RESUMEN
Primary T cell activation involves the integration of three distinct signals delivered in sequence: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing "bystander" T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4(+) T-cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy. This suppression targeted naive CD4(+) but not CD8(+) T cells and was mediated through transient suppressor of cytokine signaling-3 (SOCS3) inhibition of the STAT5b transcription factor signaling pathway. These events resulted in complete paralysis of primary CD4(+) T cell activation, affecting memory generation and induction of autoimmunity as well as impaired viral clearance. These data highlight the critical regulation of naive CD4(+) T cells during inflammatory conditions.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por Herpesviridae/terapia , Inmunoterapia/métodos , Melanoma/terapia , Muromegalovirus/inmunología , Neoplasias Cutáneas/terapia , Animales , Antígenos/inmunología , Diferenciación Celular/genética , Proliferación Celular/genética , Anergia Clonal , Femenino , Infecciones por Herpesviridae/inmunología , Humanos , Inmunidad Celular , Memoria Inmunológica , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-2/administración & dosificación , Melanoma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis por Micromatrices , Ensayos Clínicos Controlados Aleatorios como Asunto , Transducción de Señal , Neoplasias Cutáneas/inmunología , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Carga Viral/inmunologíaRESUMEN
OBJECTIVES: The relative contributions of inflammatory signalling and sequential oncogenic dysregulation driving liver cancer pathogenesis remain incompletely understood. Lymphotoxin-ß receptor (LTßR) signalling is critically involved in hepatitis and liver tumorigenesis. Therefore, we explored the interdependence of inflammatory lymphotoxin signalling and specific oncogenic pathways in the progression of hepatic cancer. DESIGN: Pathologically distinct liver tumours were initiated by hydrodynamic transfection of oncogenic V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT)/ß-catenin or AKT/Notch expressing plasmids. To investigate the relationship of LTßR signalling and specific oncogenic pathways, LTßR antagonist (LTßR-Fc) or agonist (anti-LTßR) were administered post oncogene transfection. Initiated livers/tumours were investigated for changes in oncogene expression, tumour proliferation, progression, latency and pathology. Moreover, specific LTßR-mediated molecular events were investigated in human liver cancer cell lines and through transcriptional analyses of samples from patients with intrahepatic cholangiocarcinoma (ICC). RESULTS: AKT/ß-catenin-transfected livers displayed increased expression of LTß and LTßR, with antagonism of LTßR signalling reducing tumour progression and enhancing survival. Conversely, enforced LTßR-activation of AKT/ß-catenin-initiated tumours induced robust increases in proliferation and progression of hepatic tumour phenotypes in an AKT-dependent manner. LTßR-activation also rapidly accelerated ICC progression initiated by AKT/Notch, but not Notch alone. Moreover, LTßR-accelerated development coincides with increases of Notch, Hes1, c-MYC, pAKT and ß-catenin. We further demonstrate LTßR signalling in human liver cancer cell lines to be a regulator of Notch, pAKTser473 and ß-catenin. Transcriptome analysis of samples from patients with ICC links increased LTßR network expression with poor patient survival, increased Notch1 expression and Notch and AKT/PI3K signalling. CONCLUSIONS: Our findings link LTßR and oncogenic AKT signalling in the development of ICC.
Asunto(s)
Carcinogénesis/metabolismo , Colangiocarcinoma , Neoplasias Hepáticas , Receptor beta de Linfotoxina/metabolismo , Linfotoxina beta/metabolismo , Transducción de Señal/fisiología , Animales , Proliferación Celular/fisiología , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Progresión de la Enfermedad , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Estadística como AsuntoRESUMEN
Immunosuppressive CD11b(+) Gr-1(+) myeloid-derived suppressor cells (MDSCs) accumulate in the livers of tumor-bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune-mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α-galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor-free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b(+) Gr-1(+) cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN-γ-dependent upregulation of CD40 on hepatic CD11b(+) Gr-1(+) cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor-induced CD11b(+) Gr-1(+) MDSCs as well as enhanced reactive oxygen species (ROS)-mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40(-/-) tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner.
