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1.
Nat Med ; 25(7): 1073-1081, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270502

RESUMO

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5-8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/-) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.


Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Feminino , Antígenos de Histocompatibilidade , Humanos , Camundongos , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologia
2.
J Immunol ; 203(3): 696-704, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31209101

RESUMO

Multiple sclerosis (MS) is a chronic autoimmune disease with no curative treatment. The immune regulatory properties of type I IFNs have led to the approval of IFN-ß for the treatment of relapsing-remitting MS. However, there is still an unmet need to improve the tolerability and efficacy of this therapy. In this work, we evaluated the sustained delivery of IFN-α1, either alone or fused to apolipoprotein A-1 by means of an adeno-associated viral (AAV) system in the mouse model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. These in vivo experiments demonstrated the prophylactic and therapeutic efficacy of the AAV-IFN-α or AAV-IFN-α fused to apolipoprotein A-1 vectors in experimental autoimmune encephalomyelitis, even at low doses devoid of hematological or neurologic toxicity. The sustained delivery of such low-dose IFN-α resulted in immunomodulatory effects, consisting of proinflammatory monocyte and T regulatory cell expansion. Moreover, encephalitogenic T lymphocytes from IFN-α-treated mice re-exposed to the myelin oligodendrocyte glycoprotein peptide in vitro showed a reduced proliferative response and cytokine (IL-17A and IFN-γ) production, in addition to upregulation of immunosuppressive molecules, such as IL-10, IDO, or PD-1. In conclusion, the results of the present work support the potential of sustained delivery of low-dose IFN-α for the treatment of MS and likely other T cell-dependent chronic autoimmune disorders.

3.
J Vasc Interv Radiol ; 30(7): 1098-1105, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31101416

RESUMO

PURPOSE: To evaluate the therapeutic efficacy of irreversible electroporation (IRE) combined with the intratumoral injection of the immunogenic adjuvant poly-ICLC (polyinosinic-polycytidylic acid and poly-L-lysine, a dsRNA analog mimicking viral RNA) inmediately before IRE. MATERIALS AND METHODS: Mice and rabbits bearing hepatocellular carcinoma tumors (Hepa.129 and VX2 tumor models, respectively) were treated with IRE (2 pulses of 2500V), with poly-ICLC, or with IRE + poly-ICLC combination therapy. Tumor growth in mice was monitored using a digital caliper and by computed tomography in rabbits. RESULTS: Intratumoral administration of poly-ICLC immediately before IRE elicited shrinkage of Hepa.129 cell-derived tumors in 70% of mice, compared to 30% and 26% by poly-ICLC or IRE alone, respectively (P = .0004). This combined therapy induced the shrinkage of VX-2-based hepatocellular carcinoma tumors in 40% of rabbits, whereas no response was achieved by either individual treatment (P = .045). The combined therapy activated a systemic antitumor response able to inhibit the growth of other untreated tumors. CONCLUSIONS: IRE treatment, immediately preceded by the intratumoral administration of an immunogenic adjuvant such as poly-ICLC, might enhance the antitumor effect of the IRE procedure. This combination might facilitate the induction of a long-term systemic response to prevent tumor relapses and the appearance of metastases.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Carboximetilcelulose Sódica/análogos & derivados , Carcinoma Hepatocelular/terapia , Eletroporação/métodos , Neoplasias Hepáticas Experimentais/terapia , Poli I-C/administração & dosagem , Polilisina/análogos & derivados , Animais , Carboximetilcelulose Sódica/administração & dosagem , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Injeções Intralesionais , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/patologia , Camundongos Endogâmicos C3H , Polilisina/administração & dosagem , Coelhos , Carga Tumoral
4.
Blood ; 133(22): 2401-2412, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-30975638

RESUMO

Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-κB activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-κB and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-κB-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.


