RESUMO
UBR5 is a HECT domain E3 ubiquitin ligase that is frequently amplified in breast, ovarian and prostate cancers. Heightened UBR5 expression plays a profound role in tumor growth through immune-dependent mechanisms; however, its mode of action in driving tumor metastasis has not been definitively delineated. Herein, we used a tetracycline (Tet)-inducible RNAi-mediated expression silencing cell system to investigate how UBR5 enables postsurgical mammary tumor metastatic growth in mouse lungs without the continuous influence of the primary lesion. In vitro, Ubr5 knockdown induces morphological and molecular changes characteristic of epithelial-mesenchymal transition (EMT). In vivo, UBR5 promotes lung metastasis in an E3 ubiquitin ligase-dependent manner. Moreover, doxycycline-induced UBR5 expression knockdown in metastatic cells in the lungs, following removing the primary tumors, resulted in increased apoptosis, decreased proliferation and prolonged survival, whereas silencing the expression of cell division cycle 73 (CDC73), a tumor suppressor and E3 ligase substrate of UBR5, reversed these effects. Transcriptome analyses revealed a prominent role of the p53 pathway in dovitinib-induced apoptosis of tumor cells differentially regulated by UBR5 and CDC73. In human triple-negative breast cancer (TNBC) patient specimens, a strong inverse correlation was observed between UBR5 and CDC73 protein levels, with reduced CDC73 expression at metastatic sites compared to primary lesions. Furthermore, a xenograft model of human TNBC recapitulated the metastatic properties and characteristics of the unique UBR5-CDC73 functional antagonism. This study reveals the novel and critical roles and intricate relationships of UBR5, CDC73 and p53 in postsurgical breast cancer metastasis and indicates the potential of targeting this pathway in cancer therapy.
Assuntos
Neoplasias Pulmonares , Neoplasias de Mama Triplo Negativas , Animais , Humanos , Masculino , Camundongos , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Pulmonares/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Two common γ-chain family cytokines IL-2 and IL-15 stimulate the same mammalian target of rapamycin complex-1 (mTORC1) signaling yet induce effector T (TE) and memory T (TM) cell differentiation via a poorly understood mechanism(s). Here, we prepared in vitro IL-2-stimulated TE (IL-2/TE) and IL-15-stimulated TM (IL-15/TM) cells for characterization by flow cytometry, Western blotting, confocal microscopy and Seahorse-assay analyses. We demonstrate that IL-2 and IL-15 stimulate strong and weak mTORC1 signals, respectively, which lead to the formation of CD62 ligand (CD62L)- killer cell lectin-like receptor subfamily G member-1 (KLRG)+ IL-2/TE and CD62L+KLRG- IL-15/TM cells with short- and long-term survival following their adoptive transfer into mice. The IL-15/mTORC1Weak signal activates the forkhead box-O-1 (FOXO1), T cell factor-1 (TCF1) and Eomes transcriptional network and the metabolic adenosine monophosphate-activated protein kinase-α-1 (AMPKα1), Unc-51-like autophagy-activating kinase-1 (ULK1) and autophagy-related gene-7 (ATG7) axis, increasing the expression of mitochondrial regulators aquaporin-9 (AQP9), mitochondrial transcription factor-A (TFAM), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), carnitine palmitoyl transferase-1 (CPT1α), microtubule-associated protein light chain-3 II (LC3II), Complex I and ortic atrophy-1 (OPA1), leading to promoting mitochondrial biogenesis and fatty-acid oxidation (FAO). Interestingly, AMPKα1 deficiency abrogates these downstream responses to IL-15/mTORC1Weak signaling, leading to the upregulation of mTORC1 and hypoxia-inducible factor-1α (HIF-1α), a metabolic switch from FAO to glycolysis and reduced cell survival. Taken together, our data demonstrate that IL-15/mTORC1Weak signaling controls T-cell memory via activation of the transcriptional FOXO1-TCF1-Eomes and metabolic AMPKα1-ULK1-ATG7 pathways, a finding that may greatly impact the development of efficient vaccines and immunotherapies for the treatment of cancer and infectious diseases.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Diferenciação Celular , Interleucina-15 , Interleucina-2 , Respiração , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/fisiologia , Interleucina-15/farmacologia , Mamíferos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Linfócitos TRESUMO
