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J Immunol. 2016 Jun 1;196(11):4814-31. doi: 10.4049/jimmunol.1502005. Epub 2016 May 2.

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

Author information

1
University of Paris South, U1184, 92265 Fontenay-aux-Roses, France; French Atomic Energy and Alternative Energies Commission, Organization for the Direction of Fundamental Research/Institute of Emerging Diseases and Innovative Therapies, U1184, Immunology of Viral Infections and Autoimmune Diseases, Infectious Disease Models and Innovative Therapies Infrastructure, 92265 Fontenay-aux-Roses, France; INSERM, U1184, 94276 Le Kremlin-Bicêtre, France; Vaccine Research Institute, Henri Mondor Hospital, 94010 Créteil, France;
2
ImmunoPharmacology and Biosafety Laboratory, BERTIN Pharma, French Atomic Energy and Alternative Energies Commission, 92265 Fontenay-aux-Roses, France; and.
3
Vaccine Research Institute, Henri Mondor Hospital, 94010 Créteil, France; INSERM, U955, Team 16, Clinical and Infectious Diseases Department, Hospital Henri Mondor, University of Paris East, 94010 Créteil, France.
4
University of Paris South, U1184, 92265 Fontenay-aux-Roses, France; French Atomic Energy and Alternative Energies Commission, Organization for the Direction of Fundamental Research/Institute of Emerging Diseases and Innovative Therapies, U1184, Immunology of Viral Infections and Autoimmune Diseases, Infectious Disease Models and Innovative Therapies Infrastructure, 92265 Fontenay-aux-Roses, France; INSERM, U1184, 94276 Le Kremlin-Bicêtre, France; Vaccine Research Institute, Henri Mondor Hospital, 94010 Créteil, France; anne-sophie.beignon@cea.fr.

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 CD27(hi)CD21(lo) 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.

PMID:
27183591
DOI:
10.4049/jimmunol.1502005
[Indexed for MEDLINE]
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