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1.
PLoS One ; 15(7): e0235706, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639988

RESUMO

During type 1 immune responses, CD4 T helper 1 (Th1) cells and CD8 T cells are activated via IL-12 and contribute to the elimination of intracellular pathogens through interferon gamma (IFNγ) production. In this study, we identified Placenta-specific 8 (Plac8) as a gene that is uniquely expressed in Th1 CD4 T cells relative to other CD4 T cell subsets and hypothesized that Plac8 may represent a novel therapeutic target in Th1 CD4 T cells. First, we determined that Plac8 mRNA in CD4 T cells was induced following IL-12 stimulation via an indirect route that required new protein synthesis. Upon evaluating the functional relevance of Plac8 expression in Th1 CD4 T cells, we discovered that Plac8 was important for suppressing IFNγ mRNA and protein production by CD4 T cells 24 hours after IL-12 stimulation, however Plac8 did not contribute to pathogenic CD4 T cell function during two models of intestinal inflammation. We also noted relatively high basal expression of Plac8 in CD8 T cells which could be further induced following IL-12 stimulation in CD8 T cells. Furthermore, Plac8 expression was important for establishing an optimal CD8 T cell response against influenza A virus via a T cell-intrinsic manner. Altogether, these results implicate Plac8 as a potential regulator of Th1 CD4 and CD8 T cell responses during Th1 T cell-driven inflammation.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Interferon gama/metabolismo , Proteínas/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Colite/imunologia , Colite/patologia , Modelos Animais de Doenças , Interferon gama/genética , Interleucina-12/farmacologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Proteínas/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos
2.
Nat Commun ; 11(1): 3421, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647184

RESUMO

The OX40-OX40L pathway provides crucial co-stimulatory signals for CD4 T cell responses, however the precise cellular interactions critical for OX40L provision in vivo and when these occur, remains unclear. Here, we demonstrate that provision of OX40L by dendritic cells (DCs), but not T cells, B cells nor group 3 innate lymphoid cells (ILC3s), is critical specifically for the effector Th1 response to an acute systemic infection with Listeria monocytogenes (Lm). OX40L expression by DCs is regulated by cross-talk with NK cells, with IFNγ signalling to the DC to enhance OX40L in a mechanism conserved in both mouse and human DCs. Strikingly, DC expression of OX40L is redundant in a chronic intestinal Th1 response and expression by ILC3s is necessary. Collectively these data reveal tissue specific compartmentalisation of the cellular provision of OX40L and define a mechanism controlling DC expression of OX40L in vivo.


Assuntos
Microambiente Celular , Ligante OX40/metabolismo , Células Th1/imunologia , Animais , Comunicação Celular , Sinais (Psicologia) , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Interferon gama/biossíntese , Interleucina-12/farmacologia , Intestinos/citologia , Antígeno Ki-1/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Listeria monocytogenes/fisiologia , Camundongos Endogâmicos C57BL , Receptores CXCR5/metabolismo , Receptores OX40/metabolismo , Baço/metabolismo , Regulação para Cima/efeitos dos fármacos
3.
Life Sci ; 254: 117786, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32433918

