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Cytotoxic T lymphocytes (CTLs) play a crucial role in cancer rejection. However, CTLs encounter dysfunction and exhaustion in the immunosuppressive tumor microenvironment (TME). Although the reactive oxygen species (ROS)-rich TME attenuates CTL function, the underlying molecular mechanism remains poorly understood. The nuclear factor erythroid 2-related 2 (Nrf2) is the ROS-responsible factor implicated in increasing susceptibility to cancer progression. Therefore, we examined how Nrf2 is involved in anti-tumor responses of CD8+ T and chimeric antigen receptor (CAR) T cells in the ROS-rich TME. Here, we demonstrated that tumor growth in Nrf2-/- mice was significantly controlled and was reversed by T cell depletion and further confirmed that Nrf2 deficiency in T cells promotes anti-tumor responses using an adoptive transfer model of antigen-specific CD8+ T cells. Nrf2-deficient CTLs are resistant to ROS, and their effector functions are sustained in the TME. Furthermore, Nrf2 knockdown in human CAR-T cells enhanced the survival and function of intratumoral CAR-T cells in a solid tumor xenograft model and effectively controlled tumor growth. ROS-sensing Nrf2 inhibits the anti-tumor T cell responses, indicating that Nrf2 may be a potential target for T cell immunotherapy strategies against solid tumors.
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INTRODUCTION: Cytokines of the common γ chain (γc) family are critical for the development, differentiation, and survival of T lineage cells. Cytokines play key roles in immunodeficiencies, autoimmune diseases, allergies, and cancer. Although γc is considered an assistant receptor to transmit cytokine signals and is an indispensable receptor in the immune system, its regulatory mechanism is not yet well understood. OBJECTIVE: This study focused on the molecular mechanisms that γc expression in T cells is regulated under T cell receptor (TCR) stimulation. METHODS: The γc expression in TCR-stimulated T cells was determined by flow cytometry, western blot and quantitative RT-PCR. The regulatory mechanism of γc expression in activated T cells was examined by promoter-luciferase assay and chromatin immunoprecipitation assays. NFAT1 and NFκB deficient cells generated using CRISPR-Cas9 and specific inhibitors were used to examine their role in regulation of γc expression. Specific binding motif was confirmed by γc promotor mutant cells generated using CRISPR-Cas9. IL-7TgγcTg mice were used to examine regulatory role of γc in cytokine signaling. RESULTS: We found that activated T cells significantly upregulated γc expression, wherein NFAT1 and NFκB were key in transcriptional upregulation via T cell receptor stimulation. Also, we identified the functional binding site of the γc promoter and the synergistic effect of NFAT1 and NFκB in the regulation of γc expression. Increased γc expression inhibited IL-7 signaling and rescued lymphoproliferative disorder in an IL-7Tg animal model, providing novel insights into T cell homeostasis. CONCLUSION: Our results indicate functional cooperation between NFAT1 and NFκB in upregulating γc expression in activated T cells. As γc expression also regulates γc cytokine responsiveness, our study suggests that γc expression should be considered as one of the regulators in γc cytokine signaling and the development of T cell immunotherapies. Video Abstract.
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Receptores de Citocinas , Linfócitos T , Animais , Camundongos , Citocinas , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , HumanosRESUMO
Gut microbiota was only considered as a commensal organism that aids in digestion, but recent studies revealed that the microbiome play a critical role in both physiological and pathological immune system. The gut microbiome composition is altered by environmental factors such as diet and hygiene, and the alteration affects immune cells, especially T cells. Advanced genomic techniques in microbiome research defined that specific microbes regulate T cell responses and the pathogenesis of immune-mediated disorders. Here, we review features of specific microbes-T cell crosstalk and relationship between the microbes and immunopathogenesis of diseases including in cancers, autoimmune disorders and allergic inflammations. We also discuss the limitations of current experimental animal models, cutting-edge developments and current challenges to overcome in the field, and the possibility of considering gut microbiome in the development of new drug.
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Doenças Autoimunes , Microbioma Gastrointestinal , Microbiota , Animais , Microbioma Gastrointestinal/fisiologia , Linfócitos T , InflamaçãoRESUMO
Aminoacyl-tRNA synthetases (ARSs) are emerging as important regulators in various immune diseases; however, their roles in immune cells remain unclear. In this study, using alanyl-tRNA synthetase (AARS) mutant (sti) mice with neurodegenerative disorder, we investigated the effect of translational fidelity in immune cells. Dysfunctional AARS caused disorders in immune cell responses and cellularity. The impairment was caused by dampened TCR signaling than cytokine signaling. Therefore, sti mutant inhibits TCR signaling, impeding T cell survival and responses. B cell numbers were decreased in sti mice. Despite low B cell cellularity, serum IgM, IgA, and IgE levels were higher in sti mice than in wild-type mice. Misacylation of ARS and the consequent translational infidelity induce disturbances in signaling pathways critical for immune cell survival and responses. Our findings provide a novel mechanism by which translational fidelity might play a critical role in cellular and humoral immune responses.
