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2.
Nat Immunol ; 20(7): 902-914, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209404

RESUMO

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.


Assuntos
Rim/imunologia , Nefrite Lúpica/imunologia , Biomarcadores , Biópsia , Análise por Conglomerados , Biologia Computacional/métodos , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Interferons/metabolismo , Rim/metabolismo , Rim/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Anotação de Sequência Molecular , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Célula Única , Transcriptoma
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.
Cancer Immunol Immunother ; 68(3): 421-432, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30564891

RESUMO

Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.


Assuntos
Antígeno B7-H1/genética , Imunossupressores/farmacologia , Multimerização Proteica , Processamento de RNA , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/química , Antígeno B7-H1/farmacologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Células Supressoras Mieloides/fisiologia , Placenta/metabolismo , Gravidez , Microambiente Tumoral
5.
Arthritis Res Ther ; 20(1): 139, 2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29996944

RESUMO

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 µg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.


Assuntos
Artrite Reumatoide/patologia , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Membrana Sinovial/patologia , Criopreservação , Humanos
6.
Cell Rep ; 19(13): 2853-2866, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28658630

RESUMO

Building an integrated view of cellular responses to environmental cues remains a fundamental challenge due to the complexity of intracellular networks in mammalian cells. Here, we introduce an integrative biochemical and genetic framework to dissect signal transduction events using multiple data types and, in particular, to unify signaling and transcriptional networks. Using the Toll-like receptor (TLR) system as a model cellular response, we generate multifaceted datasets on physical, enzymatic, and functional interactions and integrate these data to reveal biochemical paths that connect TLR4 signaling to transcription. We define the roles of proximal TLR4 kinases, identify and functionally test two dozen candidate regulators, and demonstrate a role for Ap1ar (encoding the Gadkin protein) and its binding partner, Picalm, potentially linking vesicle transport with pro-inflammatory responses. Our study thus demonstrates how deciphering dynamic cellular responses by integrating datasets on various regulatory layers defines key components and higher-order logic underlying signaling-to-transcription pathways.


Assuntos
Células Dendríticas/metabolismo , Receptores Toll-Like/metabolismo , Humanos , Fosforilação , Transdução de Sinais
7.
Immunity ; 46(2): 315-326, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28228285

RESUMO

Identification of human leukocyte antigen (HLA)-bound peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is poised to provide a deep understanding of rules underlying antigen presentation. However, a key obstacle is the ambiguity that arises from the co-expression of multiple HLA alleles. Here, we have implemented a scalable mono-allelic strategy for profiling the HLA peptidome. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing an application-specific spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural-network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on datasets of peptides with measured affinities. We thus demonstrate a strategy for systematically learning the rules of endogenous antigen presentation.


Assuntos
Algoritmos , Apresentação do Antígeno/imunologia , Perfilação da Expressão Gênica/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Espectrometria de Massas em Tandem/métodos , Alelos , Linhagem Celular , Cromatografia Líquida/métodos , Epitopos , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/imunologia
8.
Cell ; 162(3): 675-86, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26189680

RESUMO

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.


Assuntos
Sistemas CRISPR-Cas , Técnicas Genéticas , Imunidade Inata , Animais , Células da Medula Óssea/imunologia , Diferenciação Celular , Sobrevivência Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia
9.
Cell ; 159(2): 440-55, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25263330

RESUMO

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.


Assuntos
Adenocarcinoma/genética , Modelos Animais de Doenças , Genes Supressores de Tumor , Engenharia Genética/métodos , Neoplasias Pulmonares/genética , Oncogenes , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Dendríticas/metabolismo , Técnicas de Introdução de Genes , Vetores Genéticos , Lentivirus , Camundongos , Camundongos Transgênicos
10.
Science ; 343(6175): 1246980, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24604203

RESUMO

Little is known about how human genetic variation affects the responses to environmental stimuli in the context of complex diseases. Experimental and computational approaches were applied to determine the effects of genetic variation on the induction of pathogen-responsive genes in human dendritic cells. We identified 121 common genetic variants associated in cis with variation in expression responses to Escherichia coli lipopolysaccharide, influenza, or interferon-ß (IFN-ß). We localized and validated causal variants to binding sites of pathogen-activated STAT (signal transducer and activator of transcription) and IRF (IFN-regulatory factor) transcription factors. We also identified a common variant in IRF7 that is associated in trans with type I IFN induction in response to influenza infection. Our results reveal common alleles that explain interindividual variation in pathogen sensing and provide functional annotation for genetic variants that alter susceptibility to inflammatory diseases.


