RESUMO
Combining single-cell cytometry datasets increases the analytical flexibility and the statistical power of data analyses. However, in many cases the full potential of co-analyses is not reached due to technical variance between data from different experimental batches. Here, we present cyCombine, a method to robustly integrate cytometry data from different batches, experiments, or even different experimental techniques, such as CITE-seq, flow cytometry, and mass cytometry. We demonstrate that cyCombine maintains the biological variance and the structure of the data, while minimizing the technical variance between datasets. cyCombine does not require technical replicates across datasets, and computation time scales linearly with the number of cells, allowing for integration of massive datasets. Robust, accurate, and scalable integration of cytometry data enables integration of multiple datasets for primary data analyses and the validation of results using public datasets.
Assuntos
Tecnologia , Citometria de Fluxo/métodosRESUMO
Vi-polysaccharide conjugate vaccines are efficacious against cases of typhoid fever; however, an absolute correlate of protection is not established. In this study, we investigated the leukocyte response to a Vi-tetanus toxoid conjugate vaccine (Vi-TT) in comparison with a plain polysaccharide vaccine (Vi-PS) in healthy adults subsequently challenged with Salmonella Typhi. Immunological responses and their association with challenge outcome was assessed by mass cytometry and Vi-ELISpot assay. Immunization induced significant expansion of plasma cells in both vaccines with modest T follicular helper cell responses detectable after Vi-TT only. The Vi-specific IgG and IgM B cell response was considerably greater in magnitude in Vi-TT recipients. Intriguingly, a significant increase in a subset of IgA+ plasma cells expressing mucosal migratory markers α4ß7 and CCR10 was observed in both vaccine groups, suggesting a gut-tropic, mucosal response is induced by Vi-vaccination. The total plasma cell response was significantly associated with protection against typhoid fever in Vi-TT vaccinees but not Vi-PS. IgA+ plasma cells were not significantly associated with protection for either vaccine, although a trend is seen for Vi-PS. Conversely, the IgA- fraction of the plasma cell response was only associated with protection in Vi-TT. In summary, these data indicate that a phenotypically heterogeneous response including both gut-homing and systemic antibody secreting cells may be critical for protection induced by Vi-TT vaccination.
Assuntos
Plasmócitos/imunologia , Polissacarídeos Bacterianos/imunologia , Salmonella typhi/imunologia , Febre Tifoide/imunologia , Vacinas Tíficas-Paratíficas/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Adulto , Linfócitos B/imunologia , Linfócitos B/metabolismo , ELISPOT , Citometria de Fluxo , Humanos , Imunoglobulina A/metabolismo , Memória Imunológica , Ativação Linfocitária , Glicoproteínas de Membrana/metabolismo , Plasmócitos/metabolismo , Células T Auxiliares Foliculares/imunologia , Toxoide Tetânico/imunologia , Febre Tifoide/prevenção & controle , Vacinação , Vacinas Conjugadas/imunologiaRESUMO
Epigenetic "clocks" can now surpass chronological age in accuracy for estimating biological age. Here, we use four such age estimators to show that epigenetic aging can be reversed in humans. Using a protocol intended to regenerate the thymus, we observed protective immunological changes, improved risk indices for many age-related diseases, and a mean epigenetic age approximately 1.5 years less than baseline after 1 year of treatment (-2.5-year change compared to no treatment at the end of the study). The rate of epigenetic aging reversal relative to chronological age accelerated from -1.6 year/year from 0-9 month to -6.5 year/year from 9-12 month. The GrimAge predictor of human morbidity and mortality showed a 2-year decrease in epigenetic vs. chronological age that persisted six months after discontinuing treatment. This is to our knowledge the first report of an increase, based on an epigenetic age estimator, in predicted human lifespan by means of a currently accessible aging intervention.