Upon binding double-stranded DNA (dsDNA), cyclic GMP-AMP synthase (cGAS) is activated and initiates the cGAS-stimulator of IFN genes (STING)-type I interferon pathway. DEAD-box helicase 41 (DDX41) is a DEAD-box helicase, and mutations in DDX41 cause myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). Here, we show that DDX41-knockout (KO) cells have reduced type I interferon production after DNA virus infection. Unexpectedly, activations of cGAS and STING are affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibits ATP-dependent DNA-unwinding activity and ATP-independent strand-annealing activity. The MDS/AML-derived mutant R525H has reduced unwinding activity but retains normal strand-annealing activity and stimulates greater cGAS dinucleotide-synthesis activity than wild-type DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells results in higher type I interferon production. Our results have led to the hypothesis that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and single-stranded DNA (ssDNA), which, in turn, regulates cGAS-STING activation.
Assuntos
Infecções por Vírus de DNA , Interferon Tipo I , Leucemia Mieloide Aguda , Trifosfato de Adenosina , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Humanos , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de SinaisRESUMO
CD8+ memory T (TM) cells play a critical role in immune defense against infection. Two common γ-chain family cytokines, IL-2 and IL-7, although triggering the same mTORC1-S6K pathway, distinctly induce effector T (TE) cells and TM cells, respectively, but the underlying mechanism(s) remains elusive. In this study, we generated IL-7R-/and AMPKα1-knockout (KO)/OTI mice. By using genetic and pharmaceutical tools, we demonstrate that IL-7 deficiency represses expression of FOXO1, TCF1, p-AMPKα1 (T172), and p-ULK1 (S555) and abolishes T cell memory differentiation in IL-7R KO T cells after Listeria monocytogenesis rLmOVA infection. IL-2- and IL-7-stimulated strong and weak S6K (IL-2/S6Kstrong and IL-7/S6Kweak) signals control short-lived IL-7R-CD62L-KLRG1+ TE and long-term IL-7R+CD62L+KLRG1- TM cell formations, respectively. To assess underlying molecular pathway(s), we performed flow cytometry, Western blotting, confocal microscopy, and Seahorse assay analyses by using the IL-7/S6Kweak-stimulated TM (IL-7/TM) and the control IL-2/S6Kstrong-stimulated TE (IL-2/TE) cells. We determine that the IL-7/S6Kweak signal activates transcriptional FOXO1, TCF1, and Id3 and metabolic p-AMPKα1, p-ULK1, and ATG7 molecules in IL-7/TM cells. IL-7/TM cells upregulate IL-7R and CD62L, promote mitochondria biogenesis and fatty acid oxidation metabolism, and show long-term cell survival and functional recall responses. Interestingly, AMPKα1 deficiency abolishes the AMPKα1 but maintains the FOXO1 pathway and induces a metabolic switch from fatty acid oxidation to glycolysis in AMPKα1 KO IL-7/TM cells, leading to loss of cell survival and recall responses. Taken together, our data demonstrate that IL-7-stimulated weak strength of mTORC1-S6K signaling controls T cell memory via activation of transcriptional FOXO1-TCF1-Id3 and metabolic AMPKα1-ULK1-ATG7 pathways. This (to our knowledge) novel finding provides a new mechanism for a distinct IL-2/IL-7 stimulation model in T cell memory and greatly impacts vaccine development.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteína Forkhead Box O1/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas Inibidoras de Diferenciação/metabolismo , Interleucina-7/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Células T de Memória/imunologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Citotoxicidade Imunológica , Ácidos Graxos/metabolismo , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica , Glicólise , Fator 1-alfa Nuclear de Hepatócito/genética , Memória Imunológica , Proteínas Inibidoras de Diferenciação/genética , Interleucina-7/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-7/genética , Transdução de Sinais , Desenvolvimento de VacinasRESUMO
