Identification of vaccine-altered circulating B cell phenotypes using mass cytometry and a two-step clustering analysis

David Pejoski, Nicolas Tchitchek, Andre Rodriguez Pozo, Jamila Elhmouzi-Younes, Rahima Yousfi-Bogniaho, Christine Rogez-Kreuz, Pascal Clayette, Nathalie Dereuddre-Bosquet, Yves Lévy, Antonio Cosma, Roger Le Grand, Anne Sophie Beignon*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

21 Citations (Scopus)


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 CyTOFAbs 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 steadystate 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.

Original languageEnglish
Pages (from-to)4814-4831
Number of pages18
JournalJournal of Immunology
Issue number11
Publication statusPublished - 1 Jun 2016
Externally publishedYes


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