title: Ex vivo analysis of human memory B lymphocytes specific for A and B influenza hemagglutinin by polychromatic flow-cytometry.
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.
authors: Novartis Vaccines and Diagnostics srl, Siena, Italy.
authors: Monia
authors: M
authors: Bardelli
authors: Liliana
authors: L
authors: Alleri
authors: Francesca
authors: F
authors: Angiolini
authors: Francesca
authors: F
authors: Buricchi
authors: Simona
authors: S
authors: Tavarini
authors: Chiara
authors: C
authors: Sammicheli
authors: Sandra
authors: S
authors: Nuti
authors: Elena
authors: E
authors: Degl'Innocenti
authors: Isabelle
authors: I
authors: Isnardi
authors: Elena
authors: E
authors: Fragapane
authors: Giuseppe
authors: G
authors: Del Giudice
authors: Flora
authors: F
authors: Castellino
authors: Grazia
authors: G
authors: Galli
mesh_terms: D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D003429:Cross Reactions; D005434:Flow Cytometry / Q000379:methods; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007156:Immunologic Memory; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry* / Q000276:immunology; D053122:Influenza A Virus, H3N2 Subtype / Q000737:chemistry* / Q000276:immunology; D009981:Influenza B virus / Q000737:chemistry* / Q000276:immunology; D007252:Influenza Vaccines / Q000737:chemistry / Q000276:immunology; D007251:Influenza, Human / Q000276:immunology* / Q000821:virology; D011485:Protein Binding; D061905:Single-Domain Antibodies / Q000096:biosynthesis / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D061905:Single-Domain Antibodies
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