Irreversible electroporation (IRE) is a new cancer ablation technology, but methods to improve IRE-induced therapeutic immunity are only beginning to be investigated. We developed a mouse model bearing large primary (300 mm3) and medium distant (100 mm3) EG7 lymphomas engineered to express ovalbumin (OVA) as a nominal tumor antigen. We established experimental protocols including IRE alone and IRE combined with Toll-like receptor (TLR)3/9 agonists (poly I:C/CpG) (IRE + pIC/CpG), PD-1 blockade (IRE + PD-1 blockade), or both (IRE + Combo) to investigate therapeutic effects on primary and distant EG7 tumors and conversion-promoting effects on the immunotolerant tumor microenvironment (TME). We demonstrated that IRE alone simulated very weak OVA-specific CD8+ T cell responses and did not inhibit primary tumor growth. IRE + pIC/CpG synergistically stimulated more efficient OVA-specific CD8+ T cell responses and primary tumor growth inhibition than IRE + PD-1 blockade. IRE + pIC/CpG played a major role in the modulation of immune cell profiles but a minor role in the downregulation of PD-L1 expression in the TME and vice versa for IRE + PD-1 blockade. IRE + Combo cooperatively induced potent OVA-specific CD8+ T cell immunity and rescued exhausted intratumoral CD8+ T cells, leading to eradication of not only primary tumors but also untreated concomitant distant tumors and lung metastases. IRE + Combo efficiently modulated immune cell profiles, as evidenced by reductions in immunotolerant type-2 (M2) macrophages, myeloid-derived suppressor-cells, plasmacytoid dendritic cells, and regulatory T cells and by increases in immunogenic M1 macrophages, CD169+ macrophages, type-1 conventional dendritic cells, and CD8+ T cells, leading to conversion of immunotolerance in not only primary TMEs but also untreated distant TMEs. IRE + Combo also showed effective therapeutic effects in two breast cancer models. Therefore, our results suggest that IRE + Combo is a promising strategy to improve IRE ablation therapy in cancer.
Assuntos
Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1 , Animais , Linhagem Celular Tumoral , Eletroporação , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor 3 Toll-Like/agonistas , Receptor Toll-Like 9/agonistas , Microambiente Tumoral/imunologiaRESUMO
Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa)-inhibition of mTORC1 (RIM) promotes T-cell memory. However, the underlying pathway and the role of AMPKα1 in Rapa-induced T-cell memory remain elusive. Using genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice in vivo post Listeria monocytogenesis rLmOVA infection and in vitro transition of effector T (TE) to memory T (TM) cells. IL-2- and IL-2+Rapa-stimulated T [IL-2/T and IL-2(Rapa+)/T] cells, when transferred into mice, differentiate into short-term IL-7R-CD62L-KLRG1+ TE and long-lived IL-7R+CD62L+KLRG1- TM cells, respectively. To assess the underlying pathways, we performed Western blotting, confocal microscopy and Seahorse-assay analyses using IL-2/T and IL-2(Rapa+)/T-cells. We determined that IL-2(Rapa+)/T-cells activate transcription FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T172), pULK1(S555) and ATG7 molecules and promote mitochondrial biogenesis and fatty-acid oxidation (FAO). We found that rapamycin-treated AMPKα-deficient AMPKα1-KO IL-2(Rapa+)/TM cells up-regulate transcription factor HIF-1α and induce a metabolic switch from FAO to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient TM cells lost their cell survival capacity. Taken together, our data indicate that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis, and that AMPKα1 plays a critical role in RIM-induced T-cell memory.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Memória Imunológica/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Sirolimo/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Proteína Forkhead Box O1/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Interleucina-2/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas com Domínio T/metabolismoRESUMO