Assuntos
Envelhecimento/genética , Epigênese Genética/genética , Imunossenescência , Idoso , Voluntários Saudáveis , Humanos , Longevidade , Masculino , Pessoa de Meia-IdadeRESUMO
Mass cytometry enables the measurement of up to 50 features on single cell. This has catalyzed a shift toward multidimensional data analysis methods, rather than the manual gating strategies as traditionally for in flow cytometry data. This shift means that data scientists are involved in the analysis process to an increasing degree. As the data is analyzed in a more unbiased fashion, where noisy or uninformative observations are not easily excluded, a deeper knowledge of the origin, noise, and modalities of the data is therefore needed to embark on useful data analysis. In this primer, we introduce the idiosyncrasies of mass cytometry data with a focus on the technical properties of how data generated with the CyTOF® system, and the characteristics of protein expression in the cells of the hematopoietic continuum, specifically targeted toward data scientists. We also provide a comprehensive online repository of scripts, tutorials, and example data. © 2018 International Society for Advancement of Cytometry.
Assuntos
Citometria de Fluxo/métodos , Análise de Célula Única/métodos , Animais , Humanos , Proteínas/metabolismoRESUMO
For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments. We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods. All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values<30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.
Assuntos
Citometria de Fluxo/métodos , Leucócitos Mononucleares/fisiologia , Espectrometria de Massas/métodos , Anticorpos/metabolismo , Voluntários Saudáveis , Humanos , Imunofenotipagem , Projetos Piloto , Padrões de ReferênciaRESUMO
Mass cytometry has overcome limitations of fluorescent single cell cytometry by allowing for the measurement of up to currently â¼40 different parameters on a single cell level. However, the cellular proteome comprises many more potential analytes, and current mass cytometry instrumentation allows for theoretically up to 121 different mass detection channels. The labeling of specific probes with appropriate metal ions is a significant hurdle for exploiting more of mass cytometry's analytical capacity. To this end, we here describe the labeling of antibody with natural abundance or isotopically purified platinum as formulated in cisplatin and circumventing the use of chelator-loaded polymers. We confirm the utility of cisplatin-antibody-conjugates for surface, intracellular, and phosphoepitope-specific immunophenotyping, as well as for application in cell surface CD45-based barcoding. Cisplatin-labeling of antibody increases the analytical capacity of the CyTOF(®) platform by two channels based on available reagents, and has the potential to add a total of six channels for detection of specific probes, thus helping to better extend the analytical mass range of mass cytometers.
Assuntos
Anticorpos/química , Cisplatino/química , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Espectrometria de Massas/métodos , Linfócitos T/citologia , Expressão Gênica , Humanos , Imunoconjugados/química , Elementos da Série dos Lantanídeos/análise , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária , Espectrometria de Massas/instrumentação , Monócitos/citologia , Monócitos/imunologia , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Linfócitos T/classificação , Linfócitos T/imunologiaRESUMO
The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays.
Assuntos
Citometria de Fluxo , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores , Citocinas/metabolismo , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem/métodosRESUMO
Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well.
Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Antígenos Comuns de Leucócito , Leucócitos Mononucleares , Separação Celular/instrumentação , Citometria de Fluxo/instrumentação , Humanos , Coloração e Rotulagem/métodosRESUMO
Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOF™ (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.
RESUMO
Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.
Assuntos
Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Monócitos/imunologia , Receptores de IgG/imunologia , Transdução de Sinais/imunologia , Adulto , Anticorpos Antivirais/sangue , Células Cultivadas , Citocinas/imunologia , Feminino , Proteínas Ligadas por GPI/imunologia , Humanos , ISCOMs/imunologia , Imunoglobulina G/sangue , Masculino , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistasRESUMO
In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS. Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes. Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples. Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells. Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.
Assuntos
Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , SoftwareRESUMO
Fluorescent flow cytometry has become the method of choice for interrogation of bacterial populations at the single-cell level. However, limitations of this technique include issues of dynamic range, spectral overlap, photobleaching, and overall low signal intensity due to the small size of bacteria. The recent development of mass cytometry allows single-cell analysis with the resolution of inductively coupled plasma mass spectrometry, facilitating multiparametric analysis. Using a combination of a metal-based membrane stain and lectins conjugated to lanthanide-chelating polymers, we demonstrate that individual Escherichia coli cells can be differentiated based on their cell surface polysaccharides using mass cytometry. The model E. coli system involves evaluation of three different surface polysaccharides using element-tagged concanavalin A and wheat germ agglutinin lectins. Finally, this technique enabled experiments designed to follow the export of O-antigen substituted lipopolysaccharide in a conditional mutant. These studies revealed that the culture responds as a uniform population and that lipopolysaccharide export is approximately 10 times faster than the logarithmic bacterial doubling time.