RESUMO

AIMS: Ovarian cancer (OC) is the most lethal gynecological malignancies and many women develop chemoresistance associated with the inflammatory process. We investigated the effects of P-MAPA and IL-12 on the inflammatory and immune responses in a chemically-induced OC model. MAIN METHODS: OCs were induced with 7,12-dimethylbenz(a)anthracene into the ovarian bursa, and the animals were given P-MAPA (5 mg/kg bw., i.p., twice a week), or IL-12 (300 ng/kg bw., i.p., one a week) for 60 days, or both P-MAPA and IL-12. Immunohistochemistry, western blot, flow cytometry, and multiplex assay were used to examine the effectiveness of immunotherapies in OC. KEY FINDINGS: The combinatory therapy improved the general OC features, reducing inflammatory cells and adipocyte accumulation, in addition to revealing a soft and mobile tissue with no adherences and peritoneal implants. P-MAPA treatment increased the levels of TLR2, TLR4 and TRIF in OCs while decreasing the number of regulatory T (Treg) cells. Additionally, the association of P-MAPA with IL-12 significantly increased the number of CD4+ and CD8+ T effector cells in draining lymph nodes. Regarding the inflammatory mediators, P-MAPA enhanced the levels of the pro-inflammatory cytokine IL-17 while P-MAPA+IL-12 increased the levels of IL-1ß. Treatment with IL-12 enhanced the cytokine levels of IL-17, TNF-α, IL-1ß, and IL-2 in addition to the chemokine MIP-1α. SIGNIFICANCE: We conclude that P-MAPA upregulated TLR2 and TLR4 signaling, possibly activating the non-canonical pathway, while attenuating the tumor immunosuppression. Also, the combination of P-MAPA with IL-12 improves the antitumor immunoresponse, opening a new therapeutic approach for fighting OC.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Interleucina-12/farmacologia , Ácidos Linoleicos/farmacologia , Ácidos Oleicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adipócitos/efeitos dos fármacos , Animais , Linfócitos T CD8-Positivos/metabolismo , Quimiocina CCL3/metabolismo , Citocinas/metabolismo , Sinergismo Farmacológico , Feminino , Inflamação/tratamento farmacológico , Interleucina-12/uso terapêutico , Ácidos Linoleicos/uso terapêutico , Ácidos Oleicos/uso terapêutico , Neoplasias Ovarianas/induzido quimicamente , Neoplasias Ovarianas/metabolismo , Ratos , Linfócitos T Reguladores/efeitos dos fármacos
4.
Nat Biomed Eng ; 4(5): 531-543, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284554

RESUMO

Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in 'immunologically cold' tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however, the administration of IL-12 has been associated with immune-related adverse events. Here we show that, after intravenous administration of a collagen-binding domain fused to IL-12 (CBD-IL-12) in mice bearing aggressive mouse tumours, CBD-IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD-IL-12 induced sustained intratumoural levels of interferon-γ, substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD-IL-12 potently synergized with CPI to eradicate large established melanomas, induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD-IL-12 may potentiate CPI immunotherapy for immunologically cold tumours.


Assuntos
Colágeno/metabolismo , Inflamação/patologia , Interleucina-12/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Imunidade Inata/efeitos dos fármacos , Interleucina-12/farmacologia , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Indução de Remissão , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos
5.
PLoS Negl Trop Dis ; 14(3): e0007813, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196487

RESUMO

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by SFTS virus (SFTSV) infection. Despite a gradual increase of SFTS cases and high mortality in endemic regions, no specific viral therapy nor vaccine is available. Here, we developed a single recombinant plasmid DNA encoding SFTSV genes, Gn and Gc together with NP-NS fusion antigen, as a vaccine candidate. The viral antigens were fused with Fms-like tyrosine kinase-3 ligand (Flt3L) and IL-12 gene was incorporated into the plasmid to enhance cell-mediated immunity. Vaccination with the DNA provides complete protection of IFNAR KO mice upon lethal SFTSV challenge, whereas immunization with a plasmid without IL-12 gene resulted in partial protection. Since we failed to detect antibodies against surface glycoproteins, Gn and Gc, in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific T cell responses, might play major role in protection. Finally, we evaluated the degree of protective immunity provided by protein immunization of the individual glycoprotein, Gn or Gc. Although both protein antigens induced a significant level of neutralizing activity against SFTSV, Gn vaccination resulted in relatively higher neutralizing activity and better protection than Gc vaccination. However, both antigens failed to provide complete protection. Given that DNA vaccines have failed to induce sufficient immunogenicity in human trials when compared to protein vaccines, optimal combinations of DNA and protein elements, proper selection of target antigens, and incorporation of efficient adjuvant, need to be further investigated for SFTSV vaccine development.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos Virais/imunologia , Infecções por Bunyaviridae/prevenção & controle , Interleucina-12/administração & dosagem , Phlebovirus/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Antígenos Virais/genética , Modelos Animais de Doenças , Feminino , Imunidade Celular , Interleucina-12/farmacologia , Camundongos Knockout , Phlebovirus/genética , Plasmídeos/administração & dosagem , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
6.
Cell Immunol ; 349: 104042, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32061376

