Fig 2: Antigen-specific atBCs arise during the primary response to the PfSPZ vaccine and can be effectively recalled15 individuals were vaccinated with 3 doses of 9 × 105 PfSPZ at 8-week intervals, with blood drawn at the indicated time points.(A) Panel shows representative flow cytometry plots from a single individual of the gating of CSP-specific IgD- B cells and the CD11c, CXCR3, and CD71 expression found in these cells over time. Red “V”s indicate time points where booster immunizations were given.(B) Kinetics of the CSP-specific B cell response quantified by the number of cells of each population per million lymphocytes; mean ± SD shown.(C) The percentage of CSP-specific memory cells divided by CD11c, CXCR3, and CD71 expression over time; mean ± SD shown.(D) Proportion of CD21- CD27- cells per cell population over time; mean ± SD shown.

Fig 3: CD11c, CXCR3, and CD71 identify atBC, actBCs, and MBCs via flow cytometryPBMCs from 7 non-exp and 11 malaria-exp donors were isolated and analyzed by flow cytometry for expression of markers associated with different B cell populations.(A) Flow cytometry plots from representative individuals showing the CD11c, CXCR3, and CD71 expression on mature IgD- B cells.(B) Quantification of (A) showing the percentage of cells found in each cell type by country; bars represent mean ± SD.(C) The expression of surface markers on each cell type, measured by mean fluorescence intensity (MFI); analysis was performed using two-way ANOVA; bars represent mean ± SD.(D) Representative flow cytometry plots showing the expression of CD27 and CD21 per cell type.(E) Quantification of (D) showing the percentage of cells separated by expression of CD27 and CD21 found in each cell type; bars represent the mean proportion ± SD.Where the exact p value is not quoted, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 4: Single-cell sequencing of hemagglutinin-specific B cells to study the aged vaccine response(A) Study design. Venepuncture performed on days 0 (just prior to 2016–2017 trivalent influenza vaccine immunization), 7, and 42. Peripheral blood mononuclear cells were isolated on the day of venepuncture and cryopreserved for later index sorting experiments.(B) Hemagglutinin inhibition (HAI) assay titers from days 0, 7, and 42 are shown for 22- to 36-year-old or 67- to 86-year-old volunteers as open or grayed circles, respectively (n = 10 in both groups). Data are from one technical replicate (of two independent experiments).(C and D) PBMC ELISpot at day 7 in both age groups for antibody-secreting cells after stimulation with HA (C) or TIV (D). Data are from eight experimental runs on separate days.(E) Recombinant biotinylated hemagglutinin multimers conjugated with streptavidin-PE or streptavidin-APC allows the identification of hemagglutinin-specific B cells. Gated on live, singlet CD19+ lymphocytes. Full gating strategy is shown in Figure S1B.(F) The proportion of hemagglutinin-binding B cells increases after vaccination, for both 22- to 36-year-olds or 67- to 86-year-olds. Proportion expressed as percentage of live B cells that did not bind free streptavidin. Data are from ten experimental runs on separate days.(G) UMAP embedding of single-cell RNA sequencing from A/Cal09-specific B cells, n = 771 cells. UMAP projection based on the first 40 principal components using the features with the top 10% variance, after removal of low-quality cells (Figure S1) and size normalization by deconvolution. Louvain clustering finds five clusters. Sorts were performed on 10 days with all four conditions present. Ten library preparations were performed and sequenced as ten lanes.(H) Cell-identity assignment based on published transcriptional profiles of 29 human immune subsets including the following B cell subsets: IgD+CD27- “naive,” IgD+CD27+ “non-switched Bmem,” IgD-CD27+ “switched Bmem,” IgD-CD27- “IgD-CD27-,” IgD-CD27+CD38+ plasmablasts “PB,” and plasmacytoid DCs. The following numbers of cells were identified: naïve, 120 cells; non-switched Bmem, 165 cells; switched Bmem, 144 cells; IgD-CD27- Bmem, 334 cells; plasmablast, 3 cells; plasmacytoid DCs, 1 cell. No cells were defined as T cells or members of other lymphoid or myeloid lineages.(I–L) UMAP embedding as in (G), showing the logicle transformed index sort surface expression of IgD (I), CD27 (J), CD21 (K), and CD38 (L) proteins.In (B), p values from paired two-tailed Wilcoxon signed-rank test (on log2 transformed data) are summarized: ns, p > 0.05; *p < 0.05. In (C) and (D), groups are compared with a two-tailed Mann-Whitney test. In (F), samples from the same individual are indicated with a gray line and p values shown are from a paired two-tailed Wilcoxon signed-rank test. In (G)–(J), the scales reflect the decimal log of the logicle transformed fluorescence intensity value.