Assuntos
Linfócitos B/imunologia , Antígeno B7-H1/imunologia , Regulação Neoplásica da Expressão Gênica , Ativação Linfocitária , Linfoma Difuso de Grandes Células B/imunologia , Receptor de Morte Celular Programada 1/imunologia , Evasão Tumoral , Proteína Supressora de Tumor p53/imunologia , Animais , Linfócitos B/patologia , Antígeno B7-H1/genética , Feminino , Humanos , Imunoterapia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/terapia , Masculino , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/genética , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína Supressora de Tumor p53/genética
5.
Oncoimmunology ; 7(4): e1409321, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29632721

RESUMO

Tumor infiltrating lymphocytes have been associated with a better prognostic and with higher response rates in patients treated with checkpoint inhibiting antibodies, suggesting that strategies promoting tumor inflammation may enhance the efficacy of these currently available therapies. Our aim was thus to develop a new vaccination platform based on cold-inducible RNA binding protein (CIRP), an endogenous TLR4 ligand generated during inflammatory processes, and characterize whether it was amenable to combination with checkpoint inhibitors. In vitro, CIRP induced dendritic cell activation, migration and enhanced presentation of CIRP-bound antigens to T-cells. Accordingly, antigen conjugation to CIRP conferred immunogenicity, dependent on immunostimulatory and antigen-targeting capacities of CIRP. When applied in a therapeutic setting, vaccination led to CD8-dependent tumor rejection in several tumor models. Moreover, immunogenicity of this vaccination platform was enhanced not only by combination with additional adjuvants, but also with antibodies blocking PD-1/PD-L1, CTLA-4 and IL-10, immunosuppressive molecules usually present in the tumor environment and also induced by the vaccine. Therefore, priming with a CIRP-based vaccine combined with immune checkpoint-inhibiting antibodies rejected established B16-OVA tumors. Finally, equivalent activation and T-cell stimulatory effects were observed when using CIRP in vitro with human cells, suggesting that CIRP-based vaccination strategies could be a valuable clinical tool to include in combinatorial immunotherapeutic strategies in cancer patients.

6.
Front Immunol ; 9: 68, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29422905

RESUMO

A complex network of interactions exists between the immune, the olfactory, and the central nervous system (CNS). Inhalation of different fragrances can affect immunological reactions in response to an antigen but also may have effects on the CNS and cognitive activity. We performed an exploratory study of the immunomodulatory ability of a series of compounds representing each of the 10 odor categories or clusters described previously. We evaluated the impact of each particular odor on the immune response after immunization with the model antigen ovalbumin in combination with the TLR3 agonist poly I:C. We found that some odors behave as immunostimulatory agents, whereas others might be considered as potential immunosuppressant odors. Interestingly, the immunomodulatory capacity was, in some cases, strain-specific. In particular, one of the fragrances, carvone, was found to be immunostimulatory in BALB/c mice and immunosuppressive in C57BL/6J mice, facilitating or impairing viral clearance, respectively, in a model of a viral infection with a recombinant adenovirus. Importantly, inhalation of the odor improved the memory capacity in BALB/c mice in a fear-conditioning test, while it impaired this same capacity in C57BL/6J mice. The improvement in memory capacity in BALB/c was associated with higher CD3+ T cell infiltration into the hippocampus and increased local expression of mRNA coding for IL-1ß, TNF-α, and IL-6 cytokines. In contrast, the memory impairment in C57BL/6 was associated with a reduction in CD3 numbers and an increase in IFN-γ. These data suggest an association between the immunomodulatory capacity of smells and their impact on the cognitive functions of the animals. These results highlight the potential of studying odors as therapeutic agents for CNS-related diseases.


Assuntos
Medo/psicologia , Fatores Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Memória/efeitos dos fármacos , Monoterpenos/farmacologia , Administração por Inalação , Animais , Cognição , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Perfilação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Fatores Imunológicos/administração & dosagem , Imunomodulação/genética , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monoterpenos/administração & dosagem , Odorantes , Viroses/etiologia
7.
Haematologica ; 103(6): 1065-1072, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29191842

RESUMO

Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears to be a promising therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-/HCO3- anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic AE2 targeting peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies.


