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1.
Anticancer Res ; 39(9): 4643-4652, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519562

RESUMO

BACKGROUND/AIM: Adenoviral-mediated expression of CD40 ligand (CD40L) on dendritic cells (DCs) activates immune check point CD40/CD40L, enhancing the immunostimulation of DCs and effector cells against human renal carcinoma cells (RCC) and inducing tumor cell apoptosis in vitro. MATERIALS AND METHODS: DCs, isolated from buffy coats from healthy donors, were transduced with adenoviruses carrying human CD40L (Ad-hCD40L). Subsequently maturation marker and cytokine expression were analyzed by fluorescence-activated cell sorting and enzyme-linked immunosorbent assay. RESULTS: Adenoviral transduction induced high expression of soluble CD40L and membrane-bound CD40L, leading to a strong CD40-CD40L interaction in DCs. Interestingly, a T-helper cell type 1 shift of expressed cytokines/chemokines was observed due to the expression of membrane-bound CD40L rather than due to soIuble CD40L alone, which significantly reduced immunoactivation of DCs. However, supernatants of Ad-hCD40L-transduced DCs induced apoptosis of RCC cells. Co-culture of Ad-hCD40L DCs with cytokine-induced killer cells led to a significant stimulation of tumor-specific cytokine-induced killer cells, with increased proliferation and cytotoxicity. CONCLUSION: Use of Ad-hCD40L-transduced DCs is a promising approach to treating RCC.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Carcinoma de Células Renais/metabolismo , Células Dendríticas/metabolismo , Imunomodulação , Neoplasias Renais/metabolismo , Adenoviridae/genética , Biomarcadores , Ligante de CD40/genética , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Neoplasias Renais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética
2.
Nat Commun ; 10(1): 2352, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138793

RESUMO

Regulatory T cells (Tregs) have crucial functions in the inhibition of immune responses. Their development and suppressive functions are controlled by the T cell receptor (TCR), but the TCR signaling mechanisms that mediate these effects remain ill-defined. Here we show that CARD11-BCL10-MALT1 (CBM) signaling mediates TCR-induced NF-κB activation in Tregs and controls the conversion of resting Tregs to effector Tregs under homeostatic conditions. However, in inflammatory milieus, cytokines can bypass the CBM requirement for this differentiation step. By contrast, CBM signaling, in a MALT1 protease-dependent manner, is essential for mediating the suppressive function of Tregs. In malignant melanoma models, acute genetic blockade of BCL10 signaling selectively in Tregs or pharmacological MALT1 inhibition enhances anti-tumor immune responses. Together, our data uncover a segregation of Treg differentiation and suppressive function at the CBM complex level, and provide a rationale to explore MALT1 inhibitors for cancer immunotherapy.


Assuntos
Proteína 10 de Linfoma CCL de Células B/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Diferenciação Celular , Citocinas/imunologia , Melanoma Experimental , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo
3.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083348

RESUMO

Detrimental health consequences from exposure to space radiation are a major concern for long-duration human exploration missions to the Moon or Mars. Cellular responses to radiation are expected to be heterogeneous for space radiation exposure, where only high-energy protons and other particles traverse a fraction of the cells. Therefore, assessing DNA damage and DNA damage response in individual cells is crucial in understanding the mechanisms by which cells respond to different particle types and energies in space. In this project, we identified a cell-specific signature for radiation response by using single-cell transcriptomics of human lymphocyte subpopulations. We investigated gene expression in individual human T lymphocytes 3 h after ex vivo exposure to 2-Gy gamma rays while using the single-cell sequencing technique (10X Genomics). In the process, RNA was isolated from ~700 irradiated and ~700 non-irradiated control cells, and then sequenced with ~50 k reads/cell. RNA in each of the cells was distinctively barcoded prior to extraction to allow for quantification for individual cells. Principal component and clustering analysis of the unique molecular identifier (UMI) counts classified the cells into three groups or sub-types, which correspond to CD4+, naïve, and CD8+/NK cells. Gene expression changes after radiation exposure were evaluated using negative binomial regression. On average, BBC3, PCNA, and other TP53 related genes that are known to respond to radiation in human T cells showed increased activation. While most of the TP53 responsive genes were upregulated in all groups of cells, the expressions of IRF1, STAT1, and BATF were only upregulated in the CD4+ and naïve groups, but were unchanged in the CD8+/NK group, which suggests that the interferon-gamma pathway does not respond to radiation in CD8+/NK cells. Thus, single-cell RNA sequencing technique was useful for simultaneously identifying the expression of a set of genes in individual cells and T lymphocyte subpopulation after gamma radiation exposure. The degree of dependence of UMI counts between pairs of upregulated genes was also evaluated to construct a similarity matrix for cluster analysis. The cluster analysis identified a group of TP53-responsive genes and a group of genes that are involved in the interferon gamma pathway, which demonstrate the potential of this method for identifying previously unknown groups of genes with similar expression patterns.