Radiofrequency ablation (RFA) is the most common approach to thermal ablation for cancer therapy. Unfortunately, its efficacy is limited by incomplete ablation, and further optimization of RFA is required. Here, we demonstrate that incubation at 65 °C triggers more EG7 tumor cell death by necrosis than treatment at 45 °C, and the 65 °C-treated cells are more effective at inducing antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses after injection in mice than the 45 °C-treated ones. Dendritic cells (DCs) that phagocytose 65 °C-treated EG7 cells become mature with upregulated MHCII and CD80 expression and are capable of efficiently inducing effector CTLs in mouse tumor models. RFA (65 °C) therapy of EG7 tumors induces large areas of tumor necrosis and stimulates CTL responses. This leads to complete regression of small (~100 mm3) tumors but fails to suppress the growth of larger (~350 mm3) tumors. The administration of the Toll-like receptor-9 (TLR9) agonist unmethylated cytosine-phosphorothioate-guanine oligonucleotide (CpG) to DCs phagocytosing 65 °C-treated EG7 cells enhances the expression of MHCII and CD40 on DCs as well as DC-induced stimulation of CTL responses. Importantly, the intratumoral administration of CpG following RFA also increases the frequencies of tumor-associated immunogenic CD11b-CD11c+CD103+ DC2 and CD11b+F4/80+MHCII+ M1 macrophages and increases CD4+ and CD8+ T-cell tumor infiltration, leading to enhanced CD4+ T cell-dependent CTL responses and potent inhibition of primary RFA-treated or distant untreated tumor growth as well as tumor lung metastasis in mice bearing larger tumors. Overall, our data indicate that CpG administration, which enhances RFA-induced CTL responses and ultimately potentiates the inhibition of primary tumor growth and lung metastasis, is a promising strategy for improving RFA treatment, which may assist in optimizing this important cancer therapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Fosfatos de Dinucleosídeos/uso terapêutico , Pulmão/patologia , Ablação por Radiofrequência/métodos , Adjuvantes Imunológicos/uso terapêutico , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Células Dendríticas/metabolismo , Temperatura Alta , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Metástase Neoplásica , Transplante de Neoplasias , Receptor Toll-Like 9/agonistasRESUMO
Simulated-microgravity (SMG) promotes cell-apoptosis. We demonstrated that SMG inhibited cell proliferation/metastasis via FAK/RhoA-regulated mTORC1 pathway. Since mTORC1, NF-κB, and ERK1/2 signaling are important in cell apoptosis, we examined whether SMG-enhanced apoptosis is regulated via these signals controlled by FAK/RhoA in BL6-10 melanoma cells under clinostat-modelled SMG-condition. We show that SMG promotes cell-apoptosis, alters cytoskeleton, reduces focal adhesions (FAs), and suppresses FAK/RhoA signaling. SMG down-regulates expression of mTORC1-related Raptor, pS6K, pEIF4E, pNF-κB, and pNF-κB-regulated Bcl2, and induces relocalization of pNF-κB from the nucleus to the cytoplasm. In addition, SMG also inhibits expression of nuclear envelope proteins (NEPs) lamin-A, emerin, sun1, and nesprin-3, which control nuclear positioning, and suppresses nuclear positioning-regulated pERK1/2 signaling. Moreover, rapamycin, the mTORC1 inhibitor, also enhances apoptosis in cells under 1 g condition via suppressing the mTORC1/NF-κB pathway. Furthermore, the FAK/RhoA activator, toxin cytotoxic necrotizing factor-1 (CNF1), reduces cell apoptosis, restores the cytoskeleton, FAs, NEPs, and nuclear positioning, and converts all of the above SMG-induced changes in molecular signaling in cells under SMG. Therefore, our data demonstrate that SMG reduces FAs and alters the cytoskeleton and nuclear positioning, leading to enhanced cell apoptosis via suppressing the FAK/RhoA-regulated mTORC1/NF-κB and ERK1/2 pathways. The FAK/RhoA regulatory network may, thus, become a new target for the development of novel therapeutics for humans under spaceflight conditions with stressed physiological challenges, and for other human diseases.