RESUMO

Cytotoxic T lymphocytes (CTLs) play a major role in cancer immunotherapy. A potent tumor immunotherapy may not only require activation of anti-tumor effector cells but also rely on the use of cytokines to create a controlled environment for the development of anti-tumor T cells. In this study, we fabricated a dual-target immunonanoparticle, e.g. poly(d,l-lactide-co-glycolide) nanoparticle, by loading Interleukin-12 (IL-12) and modifying with CD8 and Glypican-3 antibodies on the surface. Our results demonstrate that the fabricated targeting immunonanoparticles bind specifically to the two target cells of interest, i.e. CD8+ T cells and HepG-2 cells via the antibody-antigen interactions and form T cell-HepG-2 cell clusters, which enhances the cytotoxicity of T cells. IL-12-containing dual-target immunonanoparticles delivered IL-12 specifically to CD8+ T cells, and favored the expansion, activation and cytotoxic activity of CD8+ T lymphocytes. These results suggest that dual-target IL-12-encapsulated nanoparticles are a promising platform for cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Imunoterapia/métodos , Interleucina-12/administração & dosagem , Nanocápsulas/administração & dosagem , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Divisão Celular/efeitos dos fármacos , Citotoxicidade Imunológica , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Células Hep G2 , Humanos , Imunofenotipagem , Testes de Liberação de Interferon-gama , Interleucina-12/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Formação de Roseta , Linfócitos T Citotóxicos/imunologia
7.
BMC Cancer ; 20(1): 26, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31914948

RESUMO

BACKGROUND: Combining anti-cancer therapies with orthogonal modes of action, such as direct cytotoxicity and immunostimulatory, hold promise for expanding clinical benefit to patients with metastatic disease. For instance, a chemotherapy agent Oxaliplatin (OXP) in combination with Interleukin-12 (IL-12) can eliminate pre-existing liver metastatic colorectal cancer and protect from relapse in a murine model. However, the underlying dynamics associated with the targeted biology and the combinatorial space consisting of possible dosage and timing of each therapy present challenges for optimizing treatment regimens. To address some of these challenges, we developed a predictive simulation platform for optimizing dose and timing of the combination therapy involving Mifepristone-induced IL-12 and chemotherapy agent OXP. METHODS: A multi-scale mathematical model comprised of impulsive ordinary differential equations was developed to describe the interaction between the immune system and tumor cells in response to the combined IL-12 and OXP therapy. An ensemble of model parameters were calibrated to published experimental data using a genetic algorithm and used to represent three different phenotypes: responders, partial-responders, and non-responders. RESULTS: The multi-scale model captures tumor growth patterns of the three phenotypic responses observed in mice in response to the combination therapy against a tumor re-challenge and was used to explore the impacts of changing the dose and timing of the mixed immune-chemotherapy on tumor growth subjected to a tumor re-challenge in mice. An increased ratio of CD8 + T effectors to regulatory T cells during and after treatment was key to improve tumor control in the responder cohort. Sensitivity analysis indicates that combined OXP and IL-12 therapy worked more efficiently in responders by increased priming of T cells, enhanced CD8 + T cell-mediated killing, and functional inhibition of regulatory T cells. In a virtual cohort that mimics non-responders and partial-responders, simulations show that an increased dose of OXP alone would improve the response. In addition, enhanced IL-12 expression alone or an increased number of treatment cycles of the mixed immune-chemotherapy can barely improve tumor control for non-responders and partial responders. CONCLUSIONS: Overall, this study illustrates how mechanistic models can be used for in silico screening of the optimal therapeutic dose and timing in combined cancer treatment strategies.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/patologia , Interleucina-12/farmacologia , Neoplasias Hepáticas/secundário , Modelos Teóricos , Oxaliplatina/farmacologia , Algoritmos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cancer Immunol Immunother ; 69(4): 501-512, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31950225

RESUMO

Obinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Interleucina-12/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de IgG/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , MicroRNAs/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Purinas/farmacologia , Quinazolinonas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
9.
Front Immunol ; 10: 2633, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31803181

