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1.
Front Immunol ; 10: 606, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984183

RESUMO

Follicular helper T cells (TFH cells), known as the primary "helpers" of the germinal center (GC) reaction, promote the humoral immune response to defend against various pathogens. Under conditions of infection by different types of pathogens, many shared transcription factors (TFs), such as Bcl-6, TCF-1, and Maf, are selectively enriched in pathogen-specific TFH cells, orchestrating TFH cell differentiation and function. In addition, TFH cells also coexpress environmentally associated TFs as their conventional T cell counterparts (such as T-bet, GATA-3, or ROR-γt, which are expressed in Th1, Th2, or Th17 cells, respectively). These features likely indicate both the lineage-specificity and environmental adaption of the TFH cell responses. However, the extent to which the TFH cell response relies on these environmentally specific TFs is not completely understood. Here, we found that T-bet was specifically expressed in Type I TFH cells but not Type II TFH cells. While dispensable for the early fate commitment of TFH cells, T-bet was essential for the maintenance of differentiated TFH cells, promoting their proliferation, and inhibiting their apoptosis during acute viral infection. Microarray analysis showed both similarities and differences in transcriptome dependency on T-bet in TFH and TH1 cells, suggesting the distinctive role of T-bet in TFH cells. Collectively, our findings reveal an important and specific supporting role for T-bet in type I TFH cell response, which can help us gain a deeper understanding of TFH cell subsets.

2.
Cell Mol Immunol ; 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842630

RESUMO

Epigenetic modifications to histones dictate the differentiation of naïve CD4+ T cells into different subsets of effector T helper (TH) cells. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been implicated in the mechanism regulating the differentiation of TH1, TH2 and regulatory T (Treg) cells. However, whether and how EZH2 regulates follicular helper T (TFH) cell differentiation remain unknown. Using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection, we observed abundant EZH2 expression and associated H3K27me3 modifications preferentially in the early committed virus-specific TFH cells compared to those in TH1 cells. Ablation of EZH2 in LCMV-specific CD4+ T cells leads to a selective impairment of early TFH cell fate commitment, but not late TFH differentiation or memory TFH maintenance. Mechanistically, EZH2 specifically stabilizes the chromatin accessibility of a cluster of genes that are important for TFH fate commitment, particularly B cell lymphoma 6 (Bcl6), and thus directs TFH cell commitment. Therefore, we identified the chromatin-modifying enzyme EZH2 as a novel regulator of early TFH differentiation during acute viral infection.

3.
Cell Physiol Biochem ; 50(1): 261-276, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30282072

RESUMO

BACKGROUND/AIMS: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically centered on paclitaxel-based chemotherapy. In this study, we aimed to evaluate whether miR-34a attenuates chemoresistance to paclitaxel by regulating target genes associated with drug resistance. METHODS: We used data from The Cancer Genome Atlas to compare miR-34a expression levels in prostate cancer (PC) tissues with normal prostate tissues. The effects of miR-34a inhibition and overexpression on PC proliferation were evaluated in vitro via Cell Counting Kit-8 (CCK-8) proliferation, colony formation, apoptosis, and cell-cycle assays. A luciferase reporter assay was employed to identify the interactions between miR-34a and specific target genes. To determine the effects of up-regulation of miR-34a on tumor growth and chemo-resistance in vivo, we injected PC cells overexpressing miR-34a into nude mice subcutaneously and evaluated the rate of tumor growth during paclitaxel treatment. We examined changes in the expression levels of miR-34a target genes JAG1 and Notch1 and their downstream genes via miR-34a transfection by quantitative reverse transcription PCR (qRT-PCR) and western blot assay. RESULTS: miR-34a served as an independent predictor of reduced patient survival. MiR-34a was down-regulated in PC-3PR cells compared with PC-3 cells. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to paclitaxel while miR-34a down-regulation resulted in chemoresistance to paclitaxel in vitro. A study of gain and loss in a series of functional assays revealed that PC cells expressing miR-34a were chemosensitive. Furthermore, the overexpression of miR-34a increased the sensitivity of PC-3PR cells to chemotherapy in vivo. The luciferase reporter assay confirmed that JAG1 and Notch1 were directly targeted by miR-34a. Interestingly, western blot analysis and qRT-PCR confirmed that miR-34a inhibited the Notch1 signaling pathway. We found that miR-34a increased the chemosensitivity of PC-3PR cells by directly repressing the TCF1/ LEF1 axis. CONCLUSION: Our results showed that miR-34a is involved in the development of chemosensitivity to paclitaxel. By regulating the JAG1/Notch1 axis, miR-34a or its target genes JAG1 or Notch1 might serve as potential predictive biomarkers of response to paclitaxel-based chemotherapy and/or therapeutic targets that will help to overcome chemoresistance at the mCRPC stage.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Proteína Jagged-1/metabolismo , MicroRNAs/metabolismo , Paclitaxel/farmacologia , Neoplasias da Próstata/patologia , Receptor Notch1/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Antagomirs/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Proteína Jagged-1/genética , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Paclitaxel/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos
4.
Cell Physiol Biochem ; 48(1): 87-98, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30001529