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Aminoacil-tRNA Sintetases/genética , Linfócitos B/imunologia , Linfócitos T/imunologia , Aminoacil-tRNA Sintetases/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocinas/farmacologia , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imunoglobulina A/sangue , Imunoglobulina E/sangue , Imunoglobulina M/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Novel engineered T cells containing chimeric antigen receptors (CAR-T cells) that combine the benefits of antigen recognition and T cell response have been developed, and their effect in the anti-tumor immunotherapy of patients with relapsed/refractory leukemia has been dramatic. Thus, CAR-T cell immunotherapy is rapidly emerging as a new therapy. However, it has limitations that prevent consistency in therapeutic effects in solid tumors, which accounts for over 90% of all cancer patients. Here, we review the literature regarding various obstacles to CAR-T cell immunotherapy for solid tumors, including those that cause CAR-T cell dysfunction in the immunosuppressive tumor microenvironment, such as reactive oxygen species, pH, O2, immunosuppressive cells, cytokines, and metabolites, as well as those that impair cell trafficking into the tumor microenvironment. Next-generation CAR-T cell therapy is currently undergoing clinical trials to overcome these challenges. Therefore, novel approaches to address the challenges faced by CAR-T cell immunotherapy in solid tumors are also discussed here.
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Processamento Alternativo/genética , Subunidade alfa de Receptor de Interleucina-7/genética , Linfócitos T/imunologia , Animais , Sequência de Bases , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SolubilidadeAssuntos
Linfócitos T CD8-Positivos/metabolismo , Imunidade Inata/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/genética , Animais , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Subpopulações de Linfócitos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismoRESUMO
OBJECTIVES: Erlotinib, a tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), has been shown to have a dramatic effect in non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, the presence of primary resistance or acquired resistance to EGFR-TKI is the most common reason for switching to other anti-cancer agents. Even though there are newer agents that have activity in the presence of the T790M mutation, identification of potential agents that could overcome resistance to EGFR-TKI is still needed for the treatment of NSCLC patients. MATERIALS AND METHODS: In this study, we used erlotinib-resistant NSCLC cell lines to investigate the effects of combination treatment with erlotinib and ampelopsin. After treatment with either single or combination, cell viability and cell death were determined with WST-1 assay, trypan blue exclusion method, colony forming assay, annexin-V staining assay and western blot assay. The content of ROS was evaluated by FACS analysis using H2DCF-staining method. To determine the effect of Nox2 and Bim on the combined treatment with erlotinib and ampelopsin-induced cell death, we transfected with Nox2 or Bim specific siRNA and performed with western blot assay for evaluation of its expression. RESULTS: Combined treatment with erlotinib and ampelopsin at non-cytotoxic concentrations significantly induced caspase-dependent cell death in erlotinib-resistant NSCLC cells. Furthermore, cell death resulted in the accumulation of reactive oxygen species (ROS) through upregulation of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) expression, a direct source of ROS. The expression level of Bim increased with combination treatment, but not with either treatment alone. CONCLUSION: Here in this study, we demonstrate that the combination of erlotinib and ampelopsin induces cell death via the Nox2-ROS-Bim pathway, and ampelopsin could be used as a novel anti-cancer agent combined with EGFR-TKI to overcome resistance to erlotinib in EGFR-mutant NSCLC.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Terapia Combinada/métodos , Cloridrato de Erlotinib/farmacologia , Flavonoides/farmacologia , NADPH Oxidase 2/genética , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib/metabolismo , Flavonoides/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismoRESUMO
The differences of serum IL-2 levels were not consistent between Behçet's Disease (BD) patients and healthy controls, however, the correlation of interleukin-2 receptor (IL-2R) and BD has not been investigated. IL-2R is composed of three subunits; alpha, beta, and gamma. The expression frequencies of IL-2R subunits were analyzed in the peripheral blood mononuclear cells, spleens, and lymph node (LN) cells. The expression of IL-2R subunits was different between BD mice and controls. IL-2R beta expressing cell frequencies were also different between BD patients and healthy controls. The IL-2/anti-mIL-2 antibody complex administration regulated the IL-2R subunits in mice. The change of expression in IL-2R was accompanied by the increase of CD8+CD44+ memory T cells, CD3-NK1.1+CD11b+CD27+ natural killer cells, and improvement of symptoms. In this study, we elucidated the role of IL-2R subunits on BD, a finding that can be connected to therapeutic strategy for patients based on the results from the treatment of BD mice.