Assuntos
Exposição à Radiação , Fator de Transcrição STAT1/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Análise por Conglomerados , Raios gama , Humanos , Imunofenotipagem , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação
4.
Nat Immunol ; 20(6): 747-755, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061531

RESUMO

Despite gathering evidence that ubiquitylation can direct non-degradative outcomes, most investigations of ubiquitylation in T cells have focused on degradation. Here, we integrated proteomic and transcriptomic datasets from primary mouse CD4+ T cells to establish a framework for predicting degradative or non-degradative outcomes of ubiquitylation. Di-glycine remnant profiling was used to reveal ubiquitylated proteins, which in combination with whole-cell proteomic and transcriptomic data allowed prediction of protein degradation. Analysis of ubiquitylated proteins identified by di-glycine remnant profiling indicated that activation of CD4+ T cells led to an increase in non-degradative ubiquitylation. This correlated with an increase in non-proteasome-targeted K29, K33 and K63 polyubiquitin chains. This study revealed over 1,200 proteins that were ubiquitylated in primary mouse CD4+ T cells and highlighted the relevance of non-proteasomally targeted ubiquitin chains in T cell signaling.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ativação Linfocitária/imunologia , Proteoma , Proteômica , Animais , Perfilação da Expressão Gênica , Ativação Linfocitária/genética , Espectrometria de Massas , Camundongos , Poliubiquitina/metabolismo , Proteômica/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcriptoma , Ubiquitinação
5.
Scand J Immunol ; 90(3): e12794, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31141185

RESUMO

Natural killer T (NKT) cells are αß T cell receptor (TCR) expressing innate-like T cells that display natural killer (NK) cell markers. Based on TCR characteristics, they are divided into two groups restricted to the MHC class I-like molecule CD1d. Type I NKT cells, most extensively studied, are identified by a semi-invariant Vα14-Jα18 (mouse, Vα24-Jα18 in humans) TCR reactive to the prototypic ligand α-galactosylceramide presented on CD1d. In contrast, type II NKT cells display diverse TCR reacting to different CD1d-presented ligands. There are no reagents that identify all type II NKT cells, limiting their exploration. Here, we searched for novel type II NKT cells by comparing Jα18-/- MHCII-/- mice that harbour type II but not type I NKT cells, and CD1d-/- MHCII-/- mice, lacking all NKT cells. We identified significantly larger populations of CD4+ and CD4- CD8- (double negative, DN) TCRß+ cells expressing NKG2D or NKG2A/C/E in Jα18-/- MHCII-/- mice compared with CD1d-/- MHCII-/- mice, suggesting that 30%-50% of these cells were type II NKT cells. They expressed CD122, NK1.1, CXCR3 and intermediate/low levels of CD45RB. Further, the CD4+ subset was CD69+ , while the DN cells were CD49b+ and CD62L+ . Both subsets expressed the NKT cell-associated promyelocytic leukaemia zinc finger (PLZF) transcription factor and Tbet, while fewer cells expressed RORγt. NKG2D+ CD4+ and DN populations were producers of IFN-γ, but rarely IL-4 and IL-17. Taken together, we identify a novel subset of primary CD4+ and DN type II NKT cells that expresses NKG2 receptors have typical NKT cell phenotypes and a TH1-like cytokine production.