Assuntos
Antineoplásicos/farmacologia , Antiportadores de Cloreto-Bicarbonato/antagonistas & inibidores , Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Peptídeos/farmacologia , Animais , Ânions/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/patologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Oncotarget ; 8(42): 71709-71724, 2017 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-29069740

RESUMO

Although T regulatory cells (Treg) are essential for the prevention of autoimmune diseases, their immunoregulatory function restrains the induction of immune responses against cancer. Thus, development of inhibitors of FOXP3, a key transcription factor for the immunosuppressive activity of Treg, might give new therapeutic opportunities. In a previous work we identified a peptide (named P60) able to enter into the cells, bind to FOXP3, and impair Treg activity in vitro and in vivo. Here we show that P60 binds to the intermediate region of FOXP3 and inhibits its homodimerization as well as its interaction with the transcription factor AML1. Alanine-scanning of P60 revealed the relevance of each position on FOXP3 binding, homodimerization, association with AML1 and inhibition of Treg activity. Introduction of alanine at positions 2, 5 and 11 improved the activity of the original P60, whereas alanine mutations at positions 1, 7, 8, 9, 10 and 12 were detrimental. Multiple mutation experiments allowed us to identify peptides with higher FOXP3 binding affinity and stronger biological activity than the original P60. Head to tail macrocyclization of peptide P60-D2A-S5A improved Treg inhibition and enhanced anti-tumor activity of anti-PD1 antibodies in a model of hepatocellular carcinoma. Introduction of a D-aminoacid at position 2 augmented significantly microsomal stability while maintained FOXP3 binding capacity and Treg inhibition in vitro. In vivo, when combined with the cytotoxic T-cell epitope AH1, it induced protection against CT26 tumor implantation. This study provides important structure-function relationships essential for further drug design to inhibit Treg cells in cancer.

9.
PLoS One ; 12(9): e0185169, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28934318

RESUMO

LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.


Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Sequência Conservada , Técnica de Seleção de Aptâmeros , Motivos de Aminoácidos , Animais , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Ativação Linfocitária , Camundongos
10.
Breast Cancer Res Treat ; 166(2): 393-405, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28756536

RESUMO

PURPOSE: Regulatory T cells (Tregs) impair the clinical benefit of cancer immunotherapy. To optimize the antitumor efficacy of therapeutic dendritic cell (DC) vaccines, we aimed to inhibit Foxp3, a transcription factor required for Treg function. METHODS: Mice bearing established syngeneic LM3 and 4T1 breast tumors were treated with antitumor DC vaccines and a synthetic peptide (P60) that has been shown to inhibit Foxp3. RESULTS: Treatment with P60 improved the therapeutic efficacy of DC vaccines in these experimental models. In addition, monotherapy with P60 inhibited tumor growth in immunocompetent as well as in immuno-compromised animals bearing established tumors. We found expression of Foxp3 in human and murine breast tumor cells. P60 inhibited IL-10 secretion in breast cancer cells that expressed Foxp3. CONCLUSIONS: Our results suggest that Foxp3 blockade improves the therapeutic efficacy of DC vaccines by inhibition of Tregs and through a direct antitumor effect. This strategy could prove useful to neutralize the immunosuppressive microenvironment and to boost antitumor immunity in breast cancer.


Assuntos
Neoplasias da Mama/terapia , Peptídeos Penetradores de Células/administração & dosagem , Células Dendríticas/transplante , Fatores de Transcrição Forkhead/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/farmacologia , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacologia , Células Dendríticas/imunologia , Feminino , Humanos , Imunoterapia , Camundongos , Linfócitos T Reguladores/imunologia , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Front Immunol ; 8: 594, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28611770

RESUMO

The CD6 lymphocyte receptor has been involved in the pathophysiology of different autoimmune disorders and is now considered a feasible target for their treatment. In vitro data show the relevance of CD6 in the stabilization of adhesive contacts between T-cell and antigen-presenting cells, and the modulation of T-cell receptor signals. However, the in vivo consequences of such a function are yet undisclosed due to the lack of suitable genetically modified animal models. Here, the in vitro and in vivo challenge of CD6-deficient (CD6-/-) cells with allogeneic cells was used as an approach to explore the role of CD6 in immune responses under relative physiological stimulatory conditions. Mixed lymphocyte reaction (MLR) assays showed lower proliferative responses of splenocytes from CD6-/- mice together with higher induction of regulatory T cells (Treg, CD4+CD25+FoxP3+) with low suppressive activity on T and B-cell proliferation. In line with these results, CD6-/- mice undergoing a lupus-like disorder induced by chronic graft-versus-host disease (cGvHD) showed higher serum titers of anti-double-stranded DNA and nucleosome autoantibodies. This occurred together with reduced splenomegaly, which was associated with lower in vivo bromodesoxyuridine incorporation of spleen cells and with increased percentages of spleen follicular B cells (B2, CD21+CD23hi) and Treg cells. Interestingly, functional analysis of in vivo-generated CD6-/- Treg cells exhibited defective suppressive activity. In conclusion, the data from MLR and cGvHD-induced lupus-like models in CD6-/- mice illustrate the relevance of CD6 in T (and B) cell proliferative responses and, even more importantly, Treg induction and suppressive function in the in vivo maintenance of peripheral tolerance.