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Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Síndrome de Behçet/imunologia , Síndrome de Behçet/metabolismo , Interleucina-2/imunologia , Receptores de Interleucina-2/metabolismo , Simplexvirus , Adulto , Animais , Anticorpos Monoclonais/administração & dosagem , Síndrome de Behçet/tratamento farmacológico , Síndrome de Behçet/virologia , Estudos de Casos e Controles , Modelos Animais de Doenças , Etanercepte/farmacologia , Etanercepte/uso terapêutico , Feminino , Humanos , Memória Imunológica , Imunofenotipagem , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/sangue , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/antagonistas & inibidores , Subunidade beta de Receptor de Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Contagem de Linfócitos , Masculino , Camundongos , Pessoa de Meia-Idade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Regulação para Cima , Adulto JovemRESUMO
BACKGROUND: The exact mechanism of the inflammatory changes occurring during the development of Behçet's disease (BD) remains unclear. OBJECTIVE: We investigated the role of classical (M1) and alternative (M2) activation of macrophages in a herpes simplex virus (HSV)-induced BD mouse model. METHODS: The classical vs. alternative activated macrophage ratio (M1/M2 ratio) was calculated by analyzing the surface markers CD16/32 and CD23 as M1 and M2 markers, respectively, by flow cytometry. mRNA expression of interferon (IFN)-γ and interleukin (IL)-6 as M1 and arginase-1, FIZZ-1, and MHC-II as M2 markers were analyzed by reverse transcription-polymerase chain reaction. Cytokine levels were assessed by enzyme-linked immunosorbent assay. RESULTS: The M1 phenotype was upregulated in BD mice, and an increased M1/M2 ratio was observed compared to that in asymptomatic BD normal and normal healthy mice. Recombinant (r)IFN-γ significantly increased the M1/M2 ratio (1.74±0.42) compared with that of rIL-4 (0.83±0.20). BD mice treated with rIL-4 showed a decreased M1/M2 ratio (1.2±0.3) compared to that of the rIFN-γ- (2.1±2.3) treated group and also showed ameliorated BD symptoms accompanied by downregulation of IL-17 and IL-6 and up-regulation of IL-4. CONCLUSION: Therefore, modulation of macrophage phenotypes could be an effective therapeutic approach for treating BD in the future.
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Síndrome de Behçet/etiologia , Inflamação/etiologia , Macrófagos/fisiologia , Simplexvirus/patogenicidade , Animais , Síndrome de Behçet/imunologia , Síndrome de Behçet/terapia , Colchicina/farmacologia , Modelos Animais de Doenças , Interferon gama/genética , Interferon gama/farmacologia , Interleucina-4/farmacologia , Interleucina-6/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Pentoxifilina/farmacologia , FenótipoRESUMO
The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. TIM-1 plays an important role in the regulation of immune responses and the development of autoimmune diseases. TIM-4 is a natural ligand of TIM-1, and the interaction of TIM-1 and TIM-4 is involved in the regulation of T helper (Th) cell responses and modulation of the Th1/Th2 cytokine balance. Behçet's disease (BD) is a chronic, multisystemic inflammatory disorder with arthritic, intestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. Tim-1 expression was lower in a herpes simplex virus-induced BD mouse model compared to that in asymptomatic BD normal (BDN) mice. Tim-4 expression was higher in BD mice than that in BDN mice. In this study, we investigated the Tim expression in a BD mouse model with BD-like symptoms. Tim-1 and Tim-4 expression was regulated by an expression vector or siRNA injected into the BD mouse model. The Tim-1 vector injected into BD mice resulted in changes in BD-like symptoms and decreased the severity score. Treatment with Tim-4 siRNA also improved BD-like symptoms and decreased the severity score accompanied by upregulation of regulatory T cells. We showed that regulating Tim-1 or Tim-4 affected BD-like symptoms in mice.
