TY - JOUR
T1 - Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry
AU - Bardelli, Monia
AU - Alleri, Liliana
AU - Angiolini, Francesca
AU - Buricchi, Francesca
AU - Tavarini, Simona
AU - Sammicheli, Chiara
AU - Nuti, Sandra
AU - Degl'Innocenti, Elena
AU - Isnardi, Isabelle
AU - Fragapane, Elena
AU - Del Giudice, Giuseppe
AU - Castellino, Flora
AU - Galli, Grazia
PY - 2013/8/15
Y1 - 2013/8/15
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0070620
DO - 10.1371/journal.pone.0070620
M3 - Article
C2 - 23976947
AN - SCOPUS:84881562064
VL - 8
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 8
M1 - e70620
ER -