12.
Nat Commun ; 8: 15424, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28548080

RESUMO

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.


Assuntos
Antineoplásicos/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cristalografia por Raios X , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interferons/imunologia , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cancer Res ; 77(13): 3672-3684, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28522749

RESUMO

Recent studies have found that tumor-infiltrating lymphocytes (TIL) expressing PD-1 can recognize autologous tumor cells, suggesting that cells derived from PD-1+ TILs can be used in adoptive T-cell therapy (ACT). However, no study thus far has evaluated the antitumor activity of PD-1-selected TILs in vivo In two mouse models of solid tumors, we show that PD-1 allows identification and isolation of tumor-specific TILs without previous knowledge of their antigen specificities. Importantly, despite the high proportion of tumor-reactive T cells present in bulk CD8 TILs before expansion, only T-cell products derived from sorted PD-1+, but not from PD-1- or bulk CD8 TILs, specifically recognized tumor cells. The fold expansion of PD-1+ CD8 TILs was 10 times lower than that of PD-1- cells, suggesting that outgrowth of PD-1- cells was the limiting factor in the tumor specificity of cells derived from bulk CD8 TILs. The highly differentiated state of PD-1+ cells was likely the main cause hampering ex vivo expansion of this subset. Moreover, PD-1 precisely identified marrow-infiltrating, myeloma-specific T cells in a mouse model of multiple myeloma. In vivo, only cells expanded from PD-1+ CD8 TILs contained tumor progression, and their efficacy was enhanced by PDL-1 blockade. Overall, our data provide a rationale for the use of PD-1-selected TILs in ACT. Cancer Res; 77(13); 3672-84. ©2017 AACR.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Receptor de Morte Celular Programada 1/biossíntese , Animais , Linhagem Celular Tumoral , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia
14.
Diabetes ; 66(2): 448-459, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28108611

RESUMO

The introduction of ß-cell autoantigens via the gut through Lactococcus lactis (L. lactis) has been demonstrated to be a promising approach for diabetes reversal in NOD mice. Here we show that a combination therapy of low-dose anti-CD3 with a clinical-grade self-containing L. lactis, appropriate for human application, secreting human proinsulin and interleukin-10, cured 66% of mice with new-onset diabetes, which is comparable to therapy results with plasmid-driven L. lactis Initial blood glucose concentrations (<350 mg/dL) and insulin autoantibody positivity were predictors of the stable reversal of hyperglycemia, and decline in insulin autoantibody positivity was an immune biomarker of therapeutic outcome. The assessment of the immune changes induced by the L. lactis-based therapy revealed elevated frequencies of CD4+Foxp3+ T cells in the pancreas-draining lymph nodes, pancreas, and peripheral blood of all treated mice, independent of metabolic outcome. Neutralization of cytotoxic T-lymphocyte antigen 4 and transforming growth factor-ß partially abrogated the suppressive function of therapy-induced regulatory T cells (Tregs). Ablation or functional impairment of Foxp3+ Tregs in vivo at the start or stop of therapy impaired immune tolerance, highlighting the dependence of the therapy-induced tolerance in mice with new-onset diabetes on the presence and functionality of CD4+Foxp3+ T cells. Biomarkers identified in this study can potentially be used in the future to tailor the L. lactis-based combination therapy for individual patients.


