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1.
Cell ; 162(1): 184-97, 2015 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-26095251

RESUMEN

Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.


Asunto(s)
Biología Computacional/métodos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/fisiopatología , Análisis de la Célula Individual/métodos , Médula Ósea/patología , Niño , Estudios de Cohortes , Heterogeneidad Genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Transcriptoma
2.
Clin Exp Allergy ; 49(5): 615-625, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30506749

RESUMEN

BACKGROUND: B cells play a critical role in the development and maintenance of food allergy by producing allergen-specific IgE. Despite the importance of B cells in IgE-mediated food allergy, the identity of sIgE-producing human B cells and how IgE is regulated are poorly understood. OBJECTIVE: To identify the immunophenotypes of circulating B cells associated with the production of galactose-alpha-1,3-galactose-specific IgE production in patients with red meat allergy. METHODS: B cells in PBMC samples obtained from 19 adults with physician-diagnosed red meat allergy and 20 non-meat allergic healthy controls were assessed by mass cytometry along with a bioinformatics analysis pipeline to identify discrete B cell phenotypes that associated with serum sIgE. Fluorescent flow cytometry was then applied to sort purify discrete B cell subsets, and B cells were functionally evaluated on an individual cell level for the production of sIgE by ELISPOT. RESULTS: Discrete B cell phenotypes abundant in meat allergic subjects compared to non-meat allergic controls were found in peripheral blood that do not share typical characteristics of classical isotype-switched memory B cells that express high levels of CD27. These B cell subsets shared higher IgD and lower IgM expression levels coupled with CXCR4, CCR6 and CD25 expression. In vitro polyclonal stimulation of purified B cell subsets from meat allergic subjects demonstrated that these subsets were enriched for cells induced to secrete sIgE. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating B cells display increased abundance of discrete B cell subsets in meat allergic subjects. This observation, coupled with the capacity of individual B cell subsets to produce sIgE following activation, implicates these novel B cell phenotypes in promoting IgE in meat allergy.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Citometría de Flujo , Hipersensibilidad a los Alimentos/inmunología , Carne Roja/efectos adversos , Adulto , Anciano , Subgrupos de Linfocitos B/metabolismo , Biomarcadores , Estudios de Casos y Controles , Análisis por Conglomerados , Manejo de la Enfermedad , Femenino , Citometría de Flujo/métodos , Hipersensibilidad a los Alimentos/sangre , Hipersensibilidad a los Alimentos/diagnóstico , Humanos , Inmunoglobulina E/inmunología , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Adulto Joven
3.
Cell Rep ; 28(3): 819-831.e4, 2019 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-31315057

RESUMEN

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery.


Asunto(s)
Ensayos Clínicos como Asunto , Inmunofenotipificación/métodos , Inmunoterapia/métodos , Monitorización Inmunológica/métodos , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Femenino , Enfermedad Injerto contra Huésped/inmunología , Humanos , Inmunofenotipificación/normas , Masculino , Persona de Mediana Edad , Monitorización Inmunológica/normas , Neoplasias/inmunología , Neoplasias/terapia , Estándares de Referencia
4.
JCI Insight ; 2(13)2017 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-28679950

RESUMEN

Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.

5.
Nat Protoc ; 10(2): 316-33, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25612231

RESUMEN

Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption and shortens instrument measurement time. Here we present an optimized MCB protocol. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3-4 h, not including sample acquisition time of ∼1 h per million cells.


Asunto(s)
Algoritmos , Citometría de Flujo/métodos , Paladio , Análisis de la Célula Individual/métodos , Programas Informáticos , Coloración y Etiquetado/métodos
6.
Science ; 332(6030): 687-96, 2011 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-21551058

RESUMEN

Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.


Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Citometría de Flujo/métodos , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Pirimidinas/farmacología , Transducción de Señal , Análisis de la Célula Individual/métodos , Tiazoles/farmacología , Algoritmos , Anticuerpos , Antígenos de Superficie/análisis , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Citocinas/metabolismo , Dasatinib , Hematopoyesis , Humanos , Inmunofenotipificación , Elementos de la Serie de los Lantanoides , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos , Subgrupos Linfocitarios/metabolismo , Espectrometría de Masas , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Elementos de Transición
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