Assuntos
Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Biomarcadores/metabolismo , Células Matadoras Naturais/imunologia , Células T Matadoras Naturais/imunologia , Animais , Feminino , Galactosilceramidas/imunologia , Galactosilceramidas/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células T Matadoras Naturais/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
6.
Nat Immunol ; 20(7): 928-942, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31061532

RESUMO

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Perfilação da Expressão Gênica , Membrana Sinovial/metabolismo , Transcriptoma , Artrite Reumatoide/patologia , Autoimunidade/genética , Biomarcadores , Biologia Computacional/métodos , Estudos Transversais , Citocinas/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Membrana Sinovial/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fluxo de Trabalho
7.
Med Sci Monit ; 25: 3032-3040, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31019190

RESUMO

BACKGROUND T follicular helper (Tfh) cells are a subgroup of activated CD4+ T cells in the germinal centers of secondary lymphoid organs, they play critical roles in the development of many chronic autoimmune inflammatory diseases. The aim of this study was to investigate whether circulating Tfh cells contribute to the development of rheumatoid arthritis (RA). MATERIAL AND METHODS Thirty patients fulfilled the diagnosis criteria that was established by the American College of Rheumatology and 30 healthy controls were recruited. The frequency of Tfh cells in patients and collagen-induced arthritis (CIA) in DBA/1J mice were analyzed by flow cytometry. The serum IL-21 level was examined by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of Blimp-1 and Bcl-6 were detected by qRT-PCR. RESULTS RA patients had more CD4⁺PD-1⁺CXCR5⁺ Tfh cells in peripheral blood compared with healthy controls, and CIA in DBA/1J mice showed similar results. Higher mRNA expression of Bcl-6 and lower Blimp-1 mRNA expression were observed in patients with RA compared to healthy controls, and the expression level of IL-21 was higher in RA patients, which was also seen in CIA mice. Furthermore, the spleen CD4⁺ICOS⁺CXCR5⁺ Tfh cells in CIA mice show significantly higher frequency than that in the control mice. The percentage of CD4⁺PD-1⁺CXCR5⁺ Tfh cells was correlated positively with the values of erythrocyte sedimentation rate (ESR) (r=0.968, P<0.001), rheumatoid factor (RF) (r=0.962, P<0.001), C-reactive protein (CRP) (r=0.953, P<0.001), and anti-cyclic citrullinated peptide antibodies (ACPA) (r=0.966, P<0.001), and the level of serum interleukin (IL)-21 in RA patients showed positive correlation with ESR (r=0.982, P<0.001), RF (r=0.959, P<0.001), CRP (r=0.951, P<0.001), and ACPA (r=0.971, P<0.001) as well. CONCLUSIONS The activated Tfh cells in the peripheral blood may be responsible for the development of RA.


Assuntos
Artrite Reumatoide/imunologia , Receptor de Morte Celular Programada 1/imunologia , Receptores CXCR5/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Idoso , Animais , Artrite Reumatoide/sangue , Autoanticorpos/imunologia , Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , Linfócitos T CD4-Positivos/imunologia , Feminino , Citometria de Fluxo , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Interleucinas/sangue , Interleucinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptores CXCR5/sangue , Fator Reumatoide/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo
8.
EBioMedicine ; 42: 109-119, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30956171

RESUMO

BACKGROUND: HIV-1-specific CD8+ T cells are required for immune suppression of HIV-1 replication and elimination of the associated viral reservoirs. However, effective induction of functional HIV-1-specific CD8+ T cells from naïve cells remains problematic in the setting of human vaccine trials. In this study, we investigated priming of functional HIV-1-specific CD8+ T cells from naïve cells. METHODS: HIV-1-specific CD8+ T cells were primed from naïve T cells of HIV-1-seronegative individuals using TLR4 ligand LPS or STING ligand 3'3'-cGAMP in vitro. We established HIV-1-specific CD8+ T cell lines from primed T cells and then investigated functional properties of these cells. FINDINGS: HIV-1-specific CD8+ T cells primed with LPS failed to suppress HIV-1. In contrast, 3'3'-cGAMP effectively primed HIV-1-specific CD8+ T cells with strong ability to suppress HIV-1. 3'3'-cGAMP-primed T cells had higher expression levels of perforin and granzyme B than LPS-primed ones. The expression levels of granzyme B and perforin and viral suppression ability of 3'3'-cGAMP-primed T cells were positively correlated with the production level of type I IFN from PBMCs stimulated with 3'3'-cGAMP. INTERPRETATION: The present study demonstrates the potential of 3'3'-cGAMP to induce HIV-1-specific CD8+ T cells with strong effector function from naïve cells via a strong type I IFN production and suggests that this STING ligand may be useful for AIDS vaccine and cure treatment.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Subpopulações de Linfócitos T/imunologia , Biomarcadores , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Citocinas/metabolismo , Humanos , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Carga Viral , Replicação Viral
9.
Ann Hematol ; 98(6): 1457-1466, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30895351