Assuntos
Anticorpos/farmacologia , Glicemia/efeitos dos fármacos , Diabetes Mellitus/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Interleucina-10/metabolismo , Lactobacillus/metabolismo , Proinsulina/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/farmacologia , Glicemia/metabolismo , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Antígeno CTLA-4/efeitos dos fármacos , Antígeno CTLA-4/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/imunologia , Teste de Tolerância a Glucose , Tolerância Imunológica/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/efeitos dos fármacos , Fator de Crescimento Transformador beta/imunologia
15.
PLoS One ; 11(8): e0161216, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27537839

RESUMO

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. C-di-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine.


Assuntos
Proteínas de Bactérias/fisiologia , GMP Cíclico/análogos & derivados , Salmonelose Animal/prevenção & controle , Vacinas contra Salmonella/uso terapêutico , Salmonella typhimurium , Fator sigma/fisiologia , Administração Oral , Animais , GMP Cíclico/deficiência , Feminino , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-17/sangue , Interleucina-2/sangue , Camundongos , Camundongos Endogâmicos BALB C , Salmonelose Animal/imunologia , Vacinas contra Salmonella/administração & dosagem , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/imunologia , Fator de Necrose Tumoral alfa/sangue , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/uso terapêutico
16.
Cancer Res ; 76(10): 2863-7, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27308833

RESUMO

The recent approval by the FDA of the combination of anti-CTLA4 and anti-PD-1 mAbs for the treatment of BRAF-unmutated unresectable or metastatic melanoma is a landmark for the development of cancer immunotherapy. On October 18 to 22, 2015, a symposium was held in Pamplona (Spain) to present and discuss the basic and clinical discoveries that have brought us to this milestone and to explore other targets and immunotherapy strategies aimed at attaining more efficacious oncology practice in the short term. Cancer Res; 76(10); 2863-7. ©2016 AACR.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno CTLA-4/imunologia , Imunomodulação/efeitos dos fármacos , Imunoterapia , Melanoma/terapia , Receptor de Morte Celular Programada 1/imunologia , Animais , Antígeno CTLA-4/antagonistas & inibidores , Humanos , Melanoma/imunologia , Melanoma/secundário , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Relatório de Pesquisa
17.
Oncoimmunology ; 5(2): e1075113, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27057445

RESUMO

Blocking antibodies against immunosuppressive molecules have shown promising results in cancer patients. However, there are not enough data to define those conditions dictating treatment efficacy. In this scenario, IL-10 is a cytokine with controversial effects on tumor growth. Thus, our aim was to characterize in which setting IL-10 blockade may potentiate the beneficial effects of a therapeutic vaccine In the IL-10-expressing B16-OVA and TC-1 P3 (A15) tumor models, therapeutic vaccination with tumor antigens plus the TLR7 ligand Imiquimod increased IL-10 production. Although blockade of IL-10 signal with anti-IL-10R antibodies did not inhibit tumor growth, when combined with vaccination it enhanced tumor rejection, associated with stronger innate and adaptive immune responses. Interestingly, a similar enhancement on immune responses was observed after simultaneous vaccination and IL-10 blockade in naive mice. However, when using vaccines containing as adjuvants the TLR3 ligand poly(I:C) or anti-CD40 agonistic antibodies, despite tumor IL-10 expression, anti-IL-10R antibodies did not provide any beneficial effect on tumor growth and antitumor immune responses. Of note, as opposed to Imiquimod, vaccination with this type of adjuvants did not induce IL-10 and correlated with a lack of in vitro IL-10 production by dendritic cells (DC). Finally, in B16-OVA-bearing mice, blockade of IL-10 during therapeutic vaccination with a multiple adjuvant combination (MAC) with potent immunostimulatory properties but still inducing IL-10 led to superior antitumor immunity and complete tumor rejection. These results suggest that for therapeutic antitumor vaccination, blockade of vaccine-induced IL-10 is more relevant than tumor-associated IL-10.

