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1.
JCI Insight ; 4(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31536480

RESUMO

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

2.
Curr Opin Immunol ; 61: 17-25, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31430664

RESUMO

Single-cell methods have revolutionized the study of T cell biology by enabling the identification and characterization of individual cells. This has led to a deeper understanding of T cell heterogeneity by generating functionally relevant measurements - like gene expression, surface markers, chromatin accessibility, T cell receptor sequences - in individual cells. While these methods are independently valuable, they can be augmented when applied jointly, either on separate cells from the same sample or on the same cells. Multimodal approaches are already being deployed to characterize T cells in diverse disease contexts and demonstrate the value of having multiple insights into a cell's function. But, these data sets pose new statistical challenges for integration and joint analysis.

3.
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
4.
Sci Transl Med ; 10(463)2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333237

RESUMO

High-dimensional single-cell analyses have improved the ability to resolve complex mixtures of cells from human disease samples; however, identifying disease-associated cell types or cell states in patient samples remains challenging because of technical and interindividual variation. Here, we present mixed-effects modeling of associations of single cells (MASC), a reverse single-cell association strategy for testing whether case-control status influences the membership of single cells in any of multiple cellular subsets while accounting for technical confounders and biological variation. Applying MASC to mass cytometry analyses of CD4+ T cells from the blood of rheumatoid arthritis (RA) patients and controls revealed a significantly expanded population of CD4+ T cells, identified as CD27- HLA-DR+ effector memory cells, in RA patients (odds ratio, 1.7; P = 1.1 × 10-3). The frequency of CD27- HLA-DR+ cells was similarly elevated in blood samples from a second RA patient cohort, and CD27- HLA-DR+ cell frequency decreased in RA patients who responded to immunosuppressive therapy. Mass cytometry and flow cytometry analyses indicated that CD27- HLA-DR+ cells were associated with RA (meta-analysis P = 2.3 × 10-4). Compared to peripheral blood, synovial fluid and synovial tissue samples from RA patients contained about fivefold higher frequencies of CD27- HLA-DR+ cells, which comprised ~10% of synovial CD4+ T cells. CD27- HLA-DR+ cells expressed a distinctive effector memory transcriptomic program with T helper 1 (TH1)- and cytotoxicity-associated features and produced abundant interferon-γ (IFN-γ) and granzyme A protein upon stimulation. We propose that MASC is a broadly applicable method to identify disease-associated cell populations in high-dimensional single-cell data.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linfócitos T CD4-Positivos/imunologia , Subpopulações de Linfócitos T/imunologia , Idoso , Proliferação de Células , Citotoxicidade Imunológica , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Células Th1/imunologia , Transcriptoma/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
5.
Curr Opin Immunol ; 49: 27-36, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28888129

RESUMO

CD4+ T cells have been long known to play an important role in the pathogenesis of rheumatoid arthritis (RA), but the specific cell populations and states that drive the disease have been challenging to identify with low dimensional single cell data and bulk assays. The advent of high dimensional single cell technologies-like single cell RNA-seq or mass cytometry-has offered promise to defining key populations, but brings new methodological and statistical challenges. Recent single cell profiling studies have revealed a broad diversity of cell types among CD4+ T cells, identifying novel populations that are expanded or altered in RA. Here, we will review recent findings on CD4+ T cell heterogeneity and RA that have come from single cell profiling studies and discuss the best practices for conducting these studies.


Assuntos
Artrite Reumatoide/imunologia , Células Sanguíneas/imunologia , Linfócitos T CD4-Positivos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Conjuntos de Dados como Assunto , Citometria de Fluxo , Humanos , Imunofenotipagem , Especificidade de Órgãos , Análise de Célula Única
6.
Nature ; 542(7639): 110-114, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28150777

RESUMO

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linfócitos B/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Artrite Reumatoide/sangue , Linfócitos B/patologia , Diferenciação Celular , Movimento Celular , Quimiocina CXCL13/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Interleucinas/metabolismo , Fatores Ativadores de Macrófagos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptores CXCR5/deficiência , Receptores CXCR5/metabolismo , Receptores de Quimiocinas/metabolismo , Proteínas Repressoras/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Líquido Sinovial/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo
7.
Nat Commun ; 6: 7147, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25962338

RESUMO

Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.


Assuntos
Coloração Cromossômica/métodos , Cromossomos/genética , Haplótipos , Hibridização in Situ Fluorescente/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Linhagem Celular , Drosophila , Biblioteca Gênica , Sondas de Oligonucleotídeos/metabolismo , Coloração e Rotulagem
8.
PLoS Genet ; 10(10): e1004646, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25340765

RESUMO

Ultraconserved elements (UCEs) are strongly depleted from segmental duplications and copy number variations (CNVs) in the human genome, suggesting that deletion or duplication of a UCE can be deleterious to the mammalian cell. Here we address the process by which CNVs become depleted of UCEs. We begin by showing that depletion for UCEs characterizes the most recent large-scale human CNV datasets and then find that even newly formed de novo CNVs, which have passed through meiosis at most once, are significantly depleted for UCEs. In striking contrast, CNVs arising specifically in cancer cells are, as a rule, not depleted for UCEs and can even become significantly enriched. This observation raises the possibility that CNVs that arise somatically and are relatively newly formed are less likely to have established a CNV profile that is depleted for UCEs. Alternatively, lack of depletion for UCEs from cancer CNVs may reflect the diseased state. In support of this latter explanation, somatic CNVs that are not associated with disease are depleted for UCEs. Finally, we show that it is possible to observe the CNVs of induced pluripotent stem (iPS) cells become depleted of UCEs over time, suggesting that depletion may be established through selection against UCE-disrupting CNVs without the requirement for meiotic divisions.


Assuntos
Sequência Conservada/genética , Variações do Número de Cópias de DNA/genética , Evolução Molecular , Neoplasias/genética , Animais , Passeio de Cromossomo , Dosagem de Genes , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas , Mamíferos , Neoplasias/patologia , Células-Tronco Pluripotentes/citologia , Deleção de Sequência
9.
Proc Natl Acad Sci U S A ; 109(52): 21301-6, 2012 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-23236188

RESUMO

A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide- and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.


Assuntos
Coloração Cromossômica/métodos , Genoma/genética , Hibridização in Situ Fluorescente/métodos , Sondas de Oligonucleotídeos/metabolismo , Animais , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Cromossomos/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Biblioteca Gênica , Humanos , Interfase/genética , Metáfase/genética , Camundongos , Ovário/citologia , Ovário/metabolismo , Coloração e Rotulagem
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