RESUMO

Natural killer (NK) cells are innate immune cells that interface with the adaptive immune system to generate a pro-inflammatory immune environment. Primary Biliary Cholangitis (PBC) is a hepatic autoimmune disorder with extrahepatic associations including systemic sclerosis, Sjogren's syndrome and thyroiditis. Immunogenetic studies have identified polymorphisms of the IL-12/STAT4 pathway as being associated with PBC. As this pathway is important for NK cell function we investigated NK cells in PBC. Circulating NK cells from individuals with PBC were constitutively activated, with higher levels of CD49a and the liver-homing marker, CXCR6, compared to participants with non-autoimmune chronic liver disease and healthy controls. Stimulation with minimal amounts of IL-12 (0.005 ng/ml) led to significant upregulation of CXCR6 (p < 0.005), and enhanced IFNγ production (p < 0.02) on NK cells from PBC patients compared to individuals with non-autoimmune chronic liver disease, indicating dysregulation of the IL-12/STAT4 axis. In RNAseq studies, resting NK cells from PBC patients had a constitutively activated transcriptional profile and upregulation of genes associated with IL-12/STAT4 signaling and metabolic reprogramming. Consistent with these findings, resting NK cells from PBC patients expressed higher levels of pSTAT4 compared to control groups (p < 0.001 vs. healthy controls and p < 0.05 vs. liver disease controls). In conclusion NK cells in PBC are sensitive to minute quantities of IL-12 and have a "primed" phenotype. We therefore propose that peripheral priming of NK cells to express tissue-homing markers may contribute to the pathophysiology of PBC.


Assuntos
Células Matadoras Naturais/imunologia , Cirrose Hepática Biliar/imunologia , Ativação Linfocitária , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Integrina alfa1/fisiologia , Interleucina-12/farmacologia , Masculino , Pessoa de Meia-Idade , Receptores CXCR6/fisiologia , Fator de Transcrição STAT4/fisiologia
10.
J Immunother Cancer ; 7(1): 154, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208461

RESUMO

BACKGROUND: Although accumulated evidence provides a strong scientific premise for using immune profiles to predict survival in patients with cancer, a universal immune profile across tumor types is still lacking, and how to achieve a survival-associated immune profile remains to be evaluated. METHODS: We analyzed datasets from The Cancer Genome Atlas to identify an immune profile associated with prolonged overall survival in multiple tumor types and tested the efficacy of tumor cell-surface vimentin-targeted interleukin 12 (ttIL-12) in inducing that immune profile and prolonging survival in both mouse and patient-derived xenograft tumor models. RESULTS: We identified an immune profile (IFNγHiCD8HiFOXP3LowCD33Low) associated with prolonged overall survival across several human tumor types. ttIL-12 in combination with surgical resection of the primary tumor transformed tumors to this immune profile. Intriguingly, this immune profile transformation led to inhibition of metastasis and to prolonged survival in both mouse and patient-derived xenograft malignant models. Wild-type IL-12 combined with surgery was significantly less effective. In the IL-12-sensitive C3H mouse strain, in fact, wild-type IL-12 and surgery resulted in shorter overall survival than in mice treated with control pDNA; this surprising result is believed to be attributable to IL-12 toxicity, which was absent in the mice treated with ttIL-12. The ttIL-12-induced immune profile associated with longer overall survival was also associated with a greater accumulation of CD8+ T cells and reduced infiltration of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. The underlying mechanism for this transformation by ttIL-12 treatment was induction of expression of CXCL9 and reduction of expression of CXCL2 and CCL22 in tumors. CONCLUSIONS: ttIL-12 when combined with surgery led to conversion to the IFNγHiCD8HiFOXP3LowCD33Low immune profile, eliminated relapse and metastasis, and prolonged overall survival.


Assuntos
Interleucina-12/farmacologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-12/imunologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Terapia de Alvo Molecular , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Osteossarcoma/genética , Osteossarcoma/imunologia , Osteossarcoma/patologia , Osteossarcoma/terapia , Análise de Sobrevida , Vimentina/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Med ; 8(10): 4753-4765, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31237116