RESUMO

BACKGROUND/AIMS: Chemoresistance is largely responsible for relapses of bladder cancer during clinical therapy. However, the molecular mechanisms involved in the chemoresistance of bladder cancer are unclear. Growing evidence supports the theory that microRNAs (miRNAs) play an important role in chemotherapeutic drug resistance because they are downregulated in many malignancies that have been implicated in the regulation of diverse processes in cancer cells. More specifically, the extent and precise mechanism of the involvement of miR-34as in chemoresistance to epirubicin (EPI) in the treatment of bladder cancer remains unclear. METHODS: In this study, real-time quantitative polymerase chain reaction (PCR) was used to analyze the expression of miR-34a in bladder cancer cell line BIU87 and its EPI chemoresistant cell line BIU87/ADR. The miR-34a profiles in bladder cancer tissues were obtained from The Cancer Genome Atlas database. The effect of miR-34a on chemosensitivity was evaluated by cell viability assays, colony formation assays, and in vivo experimentation. Apoptosis and the cell cycle were examined by flow cytometry. A luciferase reporter assay was used to assess the target genes of miR-34a. Western blot and qPCR were used to analyze the expression of target proteins and downstream molecules. RESULTS: The downregulation of miR-34a in bladder cancer serves as an independent predictor of reduced patient survival. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to EPI, while miR-34a downregulation resulted in chemoresistance to EPI in vitro. Moreover, it was found that miR-34a increased the sensitivity of BIU87/ADR cells to chemotherapy in vivo. The luciferase reporter assay ascertained that TCF1 and LEF1 are direct target genes of miR-34a. It was found that miR-34a increased chemosensitivity in BIU87/ADR cells by inhibiting the TCF1/LEF1 axis. CONCLUSIONS: The results of this study indicate that miR-34a contributes to the chemosensitivity of BIU87/ADR by inhibiting the TCF1/LEF1 axis. Consequently, miR-34a is a determinant of BIU87 chemosensitivity and may therefore serve as a potential therapeutic target in bladder cancer treatment.


Assuntos
Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , MicroRNAs/metabolismo , Neoplasias da Bexiga Urinária/patologia , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Apoptose , Linhagem Celular Tumoral , Bases de Dados Genéticas , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Epirubicina/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Fator 1-alfa Nuclear de Hepatócito/química , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Estimativa de Kaplan-Meier , Fator 1 de Ligação ao Facilitador Linfoide/química , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Metástase Neoplásica , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade
5.
Front Immunol ; 9: 1127, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29875775

RESUMO

Follicular helper CD4+ T (TFH) cells are critical for optimal B-cell-mediated humoral immunity by initiating, fueling, and sustaining germinal center reactions. The differentiation of TFH cells relies on multiple intrinsic and extrinsic factors; however, the details by which these factors are integrated to coordinate TFH differentiation are largely unknown. In this study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) viral infection, we demonstrate that mTOR complex 2 (mTORC2) kinase integrates TCR signaling and ICOS-mediated co-stimulation to promote late differentiation and functional maturation of virus-specific TFH cells. Specifically, mTORC2 functions to maintain TFH lineage specifications, including phenotypes, migratory characteristics, and functional properties. Thus, our results highlight the importance of mTORC2 in guarding TFH phenotypic and functional maturation.


Assuntos
Diferenciação Celular/imunologia , Movimento Celular/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Viroses/imunologia , Viroses/metabolismo , Animais , Biomarcadores , Complexo CD3/metabolismo , Linhagem da Célula , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Imunidade Humoral , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Viroses/virologia
6.
Cell Physiol Biochem ; 47(2): 735-746, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29794462

RESUMO

Renal cell carcinoma (RCC) is the most common kidney cancer diagnosed across the globe and has steadily increased in incidence in recent decades. Techniques for diagnosing or treating RCC are limited, and confined mostly to later stages of the disease. Almost all RCC pathological types are resistant to chemotherapeutics and radiation therapy. To this effect, new markers for diagnosis and target therapy are urgently needed. Advanced genome sequencing technologies have revealed long non-coding RNAs (lncRNAs) as a novel marker, transcribed throughout the human genome. The emergence of lncRNAs is an aberrant expression and is involved in the tumorigenesis of RCC. LncRNAs drive cancer phenotypes through their interaction with other cellular macromolecules including DNA, protein, and RNA. Recent research on lncRNA molecular mechanisms has revealed new markers to functionally annotate these cancers' associated transcripts, making them targets for effective diagnosis and therapeutic intervention in the fight against cancer. In this review, we first highlight the common mechanisms that underlie aberrant lncRNA expression in RCC. We go on to discuss the potential translational application of lncRNA research in the diagnosis, prognosis, and treatment of RCC.


Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , RNA Longo não Codificante/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Cromatina/metabolismo , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , RNA Longo não Codificante/classificação , RNA Longo não Codificante/genética , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Proteínas Supressoras de Tumor/metabolismo
7.
Cell Physiol Biochem ; 46(6): 2517-2531, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29742507

RESUMO

BACKGROUND/AIMS: Cyanidin is an anthocyanin found in many foods. Although its variable antioxidant levels are well-documented, little is known about its effects on renal cell carcinoma (RCC) tumorigenesis. This study, therefore, investigated the effects of cyanidin on the proliferation, migration, and invasion of renal cell carcinoma lines and demonstrated, for the first time, significant inhibitory effects of cyanidin on RCC tumorigenesis. METHODS: RCC cells were treated with different doses of cyanidin and the effects were tested by Cell Counting Kit-8 reagent, clone formation assay, transwell assay, and flow cytometry. Moreover, the cyanidin-mediated mechanism that curtailed tumorigenesis was analyzed by RNA sequencing (RNA-seq). Sequencing data from The Cancer Genome Atlas (TCGA) were used to compare the expression of both early growth response protein 1 (EGR1) and selenoprotein W (SEPW1) in RCC and tumor-free adjacent normal tissue samples. Real-time PCR (RT-PCR) and/or western blot were used to assess the expression of E-cadherin, cleaved-caspase3, Bcl2, p62, and ATG4. RESULTS: We found significantly greater induction of cell-cycle arrest, apoptosis, and suppression of RCC cell invasion and migration at concentrations of 25 µM and 100 µM than at a concentration of 50 µM. It was also discovered, first through RNA-seq then confirmed by RT-PCR, that cyanidin (100 µM) inhibited RCC carcinogenesis through EGR1 and SEPW1. TCGA data indicated that the expression level of EGR1 was lower and that of SEPW1 was higher in RCC tumor tissue than in normal tissues. Moreover, western blot and/or RT-PCR indicated that cleaved-caspase3 was enhanced and E-cadherin was inhibited by cyanidin treatment. Furthermore, western blot and RT-PCR also showed regulation of p62 and ATG4, which are associated with autophagy. Cyanidin in vivo significantly inhibited the growth of xenografts in nude mice. CONCLUSIONS: The results of this study showed the therapeutic potential of cyanidin for the treatment of RCC and the prevention of recurrence and metastasis.


Assuntos
Antocianinas/uso terapêutico , Antineoplásicos/uso terapêutico , Antioxidantes/uso terapêutico , Carcinogênese/efeitos dos fármacos , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Animais , Antocianinas/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Carcinogênese/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/prevenção & controle
8.
Methods Mol Biol ; 1707: 15-38, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29388097

RESUMO

B cell responses play a central role in humoral immunity, which protects an individual from invading pathogens by antigen-specific antibodies. Understanding the basic principles of the B cell responses during viral infection is of substantial importance for anti-viral vaccine development. In inbred mice, lymphocytic choriomeningitis virus (LCMV) infection elicits robust and typical T cell-dependent B cell responses, including germinal center reaction, memory B cell formation, and a long-lived plasma cell pool in bone marrow. Therefore, this system represents an ideal model to investigate anti-viral B cell responses. In this protocol, we describe how to propagate and quantify LCMV and successfully establish an acute LCMV infection in mice. This protocol also provides three different techniques to analyze B cell responses specific to an acute LCMV infection: the identification of germinal center (GC) B cells and follicular helper CD4 T (TFH) cells from the spleens and lymph nodes via flow cytometry, titration of LCMV-specific IgG in the serum after LCMV infection using an enzyme-linked immunosorbent assay (ELISA) analysis, and detection of LCMV-IgG secreted plasma cells from bone marrow with an enzyme-linked immunospot (ELISPOT) assay.