Assuntos
Citofotometria/métodos , Escherichia coli/isolamento & purificação , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Membrana Celular/química , Separação Celular , Quelantes/química , Citofotometria/instrumentação , Escherichia coli/classificação , Elementos da Série dos Lantanídeos/química , Lectinas/química , Lipopolissacarídeos/química , Espectrometria de Massas , Mutação , Ácido Pentético/químicaRESUMO
Lectins have been increasingly important in the study of glycoproteins. Here, we report a glycoprofiling method based on the covalent attachment of metal-chelating polymers to lectins for use in an ICP-MS-based assay. The labeled lectins are able to distinguish between glycoproteins covalently attached to a microtiter plate and their binding can be directly quantified by ICP-MS. Since each conjugate contains a different lanthanide, the assays can be conducted in a single or multiplex fashion, and may be readily elaborated to many different assay formats.
Assuntos
Glicoproteínas/química , Elementos da Série dos Lantanídeos/química , Lectinas/química , Espectrometria de Massas/métodos , Polímeros/química , Análise Serial de Proteínas/métodos , Animais , Quelantes/química , Modelos Moleculares , Estrutura MolecularRESUMO
The waaJ, waaT, and waaR genes encode alpha-1,2-glycosyltransferases involved in synthesis of the outer core region of the lipopolysaccharide of Escherichia coli. They belong to the glycosyltransferase CAZy family 8, characterized by the GT-A fold, DXD motifs, and by retention of configuration at the anomeric carbon of the donor sugar. Each enzyme adds a hexose residue at the same stage of core oligosaccharide backbone extension. However, they differ in the epimers for their donor nucleotide sugars, and in their acceptor residues. WaaJ is a UDP-glucose: (galactosyl) LPS alpha-1,2-glucosyltransferase, whereas WaaR and WaaT have UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase and UDP-galactose:(glucosyl) LPS alpha-1,2-galactosyltransferase activities, respectively. The objective of this work was to examine their ability to utilize alternate donors and acceptors. When expressed in the heterologous host, each enzyme was able to extend the alternate LPS acceptor in vivo but they retained their natural donor specificity. In vitro assays were then performed to test the effect of substituting the epimeric donor sugar on incorporation efficiency with the natural LPS acceptor of the enzyme. Although each enzyme could utilize the alternate donor epimer, activity was compromised because of significant decreases in k(cat) and corresponding increases in K(m)(donor). Finally, in vitro assays were performed to probe acceptor preference in the absence of the cellular machinery. The results were enzyme-dependent: while an alternate acceptor had no significant effect on the kinetic behavior of His(6)-WaaT, His(6)-WaaJ showed a significantly decreased k(cat) and increased K(m)(acceptor). These results illustrate the differences in behavior between closely related glycosyltransferase enzymes involved in the synthesis of similar glycoconjugates and have implications for glycoengineering applications.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Glicosiltransferases/metabolismo , Lipopolissacarídeos/biossíntese , Glicosiltransferases/genética , Cinética , Lipopolissacarídeos/química , Especificidade por SubstratoRESUMO
The waaJ gene encodes an alpha-1,2-glucosyltransferase involved in the synthesis of the outer core region of the lipopolysaccha-ride of some Escherichia coli and Salmonella isolates. WaaJ belongs to glycosyltransferase CAZy family 8, characterized by the GT-A fold, a DXD motif, and by retention of configuration at the anomeric carbon of the donor sugar. Detailed kinetic and structural information for bacterial family 8 glycosyltransferases has resulted from studies of Neisseria meningitidis LgtC. As many as 28 amino acids could be deleted from the C terminus of LgtC without affecting its in vitro catalytic behavior. This C-terminal domain has a high ratio of positively charged and hydrophobic residues, a feature conserved in WaaJ and some other family 8 representatives. Unexpectedly, deletion of as few as five residues from the C terminus of WaaJ resulted