Asunto(s)
Antígenos CD40/metabolismo , Hepatitis/inmunología , Células Mieloides/inmunología , Traslado Adoptivo , Alanina Transaminasa/sangre , Animales , Antígenos CD1d/biosíntesis , Arginasa/antagonistas & inhibidores , Arginasa/biosíntesis , Arginasa/metabolismo , Aspartato Aminotransferasas/sangre , Antígeno B7-1/biosíntesis , Antígeno B7-2/biosíntesis , Antígeno CD11b/metabolismo , Antígenos CD40/biosíntesis , Antígenos CD40/genética , Línea Celular , Concanavalina A/farmacología , Femenino , Galactosilceramidas/farmacología , Hepatitis/genética , Hepatocitos/inmunología , Hepatocitos/patología , Hígado/citología , Hígado/lesiones , Neoplasias Hepáticas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mitógenos/farmacología , Células Mieloides/trasplante , Especies Reactivas de Oxígeno/metabolismo , Receptores de Quimiocina/metabolismoRESUMEN
Fas ligand expression in certain tumors has been proposed to contribute to immunosuppression and poor prognosis. However, immunotherapeutic approaches may elicit the Fas-mediated elimination of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) within tumors that represent major obstacles for cancer immunotherapy. Previously, we showed that IL-2 and agonistic CD40 Ab (αCD40) elicited synergistic antitumor responses coincident with the efficient removal of Tregs and MDSCs. We demonstrate in this study in two murine tumor models that Treg and MDSC loss within the tumor microenvironment after IL-2/αCD40 occurs through a Fas-dependent cell death pathway. Among tumor-infiltrating leukocytes, CD8(+) T cells, neutrophils, and immature myeloid cells expressed Fas ligand after treatment. Fas was expressed by tumor-associated Tregs and immature myeloid cells, including MDSCs. Tregs and MDSCs in the tumor microenvironment expressed active caspases after IL-2/αCD40 therapy and, in contrast with effector T cells, Tregs significantly downregulated Bcl-2 expression. In contrast, Tregs and MDSCs proliferated and expanded in the spleen after treatment. Adoptive transfer of Fas-deficient Tregs or MDSCs into wild-type, Treg-, or MDSC-depleted hosts resulted in the persistence of Tregs or MDSCs and the loss of antitumor efficacy in response to IL-2/αCD40. These results demonstrate the importance of Fas-mediated Treg/MDSC removal for successful antitumor immunotherapy. Our results suggest that immunotherapeutic strategies that include exploiting Treg and MDSC susceptibility to Fas-mediated apoptosis hold promise for treatment of cancer.
Asunto(s)
Antineoplásicos/farmacología , Antígenos CD40/antagonistas & inhibidores , Interleucina-2/farmacología , Células Mieloides/inmunología , Neoplasias Experimentales/terapia , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Receptor fas/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/inmunología , Línea Celular Tumoral , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Células Mieloides/patología , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Linfocitos T Reguladores/patología , Microambiente Tumoral/genética , Receptor fas/genéticaRESUMEN
BACKGROUND & AIMS: Liver inflammatory diseases associated with cancer promoting somatic oncogene mutations are increasing in frequency. Preclinical cancer models that allow for the study of early tumor progression are often protracted, which limits the experimental study parameters due to time and expense. Here we report a robust inexpensive approach using Sleeping Beauty transposition (SBT) delivery of oncogenes along with Gaussia Luciferase expression vector GLuc, to assess de novo liver tumor progression, as well as the detection of innate immune responses or responses induced by therapeutic intervention. METHODS: Tracking de novo liver tumor progression with GLuc was demonstrated in models of hepatocellular carcinoma (HCC) or adenoma (HCA) initiated by hydrodynamic delivery of SBT oncogenes. RESULTS: Rising serum luciferase levels correlated directly with increasing liver tumor burden and eventual morbidity. Early detection of hepatocyte apoptosis from mice with MET+CAT transfected hepatocytes was associated with a transient delay in HCC growth mediated by a CD8(+) T-cell response against transformed hepatocytes. Furthermore, mice that lack B cells or macrophages had an increase in TUNEL(+) hepatocytes following liver MET transfection demonstrating that these cells provide protection from MET-induced hepatocyte apoptosis. Treatment with IL-18+IL-12 of mice displaying established HCC decreased tumor burden which was associated with decreased levels of serum luciferase. CONCLUSIONS: Hydrodynamic delivery of the SBT vector GLuc to hepatocytes serves as a simple blood-based approach for real-time tracking of pathologically distinct types of liver cancer. This revealed tumor-induced immunologic responses and was beneficial in monitoring the efficacy of therapeutic interventions.
