RESUMEN
BACKGROUND: Lymphopenia promotes naïve T-cell homeostatic proliferation and adoptive effector T-cell survival and memory formation. IL-7 plays a critical role in homeostatic proliferation, survival and memory formation of naïve T-cells in lymphopenia, and its underlying molecular mechanism has also been well studied. However, the mechanism for adoptively transferred effector T-cell survival and memory formation is not fully understood. Here, we transferred in vitro-activated transgenic OT-I CD8(+) effector T-cells into irradiation (600 rads)-induced lymphopenic C57BL/6, IL-7 knockout (KO) and IL-15 KO mice, and investigated the survival and memory formation of transferred T-cells in lymphopenia. RESULTS: We demonstrate that transferred T-cells prolong their survival and enhance their memory in lymphopenic mice, in a manner that depends on IL-15 signaling, but not IL-7. We determine that in vitro stimulation of naïve or effector T-cells with IL-7 and IL-15 reduces IL-7Rα, and increases and/or maintains IL-15Rß expression, respectively. Consistent with these findings, the expression of IL-7Rα and IL-15Rß is down- and up-regulated, respectively, in vivo on transferred T-cells in an early phase post T-cell transfer in lymphopenia. We further show that in vitro IL-15 restimulation-induced memory T-cells (compared to IL-2 restimulation-induced effector T-cells) and in vivo transferred T-cells in irradiated IL-15-sufficient C57BL/6 mice (compared to IL-15-deficient IL-15 KO mice) have increased mitochondrial content, but less NADH and lower mitochondrial potential (ΔΨm), and demonstrate greater phosphorylation of signal transducers and activators of transcription-5 (STAT5) and Unc-51-like kinase-1 (ULK1), and higher expression of B-cell leukemia/lymphoma-2 (Bcl2) and memory-, autophagy- and mitochondrial biogenesis-related molecules. CONCLUSION: Irradiation-induced lymphopenia promotes effector T-cell survival via IL-15 signaling the STAT5/Bcl2 pathway, enhances T-cell memory formation via IL-15 activation of the forkhead-box family of transcription factor (FOXO)/eomesodermin (Eomes) memory and ULK1/autophagy-related gene-7 (ATG7) autophagy pathways, and via IL-15 activation of the mitochondrial remodeling. Our data thus identify some important targets to consider when designing potent adoptive T-cell immunotherapies of cancer.
RESUMEN
LFA-1 signaling is required for the generation of central memory CD8(+) T cells in priming phase. However, its role for effector and memory CD8(+) T cell survival in transition and maintenance phases is elusive. We transferred effector and memory CD8(+) T cells into C57BL/6 and CD54(-/-) mice, which were generated by cultivation of ovalbumin (OVA)-pulsed dendritic cells (DC(OVA)) with naïve CD8(+) T cells derived from transgenic OT I mice and purified from effector CD8(+) T cell-transferred C57BL/6 mice, respectively. We then assessed kinetics of T cell survival using PE-H2-K(b)/OVAI tetramer and FITC-CD8 staining by flow cytometry. We found that survival of transferred effector and memory CD8(+) T cells in CD54(-/-) mice significantly decreased (p<0.05) compared to that in C57BL/6 mice due to an increased T cell apoptosis, which is mediated via downregulation of proapoptotic Bid, anti-apoptotic Bcl-2, Bcl-X(L) and pro-Caspase-8, and up-regulation of apoptotic Bax and cleaved Caspase-3 and -7 by RNA array and Western blotting analyses. Decreased expression of CD27 and IL-15R on transferred CD8(+) T cells with less survival was found to be associated with increased T cell apoptosis, which was confirmed by silencing CD27 with siRNA transfection or using CD8(+) (IL-15R(-/-))T cells for adoptive transfer into C57BL/6 mice. These data indicate that LFA-1 signal defect-induced CD8(+) T cell apoptosis is associated with reduced CD27 costimulation and IL-15R survival signal. Therefore, our study provides important evidence on and elucidates the molecular mechanism associated with the role LFA-1 signaling plays in effector and memory CD8(+) T cell survival.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Caspasas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Interleucina-15/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Traslado Adoptivo , Animales , Apoptosis/inmunología , Apoptosis/fisiología , Secuencia de Bases , Linfocitos T CD8-positivos/citología , Células Dendríticas/inmunología , Regulación hacia Abajo , Femenino , Memoria Inmunológica , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Inmunológicos , ARN Interferente Pequeño/genética , Receptores de Interleucina-15/deficiencia , Receptores de Interleucina-15/genética , Transducción de Señal , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidores , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
