Assessment of B-Cell and T-Cell Responses

To assess the overall acute vaccine responses, PBMCs collected on day 0 and on day 7 after vaccination were analyzed for frequencies of plasmablasts (PBs; CD3⁻CD20⁻CD27^hiCD38^hi), memory B-cell (MBC) subsets (class-switched MBCs: CD20⁺CD27⁺IgD⁺; IgM MBCs: CD20⁺CD27⁺IgD⁻), and peripheral follicular helper T cells (pT_FH: CD4⁺CD45RA⁻CXCR5⁺PD1⁺ICOS⁺) by flow cytometry (Supplementary Figure 1). Virus-specific PBs, also known as Ab-secreting cells (ASCs), were evaluated by an ex vivo enzyme-linked immunospot (ELISPOT) assay as previously described [15]. Briefly, day 0 and day 7 paired sets of PBMCs were directly placed onto A(H3N2) virus–coated ELISPOT plates (100 hemagglutinin units/well) overnight, and ASCs were detected through enzymatic reaction. T-cell responses were measured by flow cytometry, as previously described [15], for cytokine-secreting CD4⁺ or CD8⁺ T cells in response to in vitro stimulation with Tex/50 or Neb/04 viruses on day 0 and on days 7 and 21 after vaccination. The level of activated, cytokine-expressing T cells was expressed as percentage of cytokine-expressing cells among either CD4⁺CD69⁺ T cells and CD8⁺CD69⁺ T cells (Supplementary Figure 2). The limit of detection was 0.01%. Induction of antigen-specific memory B cells was evaluated by an ELISPOT assay, using a paired set of day 0 and day 21 PBMCs stimulated in vitro for 5 days with polyclonal stimuli as previously described [15].