RESUMO

The exact role of regulatory T cells (Tregs) in multiple myeloma (MM) has not been yet determined. Data regarding alterations of Tregs during therapy with novel agents (NA), i.e., bortezomib and lenalidomide are conflicted and limited. We evaluated prospectively alterations of Tregs and searched for correlations with disease characteristics, response, and outcome in 29 patients with active MM treated with either bortezomib-dexamethasone (BD; 11 patients) or lenalidomide-dexamethasone (LenDex, 18 patients). Additionally, we recorded changes of lymphocytes subsets and cytokines related to Tregs function and MM biology, i.e., interleukin (IL) 6, 2, 17, and TGF-ß. Compared with controls, patients had significantly higher median levels of Tregs%, IL-6, and IL-17 (p < 0.001). Median CD4 T and B cells frequencies were significantly lower, whereas CD8 T and natural killers were increased compared to controls. In BD group, no significant alterations of Tregs% were observed. Patients treated with LenDex, displayed a significant reduction of Tregs% (p < 0.001) especially those who achieved at least very good partial response (≥vgPR) (p = 0.04). Lymphocyte subsets or cytokines did not significantly change during therapy. In summary, Tregs% are higher in patients with active MM compared with controls, and they significantly decrease after treatment with LenDex but not with BD; After therapy with LenDex, Tregs reduction between baseline and major response correlated with achievement of ≥vgPR suggesting a possible predictive role, that may contribute to therapeutic strategy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mieloma Múltiplo/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bortezomib/administração & dosagem , Bortezomib/farmacologia , Citocinas/sangue , Dexametasona/administração & dosagem , Dexametasona/farmacologia , Feminino , Humanos , Lenalidomida/administração & dosagem , Lenalidomida/farmacologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/tratamento farmacológico , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/análise , Resultado do Tratamento
10.
Immunity ; 50(4): 1043-1053.e5, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30902636

RESUMO

Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this "inside-out" triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during "outside" signaling, as measured by single-cell force microscopy. Our findings provide insight into the "inside-out" triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.


Assuntos
Antígenos CD/química , Butirofilinas/química , Ativação Linfocitária , Organofosfatos/metabolismo , Subpopulações de Linfócitos T/imunologia , Antígenos CD/metabolismo , Sítios de Ligação , Butirofilinas/metabolismo , Cristalografia por Raios X , Dimerização , Desenho de Drogas , Humanos , Ligações de Hidrogênio , Imunoterapia , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos T gama-delta , Análise de Célula Única , Relação Estrutura-Atividade , Subpopulações de Linfócitos T/metabolismo
11.
BMC Immunol ; 20(1): 10, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30832584

