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

Pejoski, D., Tchitchek, N., Rodriguez, P. A. et al.

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. 

Citation

Pejoski, D., Tchitchek, N., Rodriguez, P. A. et al. "Identification of vaccine-altered circulating B cell phenotypes using mass cytometry and a two-step clustering analysis" The Journal of Immunology (2016): 4,814–31