Assuntos
Citoesqueleto/metabolismo , MAP Quinases Reguladas por Sinal Extracelular , Adesões Focais , Simulação de Ausência de Peso/métodos , Animais , Apoptose , Núcleo Celular , Proliferação de Células , Células Cultivadas , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Simulated microgravity (SMG) was reported to affect tumor cell proliferation and metastasis. However, the underlying mechanism is elusive. In this study, we demonstrate that clinostat-modelled SMG reduces BL6-10 melanoma cell proliferation, adhesion and invasiveness in vitro and decreases tumor lung metastasis in vivo. It down-regulates metastasis-related integrin α6ß4, MMP9 and Met72 molecules. SMG significantly reduces formation of focal adhesions and activation of focal adhesion kinase (FAK) and Rho family proteins (RhoA, Rac1 and Cdc42) and of mTORC1 kinase, but activates AMPK and ULK1 kinases. We demonstrate that SMG inhibits NADH induction and glycolysis, but induces mitochondrial biogenesis. Interestingly, administration of a RhoA activator, the cytotoxic necrotizing factor-1 (CNF1) effectively converts SMG-triggered alterations and effects on mitochondria biogenesis or glycolysis. CNF1 also converts the SMG-altered cell proliferation and tumor metastasis. In contrast, mTORC inhibitor, rapamycin, produces opposite responses and mimics SMG-induced effects in cells at normal gravity. Taken together, our observations indicate that SMG inhibits focal adhesions, leading to inhibition of signaling FAK and RhoA, and the mTORC1 pathway, which results in activation of the AMPK pathway and reduced melanoma cell proliferation and metastasis. Overall, our findings shed a new light on effects of microgravity on cell biology and human health.
Assuntos
Adesões Focais , Melanoma/patologia , Transdução de Sinais , Simulação de Ausência de Peso , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Citoesqueleto/metabolismo , Ativação Enzimática , Quinase 1 de Adesão Focal/metabolismo , Glicólise , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mitocôndrias/metabolismo , NAD/metabolismo , Metástase Neoplásica , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
DNA vaccines composed of heterologous human HER2 and rat neu sequences induce stronger antibody response and protective antitumor immunity than either HER2 or neu DNA vaccines in transgenic mice. We previously developed HER2-specific exosome-targeted T-cell vaccine HER2-TEXO capable of stimulating HER2-specific CD8+ T-cell responses, but only leading to partial protective immunity in double-transgenic HLA-A2/HER2 mice with self-immune tolerance to HER2. Here, we constructed an adenoviral vector AdVHuRt expressing HuRt fusion protein composed of NH2-HER21-407 (Hu) and COOH-neu408-690 (Rt) fragments, and developed a heterologous human/rat HER2-specific exosome-targeted T-cell vaccine HuRt-TEXO using polyclonal CD4+ T-cells uptaking exosomes released by AdVHuRt-transfected dendritic cells. We found that the HuRt-TEXO vaccine stimulates enhanced CD4+ T-cell responses leading to increased induction of HER2-specific antibody (â¼70⯵g/ml) compared to that (â¼40⯵g/ml) triggered by the homologous HER2-TEXO vaccine. By using PE-H-2Kd/HER223-71 tetramer, we determined that HuRt-TEXO stimulates stronger HER2-specific CD8+ T-cell responses eradicating 90% of HER2-specific target cells, while HER2-TEXO-induced CD8+ T-cell responses only eliminating 53% targets. Furthermore, HuRt-TEXO, but not HER2-TEXO vaccination, is capable of suppressing early stage-established HER2-expressing 4T1HER2 breast cancer in its lung metastasis or subcutaneous form in BALB/c mice, and of completely protecting transgenic HLA-A2/HER2 mice from growth of HLA-A2/HER2-expressing BL6-10A2/HER2 melanoma. HuRt-TEXO-stimulated HER2-specific CD8+ T-cells not only are cytolytic to trastuzumab-resistant HLA-A2/HER2-expressing BT474/A2 breast tumor cells in vitro but also eradicates pre-established BT474/A2 tumors in athymic nude mice. Therefore, our novel heterologous human/rat HER2-specific T-cell vaccine HuRt-TEXO, circumventing HER2 tolerance, may provide a new therapeutic alternative for patients with trastuzumab-resistant HER2+ breast tumor.