RESUMO

BACKGROUND: Previously, we demonstrated up-regulated activated CD4+ and CD8+ T lymphocytes as well as up-regulated cytotoxic NK cells in the blood of patients with idiopathic recurrent miscarriage. In the present study, we tried to identify deficiencies in counter-regulating immune mechanisms of these patients. METHOD: Cytokines were determined in NK cells and in plasma samples of 35 healthy controls, 33 patients with idiopathic recurrent miscarriage, 34 patients with end stage renal disease, 10 transplant patients early and 37 transplant patients late post-transplant using flow-cytometry and luminex. In addition, cytokines were studied in supernatants of cell cultures with peripheral blood mononuclear cells stimulated in-vitro with tumor cell line K562. RESULTS: Patients with idiopathic recurrent miscarriage exhibited the highest absolute cell counts of circulating TGFß1+ NK, NKT and T lymphocytes and the lowest TGFß1 plasma levels of all study groups (for all p < 0.050). In-vitro, peripheral blood mononuclear cells of patients with idiopathic recurrent miscarriage showed high spontaneous TGFß1 production that could not be further increased by stimulation with K562, indicating increased consumption of TGFß1 by activated cells in the cell culture. Moreover, patients with idiopathic recurrent miscarriage had abnormally high IL4+ as well as abnormally high IFNy+ NK cells (p < 0.010) but similar IL10+ NK cell numbers as female healthy controls and showed the lowest plasma levels of IL10, TGFß3, IL1RA, IL1ß, IL5, IL6, IL8, IL17, TNFα, GM-CSF, TPO and VEGF and the highest plasma levels of G-CSF, FGF-basic, CCL3 and CXCL5 as compared to female HC and female transplant recipients (for all p < 0.050). CONCLUSIONS: Patients with idiopathic recurrent miscarriage show an activated immune system that can hardly be stimulated further and cannot be efficiently down-regulated by up-regulated TGFß1+ and IL4+ NK, NKT and T lymphocytes which are present concomitantly in these patients. The strongly decreased TGFß and IL10 plasma levels indicate deficient down-regulation and reflect a dysbalance of the immune system in patients with idiopathic recurrent miscarriage. These findings may be relevant for explaining the pathogenesis of idiopathic recurrent miscarriage.


Assuntos
Aborto Habitual/sangue , Aborto Habitual/imunologia , Células Matadoras Naturais/imunologia , Contagem de Linfócitos , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Fator de Crescimento Transformador beta/sangue , Biomarcadores , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Células Matadoras Naturais/metabolismo , Células T Matadoras Naturais/metabolismo , Receptores de Citocinas/metabolismo , Subpopulações de Linfócitos T/metabolismo
12.
Mol Med Rep ; 19(5): 4195-4204, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896872

RESUMO

Regulatory T cells (Tregs) maintain immune homeostasis and modulate tumor­induced neovascularization. However, the mechanisms underlying the role of Tregs in acute lymphoblastic leukemia (ALL) remain to be elucidated. Helios, combined with forkhead box P3, is considered a suitable marker for discriminating functional Tregs. In the present study, a microenvironment was created with a high proportion of Helios+ Tregs in T cell­deficient nude mice to determine the mechanism underlying Tregs expressing Helios in ALL. It was revealed that umbilical cord blood­derived Helios+ Tregs had proliferation and immunosuppression abilities similar to those of normal pediatric Tregs. The accumulation of Helios+ Tregs accelerated leukemogenesis and the infiltration of leukemic cells into the bone marrow. Importantly, a high expression of Helios in Tregs promoted angiogenesis in the bone marrow via the vascular endothelial growth factor (VEGF)A/VEGF receptor 2 (VEGFR2) pathway. Furthermore, the expression of chemokine CC­chemokine ligand 22 (CCL22) in the bone marrow and serum of ALL mice infused with Helioshigh Treg cells was increased. The results demonstrated that Helios promotes the secretion of chemokine CCL22, which may recruit more Tregs into the bone marrow. Increased Helios+ Treg cells promoted angiogenesis in the bone marrow of ALL mice via the VEGFA/VEGFR2 pathway. Therefore, Helios may be a target to manipulate Treg activity in clinical settings.


Assuntos
Quimiocina CCL22/metabolismo , Sangue Fetal/citologia , Neovascularização Patológica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Linfócitos T Reguladores/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Medula Óssea/patologia , Humanos , Camundongos , Neovascularização Patológica/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia
13.
Mol Med Rep ; 19(4): 3217-3229, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816506