Previous studies have shown that interleukin-17F (IL-17F) can markedly inhibit the angiogenesis of endothelial cells, implying that it may play a role in antiangiogenic therapy for tumors. To explore its effect on antiangiogenic therapy for hepatocellular carcinoma (HCC), we constructed a recombinant retrovirus vector RV-IL-17F expressing IL-17F, transfected SMMC-7721 human hepatocarcinoma cells with RV-IL-17F, and investigated the effect of transgene IL-17F expression on human hepatocarcinoma cells in vitro and in vivo in animal model. We demonstrated that IL-17F expression exerted no direct effect on in vitro proliferation and cell cycle of SMMC-7721 hepatocarcinoma cells, while it downregulated IL-6, IL-8, and VEGF expression in SMMC-7721 cells at both protein and mRNA levels and IL-17F-expressing supernatant from SMMC-7721/RV-IL-17F directly inhibited ECV304 vascular endothelial cell growth. Moreover, SMMC-7721/RV-IL-17F exhibited a significant decrease in tumor size and microvessel density as compared to the SMMC-7721/RV control when transplanted in nude mice. This retarded tumor growth in vivo elicited by IL-17F was associated with direct suppression of vascular endothelial cells and reduced expression of proangiogenic factors IL-6, IL-8, and VEGF leading to the inhibition of tumor angiogenesis. Thus, our results indicate that IL-17F, a novel antiangiogenic factor, may be useful in antiangiogenic therapy for HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proliferación Celular , Interleucina-17/metabolismo , Neoplasias Hepáticas/metabolismo , Neovascularización Patológica/prevención & control , Animales , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Ciclo Celular , Línea Celular , Regulación hacia Abajo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-17/genética , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Microvasos/metabolismo , Microvasos/patología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , ARN Mensajero/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Previous studies have demonstrated that interleukin-24 [IL-24; originally called melanoma differentiation associated gene-7 (mda-7)] as a novel tumor suppressor gene has tumor-suppressive activity against a broad spectrum of human cancers. However, the therapeutic effect of the recombinant human IL-24 (rhIL-24) protein purified from prokaryotic cells on gastric cancer has not been reported. In this study, we purified soluble rhIL-24 using Q-Sepharose column after the denaturing and renaturing process from the protein of Escherichia coli BL21 transfected with pET-21a(+)-hIL-24 vector and treated by isopropyl-beta-D-1-thiogalactopyranoside (IPTG) for enhanced expression of transgene rhIL-24. We demonstrated that rhIL-24 was capable of inducing in vitro apoptosis of SGC7901 gastric cancer cells and activating peripheral blood mononuclear cellsto secrete cytokines such as IL-6, TNF-alpha, and IFN-gamma. We also showed that rhIL-24 was able to inhibit formation of blood capillaries on chicken embryonic allantois and in vivo tumor angiogenesis leading to suppressing SGC7901 gastric cancer cell growth in vitro and in vivo possibly due to its downregulation of Bcl-2/Bax ratio, VEGF (vascular endothelial growth factor), and CD34. Therefore, our results indicate that rhIL-24 has potent suppressive effect on human SGC7901 gastric carcinoma cell line and warrant its further investigation for therapeutic application against gastric cancer.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma/tratamiento farmacológico , Interleucinas/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Animales , Antígenos CD34/metabolismo , Apoptosis/efectos de los fármacos , Carcinoma/irrigación sanguínea , Carcinoma/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Clonación Molecular , Citocinas/metabolismo , Humanos , Interleucinas/genética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Ratones Desnudos , Neovascularización Patológica/patología , Neovascularización Patológica/prevención & control , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Recombinantes/farmacología , Neoplasias Gástricas/irrigación sanguínea , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Recent studies have demonstrated that ING4, as a novel member of the ING (inhibitor of growth) family, has a potential effect on tumor inhibition via multiple pathways. However, adenovirus-mediated ING4 expression in the application of gene therapy for pancreatic carcinoma has not been reported. To explore its therapeutic effect on human pancreatic carcinoma, we constructed a recombinant adenoviral vector, Ad-ING4, expressing the green fluorescent protein (GFP) marker gene and the tumor-suppressor gene, humanized ING4 derived from murine ING4 with two amino-acid modifications at residues 66 (Arg to Lys) and 156 (Ala to Thr) by site-directed mutagenesis. We demonstrated that Ad-ING4-mediated transfection of PANC-1 human pancreatic carcinoma cells inhibited cell growth, altered the cell cycle with S-phase reduction and G2/M phase arrest, induced apoptosis, and downregulated interleukin (IL)-6 and IL-8 expression of transfected tumor cells. In athymic mice bearing the PANC-1 human pancreatic tumors, intratumoral injections of Ad-ING4 suppressed the tumor growth, downregulated CD34 expression, and reduced the tumor microvessel formation. Therefore, this study will provide a framework for future clinical application of Ad-ING4 in human pancreatic carcinoma gene therapy.