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Síndrome de Behçet/imunologia , Proteínas de Membrana/fisiologia , Animais , Citocinas/fisiologia , Modelos Animais de Doenças , Receptor Celular 1 do Vírus da Hepatite A , Subunidade beta de Receptor de Interleucina-2/análise , Masculino , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos ICR , RNA Interferente Pequeno/genética , Simplexvirus/patogenicidade , Linfócitos T Reguladores/imunologiaRESUMO
Interleukin-6 (IL-6) is considered to be an early marker of severe sepsis that is associated with increased morbidity and mortality. Therefore, we pretreated male ICR mice with IL-6 small interfering RNA (siRNA) before cecal ligation and puncture (CLP) and observed the changes in their survival in response to down regulation of IL-6, as well as the role of Th subsets during sepsis. In addition, sham and CLP operated mice were sacrificed at different time points to determine the serum IL-6 levels during early and late sepsis. IL-6 siRNA pretreated septic mice showed markedly extended survival (23.3%) up to 10 days and significantly reduced serum IL-6 levels at day 5 (209.90 ± 0.50 pg/ml; P<0.0001) when compared to CLP mice at day 1. Furthermore, IL-6 mRNA and protein were highly expressed during early sepsis in CLP mice at day 1 and mRNA was not detected in IL-6 siRNA treated CLP mice at days 1 or 5 and serum level of IL-6 was also decreased significantly (P<0.01). In addition, the mRNA expression of C5aR, ROR-γt, PU.1 and protein expression of IL-17 were high at day 5 in IL-6 siRNA treated mice. Taken together, the results of the present study demonstrate that pretreatment with IL-6 siRNA improved CLP induced septic mice survival. Furthermore, the IL-6 level was down-regulated and the transcription factors ROR-γt and PU.1 were up-regulated by IL-6 siRNA at late sepsis. The results presented herein also suggest that IL-6 siRNA could be a potential molecular therapeutic strategy for the treatment of sepsis.
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Ceco/lesões , Ceco/microbiologia , Regulação para Baixo/genética , Interleucina-6/deficiência , Interleucina-6/genética , Punções/efeitos adversos , RNA Interferente Pequeno/genética , Alanina Transaminase/metabolismo , Animais , Antibacterianos/farmacologia , Nitrogênio da Ureia Sanguínea , Ceco/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Interleucina-6/biossíntese , Ligadura/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Receptores de Complemento/genética , Sepse/etiologia , Sepse/metabolismo , Sepse/microbiologia , Análise de Sobrevida , Taxa de Sobrevida , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de TempoRESUMO
Galectin-9 (Gal-9) has been identified as a Tim-3 ligand (L). The Tim-3-Tim-3L interaction serves as a specific down-regulator of the Th1 immune response. It has been reported that Tim-3 expression is higher in patients with inflammatory disorders such as rheumatoid arthritis compared to controls. In a herpes simplex virus-induced Behcet's disease (BD) mouse model, Tim-3 was expressed in a similarly high level. The expression of Gal-9 in macrophages from BD-like mice was lower than in asymptomatic BD normal mice; therefore, we injected 100 µg of Gal-9 into BD-like mice five times at 3 day intervals and subsequently observed changes in symptoms over 15 days. Gal-9 improved the symptoms of inflammation, decreased the severity score, and increased regulatory T cell expression in treated mice. Moreover, pro-inflammatory cytokine levels were lower in the Gal-9-treated group compared to the control group. Therefore, in the present study, Tim-3-Tim-3L interaction was found to influence inflammatory symptoms in BD-like mice.
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Síndrome de Behçet/tratamento farmacológico , Galectinas/uso terapêutico , Herpes Simples/tratamento farmacológico , Inflamação/tratamento farmacológico , Receptores Virais/metabolismo , Animais , Apoptose , Síndrome de Behçet/complicações , Síndrome de Behçet/imunologia , Síndrome de Behçet/virologia , Células Cultivadas , Modelos Animais de Doenças , Galectinas/administração & dosagem , Expressão Gênica , Receptor Celular 2 do Vírus da Hepatite A , Herpes Simples/complicações , Herpes Simples/imunologia , Herpes Simples/virologia , Inflamação/complicações , Inflamação/imunologia , Inflamação/virologia , Injeções Intraperitoneais , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Receptores Virais/genética , Receptores Virais/imunologia , Simplexvirus/efeitos dos fármacos , Simplexvirus/fisiologiaRESUMO
BACKGROUND: It has been suggested that the HLA-G molecule is a genetic risk factor for Behcet's disease (BD). In this study, we evaluated the level of Qa-2, a murine nonclassical class I MHC molecule and possible functional homolog of HLA-G, to determine if it was associated with various symptoms of BD-like mice. In addition, we investigated siRNA (small interfering RNA) treatment to determine if it inhibited Qa-2 expression, thereby changing the symptoms of mice. METHODS: RNA interference (RNAi) and vector transfection were employed to manipulate gene expression in vivo in mice. siRNA (small interfering RNA) or Qa-2 expression vector was applied to inhibit or up-regulate Qa-2 expression, respectively. RESULTS: The Qa-2 levels in granulocytes were lower in BD-like mice than in normal controls. The silencing of Qa-2 by intravenous injection of siRNA (500 nmol/mouse, 4 times at 3-day intervals) specifically reduced the Qa-2 levels and worsened the BD-like symptoms. CONCLUSIONS: Silencing Qa-2 by injecting siRNA into mice resulted in deterioration of symptoms in BD-like mice.