Figure 2.

Binding domains and isotypes of HA-trimer H1N1_pdm2009 specific B cells. The antibody responses of six healthy donors were evaluated on antibody level by ELISA and on B cell level by flow cytometry. (A) The IgG antibody responses in plasma of six healthy donors (HD) to HA-trimer RBS-mut (blue), trimeric stem (purple), or monomeric head RBS-mut (orange) were measured in a Strep-Tactin XT ELISA. (B) A schematic representation of the triple-probe flow cytometry strategy used in this study. The HA-trimer specific cells (CD20+/HA-trimer+) are depicted in the left panel together with HA-negative B cells (in green). The HA-trimer specific population was analyzed as shown in the right panel together with representative flow cytometry plot, with monomeric head+ cells (orange), trimeric stem+ cells (purple) and the cells that are not recognizing the head or stem probe (blue). (C) Frequencies of HA-trimer specific B cells among total B cells in six healthy donors. (D) Frequencies of IgA, IgM and IgG positive B cells among total B cells (blue) and HA-trimer specific B cells (yellow). B cells that were double/triple positive or negative for all three isotypes were unclassified. (E) Frequencies of probe positive cells among the population of HA-trimer specific B cells.