Asunto(s)
Apoptosis/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Homeodominio/genética , Neoplasias Pancreáticas/terapia , Proteínas Supresoras de Tumor/genética , Adenoviridae/genética , Animales , Antígenos CD34/biosíntesis , Proteínas Portadoras/biosíntesis , Ciclo Celular/genética , Proteínas de Ciclo Celular/biosíntesis , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica , Proteínas de Homeodominio/biosíntesis , Humanos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Mutagénesis Sitio-Dirigida , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neovascularización Patológica/terapia , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección/métodos , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
CD4+ helper T (Th) cells play crucial role in priming, expansion and survival of CD8+ cytotoxic T lymphocytes (CTLs). However, how CD4+ Th cell's help is delivered to CD8+ T cells in vivo is still unclear. We previously demonstrated that CD4+ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DC(OVA)) stimulation, and then stimulate OVA-specific CD8+ CTL responses in C57BL/6 mice. In this study, we further investigated CD4+ Th cell's effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4+ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DC(OVA)-activated CD4+ Th cells (3 x 10(6) cells/mouse) can directly stimulate OVA-specific CD8+ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4+ T cells. A large amount of CD4+ Th cells (12 x 10(6) cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8+ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4+ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4+ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Diabetes Mellitus/inmunología , Islotes Pancreáticos/inmunología , Activación de Linfocitos , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Autoinmunidad , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Células Dendríticas/metabolismo , Genes MHC Clase II , Antígenos de Histocompatibilidad Clase II/inmunología , Tolerancia Inmunológica , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Interleukin-24 (IL-24) has been shown to be a tumor-suppressor gene and the protein product found to be constitutively expressed by melanocytes, nerve cells, and some primary melanomas. The potential effect of adenovirus (AdV)-mediated IL-24 gene therapy was explored on human pancreatic carcinoma by using a pancreatic carcinoma cell line, patu8988. A recombinant adenovirus, AdVGFP/IL-24, expressing the marker, green fluorescent protein (GFP), and the tumor-suppressor gene, IL-24, was constructed. AdVGFP/IL-24 treatment of pancreatic carcinoma cells in vitro significantly induced pancreatic carcinoma cell cytotoxicity and apoptosis, compared with AdVGFP without IL-24 expression. In nude mice bearing patu8988 tumors, intratumoral injections of AdVGFP/IL-24 significantly inhibited pancreatic carcinoma growth. In addition, the molecular mechanism of tumor suppression was elucidated by downregulating the expression of vascular endothelial growth factor, CD34, and Bcl-2, as well as inhibiting tumor angiogenesis. Therefore, AdVGFP/IL-24 has the potential to serve as a novel tool for pancreatic carcinoma gene therapy.
Asunto(s)
Adenoviridae/genética , Terapia Genética/métodos , Interleucinas/genética , Neoplasias Pancreáticas/terapia , Animales , Antígenos CD34/metabolismo , Apoptosis/genética , Apoptosis/fisiología , Línea Celular Tumoral , Proliferación Celular , Expresión Génica , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Interleucinas/metabolismo , Interleucinas/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Previous studies demonstrated that ING4 as a novel member of ING (inhibitor of growth) family has potential effect on tumor inhibition via multiple pathways. However, adenovirus-mediated ING4 expression in inhibition of human tumors has not been reported. To explore its therapeutic effect on human lung carcinoma, we constructed a recombinant adenoviral vector Ad-ING4 expressing the humanized ING4 gene derived from murine ING4 with two amino acid modifications at residue 66 (Arg to Lys) and 156 (Ala to Thr) by site-directed mutagenesis. We demonstrated that Ad-ING4-mediated transfection of A549 human lung carcinoma cells induced cell apoptosis, altered cell cycle with S phase reduction and G2/M phase arrest, suppressed cell invasiveness, and down-regulated IL-6, IL-8, MMP-2, and MMP-9 expression of transfected tumor cells. In athymic mice bearing A549 lung tumors, intratumoral injections of Ad-ING4 suppressed the tumor growth and reduced the tumor microvessel formation. Therefore, Ad-ING4 may be useful in gene therapy of human lung carcinoma.