Assuntos
Células Dendríticas/imunologia , Interação Gene-Ambiente , Interações Hospedeiro-Patógeno/genética , Fator Regulador 7 de Interferon/genética , Fatores de Transcrição STAT/genética , Adulto , Doenças Autoimunes/genética , Doenças Transmissíveis/genética , Células Dendríticas/efeitos dos fármacos , Escherichia coli , Feminino , Loci Gênicos , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Vírus da Influenza A , Interferon beta/farmacologia , Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Transcriptoma , Adulto Jovem
11.
Cell ; 147(4): 853-67, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-22078882

RESUMO

Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small-molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.


Assuntos
Células Dendríticas/imunologia , Transdução de Sinais , Receptores Toll-Like/metabolismo , Vírus , Animais , Células Dendríticas/metabolismo , Feminino , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo
12.
J Immunol ; 184(2): 685-93, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20018615

RESUMO

Functional studies of human primary immune cells have been hampered by the lack of tools to silence gene functions. In this study, we report the application of a lentiviral RNA interference library in primary human T cells. Using a subgenomic short hair RNA library targeting approximately 1000 signaling genes, we identified novel genes that control the levels of IL-10 produced. IL-10 is a potent anti-inflammatory cytokine secreted by several cell types, including T regulatory type 1 cells, a subset of T regulatory cells that exert their suppressive activity through IL-10 secretion. FLT3, a known hematopoeitic growth factor, was found to be a negative regulator of IL-10 levels in activated T cells. This was based on several observations. First, FLT3 and its ligand (FL) were both induced by T cell activation. Second, silencing of FLT3 led to increased IL-10 levels, whereas addition of FL suppressed IL-10 secretion and increased FLT3 surface levels. Third, engagement of CD46, a known inducer of T regulatory type 1 cells, upregulated surface FLT3, and secreted FL, which then inhibited IL-10 production by T cells. Hence, FL and FLT3 form a novel regulatory feedback loop that limits IL-10 production in T cells. Our results identified FLT3 as a new regulator of T cell function and offer a strategy to genetically dissect specific pathways in T cells.


Assuntos
Interleucina-10/metabolismo , RNA Interferente Pequeno/farmacologia , Linfócitos T/imunologia , Tirosina Quinase 3 Semelhante a fms/imunologia , Células Cultivadas , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Biblioteca Gênica , Humanos , Fatores Imunológicos , Interleucina-10/genética , Lentivirus/genética , Ligantes , Ativação Linfocitária , Proteína Cofatora de Membrana/metabolismo , Interferência de RNA/imunologia
13.
Cell ; 124(6): 1283-98, 2006 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-16564017

RESUMO

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.


Assuntos
Biblioteca Gênica , Engenharia Genética/métodos , Vetores Genéticos , Lentivirus/genética , RNA Interferente Pequeno/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Células Cultivadas , Humanos , Bibliotecas , Camundongos , Análise em Microsséries
14.
J Biol Chem ; 278(33): 30975-84, 2003 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-12773540

RESUMO

Rho GTPases control fundamental cellular processes, including cytoskeletal reorganization and transcription. Rho guanine nucleotide exchange factors (GEFs) compose a large (>65) and diverse family of related proteins that activate Rho GTPases. Lsc/p115-RhoGEF is a Rho-specific GEF required for normal B and T lymphocyte function. Despite its essential role in lymphocytes, Lsc/p115-RhoGEF signaling in vivo is not well understood. To define Lsc/p115-RhoGEF signaling pathways in vivo, we set out to identify proteins that interact with regulatory regions of Lsc. The 146-amino acid C terminus of Lsc contains a predicted coiled-coil domain, and we demonstrated that deletion of this C terminus confers a gain of function in vivo. Surprisingly, a yeast two-hybrid screen for proteins that interact with this regulatory C terminus isolated a larger C-terminal fragment of Lsc itself. Co-immunoprecipitation experiments in mammalian cells demonstrated that Lsc specifically homo-oligomerizes and that the coiled-coil domain in the C terminus is required for homo-oligomerization. Mutagenesis experiments revealed that homo-oligomerization and negative regulation are distinct functions of the C terminus. Two novel isoforms of Lsc found in the spleen lack portions of this C terminus, including the coiled-coil domain. Importantly, the C termini of both isoforms confer a gain of function and eliminate homo-oligomerization. These results define two important features of Lsc signaling. First, Lsc homo-oligomerizes and is negatively regulated through domains in its C terminus; and second, functionally distinct isoforms of Lsc lacking these domains are present in the spleen.


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Baço/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Células COS , Isomerismo , Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Fatores de Troca de Nucleotídeo Guanina Rho , Técnicas do Sistema de Duplo-Híbrido , Proteínas rho de Ligação ao GTP/metabolismo
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