in substantially reduced in vivo activity. With deletions of 15 residues or less, activity was only detected when levels of expression were elevated. No in vivo activity was detected after the removal of 20 amino acids, regardless of expression levels. Longer deletions (20 residues and greater) compromised the ability of WaaJ to associate with the membrane. However, the reduced in vivo activity in enzymes lacking 5-12 C-terminal residues also reflected a dramatic drop in catalytic activity in vitro (a 294-fold decrease in the apparent kcat/Km,LPS). Deletions removing 20 or more residues resulted in a protein showing no detectable in vitro activity. Therefore, the C-terminal domain of WaaJ plays a critical role in enzyme function.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Catálise , Domínio Catalítico , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Hexosiltransferases/genética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transferases/química , Transferases/genéticaRESUMO
8-Oxo-7,8-dihydroguanine (OG) is susceptible to further oxidation in vitro to form two secondary oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). Previous work from this laboratory has shown that OG, Gh, and Sp are recognized and excised from duplex DNA substrates by the Escherichia coli DNA repair enzyme Fpg. In this report, we extend these studies to the functionally related eukaryotic OG glycosylases (OGG) from yeast and humans: yOGG1, yOGG2, and hOGG1. The hOGG1 enzyme was active only toward the removal of 8-oxoguanine, exhibiting a 1000-fold faster rate of removal of 8-oxoguanine from OG.C-containing duplexes relative to their OG.A counterparts. Duplexes containing Gh or Sp opposite any of the four natural bases were not substrates for the hOGG1 enzyme. In contrast, both yOGG1 and yOGG2 enzymes removed Gh and Sp in a relatively efficient manner from an 18 bp duplex. No significant difference was observed in the rate of reaction of Gh- and Sp-containing duplexes with yOGG1. However, yOGG2 removed Sp at a faster rate than Gh. Both yOGG enzymes exhibit a negligible dependence on the base opposite the lesion, suggesting that the activity of these enzymes may be promutagenic. Surprisingly, in the 18 bp sequence context, both yOGG enzymes did not exhibit OG removal activity. However, both removed OG in a 30 bp duplex with a different sequence surrounding the OG. The wide range of repair efficiencies observed by these enzymes with different substrates in vitro suggests that this could greatly affect the mutagenicity of these lesions in vivo. Indeed, the greater efficiency of the yOGG proteins for removal of the further oxidized products, Gh and Sp, over their 8-oxoguanine parent, suggests that these lesions may be the preferred substrates in vivo.
Assuntos
DNA Glicosilases/metabolismo , DNA Ligases/metabolismo , Reparo do DNA , DNA/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Escherichia coli/metabolismo , Guanina/química , Humanos , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Oxirredução , Saccharomyces cerevisiae/enzimologia , Especificidade por SubstratoRESUMO
In vitro work in this laboratory has identified new DNA lesions resulting from further oxidation of a common biomarker of oxidative damage, 8-oxo-7,8-dihydroguanine (OG). The major product of oxidation of OG in a nucleoside, nucleotide, or single-stranded oligodeoxynucleotide using metal ions that act as one-electron oxidants is the new nucleoside derivative spiroiminodihydantoin (Sp). In duplex DNA an equilibrating mixture of two isomeric products, guanidinohydantoin (Gh) and iminoallantoin (Ia), is produced. These products are also formed by the overall four-electron oxidation of guanosine by photochemical processes involving O(2). DNA template strands containing either Sp or Gh/Ia generally acted as a block to DNA synthesis with the Klenow exo(-) fragment of pol I. However, when nucleotide insertion did occur opposite the lesions, only 2'-deoxyadenosine 5-triphosphate and 2'-deoxyguanine 5-triphosphate were used for primer extension. The Escherichia coli DNA repair enzyme Fpg was able to remove the Sp and Gh/Ia lesions from duplex DNA substrates, although the efficiency was depended on the base opposite the lesion.