Assuntos
Vacinas Anticâncer/imunologia , Exossomos , Tolerância Imunológica , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Vacinas Anticâncer/genética , Linhagem Celular , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Ordem dos Genes , Vetores Genéticos/genética , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Memória Imunológica , Camundongos , Camundongos Transgênicos , Ratos , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusão/genética , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Expansion of PD-1-expressing CD8+ cytotoxic T lymphocytes (CTLs) and associated CTL exhaustion are chief issues for ineffective virus-elimination in chronic infectious diseases. PD-1 blockade using antagonistic anti-PD-L1 antibodies results in a moderate conversion of CTL exhaustion. We previously demonstrated that CD40L signaling of ovalbumin (OVA)-specific vaccine, OVA-Texo, converts CTL exhaustion via the activation of the mTORC1 pathway in OVA-expressing adenovirus (AdVova)-infected B6 mice showing CTL inflation and exhaustion. Here, we developed AdVova-infected B6 and transgenic CD11c-DTR (termed AdVova-B6 and AdVova-CD11c-DTR) mice with chronic infection, and assessed a potential effect of CD40 agonist on the conversion of CTL exhaustion and on a potential enhancement of PD-1 antagonist action in rescuing exhausted CTLs in our chronic infection models. We demonstrate that a single dose of anti-CD40 alone can effectively convert CTL exhaustion by activating the mTORC1 pathway, leading to CTL proliferation, up-regulation of an effector-cytokine IFN-γ and the cytolytic effect in AdVova-B6 mice. Using anti-CD4 antibody and diphtheria toxin (DT) to deplete CD4+ T-cells and dendritic cells (DCs), we discovered that the CD40 agonist-induced conversion in AdVova-B6 and AdVova-CD11c-DTR mice is dependent upon host CD4+ T-cell and DC involvements. Moreover, CD40 agonist significantly enhances PD-1 antagonist effectiveness in rescuing exhausted CTLs in chronic infection. Taken together, our data demonstrate the importance of CD40 signaling in the conversion of CTL exhaustion and its ability to enhance PD-1 antagonist action in rescuing exhausted CTLs in chronic infection. Therefore, our findings may positively impact the design of new therapeutic strategies for chronic infectious diseases.