Asunto(s)
Adenoma de Células Hepáticas/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/inmunología , Inmunidad Celular , Neoplasias Hepáticas Experimentales/inmunología , Luciferasas/sangre , Proteínas Recombinantes/uso terapéutico , Adenoma de Células Hepáticas/tratamiento farmacológico , Adenoma de Células Hepáticas/patología , Animales , Apoptosis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Progresión de la Enfermedad , Hepatocitos/patología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Interleucina-12/uso terapéutico , Interleucina-18/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/patología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8(+) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8(+) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8(+) T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8(+) CD25(-) cells within the tumor site. These findings demonstrate that memory CD8(+) T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.
Asunto(s)
Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/uso terapéutico , Inmunoterapia/métodos , Neoplasias/terapia , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Animales , Células Cultivadas , Método Doble Ciego , Humanos , Memoria Inmunológica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/inmunología , Placebos , Ensayos Clínicos Controlados Aleatorios como Asunto , Especificidad del Receptor de Antígeno de Linfocitos T/fisiología , Factores de TiempoRESUMEN
Protective cell-mediated immune responses in cancer are critically dependent on T-helper type 1 (T(H)1) cytokines such as interferon-gamma (IFN-gamma). We have previously shown that the combination of CD40 stimulation and interleukin-2 (IL-2) leads to synergistic antitumor responses in several models of advanced metastatic disease. We now report that after this treatment and other immunotherapy regimens, the CD4+ T-cell population, in contrast to CD8+ T cells, did not significantly increase but rather exhibited a substantial level of apoptosis that was dependent on IFN-gamma. Mice immunized with tumor cells and treated with an immunotherapy regimen that was initially protective were later unable to mount effective memory responses compared with immunized mice not receiving immunotherapy. Immunotherapy given to tumor-bearing Ifngr-/- mice resulted in restoration of secondary responses. Thus, although immunotherapeutic regimens inducing strong IFN-gamma responses can lead to successful early antitumor efficacy, they may also impair the development of durable antitumor responses.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Carcinoma de Células Renales/inmunología , Memoria Inmunológica , Inmunoterapia Activa , Interferón gamma/fisiología , Neoplasias Renales/inmunología , Melanoma Experimental/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/prevención & control , Línea Celular , Células Cultivadas , Supresión Clonal/genética , Supresión Clonal/inmunología , Femenino , Memoria Inmunológica/genética , Neoplasias Renales/patología , Neoplasias Renales/prevención & control , Melanoma Experimental/patología , Melanoma Experimental/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Mutant KRAS in lung cancers induces molecular pathways that regulate cellular proliferation, survival and inflammation, which enhance tumorigenesis. Inducible nitric oxide synthase (NOS2) upregulation and sustained nitric oxide generation are induced during the inflammatory response and correlate positively with lung tumorigenesis. To explore the mechanistic contribution of NOS2 to KRAS-induced lung tumorigenesis and inflammation, we