Asunto(s)
Adenoviridae/fisiología , Apoptosis/fisiología , Proteínas de Ciclo Celular/genética , Ciclo Celular/fisiología , División Celular/fisiología , Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Neoplasias Pulmonares/patología , Invasividad Neoplásica , Neovascularización Patológica , Proteínas Supresoras de Tumor/genética , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Neoplasias Pulmonares/irrigación sanguínea , Ratones , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previous studies have shown that interleukin-24 (IL-24; mda-7) as a novel tumor suppressor gene has tumor-suppressive activity against a broad spectrum of human cancers. However, the therapeutic effect of the recombinant human IL-24 (rhIL-24) protein purified from prokaryotic cells on human lung cancers has not been reported. In this study, we cloned the human gene coding for IL-24 from lipopolysaccharide-activated human peripheral blood mononuclear cells (PBMCs) by reverse-transcriptase polymerase chain reaction and constructed an expression vector pBV220-IL-24. We then transfected Escherichia coli DH5alpha with pBV220-IL-24. The soluble rhIL-24 was obtained from purified insoluble inclusion bodies of transfected cells by a denaturing and renaturing process. We demonstrated that the purified soluble rhIL-24 protein with 18.5 kappaDa was capable of (1) inducing in vitro apoptosis of A549 lung carcinoma cells; (2) activating PBMCs to secrete cytokines such as IL-6, tumor necrosis factor-alpha, and interferon-gamma; (3) inhibiting the formation of blood capillaries on chicken embryonic allantois and in vivo tumor angiogenesis; and (4) inhibiting A549 lung tumor cell growth in vitro and in vivo. Therefore, our results indicate its potent suppressive effect on human lung carcinoma cell line and warrant its further investigation for therapeutic application against human lung cancer.
Asunto(s)
Apoptosis , Carcinoma/tratamiento farmacológico , Interleucinas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neovascularización Patológica , Proteínas Recombinantes/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular , Embrión de Pollo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/metabolismo , RatonesRESUMEN
IL-12 priming plays an important role in stimulation of CD8+ effector T cells and development of CD8+ memory T (Tm) cells. However, the functional alteration of CD8+ Tm cells developed in the absence of IL-12 priming is elusive. In this study, we investigated the capacity of secondary expansion of CD8+ Tm cells developed from transgenic OT I CD8+ T cells. The latter cells were in vitro and in vivo stimulated by ovalbumin (OVA)-pulsed dendritic cells [DCOVA and (IL-12-/-)DCOVA] derived from wild-type C57BL/6 and IL-12 gene knockout mice, respectively. We demonstrated that IL-12 priming is important not only in CD8+ T cell clonal expansion, but also in generation of CD8+ Tm cells with the capacity of secondary expansion upon antigen re-encounter. However, IL-12 signaling is not involved in CD8+ Tm cell survival and recall responses. Therefore, this study provides useful information for vaccine design and development.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Animales , Proliferación Celular , Supervivencia Celular/inmunología , Interleucina-12/inmunología , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Selectina L/genética , Selectina L/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8(+) CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4(+) and CD8(+) T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia(b-/-) gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC(OVA))-stimulated CD8(+) CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8(+) T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4(+) T cell help, and (iii) increasing CD4(+) and CD8(+) T cell precursors overcomes immune suppression to DC(OVA)-stimulated CD8(+) CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.