RESUMO

BACKGROUND AIMS: Chimeric antigen receptor T cells (CAR-T cells) have been successfully used in treatments of hematological tumors, however, their anti-tumor activity in solid tumor treatments was limited. As IL-12 increases T-cell immune functions, we designed carcinoembryonic antigen (CEA) specific CAR-T (CEA-CAR-T) cells and, for the first time, used them in combination with recombinant human IL-12 (rhIL-12) to treat several types of solid tumors. METHODS: In vitro anti-tumor activity of CEA-CAR-T cells in combination with rhIL-12 was confirmed by evaluation of CEA-CAR-T cell activation, proliferation, and cytotoxicity after co-incubation with CEA-positive or CEA-negative human tumor cells. In vivo anti-tumor activity of CEA-CAR-T cells in combination with rhIL-12 was confirmed in a xenograft model in nude mice for treatments of several types of solid tumors. RESULTS: In vitro experiments confirmed that rhIL-12 significantly increased the activation, proliferation, and cytotoxicity of CEA-CAR-T cells. Similarly, in vivo experiments found that CEA-CAR-T cells in combination with rhIL-12 had significantly enhanced anti-tumor activity than CEA-CAR-T cells in growth inhibition of newly colonized colorectal cancer cell HT-29, pancreatic cancer cell AsPC-1, and gastric cancer cell MGC803. CONCLUSIONS: These works confirmed that simultaneous use of cytokines, for example, rhIL-12, can increase the anti-tumor activity of CAR-T cells, especially for treatments of several types of solid tumors.


Assuntos
Antígeno Carcinoembrionário/imunologia , Imunoterapia Adotiva/métodos , Interleucina-12/administração & dosagem , Neoplasias/terapia , Receptores de Antígenos Quiméricos/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HT29 , Humanos , Interleucina-12/farmacologia , Camundongos , Camundongos Nus , Neoplasias/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Gene Ther ; 26(7-8): 216-233, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30622324

RESUMO

Interleukin 12 is a promising anti-cancer agent; however, IL12 systemic administration is hampered by side-effects. Although intratumoral administration of IL12 is giving promising results in clinical trials, only a small percentage of patients show a complete therapeutic response. This outcome could be improved by controlling the IL12 expression window. In this work we have tested the efficacy of a self-processing P2A and codon optimized murine IL12 (mIL12Pop) using inflammation-regulated lentivectors in a syngeneic tumor model. Our results show that implantation of cells expressing mIL12Pop employing either the strong constitutive SFFV promoter or a NFkB-based promoter reduced tumor growth, caused CD8+ T cell activation and increased IFNγ production. Importantly, the use of NFkBp-mIL12Pop increased the number of CD8+ TILs and improved the remission rate without increasing IL12-serum concentration. Further experiments suggest that there is a threshold intratumoral IL12 concentration that must be reached to trigger an efficient antitumor response and a limit that once surpassed causes detrimental systemic side effects. Altogether, these results demonstrate that using NFKBp-mIL12Pop significantly increases the overall survival of the mice. In summary, this new inflammation-regulated expression system might be useful for the development of new IL12 delivery systems with improved anti-tumor activity and limited toxicity.


Assuntos
Interleucina-12/uso terapêutico , NF-kappa B/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Interleucina-12/farmacologia , Masculino , Camundongos
13.
J Biol Chem ; 294(12): 4412-4424, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670588

RESUMO

Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC50 value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.


Assuntos
Heparina/farmacologia , Interleucina-12/farmacologia , Animais , Fenômenos Biofísicos , Calorimetria , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Células HEK293 , Heparina/química , Heparitina Sulfato/metabolismo , Humanos , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
14.
Gynecol Oncol ; 153(1): 149-157, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30658847

RESUMO

OBJECTIVE: Natural killer (NK) cells are lymphocytes well suited for adoptive immunotherapy. Attempts with adoptive NK cell immunotherapy against ovarian cancer have proven unsuccessful, with the main limitations including failure to expand and diminished effector function. We investigated if incubation of NK cells with interleukin (IL)-12, IL-15, and IL-18 for 16h could produce cytokine-induced memory-like (CIML) NK cells capable of enhanced function against ovarian cancer. METHODS: NK cells were preactivated briefly with IL-12, IL-15, and IL-18, rested, then placed against ovarian cancer targets to assess phenotype and function via flow cytometry. Real-time NK-cell-mediated tumor-killing was evaluated. Using ascites cells and cell-free ascites fluid, NK cell proliferation and function within the immunosuppressive microenvironment was evaluated in vitro. Finally, CIML NK cells were injected intraperitoneal (IP) into an in vivo xenogeneic mouse model of ovarian cancer. RESULTS: CIML NK cells demonstrate enhanced cytokine (IFN-γ) production and NK-cell-mediated killing of ovarian cancer. NK cells treated overnight with cytokines led to robust activation characterized by temporal shedding of CD16, induction of CD25, and enhanced proliferation. CIML NK cells proliferate more with enhanced effector function compared to controls in an immunosuppressive microenvironment. Finally, human CIML NK cells exhibited potent antitumor effects within a xenogeneic mouse model of ovarian cancer. CONCLUSIONS: CIML NK cells have enhanced functionality and persistence against ovarian cancer in vitro and in vivo, even when exposed to ascites fluid. These findings provide a strategy for NK cell-based immunotherapy to circumvent the immunosuppressive nature of ovarian cancer.