18.
Oncotarget ; 7(17): 23182-96, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-26992239

RESUMO

In this work we show a clinically feasible strategy to convert in situ the own tumor into an endogenous vaccine by coating the melanoma cancerous cells with CD28 costimulatory ligands. This therapeutic approach is aimed at targeting T-cell costimulation to chemotherapy-resistant tumors which are refractory and been considered as untreatable cancers. These tumors are usually defined by an enrichment of cancer stem cells and characterized by the higher expression of chemotherapy-resistant proteins. In this work we develop the first aptamer that targets chemotherapy-resistant tumors expressing MRP1 through a novel combinatorial peptide-cell SELEX. With the use of the MRP1 aptamer we engineer a MRP1-CD28 bivalent aptamer that is able to bind MRP1-expressing tumors and deliver the CD28 costimulatory signal to tumor-infiltrating lymphocytes. The bi-specific aptamer is able to enhance costimulation in chemotherapy-resistant tumors. Melanoma-bearing mice systemically treated with MRP1-CD28 bivalent aptamer show reduced growth, thus proving an improved mice survival.Besides, we have designed a technically feasible and translational whole-cell vaccine (Aptvax). Disaggregated cells from tumors can be directly decorated with costimulatory ligand aptamers to generate the vaccine Aptvax. CD28Aptvax made of irradiated tumor cells coated with the CD28-agonistic aptamer attached to MRP1 elicits a strong tumor- cell immune response against melanoma tumors reducing tumor growth.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Antígenos CD28/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Células-Tronco Neoplásicas/imunologia , Animais , Apoptose , Antígenos CD28/genética , Proliferação de Células , Feminino , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células Tumorais Cultivadas
19.
Biomaterials ; 91: 73-80, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26999456

RESUMO

The specific inhibition of Treg function has long been a major technical challenge in cancer immunotherapy. So far no single cell-surface marker has been identified that could be used to distinguish Treg cells from other lymphocytes. The only available specific marker mostly expressed in Treg is Foxp3, which is an intracellular transcription factor. A targeting molecule able to penetrate the membrane and inhibit Foxp3 within the cell is needed. P60-peptide is able to do that, but due to lack of target specificity, the doses are extremely high. In this study we have shown as a proof of concept that P60 Foxp3 inhibitor peptide can be conjugated with a CD28 targeting aptamer to deliver the peptide to CD28-expressing cells. The AptCD28-P60 construct is a clinically feasible reagent that improves the efficacy of the unconjugated P60 peptide very significantly. This approach was used to inhibit Treg function in a vaccination context, and it has shown a significant improvement in the induced immune response, entailing a lower tumor load in an antigen-specific cancer vaccine protocol.


Assuntos
Vacinas Anticâncer/administração & dosagem , Fatores de Transcrição Forkhead/antagonistas & inibidores , Neoplasias/prevenção & controle , Peptídeos/administração & dosagem , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Animais , Aptâmeros de Nucleotídeos/imunologia , Antígenos CD28/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Fatores de Transcrição Forkhead/imunologia , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/patologia , Peptídeos/imunologia , Peptídeos/uso terapêutico , Linfócitos T Reguladores/patologia , Vacinação
20.
J Med Virol ; 88(5): 843-51, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26447929

RESUMO

Viral clearance during acute hepatitis C virus (HCV) infection is associated with the induction of potent antiviral T-cell responses. Since dendritic cells (DC) are essential in the activation of primary T-cell responses, gene expression was analyzed in DC from patients during acute HCV infection. By using microarrays, gene expression was compared in resting and activated peripheral blood plasmacytoid (pDC) and myeloid (mDC) DC from acute HCV resolving patients (AR) and from patients who become chronically infected (ANR), as well as in healthy individuals (CTRL) and chronically-infected patients (CHR). For pDC, a high number of upregulated genes was found in AR patients, irrespective of DC stimulation. However, for mDC, most evident differences were detected after DC stimulation, again corresponding to upregulated genes in AR patients. Divergent behavior of ANR was also observed when analyzing DC from CTRL and CHR, with ANR patients clustering again apart from these groups. These differences corresponded to metabolism-associated genes and genes belonging to pathways relevant for DC activation and cytokine responses. Thus, upregulation of relevant genes in DC during acute HCV infection may determine viral clearance, suggesting that dysfunctional DC may be responsible for the lack of efficient T-cell responses which lead to chronic HCV infection.


Assuntos
Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Hepatite C/imunologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade
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