Asunto(s)
Inmunidad Innata/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
CD8+ cytotoxic T (Tc) cells play a crucial role in host immune responses to cancer, and in this context, adoptive CD8+ Tc cell therapy has been studied in numerous animal tumor models. Its antitumor efficacy is, to a large extent, determined by the ability of Tc cells to survive and infiltrate tumors. In clinical trials, such in vitro-activated T cells often die within hours to days, and this greatly limits their therapeutic efficacy. CD8+ Tc cells fall into two subpopulations based upon their differential cytokine secretion. In this study, we in vitro generated that ovalbumin (OVA)-pulsed dendritic cell (DCOVA)-activated CD8+ type 1 Tc (Tc1) cells secreting IFN-gamma, and CD8+ type 2 Tc (Tc2) cells secreting IL-4, IL-5 and IL-10, which were derived from OVA-specific T cell receptor (TCR) transgenic OT I mice. We then systemically investigated the in vitro and in vivo effector function and survival of Tc1 and Tc2 cells, and then assessed their survival kinetics after adoptively transferred into C57BL/6 mice, respectively. We demonstrated that, when compared to CD8+ Tc2, Tc1 cells were significantly more effective in perforin-mediated cytotoxicity to tumor cells, had a significantly higher capacity for in vivo survival after the adoptive T cell transfer, and had a significantly stronger therapeutic effect on eradication of well-established tumors expressing OVA in animal models. In addition, CD8+ Tc1 and Tc2 cells skewed the phenotype of CD4+ T cells toward Th1 and Th2 type, respectively. Therefore, the information regarding the differential effector function, survival and immune modulation of CD8+ Tc1 and Tc2 cells may provide useful information when preparing in vitro DC-activated CD8+ T cells for adoptive T cell therapy of cancer.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Supervivencia Celular , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias Experimentales , Ovalbúmina/farmacología , Fenotipo , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Cytokine and costimulation signals determine CD8(+) T cell responses in proliferation phase. In this study, we assessed the potential effect of cytokines and costimulations to CD8(+) T cell survival in transition phase by transferring in vitro ovalbumin (OVA)-pulsed dendritic cell-activated CD8(+) T cells derived from OVA-specific T cell receptor transgenic OT I mice into wild-type C57BL/6 mice or mice with designated gene knockout. We found that deficiency of IL-10, IL-12, IFN-gamma, CD28, CD40, CD80, CD40L, and 41BBL in recipients did not affect CD8(+) T cell survival after adoptive transfer. In contrast, TNF-alpha deficiency in both recipients and donor CD8(+) effector T cells significantly reduced CD8(+) T cell survival. Therefore, our data demonstrate that the host- and T cell-derived TNF-alpha signaling contributes to CD8(+) effector T cell survival and their transition to memory T cells in the transition phase, and may be useful information when designing vaccination.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Traslado Adoptivo , Animales , Antígenos CD/genética , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/trasplante , Supervivencia Celular/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Inmunofenotipificación , Interleucinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Previous studies have shown that interleukin (IL)-24 as a novel tumor suppressor gene has tumor-suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo. In this study, we explored the potential effect of adenovirus-mediated IL-24 gene therapy on human hepatocellular carcinoma (HCC) by using a HCC cell line, SMMC-7721. We constructed a recombinant adenovirus, AdVGFP/IL-24 expressing the marker green fluorescent protein (GFP) and the tumor-suppressor gene, IL-24. We demonstrated that AdVGFP/IL-24 treatment of SMMC-7721 cells in vitro significantly induced HCC cell cytotoxicity and apoptosis, and altered HCC cell cycling with an S-phase reduction and G2/M phase arrest, compared with AdVGFP, without IL-24 expresssion (p < 0.05). Furthermore, we also showed that the treatment of SMMC-7721 tumors by an intratumoral injection of AdVGFP/IL-24 significantly suppressed in vivo HCC growth in athymic nude mice, compared with AdVGFP treatment (p < 0.05). In addition, we also elucidated the molecular mechanism responsible for AdVGFP/IL-24-associated tumor suppression. These include: (1) upregulation of p53-independent apoptosis-associated caspase-3 and (2) downregulation of angiogenesis-associated vascular endothelial growth factor and CD34. Therefore, this study will provide a framework for future clinical applications of AdVGFP/IL-24 in HCC gene therapy.