Assuntos
Carcinoma Epitelial do Ovário/terapia , Células Matadoras Induzidas por Citocinas/imunologia , Células Matadoras Induzidas por Citocinas/transplante , Imunoterapia Adotiva/métodos , Interleucinas/farmacologia , Animais , Carcinoma Epitelial do Ovário/imunologia , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Memória Imunológica/imunologia , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-12/farmacologia , Interleucina-15/imunologia , Interleucina-15/farmacologia , Interleucina-18/imunologia , Interleucina-18/farmacologia , Interleucinas/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Eur Cytokine Netw ; 30(4): 160-167, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32096478

RESUMO

OBJECTIVE: Stage II melanoma patients have high risk for regional and distant metastases and may benefit from novel therapeutic strategies. To clarify the role of NK cells in Stage II melanoma, we characterized the cytotoxic activity of NK cells and the expression of various activating and inhibitory receptors in high-risk cutaneous melanoma patients (Stages IIB and IIC) compared to low-risk patients (Stage IA). MATERIALS AND METHODS: Native and cytokine-treated peripheral blood mononuclear cells were used for functional and phenotypical analyses. RESULTS: Compared to Stage IA-B patients, Stage IIB-C patients showed significantly decreased NK cell activity, as well as decreased expression of the activating NKG2D and CD161 receptors, most likely due to increased serum levels of the immunosuppressive cytokine TGF-ß1 in these patients. Interestingly, treatment of periperal blood mononuclear cells with IFN-α, IL-2, IL-12 or the combination of IL-12 and IL-18 significantly induced NK cell activity for both groups of melanoma patients. However, only low-risk patients had a significant increase in the expression of the NKG2D receptor after in vitro treatment with IFN-α, as well as an significant increase in the expression of CD161 after treatment with IFN-α or IL-12. Although IL-2 induced the expression of NKG2D in both groups of patients, this increase was significantly lower in high-risk melanoma. CONCLUSION: NK cell parameters may be useful as biomarkers of disease progression in localized melanoma patients. Our results further suggest that the use of NK cell-activating cytokines in combination with inhibitors of immunosuppressive factors like TGF-ß1 could be a therapeutic option for the treatment of high-risk cutaneous melanoma patients.


Assuntos
Regulação Neoplásica da Expressão Gênica/imunologia , Células Matadoras Naturais/imunologia , Melanoma/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neoplasias Cutâneas/imunologia , Idoso , Citotoxicidade Imunológica , Feminino , Humanos , Interferon-alfa/farmacologia , Interleucina-12/farmacologia , Interleucina-18/farmacologia , Interleucina-2/farmacologia , Células K562 , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Masculino , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Subfamília B de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Estadiamento de Neoplasias , Cultura Primária de Células , Medição de Risco , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia
16.
Cancer Immunol Immunother ; 68(3): 395-405, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30552459

RESUMO

Optimal ex vivo expansion protocols for adoptive cell therapy (ACT) must yield T cells able to effectively home to tumors and survive the inhospitable conditions of the tumor microenvironment (TME), while simultaneously exerting persistent anti-tumor effector functions. Our previous work has shown that ex vivo activation in the presence of IL-12 can induce optimal expansion of murine CD8+ T cells, thus resulting in significant tumor regression after ACT mostly via sustained secretion of IFN-γ. In this report, we further elucidate the mechanism of this potency, showing that IL-12 additionally counteracts the negative regulatory effects of autocrine IFN-γ. IL-12 not only downregulates PD-1 expression by T cells, thus minimizing the effects of IFN-γ-induced PD-L1 upregulation by tumor stromal cells, but also inhibits IFNγR2 expression, thereby protecting T cells from IFN-γ-induced cell death. Thus, the enhanced anti-tumor activity of CD8+ T cells expanded ex vivo in the presence of IL-12 is due not only to the ability of IL-12-stimulated cells to secrete sustained levels of IFN-γ, but also to the additional capacity of IL-12 to counter the negative regulatory effects of autocrine IFN-γ.


Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Interferon gama/fisiologia , Interleucina-12/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Humanos , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interferon/análise , Receptores de Interferon/fisiologia
17.
Hepatol Int ; 13(1): 75-83, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30467624

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is common, but remains difficult to treat. Natural killer (NK) cells are cells of the innate immune system that have potent anti-cancer activity. Recent work has shown that stimulation with IL-12/15/18 leads to the generation of NK cells with enhanced functional and putative "memory" properties. We have investigated the activity of these NK cells against HCC cell lines in vitro and in a mouse model. METHODS: NK cells from healthy donors or individuals with HCC were activated with IL-12/15/18 in vitro and tested for cytotoxic activity against a panel of human HCC cell lines. IL-12/15/18 primed murine NK cells were then infused into a murine model of spontaneously arising HCC to test for anti-tumor activity. RESULTS: NK cells from patients and healthy controls had similar expression levels of activating and inhibitory NK cell receptors. However, proliferation of NK cells from HCC patients was weaker than healthy controls in response to IL-12/15/18 and IL-2 (p < 0.001 at day 9). In vitro, NK cells from both groups of individuals killed HCC targets to similar levels and this was unrelated to NKG2D expression. In a spontaneous model of HCC, IL-12/15/18 activated NK cells trafficked to the liver and resulted in lower levels of spontaneous HCC formation (p < 0.01). CONCLUSION: Cytokine-primed NK cells from patients with HCC have similar levels of activity against HCC cell lines as those from healthy controls. This type of activated NK cell has immunotherapeutic potential against hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/terapia , Imunoterapia/métodos , Células Matadoras Naturais/fisiologia , Neoplasias Hepáticas/terapia , Idoso , Animais , Linhagem Celular Tumoral , Citocinas/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Inata , Interleucina-12/farmacologia , Interleucina-15/farmacologia , Interleucina-18/farmacologia , Masculino , Camundongos Transgênicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo
18.
Cancer Immunol Immunother ; 67(9): 1425-1436, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30019146

RESUMO

There has recently been a paradigm shift in the field of dendritic cell (DC)-based immunotherapy, where several clinical studies have confirmed the feasibility and advantageousness of using directly isolated human blood-derived DCs over in vitro differentiated subsets. There are two major DC subsets found in blood; plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), and both have been tested clinically. CD1c+ mDCs are highly efficient antigen-presenting cells that have the ability to secrete IL-12p70, while pDCs are professional IFN-α-secreting cells that are shown to induce innate immune responses in melanoma patients. Hence, combining mDCs and pDCs poses as an attractive, multi-functional vaccine approach. However, type I IFNs have been reported to inhibit IL-12p70 production and mDC-induced T-cell activation. In this study, we investigate the effect of IFN-α on mDC maturation and function. We demonstrate that both recombinant IFN-α and activated pDCs strongly enhance mDC maturation and increase IL-12p70 production. Co-cultured mDCs and pDCs additionally have beneficial effect on NK and NKT-cell activation and also enhances IFN-γ production by allogeneic T cells. In contrast, the presence of type I IFNs reduces the proliferative T-cell response. The mere presence of a small fraction of activated pDCs is sufficient for these effects and the required ratio between the subsets is non-stringent. Taken together, these results support the usage of mDCs and pDCs combined into one immunotherapeutic vaccine with broad immunostimulatory features.