used a genetic strategy of crossing NOS2 knockout (NOS2KO) C57BL6 inbred mice with a KRAS(G12D)-driven mouse lung cancer model. KRAS(G12D);NOS2KO mice exhibited delayed lung tumorigenesis and a longer overall survival time compared to that of KRAS(G12D);NOS2WT (wild-type) controls. Correspondingly, tumors in KRAS(G12D);NOS2KO mice had reduced tumor cell proliferation in adenomas and carcinomas. NOS2 deficiency also led to markedly suppressed inflammatory response by attenuation of macrophage recruitment into alveoli and within tumor foci. In contrast, FOXP3+ regulatory T cells were increased in tumors from KRAS(G12D) ;NOS2KO mice. We further analyzed the expression of microRNA-21 (miR-21), an oncogenic noncoding RNA involved in oncogenic Ras signaling, by quantitative reverse-transcription polymerase chain reaction and in situ hybridization. Lung carcinomas dissected from KRAS(G12D);NOS2KO mice showed a significantly reduced miR-21 expression along with decreased tumor cell proliferation, suggesting that NOS2 deficiency could attenuate RAS signaling pathways that transactivate miR-21 expression. Therefore, deletion of NOS2 decreases lung tumor growth as well as inflammatory responses initiated by oncogenic KRAS, suggesting that both KRAS and NOS2 cooperate in driving lung tumorigenesis and inflammation. Inhibition of NOS2 may have a therapeutic value in lung cancers with oncogenic KRAS mutations.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , MicroARNs/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Procesos de Crecimiento Celular/fisiología , Transformación Celular Neoplásica/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Neoplasias Pulmonares/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Mutación/genética , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Linfocitos T Reguladores/metabolismoRESUMEN
Sustained hepatic inflammation, driven by alcohol consumption, nonalcoholic fatty liver disease, and/or chronic viral hepatitis (hepatitis B and C), results in damage to parenchyma, oxidative stress, and compensatory regeneration/proliferation. There is substantial evidence linking these inflammation-associated events with the increased incidence of hepatocellular carcinogenesis. Although acute liver inflammation can play a vital and beneficial role in response to liver damage or acute infection, the effects of chronic liver inflammation, including liver fibrosis and cirrhosis, are sufficient in a fraction of individuals to initiate the process of transformation and the development of hepatocellular carcinoma. This review highlights immune-dependent mechanisms that may be associated with hepatocellular oncogenesis, including critical transformative events/pathways in the context of chronic inflammation and subverted tolerogenesis.
Asunto(s)
Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/inmunología , Hepatitis Viral Humana/patología , Neoplasias Hepáticas/patología , Carcinoma Hepatocelular/inmunología , Transformación Celular Neoplásica/patología , Hígado Graso/inmunología , Hígado Graso/patología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/patología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/patología , Hepatitis Crónica/inmunología , Hepatitis Crónica/patología , Hepatitis Viral Humana/inmunología , Humanos , Cirrosis Hepática/inmunología , Cirrosis Hepática/patología , Neoplasias Hepáticas/inmunología , Enfermedad del Hígado Graso no Alcohólico , Estrés Oxidativo/inmunología , Estrés Oxidativo/fisiología , Lesiones Precancerosas/patología , PronósticoRESUMEN
The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.
