Multicolor Flow Cytometry and High-Dimensional Data Analysis to Probe Complex Questions in Vaccinology

Methods Mol Biol. 2022;2414:433-447. doi: 10.1007/978-1-0716-1900-1_23.


Vaccines induce a highly complex immune reaction in secondary lymphoid organs to generate immunological memory against an antigen or antigens of interest. Measurement of post immunization immune responses generated by specialized lymphocyte subsets requires time-dependent sampling, usually of the blood. Several T and B cell subsets are involved in the reaction, including CD4 and CD8 T cells, T follicular helper cells (Tfh), and germinal center B cells alongside their circulating (c) counterparts; cTfh and antibody secreting cells. Multicolor flow cytometry of peripheral blood mononuclear cells (PBMC) coupled with high-dimensional analysis offers an opportunity to study these cells in detail. Here we demonstrate a method by which such data can be generated and analysed using software that renders multidimensional data on a two dimensional map to identify rare vaccine-induced T and B cell subsets.

Keywords: Flow cytometry; FlowJoTM; FlowSOM; High-dimensional data analysis; T cells; Vaccine; immunophenotyping; t-SNE.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Data Analysis
  • Flow Cytometry*
  • Leukocytes, Mononuclear*
  • T-Lymphocytes, Helper-Inducer
  • Vaccinology