Assuntos
Células Dendríticas/imunologia , Interferon Tipo I/farmacologia , Interleucina-12/biossíntese , Células Mieloides/imunologia , Antígenos CD1/imunologia , Antígenos CD1/farmacologia , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Glicoproteínas/imunologia , Glicoproteínas/farmacologia , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Interferon alfa-2 , Interferon-alfa/imunologia , Interferon-alfa/farmacologia , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-12/farmacologia , Ativação Linfocitária , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Quinolinas/farmacologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
19.
J Infect Dis ; 218(suppl_5): S519-S527, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30053157

RESUMO

In previous studies, we showed that deoxyribonucleic acid (DNA) vaccines expressing codon-optimized filovirus envelope glycoprotein genes protect mice and nonhuman primates from viral challenge when delivered by intramuscular (IM) electroporation (EP). To determine whether we could achieve equivalent immunogenicity and protective efficacy by a simplified delivery method, we generated DNA vaccine plasmids expressing genetic adjuvants to potentiate immune responses. We tested the Th1-inducing cytokine interleukin-12 and the granulocyte growth factor granulocyte-macrophage colony stimulating factor, both of which have demonstrated significant adjuvant effect when included in clinical DNA vaccine formulations. In addition, because interferon (IFN)-αß is required for DNA vaccine-induced immunity, we tested inclusion of a potent stimulator of the IFN-αß pathway. Our data suggest that IM vaccination of mice with plasmid DNA encoding genetic adjuvants enhances vaccine immunogenicity, resulting in increased anti-Ebola virus (EBOV) immunoglobulin G and T-cell responses. Codelivery of genetic adjuvants also improved EBOV neutralizing capability compared with vaccine alone. Finally, IM vaccination with plasmid EBOV and genetic adjuvants provided complete protection against EBOV challenge. Overall, our data suggest that codelivery of genetic adjuvants with filovirus DNA vaccines using IM delivery can provide comparable efficacy to the same DNA vaccines when delivered using IM-EP devices.


Assuntos
Vacinas contra Ebola/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-12/farmacologia , Vacinas de DNA/imunologia , Animais , Células COS , Chlorocebus aethiops , Vacinas contra Ebola/administração & dosagem , Eletroporação , Feminino , Glicoproteínas/genética , Imunogenicidade da Vacina , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Vacinas de DNA/administração & dosagem
20.
Ann Rheum Dis ; 77(7): 1070-1077, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29475858

RESUMO

OBJECTIVES: Genetic variants in the transcription factor STAT4 are associated with increased susceptibility to systemic lupus erythematosus (SLE) and a more severe disease phenotype. This study aimed to clarify how the SLE-associated intronic STAT4 risk allele rs7574865[T] affects the function of immune cells in SLE. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 52 genotyped patients with SLE. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) in response to interferon (IFN)-α, IFN-γ or interleukin (IL)-12, total levels of STAT4, STAT1 and T-bet, and frequency of IFN-γ+ cells on IL-12 stimulation were determined by flow cytometry in subsets of immune cells before and after preactivation of cells with phytohaemagglutinin (PHA) and IL-2. Cellular responses and phenotypes were correlated to STAT4 risk allele carriership. Janus kinase inhibitors (JAKi) selective for TYK2 (TYK2i) or JAK2 (JAK2i) were evaluated for inhibition of IL-12 or IFN-γ-induced activation of SLE PBMCs. RESULTS: In resting PBMCs, the STAT4 risk allele was neither associated with total levels of STAT4 or STAT1, nor cytokine-induced pSTAT4 or pSTAT1. Following PHA/IL-2 activation, CD8+ T cells from STAT4 risk allele carriers displayed increased levels of STAT4 resulting in increased pSTAT4 in response to IL-12 and IFN-α, and an augmented IL-12-induced IFN-γ production in CD8+ and CD4+ T cells. The TYK2i and the JAK2i efficiently blocked IL-12 and IFN-γ-induced activation of PBMCs from STAT4 risk patients, respectively. CONCLUSIONS: T cells from patients with SLE carrying the STAT4 risk allele rs7574865[T] display an augmented response to IL-12 and IFN-α. This subset of patients may benefit from JAKi treatment.


Assuntos
Regulação da Expressão Gênica , Interleucina-12/farmacologia , Lúpus Eritematoso Sistêmico/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT4/genética , Adulto , Alelos , Células Cultivadas , Feminino , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Masculino , Fosforilação , Sensibilidade e Especificidade , Transdução de Sinais
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