Asunto(s)
Diferenciación Celular/inmunología , Hígado/inmunología , Hígado/metabolismo , Activación de Linfocitos/inmunología , Depleción Linfocítica , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Animales , Caspasa 3/metabolismo , Galactosilceramidas/fisiología , Inmunofenotipificación , Interleucina-12/fisiología , Interleucina-18/fisiología , Hígado/citología , Depleción Linfocítica/métodos , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
A model of immune evasion mediated by tumors expressing constitutively activated Stat3 was recently proposed in Nature Medicine by Wang et al., suggesting opportunities for a new therapeutic approach for cancer.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Dendríticas/inmunología , Genes MHC Clase II/fisiología , Linfocitos T/inmunología , Transactivadores/metabolismo , División Celular , Quimiocina CCL5/inmunología , Quimiocina CCL5/metabolismo , Células Dendríticas/metabolismo , Regulación Neoplásica de la Expresión Génica/inmunología , Regulación Neoplásica de la Expresión Génica/fisiología , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Modelos Moleculares , Neoplasias/inmunología , Neoplasias/metabolismo , Fosfotirosina , Factor de Transcripción STAT3 , Transducción de Señal , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Treatment of mice bearing orthotopic, metastatic tumors with anti-CD40 antibody resulted in only partial, transient anti-tumor effects whereas combined treatment with IL-2/anti-CD40, induced tumor regression. The mechanisms for these divergent anti-tumor responses were examined by profiling tumor-infiltrating leukocyte subsets and chemokine expression within the tumor microenvironment after immunotherapy. IL-2/anti-CD40, but not anti-CD40 alone, induced significant infiltration of established tumors by NK and CD8(+) T cells. To further define the role of chemokines in leukocyte recruitment into tumors, we evaluated anti-tumor responses in mice lacking the chemokine receptor, CCR2. The anti-tumor effects and leukocyte recruitment mediated by anti-CD40 alone, were completely abolished in CCR2(-/-) mice. In contrast, IL-2/anti-CD40-mediated leukocyte recruitment and reductions in primary tumors and metastases were maintained in CCR2(-/-) mice. Treatment of mice with IL-2/anti-CD40, but not anti-CD40 alone, also caused an IFN-gamma-dependent increase in the expression of multiple Th1 chemokines within the tumor microenvironment. Interestingly, although IL-2/anti-CD40 treatment increased Tregs in the spleen, it also caused a coincident IFN-gamma-dependent reduction in CD4(+)/FoxP3(+) Tregs, myeloid-derived suppressor cells and Th2 chemokine expression specifically within the tumor microenvironment that was not observed after treatment with anti-CD40 alone. Similar effects were observed using IL-15 in combination with anti-CD40. Taken together, our data demonstrate that IL-2/anti-CD40, but not anti-CD40 alone, can preferentially reduce the overall immunosuppressive milieu within the tumor microenvironment. These results suggest that the use of anti-CD40 in combination with IL-2 or IL-15 may hold substantially more promise for clinical cancer treatment than anti-CD40 alone.
Asunto(s)
Anticuerpos/uso terapéutico , Antígenos CD40/agonistas , Terapia de Inmunosupresión/métodos , Interleucina-2/uso terapéutico , Neoplasias/terapia , Animales , Arginasa/biosíntesis , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Quimiocina CCL5/biosíntesis , Quimiocina CXCL9/biosíntesis , Quimiocinas/biosíntesis , Quimiocinas CC/biosíntesis , Sinergismo Farmacológico , Linfocitos Infiltrantes de Tumor/inmunología , Proteínas Inflamatorias de Macrófagos/biosíntesis , Ratones , Ratones Endogámicos BALB C , Neoplasias/inmunología , Receptores CCR2/biosíntesis , Receptores CCR2/genética , Receptores de Citocinas/biosíntesisRESUMEN
Immune deficiency immediately following bone marrow transplantation (BMT) increases susceptibility to opportunistic infections as well as tumor relapse. Natural Killer (NK) cells play important roles in the resistance to virally infected and transformed cells. Interleukin (IL)-15 has been shown to be essential for NK cell development and survival. We administered human (h) IL-15 cDNA (pIL-15) via hydrodynamic delivery to murine recipients undergoing congenic BMT to determine its effects on NK cell reconstitution. Hydrodynamic pIL-15 delivery resulted in high levels of hIL-15 protein in the serum that lasted for several days and then quickly declined. The appearance of hIL-15 was followed by a significant increase of mature donor-derived NK cells within the bone marrow, spleens, and livers of the treated recipients. No accumulation of immature NK cell progenitors was observed. The NK cells from IL-15-treated recipients displayed an activated phenotype and were lytically active toward tumor targets in vitro to a similar degree as did those cells from recipients treated with control plasmid. This suggests that the predominant effect of IL-15 was a quantitative increase in total NK cell numbers and not qualitative changes in NK cell functions. No toxicities or adverse effects were observed. Studies performed in transplanted mice bearing renal carcinoma tumors demonstrated that this mode of hIL-15 gene delivery resulted in increased antitumor responses. These results support the use of cytokine gene transfer-based regimens as a platform to augment NK cell recovery after BMT.