RESUMO

Our previous studies suggested that paeonol, the active constituent of the traditional Chinese medicine Cortex Moutan, may be an effective treatment for inflammatory disorders. In the present study, the therapeutic potential of paeonol on atopic dermatitis (AD) was investigated using animal and cell experiments. AD­like lesions were induced by repeated application of 1­chloro­2,4­dinitrobenzene (DNCB) to the shaved dorsal skin of BALB/c mice, and P815 cells were used for in vitro assays. The skin lesions, serum and spleens of the mice were analyzed using lesion severity scoring, histological analysis, flow cytometry, reverse transcription­quantitative polymerase chain reaction, western blotting and ELISA, in order to investigate the anti­AD effects of paeonol. In addition, western blotting and ELISA were conducted for in vitro analysis of P815 cells. The results demonstrated that oral administration of paeonol inhibited the development of DNCB­induced AD­like lesions in the BALB/c mice by reducing severity of the lesions, epidermal thickness and mast cell infiltration; this was accompanied by reduced levels of immunoglobulin E and inflammatory cytokines [interleukin (IL)­4, histamine, IL­13, IL­31 and thymic stromal lymphopoietin], along with regulation of the T helper (Th) cell subset (Th1/Th2) ratio. Application of paeonol also reduced the protein expression levels of phosphorylated (p)­p38 and p­extracellular signal­regulated kinase (ERK) in skin lesions. In vitro, paeonol reduced the expression levels of tumor necrosis factor­α and histamine in P815 cells, and inhibited p38/ERK/mitogen­activated protein kinase signaling. The present findings indicated that paeonol may relieve dermatitis by acting on cluster of differentiation 4+ T and mast cells; therefore, paeonol may represent a potential therapeutic strategy for the treatment of allergic inflammatory conditions via immunoregulation.


Assuntos
Acetofenonas/farmacologia , Dermatite Atópica/etiologia , Dermatite Atópica/metabolismo , Dinitroclorobenzeno/efeitos adversos , Mastócitos/imunologia , Mastócitos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Modelos Animais de Doenças , Feminino , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Mediadores da Inflamação/metabolismo , Camundongos , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
14.
Iran J Immunol ; 16(1): 1-10, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30864551

RESUMO

BACKGROUND: All-trans retinoic acid (ATRA) potentiates TGF-ß-dependent regulatory T cells (Treg) induction, while it inhibits pro-inflammatory interleukin-17-producing T helper cells (Th17) differentiation. Combined application of ATRA and TGF-ß may shift Treg-Th17 balance towards Treg. OBJECTIVE: To investigates the effect of ATRA on the regulation of Th17-Treg balance through ERK and p38 signaling pathway. METHODS: Mice naive CD4+T cells were isolated and co-cultured with 100 nmol/ml ATRA and 5 ng/ml TGF-ß. The effect of ATRA on the phosphorylation of ERK and P38 was evaluated. The induction of Treg and Th17 was investigated before and after the application of the inhibitor of ERK and P38. RESULTS: The expression of p-ERK1 and p-ERK2 increased significantly when the cells were incubated for 3 days with both TGF-ß and ATRA. The upregulated expression of p38 was found after incubation for 1 day. The inhibition of ERK prohibited Treg induction and promoted Th17 development. However, the inhibition of p38 only had inhibitory effect on Treg induction. CONCLUSIONS: ERK and p38 pathways participated in ATRA-activated Treg-Th17 balance adjustment.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismo , Tretinoína/farmacologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Ativação Linfocitária , Masculino , Camundongos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
PLoS Pathog ; 15(3): e1007633, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30875408

RESUMO

Memory CD8+ T cells in the circulation rapidly infiltrate non-lymphoid tissues following infection and provide protective immunity in an antigen-specific manner. However, the subsequent fate of memory CD8+ T cells after entering non-lymphoid tissues such as the skin during a secondary infection is largely unknown. Furthermore, because expression of CD62L is often used to identify the central memory (TCM) CD8+ T cell subset, uncoupling the physical requirement for CD62L-mediated lymph node homing versus other functional attributes of TCM CD8+ T cells remains unresolved. Here, we show that in contrast to naïve CD8+ T cells, memory CD8+ T cells traffic into the skin independent of CD62L-mediated lymph node re-activation and provide robust protective immunity against Vaccinia virus (VacV) infection. TCM, but not effector memory (TEM), CD8+ T cells differentiated into functional CD69+/CD103- tissue residents following viral clearance, which was also dependent on local recognition of antigen in the skin microenvironment. Finally, we found that memory CD8+ T cells expressed granzyme B after trafficking into the skin and utilized cytolysis to provide protective immunity against VacV infection. Collectively, these findings demonstrate that TCM CD8+ T cells become cytolytic following rapid infiltration of the skin to protect against viral infection and subsequently differentiate into functional CD69+ tissue-residents.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Memória Imunológica/fisiologia , Pele/imunologia , Animais , Antígenos CD/metabolismo , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos de Diferenciação de Linfócitos T/fisiologia , Linfócitos T CD8-Positivos/virologia , Feminino , Selectina L/metabolismo , Lectinas Tipo C/metabolismo , Lectinas Tipo C/fisiologia , Linfonodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pele/virologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/fisiologia , Vírus Vaccinia/imunologia , Vírus Vaccinia/patogenicidade
16.
Med Sci Monit ; 25: 2206-2210, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30908476

