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

Abstract

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.

NK cell immune responses differ after prime and boost vaccination

“…We vaccinated 4 adult male cynomolgus macaques with a recombinant MVA‐based vaccine following the homologous prime‐boost strategy described in Fig. A . Blood samples were taken before and at various time points during the vaccination time course and fixed extemporaneously without ex vivo restimulation with the vaccine.…”

“…Also, functional analyses are required to define the exact enhanced functions of the phenotypically distinct NK cells responding to the boost. Deep phenotyping analyses were performed on these animals on different cell compartments, not only in this paper but also elsewhere . As a consequence, the number of blood samples left available was too limited to assess NK cell functions at relevant time points.…”

CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets

“…Thus, the classical regression framework allows also to flexibly test situations well beyond two-group differences. Of course, alternatives exist for two group comparisons, such as the nonparametric Mann-Whitney-Wilcoxon test ( Hartmann et al , 2016), which makes no assumptions about normality of the data, or the Student’s t-test ( Pejoski et al , 2016) and its variations, such as the paired t-test.…”

“…A common strategy, which we will refer to through this workflow as the “classic” approach, is to first identify cell populations of interest by manual gating or automated clustering ( Hartmann et al , 2016; Pejoski et al , 2016). Second, using statistical tests, one can determine which of the cell subpopulations or protein markers are associated with a phenotype (e.g.…”

SPADEVizR: an R package for visualization, analysis and integration of SPADE results

“…SPADEVizR also has features allowing the quantification and the visualization of the quality of clustering results and can be used with results generated by algorithms different from SPADE. We illustrate the capabilities of our package using a mass cytometry dataset (Pejoski et al , 2016), obtained in a macaque vaccination study (Supplementary Fig. S1, and user tutorial).…”