Asunto(s)
Trasplante de Médula Ósea/métodos , ADN Complementario/administración & dosificación , Interleucina-15/genética , Células Asesinas Naturales/inmunología , Animales , Trasplante de Médula Ósea/inmunología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/terapia , ADN Complementario/genética , ADN Complementario/inmunología , Modelos Animales de Enfermedad , Femenino , Terapia Genética/métodos , Humanos , Interleucina-15/biosíntesis , Interleucina-15/sangre , Interleucina-15/inmunología , Neoplasias Renales/genética , Neoplasias Renales/inmunología , Neoplasias Renales/terapia , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Previously, we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3(+) cells present in CD25(high), CD25(low) and even CD25(-) subsets of CD4(+) cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4(+)CD25(+) Treg included all of the FOXP3(+) cells present in the CD4(+)CD25(high) subset as well as a substantial proportion of the FOXP3(+) cells present in the CD4(+)CD25(low) subset. Flow cytometric analysis of PB identified five-fold more Treg, determined by FOXP3 expression, in the CD4(+)CD25(+)TNFR2(+) subset than in the CD4(+)CD25(high) subset. In addition, similar levels of FOXP3(+) cells were identified in both the CD4(+)CD25(+)TNFR2(+) and CD4(+)CD25(+)CD127(low/-) subsets. Furthermore, the CD4(+)CD25(+)TNFR2(+) subset expressed high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4(+)CD25(+)TNFR2(+) cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T-responder cells. In contrast, CD4(+)CD25(+)TNFR2(-) and CD4(+)CD25(-)TNFR2(+) T cells did not show inhibitory activity. As some non-Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.
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Factores de Transcripción Forkhead/análisis , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Receptores Tipo II del Factor de Necrosis Tumoral/biosíntesis , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/clasificación , Adulto , Presentación de Antígeno , Antígenos CD/análisis , Antígeno CTLA-4 , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Técnicas de Cocultivo , Citometría de Flujo , Humanos , Inmunofenotipificación , Interferón gamma/biosíntesis , Subunidad alfa del Receptor de Interleucina-2/análisis , Subunidad alfa del Receptor de Interleucina-7/análisis , Antígenos Comunes de Leucocito/análisis , Activación de Linfocitos/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/análisis , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Natural killer (NK) and NKT cells are a first line of defense against pathogens and transformed cells. However, dysregulation of their function can lead to autoimmune disease. A better understanding of the mechanisms controlling NK and NKT effector function should lead to the development of improved strategies for the treatment of many diseases. The site in which NK and NKT cells reside should be taken into account, because accumulating evidence suggests that the tissue microenvironment strongly influences their function. In this regard, the liver represents a unique immunologic organ in which the balance between the need for tolerance and the ability to respond rapidly to pathogens and tissue injury is tightly regulated. NK cells in the liver have augmented cytolytic activity as compared to other organs, which is consistent with a role for liver-associated NK cells in being critical effector cells for inhibiting tumor metastasis in the liver. Several studies also suggest that hepatic NKT cells have different functions than those in other organs. Whereas splenic and thymic NKT cells have been shown to suppress diabetes development, facilitate the induction of systemic tolerance and are regulated by IL-4 and other Th2 cytokines, certain subsets of NKT cells in the liver are important sources of Th1 cytokines such as Interferon gamma, and are the primary mediators of anti-tumor responses. The unique properties and roles as critical effector cells make NK and NKT cells within the liver microenvironment attractive targets of immunotherapeutic approaches that have the goal of controlling tumor metastasis in the liver.
