3, JNK, and PTEN. Additionally, we identify bystander cell killing activated by exposure of pancreatic tumor cells to secreted human MDA-7 protein. In pancreatic tumor cells, exogenous MDA-7 protein activates STAT3 and kills cells via engagement of IL-20 receptors. The specificity of bystander killing is demonstrated using neutralizing anti-MDA-7 antibodies and anti-receptor antibodies, which inhibit the apoptotic effects. In sum, we show that Ad-mda7 is able to induce growth inhibition and apoptosis in pancreatic cancer cells via inhibition of the Wnt/PI3K pathways and identify a novel bystander mechanism of MDA-7 killing in pancreatic cancer that functions via IL-20 receptors.
Asunto(s)
Efecto Espectador , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interleucinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Interleucina/metabolismo , Apoptosis , Proteínas del Citoesqueleto/metabolismo , Genes Supresores de Tumor , Vectores Genéticos/genética , Humanos , Interleucinas/genética , Neoplasias Pancreáticas/genética , Transducción de Señal , Factores de Tiempo , Transactivadores/metabolismo , Proteínas Wnt , beta CateninaRESUMEN
The melanoma differentiation-associated gene-7 (mda-7/IL24) is a unique member of the IL-10 family of cytokines, with ubiquitous tumor cell proapoptotic activity. Transduction of tumor or normal cells with the mda-7 gene results in secretion of glycosylated MDA-7 protein. Recent data indicate that secreted MDA-7 protein functions as a pro-Th1 cytokine and as a potent antiangiogenic molecule. MDA-7 protein binds two distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2 (type 1 IL-20R) and IL-22R1/IL-20R2 (type 2 IL-20R). In this study we analyzed the activity of glycosylated secreted MDA-7 against human melanoma cells. MDA-7 protein induces phosphorylation and nuclear translocation of STAT3 in melanoma cells via both type 1 and type 2 IL-20R. MDA-7 induces dose-dependent cell death in melanoma tumor cells. MDA-7 receptor engagement results in up-regulation of BAX and subsequent apoptosis induction; this effect is mediated by STAT3-independent signaling. Additional IL-10 family members (IL-10, -19, -20, and -22) also activate STAT3; however, these ligands do not activate death pathways in melanoma. In normal cells, MDA-7 can bind to its cognate receptors and induce phosphorylation of STAT3, without cytotoxic sequelae. This study defines a tumor-selective cytotoxic bystander role for secreted MDA-7 protein and identifies a novel receptor-mediated, STAT3-independent, and PKR-independent death pathway.
Asunto(s)
Adenoviridae/genética , Apoptosis/genética , Efecto Espectador/fisiología , Proteínas de Unión al ADN/metabolismo , Interleucinas/metabolismo , Melanoma/patología , Receptores de Interleucina/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Ciclo Celular , Línea Celular Tumoral , Genes Supresores de Tumor , Humanos , Interleucina-10/clasificación , Interleucina-10/farmacología , Interleucinas/química , Interleucinas/genética , Melanoma/metabolismo , Fosforilación , Factor de Transcripción STAT3 , eIF-2 Quinasa/metabolismoRESUMEN
The melanoma differentiation-associated gene 7 (mda-7) has been studied primarily in the context of its tumor suppressor activity. Although mda-7 has been designated as IL-24 based on its gene location in the IL-10 locus and its mRNA expression in leukocytes, no functional evidence supporting this cytokine designation exists. To further characterize MDA-7/IL-24 expression patterns in the human immune system, MDA-7/IL-24 protein levels were examined in human PBMC. MDA-7/IL-24 was detected in PHA- and LPS-stimulated whole PBMC lysate by Western blot and in PHA-activated CD56 and CD19 subsets by immunohistochemistry. The biological function of MDA-7/IL-24, secreted from Ad-MDA7-transfected HEK 293 cells, was assessed by examining the effect of MDA-7/IL-24 on the cytokine secretion profile of PBMC. Within 48 h MDA-7/IL-24 induced secretion of high levels of IL-6, TNF-alpha, and IFN-gamma and low levels of IL-1beta, IL-12, and GM-CSF from human PBMC as measured by ELISA. The MDA-7/IL-24-mediated induction of these Th1-type cytokines was inhibited by the addition of IL-10 to the PBMC cultures, suggesting that these two related protein family members may provide antagonistic functions. Therefore, because human blood leukocytes can be stimulated to produce MDA-7/IL-24, as well as respond to MDA-7/IL-24 by expressing secondary cytokines, MDA-7/IL-24 has the expression profile and major functional attributes that justify its designation as an IL.