RESUMO

BACKGROUND It can be difficult to distinguish between bronchial asthma and chronic obstructive pulmonary disease (COPD) clinically, although these conditions are associated with different profiles of inflammatory cytokines and immune cells. This study aimed to compare T-lymphocyte subsets and inflammatory cytokines in the serum and sputum of patients with bronchial asthma and COPD who had respiratory function testing. MATERIAL AND METHODS The study included 42 patients with bronchial asthma, 48 patients with COPD, and 45 patients with bronchial asthma complicated with COPD. The percentage predicted values of the forced expiratory volume in one second (FEV1), the forced vital capacity (FVC), and the peak expiratory flow (PEF) rate were measured. Serum and sputum levels of interleukin (IL)-4, IL-5, IL-9, IL-13, IL-1ß, IL-6 and tumor necrosis factor-alpha (TNF-alpha) were measured using an enzyme-linked immunosorbent assay (ELISA). Flow cytometry measured the CD4 and CD8 T-lymphocyte subsets, and the CD4: CD8 ratio was calculated. RESULTS The FEV1, FVC, and PEF were significantly lower in patients with COPD compared with the other two patient groups. Serum and sputum levels of IL-4, IL-5, IL-9 and IL-13 were significantly increased in the COPD patient group, and levels of TNF-alpha, IL-1ß and IL-6 were significantly increased in the bronchial asthma patient group. The CD4: CD8 ratio in sputum was lowest in bronchial asthma patient group and highest in COPD patient group. CONCLUSIONS The detection of serum and sputum inflammatory cytokines and T-lymphocyte subsets may distinguish between bronchial asthma and COPD.


Assuntos
Asma/imunologia , Citocinas/metabolismo , Doença Pulmonar Obstrutiva Crônica/imunologia , Subpopulações de Linfócitos T/metabolismo , Adulto , Idoso , Asma/sangue , Asma/fisiopatologia , Citocinas/sangue , Citocinas/imunologia , Feminino , Volume Expiratório Forçado , Humanos , Interleucinas/sangue , Interleucinas/imunologia , Interleucinas/metabolismo , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Testes de Função Respiratória , Escarro/imunologia , Subpopulações de Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Capacidade Vital
17.
Clin Adv Hematol Oncol ; 17(2): 109-119, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30845114

RESUMO

Immunotherapy with checkpoint blockade of pro-grammed death 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) has substantially increased the number of anticancer agents in our arsenal. However, these therapies are not effective in all cancer types, benefitting only a subset of patients with susceptible, immunogenic cancers. This problem is especially significant in gastrointestinal malignancies, which infrequently respond to immunotherapy. Although we clearly need more accurate biomarkers to predict response to immune checkpoint inhibition in gastrointestinal cancers, the established markers of mismatch repair deficiency, microsatellite instability, programmed death ligand 1 (PD-L1) expression, and tumor mutational burden are good starting points to identify patients who may benefit. Tumor-infiltrating lymphocytes, Epstein-Barr virus, and the stool microbiome are candidates for future immuno-oncology biomarkers in gastrointestinal malignancies. The availability of better biomarkers will improve patient selection for immunotherapy; it will also improve the design of clinical trials of agents intended for this population of patients, who require more effective treatment options.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Neoplasias Gastrointestinais/imunologia , Neoplasias Gastrointestinais/terapia , Imunoterapia , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Antígeno B7-H1/metabolismo , Antígeno CTLA-4/metabolismo , Reparo de Erro de Pareamento de DNA , Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/genética , Humanos , Imunomodulação/efeitos dos fármacos , Instabilidade de Microssatélites , Terapia de Alvo Molecular , Mutação , Receptor de Morte Celular Programada 1/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
18.
Int J Mol Sci ; 20(6)2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30909402