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Citotoxicidad Inmunológica , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Neoplasias Hepáticas/terapia , Hígado/inmunología , Células T Asesinas Naturales/inmunología , Animales , Citocinas/inmunología , Humanos , Vigilancia Inmunológica , Hígado/patología , Neoplasias Hepáticas/secundario , Metástasis de la NeoplasiaRESUMEN
The use of interleukin-18 (IL-18) together with IL-12 induced high levels of IFN-gamma in tumor-bearing mice and regression of liver tumors that was abolished in IFN-gamma((-/-)) mice. Natural killer (NK) and NKT cells were the major producers of IFN-gamma in the livers of mice treated with IL-18 and/or IL-12. Liver NK cells were significantly increased by treatment with IL-18/IL-12, whereas the degree of liver NKT cell TCR detection was diminished by this treatment. Reduction of NK cells with anti-asGM1 decreased the antitumor activity of IL-18/IL-12 therapy and revealed NK cells to be an important component for tumor regression in the liver. In contrast, the antitumor effects of both IL-18 and IL-12 were further increased in CD1d((-/-)) mice, which lack NKT cells. Our data, therefore, show that the antitumor activity induced in mice by IL-18/IL-12 is NK and IFN-gamma dependent and is able to overcome an endogenous immunosuppressive effect of NKT cells in the liver microenvironment. These results suggest that immunotherapeutic approaches that enhance NK cell function while eliminating or altering NKT cells could be effective in the treatment of cancer in the liver.
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Interleucina-12/farmacología , Interleucina-18/farmacología , Células Asesinas Naturales/inmunología , Hígado/inmunología , Linfocitos T/inmunología , Animales , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Células Asesinas Naturales/efectos de los fármacos , Hígado/citología , Hígado/efectos de los fármacos , Neoplasias Hepáticas Experimentales/inmunología , Neoplasias Hepáticas Experimentales/secundario , Neoplasias Hepáticas Experimentales/terapia , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacosRESUMEN
Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are clinically disparate primary liver cancers with etiological and biological heterogeneity. We identified common molecular subtypes linked to similar prognosis among 199 Thai ICC and HCC patients through systems integration of genomics, transcriptomics, and metabolomics. While ICC and HCC share recurrently mutated genes, including TP53, ARID1A, and ARID2, mitotic checkpoint anomalies distinguish the C1 subtype with key drivers PLK1 and ECT2, whereas the C2 subtype is linked to obesity, T cell infiltration, and bile acid metabolism. These molecular subtypes are found in 582 Asian, but less so in 265 Caucasian patients. Thus, Asian ICC and HCC, while clinically treated as separate entities, share common molecular subtypes with similar actionable drivers to improve precision therapy.
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Pueblo Asiatico/genética , Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/diagnóstico , Colangiocarcinoma/diagnóstico , Análisis por Conglomerados , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/diagnóstico , Pronóstico , TranscriptomaRESUMEN
Recent reports have suggested that the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may partially limit the formation of hepatic metastases of a variety of mouse tumors, and the major source of TRAIL in the liver was shown to be local natural killer cells. We isolated a clone (R331) of the murine renal cancer cell line Renca that was strikingly more sensitive to both Fas and TRAIL death receptor-mediated apoptosis in vitro. R331 grew in tissue culture in vitro at a rate similar to that of the parental Renca cell line but formed larger and more numerous colonies than parental Renca in soft agar. After s.c. implantation, R331 tumors progressed more rapidly than parental Renca tumors. However, R331 formed far fewer lung and liver metastases in wild-type (WT) BALB/c mice. Administration of antibodies that neutralized TRAIL dramatically increased the number of R331 liver metastases. Furthermore, numbers of R331 liver metastases were much greater in TRAIL(-/-) than in WT BALB/c mice. In contrast, no difference was seen in numbers of lung metastases when comparing TRAIL(-/-) and WT mice, suggesting that the antitumor effects of TRAIL in vivo were compartment specific. Transfection of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein into R331 increased the numbers of liver metastases in BALB/c mice by up to 10-fold, indicating that local TRAIL in the liver was directly mediating tumor cell apoptosis. These organ-specific differences in the endogenous levels of death ligands may apply different selective pressures on the development of liver or lung metastases. Consequently, the efficacy of TRAIL therapy may vary depending on the location of the tumor metastases.