Assuntos
Anticorpos Antivirais/imunologia , Células da Medula Óssea/imunologia , Imunoglobulina G/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Plasmócitos/imunologia , Animais , Células da Medula Óssea/patologia , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Coriomeningite Linfocítica/patologia , Camundongos , Plasmócitos/patologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia
9.
Immunity ; 47(3): 538-551.e5, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28930662

RESUMO

Follicular regulatory T (Tfr) cells differentiate from conventional regulatory T (Treg) cells and suppress excessive germinal center (GC) responses by acting on both GC B cells and T follicular helper (Tfh) cells. Here, we examined the impact of mTOR, a serine/threonine protein kinase that senses and integrates diverse environmental cues, on the differentiation and functional competency of Tfr cells in response to protein immunization or viral infection. By genetically deleting Rptor or Rictor, essential components for mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), respectively, we found that mTORC1 but not mTORC2 is essential for Tfr differentiation. Mechanistically, mTORC1-mediated phosphorylation of the transcription factor STAT3 induced the expression of the transcription factor TCF-1 by promoting STAT3 binding to the Tcf7 5'-regulatory region. Subsequently, TCF-1 bound to the Bcl6 promoter to induce Bcl6 expression, which launched the Tfr cell differentiation program. Thus, mTORC1 initiates Tfr cell differentiation by activating the TCF-1-Bcl-6 axis during immunization or infection.


Assuntos
Imunomodulação , Complexos Multiproteicos/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Biomarcadores , Diferenciação Celular/imunologia , Análise por Conglomerados , Perfilação da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Imunização , Imunofenotipagem , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/citologia , Serina-Treonina Quinases TOR/genética
11.
Nature ; 537(7620): 412-428, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27501245

RESUMO

During chronic viral infection, virus-specific CD8(+) T cells become exhausted, exhibit poor effector function and lose memory potential. However, exhausted CD8(+) T cells can still contain viral replication in chronic infections, although the mechanism of this containment is largely unknown. Here we show that a subset of exhausted CD8(+) T cells expressing the chemokine receptor CXCR5 has a critical role in the control of viral replication in mice that were chronically infected with lymphocytic choriomeningitis virus (LCMV). These CXCR5(+) CD8(+) T cells were able to migrate into B-cell follicles, expressed lower levels of inhibitory receptors and exhibited more potent cytotoxicity than the CXCR5(-) [corrected] subset. Furthermore, we identified the Id2-E2A signalling axis as an important regulator of the generation of this subset. In patients with HIV, we also identified a virus-specific CXCR5(+) CD8(+) T-cell subset, and its number was inversely correlated with viral load. The CXCR5(+) subset showed greater therapeutic potential than the CXCR5(-) [corrected] subset when adoptively transferred to chronically infected mice, and exhibited synergistic reduction of viral load when combined with anti-PD-L1 treatment. This study defines a unique subset of exhausted CD8(+) T cells that has a pivotal role in the control of viral replication during chronic viral infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Centro Germinativo/citologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptores CXCR5/metabolismo , Transferência Adotiva , Animais , Linfócitos B/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/transplante , Diferenciação Celular , Doença Crônica , Feminino , Centro Germinativo/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Proteína 2 Inibidora de Diferenciação/metabolismo , Vírus da Coriomeningite Linfocítica/crescimento & desenvolvimento , Masculino , Camundongos , Receptores CXCR5/deficiência , Transdução de Sinais , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Carga Viral/imunologia , Replicação Viral/imunologia
12.
Nat Immunol ; 16(9): 991-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26214740

RESUMO

Induction of the transcriptional repressor Bcl-6 in CD4(+) T cells is critical for the differentiation of follicular helper T cells (T(FH) cells), which are essential for B cell-mediated immunity. In contrast, the transcription factor Blimp1 (encoded by Prdm1) inhibits T(FH) differentiation by antagonizing Bcl-6. Here we found that the transcription factor TCF-1 was essential for both the initiation of T(FH) differentiation and the effector function of differentiated T(FH) cells during acute viral infection. Mechanistically, TCF-1 bound directly to the Bcl6 promoter and Prdm1 5' regulatory regions, which promoted Bcl-6 expression but repressed Blimp1 expression. TCF-1-null T(FH) cells upregulated genes associated with non-T(FH) cell lineages. Thus, TCF-1 functions as an important hub upstream of the Bcl-6-Blimp1 axis to initiate and secure the differentiation of T(FH) cells during acute viral infection.


Assuntos
Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/imunologia , Fator 1-alfa Nuclear de Hepatócito/imunologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Fatores de Transcrição/imunologia , Animais , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Vírus da Influenza A , Camundongos , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-6 , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Transcrição/genética
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