Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry

Monia Bardelli, Liliana Alleri, Francesca Angiolini, Francesca Buricchi, Simona Tavarini, Chiara Sammicheli, Sandra Nuti, Elena Degl'Innocenti, Isabelle Isnardi, Elena Fragapane, Giuseppe Del Giudice, Flora Castellino, Grazia Galli

Research output: Contribution to journalArticlepeer-review

Abstract

Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA+ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.

Original languageEnglish
Article numbere70620
JournalPLoS One
Volume8
Issue number8
DOIs
Publication statusPublished - Aug 15 2013

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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