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Apoptosis/fisiología , Carcinoma de Células Renales/secundario , Inmunidad Innata/fisiología , Neoplasias Renales/patología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Glicoproteínas de Membrana/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Carcinoma de Células Renales/patología , Caspasa 3 , Caspasas/metabolismo , División Celular , Citometría de Flujo , Inmunidad Innata/genética , Neoplasias Hepáticas/patología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Inflammation influences the development of cancer. The nitric oxide synthase (NOS2) is induced by inflammatory cytokines, e.g., tumor necrosis factor alpha and interleukin 1beta, and produces nitric oxide (NO*), a critical mediator of the inflammatory response. Because p53 governs NO* production by transcriptionally transrepressing NOS2, we used a genetic strategy to determine whether NO* and p53 cooperatively regulate tumorigenesis. Lymphomas developed more rapidly in p53-/-NOS2-/- or p53-/-NOS2+/- mice than in p53-/-NOS2+/+ mice that were cross-bred into a >95% C57BL6 background and maintained in a pathogen-free condition. Likewise, sarcomas and lymphomas developed faster in p53+/-NOS2-/- or p53+/-NOS2+/- than in p53+/-NOS2+/+ mice. When compared with the double knockout mice, p53-/-NOS2+/+ mice showed a higher apoptotic index and a decreased proliferation index with an increased expression of death receptor ligands, CD95-L and tumor necrosis factor-related apoptosis-inducing ligand, and the cell cycle checkpoint protein, p21(waf1), in the spleen and thymus before tumor development. Furthermore, mice deficient in both p53 and NOS2 produced a high level of anti-inflammatory interleukin 10 when compared with p53-deficient mice. These studies provide genetic and mechanistic evidence that NO* can suppress tumorigenesis.
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Mediadores de Inflamación/metabolismo , Linfoma de Células T/metabolismo , Óxido Nítrico/metabolismo , Sarcoma Experimental/metabolismo , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Modelos Animales de Enfermedad , Proteína Ligando Fas , Femenino , Endogamia , Interleucina-10/biosíntesis , Antígeno Ki-67/biosíntesis , Linfoma de Células T/enzimología , Linfoma de Células T/patología , Masculino , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Sarcoma Experimental/enzimología , Sarcoma Experimental/patología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/biosíntesis , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Chemokines and chemokine receptors have critical roles in cancer metastasis and have emerged as one of the targeting options in cancer therapy. However, the treatment efficacy on both tumor and host compartments needs to be carefully evaluated. Here we report that targeting CXCR3 decreased tumor cell migration and at the same time improved host anti-tumor immunity. We observed an increased expression of CXCR3 in metastatic tumor cells compared to those from non-metastatic tumor cells. Knockdown (KD) of CXCR3 in metastatic tumor cells suppressed tumor cell migration and metastasis. Importantly, CXCR3 expression in clinical breast cancer samples correlated with progression and metastasis. For the host compartment, deletion of CXCR3 in all host cells in 4T1 mammary tumor model significantly decreased metastasis. The underlying mechanisms involve a decreased expression of IL-4, IL-10, iNOs, and Arg-1 in myeloid cells and an increased T cell response. IFN-γ neutralization diminished the metastasis inhibition in the CXCR3 knockout (KO) mice bearing 4T1 tumors, suggesting a critical role of host CXCR3 in immune suppression. Consistently, targeting CXCR3 using a small molecular inhibitor (AMG487) significantly suppressed metastasis and improved host anti-tumor immunity. Our findings demonstrate that targeting CXCR3 is effective in both tumor and host compartments, and suggest that CXCR3 inhibition is likely to avoid adverse effects on host cells.