Asunto(s)
Adenoviridae/genética , Apoptosis , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Interleucinas/metabolismo , Animales , Antígenos CD34/metabolismo , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/genética , Caspasa 3/metabolismo , Línea Celular , Regulación hacia Abajo , Terapia Genética , Humanos , Interleucinas/genética , Ratones , Ratones Desnudos , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The acquisition of dendritic cell (DC) molecules by T cells has been previously reported. However, it remains unclear whether the transfer is only mono- or bidirectional. In this study, we incubated CMFDA-labeled ovalbumin (OVA)-pulsed DC2.4 (DC2.4(OVA)) cells with Dil-labeled OT II CD4(+) T cells and analyzed the potential bidirectional molecule transfer. We also assessed the distribution of internalized membrane using two engineered DC2.4/Ia(b)GFP and MF4/TCRCFP DC lines. Our findings showed that membrane molecule transfer is bidirectional. CD4(+) T cells acquired Ia(b), CD11c, CD40, and CD80 from DC2.4(OVA) cells, and conversely DC2.4(OVA) cells took up CD4, CD25, CD69, and T cell receptor from T cells. The internalized molecules acquired by T cells and DCs mostly localized in endosomes and lysosomes, respectively. Taken together, this study demonstrated a novel phenomenon of bidirectional membrane molecule transfer between DCs and T cells.
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Células Dendríticas/metabolismo , Ovalbúmina/metabolismo , Linfocitos T/metabolismo , Antígeno B7-1/biosíntesis , Antígeno CD11c/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD40/biosíntesis , Línea Celular , Células Dendríticas/citología , Endosomas/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisosomas/metabolismo , Microscopía Confocal , FenotipoRESUMEN
The cooperative role of CD4+ helper T (Th) cells has been reported for CD8+ cytotoxic T (Tc) cells in tumor eradication. However, its molecular mechanisms have not been well elucidated. We have recently demonstrated that CD4+ Th cells can acquire major histocompatibility complex/peptide I (pMHC I) complexes and costimulatory molecules by dendritic cell (DC) activation, and further stimulate naïve CD8+ T cell proliferation and activation. In this study, we used CD4+ Th1 and CD8+ Tc1 cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice to study CD4+ Th1 cell's help effects on active CD8+ Tc1 cells and the molecular mechanisms involved in CD8+ Tc1-cell immunotherapy of OVA-expressing EG7 tumors. Our data showed that CD4+ Th1 cells with acquired pMHC I by OVA-pulsed DC (DCOVA) stimulation are capable of prolonging survival and reducing apoptosis formation of active CD8+ Tc1 cells in vitro, and promoting CD8+ Tc1 cell tumor localization and memory responses in vivo by 3-folds. A combined adoptive T-cell therapy of CD8+ Tc1 with CD4+ Th1 cells resulted in regression of well-established EG7 tumors (5 mm in diameter) in all 10/10 mice. The CD4+ Th1's help effect is mediated via the helper cytokine IL-2 specifically targeted to CD8+ Tc1 cells in vivo by acquired pMHC I complexes. Taken together, these results will have important implications for designing adoptive T-cell immunotherapy protocols in treatment of solid tumors.
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Genes MHC Clase I/inmunología , Interleucina-2/inmunología , Neoplasias Experimentales/terapia , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Animales , Proliferación Celular , Supervivencia Celular/inmunología , Células Dendríticas/inmunología , Femenino , Memoria Inmunológica , Inmunoterapia Adoptiva/métodos , Cooperación Linfocítica/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales/inmunología , Ovalbúmina/inmunologíaRESUMEN
Interleukin-10 (IL-10) has been identified as a key immunomodulatory cytokine on T cells. However, both immunosuppressive and immunostimulatory effects of IL-10 on T cells also have been reported. The discrepancy between these in vitro effects of IL-10 may be due to the different T cells (naive vs. active or resting active T cells) used under various experimental conditions in these studies. Therefore, it is necessary to clearly define the IL-10 effect on T cell subsets in their different statuses. In this study, we used a molecularly defined T cell system, the ovalbumin (OVA)-specific CD4(+) and CD8(+) T cells from transgenic OT-I and OT-II mice expressing OVA-specific T cell receptor (TCR). We investigated the effect of IL-10 on these OVA-specific T cell subsets in their different statuses (i.e., naive and active T cells). Our data demonstrate that IL-10 has distinct immunoregulatory effects on naive and active T cell subsets. IL-10 inhibits active CD4(+) T cell proliferation, whereas it stimulates and suppresses active CD8(+) T cell proliferation and cytotoxicity, respectively. IL-10-treated dendritic cells (DCs) stimulate anergic cytotoxic T lymphocyte-associated molecule-4 (CTLA)-4-expressing CD4(+) T cell responses possibly through downregulation of major histocompetibility complex (MHC) class II and costimulatory molecule expression on DCs. The anergic CD4(+) T cells suppress T cell proliferation mainly through a CTLA-4-mediated pathway. The distinct role of IL-10 on T cell subsets may be useful in designing T cell-based immunotherapy of cancer and infectious diseases.