Assuntos
Antígenos CD40/imunologia , Infecções/imunologia , Infecções/patologia , Complexos Multiproteicos/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Citotóxicos/imunologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Antígenos CD40/agonistas , Células Cultivadas , Doença Crônica , Ativação Linfocitária/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Compared with CD4+25+ regulatory T cells (Tregs), the mechanisms for natural, polyclonal CD8+25+ Treg immune suppression have been significantly less studied. We previously showed that polyclonal T cells can acquire antigen-specific targeting activity through arming with exosomal peptide-MHC (pMHC). In this study, we assessed the suppressive effect of CD8+25+ Tregs or CD8+25+ Tregs armed with ovalbumin (OVA)-specific exosomes on other immune cells and OVA-specific dendritic cell (DCOVA)-stimulated antitumor immunity. We demonstrate that CD8+25+ Tregs inhibit T cell proliferation in vitro in a cell contact-dependent fashion but independent of the expression of immunosuppressive IL-10, TGF-ß, and CTLA-4. CD8+25+ Tregs anergize naïve T cells upon stimulation by up-regulating T cell anergy-associated Egr2 and down-regulating IL-2 production. Tregs also anergize DCs by preventing DC maturation through the down-regulation of Iab, CD80, CD86, and inflammatory cytokines, leading to defects in T cell stimulation. Moreover, CD8+25+ Tregs inhibit CTLs through inducing CTL death via perforin-mediated apoptosis and through reducing effector CTL cytotoxic activity via down-regulating CTL perforin-production and degranulation. In addition, we show that CD8+25+ Tregs suppress DCOVA-stimulated CTL responses in priming and effector phases and inhibit immunity against OVA-expressing CCLOVA lung cancer. Remarkably, polyclonal CD8+25+ Tregs armed with OVA-specific exosomal pMHC class-II (pMHC-II), or pMHC class-I (pMHC-I) complexes exert their enhanced inhibition of CTL responses in the priming and the effector phases, respectively. Taken together, our investigation reveals that assigning antigen specificity to nonspecific polyclonal CD8+25+ Tregs for enhanced immune suppression can be achieved through exosomal pMHC arming. This principle may have a great effect on Treg-mediated immunotherapy of autoimmune diseases.
Assuntos
Células Dendríticas/imunologia , Exossomos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Proliferação de Células , Anergia Clonal , Citotoxicidade Imunológica , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Exossomos/química , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Ovalbumina/farmacologia , Cultura Primária de Células , Transdução de Sinais , Análise de Sobrevida , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/patologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
CD8+ cytotoxic T lymphocyte (CTL) exhaustion is a chief issue for ineffective virus elimination in chronic infectious diseases. We generated novel ovalbumin (OVA)-specific OVA-Texo and HIV-specific Gag-Texo vaccines inducing therapeutic immunity. To assess their therapeutic effect in chronic infection, we developed a new chronic infection model by i.v. infecting C57BL/6 mice with the OVA-expressing adenovirus AdVova. During chronic AdVova infection, mouse CTLs were found to express the inhibitory molecules programmed cell-death protein-1 (PD-1) and lymphocyte-activation gene-3 (LAG-3) and to be functionally exhausted, showing a significant deficiency in T-cell proliferation, IFN-γ production and cytolytic effects. Naive CD8+ T cells upregulated inhibitory PD-ligand 1 (PD-L1), B- and T-lymphocyte attenuator and T-cell anergy-associated molecules (Grail and Itch) while down-regulating the proliferative response upon stimulation in mice with chronic infection. Remarkably, the OVA-Texo vaccine counteracted T-cell anergy and converted CTL exhaustion. The latter was associated with (i) the upregulation of a marker for CTL functionality, diacetylated histone-H3 (diAcH3), (ii) a fourfold increase in CTLs, occurring independent of host DCs or CD4+ T cells, and (iii) the restoration of CTL IFN-γ production and cytotoxicity. In vivo OVA-Texo-stimulated CTLs upregulated the activities of the mTORC1 pathway-related molecules Akt, S6, eIF4E and T-bet, and treatment of the CTLs with an mTORC1 inhibitor, rapamycin, significantly reduced the OVA-Texo-induced increase in CTLs. Interestingly, OVA-Texo-mediated CD40L signaling played a critical role in the observed immunological effects. Importantly, the Gag-Texo vaccine induced Gag-specific therapeutic immunity in chronic infection. Therefore, this study should have a serious impact on the development of new therapeutic vaccines for human immunodeficiency virus (HIV-1) infection.
