Methods for discovery and characterization of cell subsets in high dimensional mass cytometry data

Diggins, Kirsten E.; Ferrell, P. Brent; Irish, Jonathan M.

doi:10.1016/j.ymeth.2015.05.008

Cited by 125 publications

(154 citation statements)

References 58 publications

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“…SPADE and hierarchical clustering of SPADE clusters and markers are proving to be useful analytical approaches for identifying correlations of marker expression (27,92), and which was confirmed with ACCENSE. As previously reported (25), CD21, a complement receptor involved in B cell Ag processing (93), showed patterns of coexpression with CD62L.…”

Section: Discussionmentioning

confidence: 65%

Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis

Pejoski¹,

Tchitchek²,

Pozo³

et al. 2016

The Journal of Immunology

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Broadening our understanding of the abundance and phenotype of B cell subsets that are induced or perturbed by exogenous Ags will improve the vaccine evaluation process. Mass cytometry (CyTOF) is being used to increase the number of markers that can be investigated in single cells, and therefore characterize cell phenotype at an unprecedented level. We designed a panel of CyTOF Abs to compare the B cell response in cynomolgus macaques at baseline, and 8 and 28 d after the second homologous immunization with modified vaccinia virus Ankara. The spanning-tree progression analysis of density-normalized events (SPADE) algorithm was used to identify clusters of CD20+ B cells. Our data revealed the phenotypic complexity and diversity of circulating B cells at steady-state and significant vaccine-induced changes in the proportions of some B cell clusters. All SPADE clusters, including those altered quantitatively by vaccination, were characterized phenotypically and compared using double hierarchical clustering. Vaccine-altered clusters composed of previously described subsets including CD27hiCD21lo activated memory and CD27+CD21+ resting memory B cells, and subphenotypes with novel patterns of marker coexpression. The expansion, followed by the contraction, of a single memory B cell SPADE cluster was positively correlated with serum anti-vaccine Ab titers. Similar results were generated by a different algorithm, automatic classification of cellular expression by nonlinear stochastic embedding. In conclusion, we present an in-depth characterization of B cell subphenotypes and proportions, before and after vaccination, using a two-step clustering analysis of CyTOF data, which is suitable for longitudinal studies and B cell subsets and biomarkers discovery.

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Section: Discussionmentioning

confidence: 65%

Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis

Pejoski¹,

Tchitchek²,

Pozo³

et al. 2016

The Journal of Immunology

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“…Biomarkers that, first, identify AA patients from HDs and, second, identify at time of diagnosis who are less likely to respond to IST, have as yet not been identified. It is now possible to comprehensively characterize rare, complex populations of cells with minimal bias 15,32 using CyTOF to measure the expression level of more than 40 parameters at the singlecell level. [32][33][34] The complexity of Treg subsets in HDs has been previously demonstrated by mass cytometry on sorted Tregs 35 ; however, their biological importance has not been investigated.…”

Section: Discussionmentioning

confidence: 99%

“…Acquired data were normalized based on normalization beads (Ce 140, Eu151, Eu153, Ho165, and Lu175). 15 Automated clustering was performed on a subset of 800 000 cells sampled from all individuals. The number of cells sampled from each individual was proportional to the total number of cells in that sample.…”

Section: Antibodies and Cell Stainingmentioning

confidence: 99%

Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment

Kordasti

Costantini

Seidl

et al. 2016

Blood

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119

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Key Points• hi , CD127 lo Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro-expanded Tregs become functional and take on the characteristics of Treg B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2-sensitive and expandable in vitro, suggesting novel therapeutic approaches such as low-dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration. (Blood. 2016;128(9):1193-1205

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“…Going forward, combinations of high-dimensional single-cell approaches, such as mass cytometry, and machine-learning computational analysis tools are expected to systematically reveal and characterize clinically relevant alterations in cancer cell signaling. [66][67][68] …”

Section: Discussionmentioning

confidence: 99%

Distinct patterns of B-cell receptor signaling in non-Hodgkin lymphomas identified by single-cell profiling

Myklebust

Brody

Kohrt

et al. 2017

Blood

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• Contrasting patterns of basal phosphorylation levels and a-BCR-induced signaling between CLL and MCL tumors.• Direct association between BCR-induced signaling strength and CD79B level, but inverse association with BTK and SYK inhibitor efficacy.Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large Bcell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cg), but had low a-BCR-induced signaling. This contrasted MCL tumors, where a-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, a-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors. (Blood. 2017;129(6):759-770)

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Methods for discovery and characterization of cell subsets in high dimensional mass cytometry data

Cited by 125 publications

References 58 publications

Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis

Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis

Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment

Distinct patterns of B-cell receptor signaling in non-Hodgkin lymphomas identified by single-cell profiling

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