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Interleucina-10/inmunología , Activación de Linfocitos , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4 , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunización , Ratones , Ratones Transgénicos , Ovalbúmina/inmunología , Receptores de Interleucina-10/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
Study effect and mechanisms of growth-suppression of hepatocelluar carcinoma (HCC) in nude mice. The construction of the pAdeasy-1-pTrack-CMV-hIL-24 recombined adenovirus vector (Ad-hIL-24) was completed and lineared with PacI. Ad-hIL-24 were transfected into QBI-293 cells and obtained. 16 nude mice of the subcutaneous tumor models were established with SMMC-7721 HCC and were randomly divided into NS, 5-Fu, Ad and Ad-hIL-24 groups. Then 100 microL NS, Ad (10(7) pfu) and Ad-hIL-24 (10(7) pfu) for each one were given respectively QOD, and 5-Fu (20 microg/kg) were injected Q.D., for 5 times, with intratumor injections. After 15 d, 16 mice were sacrificed and subcutaneous tumors were taken out. The volumes (before administration, 1 week and 2weeks after administration) were measured and the weights of tumor were weighed and ratios of tumor-suppression were calculated. The morphological changes of apoptotic tumor cells were observed under microscope. Caspase3, P53 and P27, CD34 and VEGF were tested in immunohistochemistry. In tumor subcutaneous model, compared with NS group, the ratios of tumor-suppression of Ad-hIL-24 group and 5-Fu group were 68.52% (P < 0.01) and 65.64 (P < 0.01), respectively. Caspase3 protein in Ad-hIL-24 group was higher than other 3 groups significantly (P < 0.01). The expression of P27 also differed from NS group (P < 0.01). CD34 and VEGF protein in Ad-hIL-24 group can inhibit neovascularization obviously (P < 0.001), compared with NS and Ad groups. Ad-hIL-24 inhibits the growth of SMMC-7721 HCC on nude mice's. The mechanisms of tumor-suppression may be multi-pathways such as the induction of caspase3 pathway, P27 activities and the antiangiogenic mechanism.
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Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Adenoviridae/genética , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Interleucinas/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Plásmidos/genéticaRESUMEN
Oncolytic adenovirus (rAd)-mediated E1A gene therapy of cancer has become a novel therapeutic modality. In this study, we constructed a recombinant oncolytic adenovirus (rAd-E1A) expressing the tumor suppressor E1A gene. We demonstrated that the rAd-E1A replicated in HepG2 and SMMC-7721 human hepatocellular carcinoma (HCC) cells but attenuated in the normal liver cell line HL-7702. It induced HCC cell apoptosis through upregulation of apoptosis-associated Bax, caspase-3, and Fas and downregulation of survivin and Bcl-2 in a p53-dependent pathway. It also downregulated the expression of angiogenesis- associated vascular endothelial growth factor (VEGF) and CD34 genes and reduced tumor vessel formation and angiogenesis. In mice bearing SMMC-7721 tumors, intratumoral injections of rAd- E1A significantly inhibited HCC growth. Therefore, the oncolytic adenovirus-mediated E1A gene therapy may be a useful therapeutic approach for HCC treatment.
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Proteínas E1A de Adenovirus/genética , Apoptosis , Carcinoma Hepatocelular/terapia , Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Neoplasias/patología , Neovascularización Patológica , Virus Oncolíticos/genética , Animales , Línea Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/metabolismoRESUMEN
The E1A gene was obtained by PCR with QBI-293A cell genome DNA as template. After enzyme digestion, the E1A gene was ligated to transfer vector pAdTrack-CMV. The positive clone pAdTrack-CMV-E1A were lineared by PmeI and co-transformed with pAdEasy-1 in BJ5183 E. coli. The recombinant adenovirus vector pAdEasy-1-pAdTrack-CMV-E1A were digested by PacI and transfected into QBI-293A cells with liposomes. The oncolytic recombinant adenovirus Ad-E1A was obtained after 7 days. The results showed that this oncolytic adenovirus Ad-E1A can replicate in ECV304 cells and inhibit growth of ECV304 cell. In addition, it also decreased the secretion of VEGF and expression of NF-kappaB of ECV304 cells, indicating that Ad-E1A have potential of inhibition of tumor metastasis.