Assuntos
Neoplasias Experimentais/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Virais/imunologia , Viroses/imunologia , Animais , Ligante de CD40/metabolismo , Antígenos CD8/metabolismo , Doença Crônica , Anergia Clonal , Exossomos/metabolismo , Humanos , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de SinaisRESUMO
Vascular endothelial growth factor (VEGF) plays an important role in the progression of various cancers. The VEGF-specific antibody bevacizumab combined with chemotherapy was shown to significantly improve progression-free survival in certain cancers. However, repeated administration is necessary for effective suppression of VEGF, thereby making the therapy expensive and cumbersome. Thus, it is urgent to develop alternative reagents such as VEGF vaccines. Here we report that DTT-VEGF, a VEGF-based antigen consisting of the receptor-binding domain of VEGF and diphtheria toxin T domain (DTT), not only stimulated neutralizing antibody response, but also induced type 1 immune response as well as anti-tumor cytotoxic T lymphocytes in mice when administered with aluminum hydroxide adjuvant. The antibodies triggered by DTT-VEGF immunization inhibited the binding of VEGF to VEGF receptor and downregulated the serum VEGF levels in tumor-bearing mice. VEGF-specific IgG2a and IgG2b antibodies as well as type 1 cytokines were stimulated by DTT-VEGF vaccination. The splenocytes from DTT-VEGF-immunized mice showed cytotoxic activity against B16-F10 cells expressing VEGF. Extensive necrosis with severe hemorrhage and enhanced CD8+ T cell infiltration were observed in tumors from DTT-VEGF-immunized mice. The percentages of CD31+ vascular areas in the tumor sections from DTT-VEGF-immunized mice were significantly lower than those of control mice. DTT-VEGF significantly inhibited tumor growth in preventive and therapeutic vaccination settings in mouse models. Our data suggest that DTT is an effective antigen carrier to break immune self-tolerance and our vaccine design has potential to be used for human cancer therapy.
Assuntos
Vacinas Anticâncer/imunologia , Toxoide Diftérico/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Vacinas Anticâncer/química , Vacinas Anticâncer/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/terapia , Toxoide Diftérico/química , Toxoide Diftérico/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos , Fator A de Crescimento do Endotélio Vascular/químicaAssuntos
Vacinas contra a AIDS/imunologia , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Exossomos/imunologia , Vacinas contra a AIDS/uso terapêutico , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Vacinas Anticâncer/uso terapêutico , Infecções por HIV/imunologia , Infecções por HIV/terapia , Humanos , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
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.
RESUMO
BACKGROUND: Adoptive transfer of CD8(+) T-cells specific for tumor-antigens is an attractive strategy for anti-tumor therapy. In the present study, the subsets TA and TB were used to represent the population of CD8(+) T cells generated by culturing the respective cells with irradiated dendritic cells (DCs) pulsed with ovalbumin (OVA) protein and transfected with adenoviral vector constructs as described. METHODS: Naïve OVA specific CD8(+) T cells were isolated from the spleen of OVA-specific T-cell receptor transgenic OTI mice. The subsets TA and TB were then generated by in vitro activating the population of CD8(+) T cells with OVA-pulsed DCs transfected with IL-6-expressing adenoviral vector (AdVIL-6 ) or the control vector (AdVNull ). To assess their in vivo immunotherapeutic effects, TA - or TB -cells were intravenously transferred into C57BL/6 mice bearing EG7 thymoma (6-8 mm in diameter). RESULTS: TA -cells displayed a higher level of expression of CD62 l, IL-7R, FasL, perforin and CCR6, and also exhibited more potent in vitro cytotoxicity to OVA-expressing EG7 thymoma cells via perforin- and Fas/FasL-mediated apoptosis than TB -cells. CD8(+) T-cell survival was kinetically analyzed in C57BL/6 mice transferred with TA - or TB -cells by flow cytometry. We found that the adoptively transferred TA -cells had prolonged survival and enhanced T-cell memory development compared to TB -cells. In addition, TA -, but not TB -cells were able to eradicate well-established EG7 thymomas in all eight tumor-bearing mice. CONCLUSIONS: Our data suggest that AdVIL-6 -transfected DC-stimulated CD8(+) T cells with potent cytotoxicity and survival advantage may serve as an effective adoptive CD8(+) T-cell immunotherapy strategy for anti-tumor treatment.