RESUMO

On healthy cells the non-classical HLA class Ib molecule HLA-E displays the cognate ligand for the NK cell receptor NKG2A/CD94 when bound to HLA class I signal peptide sequences. In a pathogenic situation when HLA class I is absent, HLA-E is bound to a diverse set of peptides and enables the stimulatory NKG2C/CD94 receptor to bind. The activation of CD8⁺ T cells by certain p:HLA-E complexes illustrates the dual role of this low polymorphic HLA molecule in innate and adaptive immunity. Recent studies revealed a shift in the HLA-E peptide repertoire in cells with defects in the peptide loading complex machinery. We recently showed that HLA-E presents a highly diverse set of peptides in the absence of HLA class Ia and revealed a non-protective feature against NK cell cytotoxicity mediated by these peptides. In the present study we have evaluated the molecular basis for the impaired NK cell inhibition by these peptides and determined the cell surface stability of individual p:HLA-E complexes and their binding efficiency to soluble NKG2A/CD94 or NKG2C/CD94 receptors. Additionally, we analyzed the recognition of these p:HLA-E epitopes by CD8⁺ T cells. We show that non-canonical peptides provide stable cell surface expression of HLA-E, and these p:HLA-E complexes still bind to NKG2/CD94 receptors in a peptide-restricted fashion. Furthermore, individual p:HLA-E complexes elicit activation of CD8⁺ T cells with an effector memory phenotype. These novel HLA-E epitopes provide new implications for therapies targeting cells with abnormal HLA class I expression.


Assuntos
Imunidade Adaptativa , Imunidade Inata , Imunomodulação , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/química , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/química , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica , Multimerização Proteica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
19.
Methods Mol Biol ; 1951: 209-216, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825155

RESUMO

Plasma membrane lipid rafts are highly ordered membrane microdomains enriched for glycosphingolipids and cholesterol, which play an important role during T-cell antigen receptor (TCR) signaling. Our previous work has demonstrated that plasma membrane lipid composition is an important determinant of human CD4+ T-cell function and that defects in lipid raft expression contribute to CD4+ dysfunction in patients with autoimmunity. In this chapter we share three flow cytometry-based methods to quantitatively analyze plasma membrane lipid composition in primary human CD4+ T cells. We describe the quantification of glycosphingolipid expression using cholera toxin subunit B, cholesterol expression using filipin staining, and membrane "lipid order" using di-4-ANEPPDHQ. These methods can easily be adapted to analyze different cell types.


Assuntos
Membrana Celular/metabolismo , Citometria de Fluxo , Lipídeos de Membrana/metabolismo , Linfócitos T/metabolismo , Toxina da Cólera/metabolismo , Filipina/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Microdomínios da Membrana/metabolismo , Subpopulações de Linfócitos T/metabolismo
20.
Nat Commun ; 10(1): 687, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737409

RESUMO

How innate T cells (ITC), including invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, maintain a poised effector state has been unclear. Here we address this question using low-input and single-cell RNA-seq of human lymphocyte populations. Unbiased transcriptomic analyses uncover a continuous 'innateness gradient', with adaptive T cells at one end, followed by MAIT, iNKT, γδ T and natural killer cells at the other end. Single-cell RNA-seq reveals four broad states of innateness, and heterogeneity within canonical innate and adaptive populations. Transcriptional and functional data show that innateness is characterized by pre-formed mRNA encoding effector functions, but impaired proliferation marked by decreased baseline expression of ribosomal genes. Together, our data shed new light on the poised state of ITC, in which innateness is defined by a transcriptionally-orchestrated trade-off between rapid cell growth and rapid effector function.


Assuntos
Proliferação de Células/fisiologia , Linfócitos/metabolismo , Feminino , Ontologia Genética , Humanos , Imunidade Inata/fisiologia , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Células T Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/metabolismo
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