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: Influenza-specific atBCs expand following vaccination9 individuals were vaccinated with inactivated influenza vaccine (IIV) with blood drawn at baseline and 14 or 28 days later.(A) Panel shows representative flow cytometry plots showing the number of either B/Phuket or H1-specific IgD− B cells and the CD11c, CXCR3, and CD71 expression found in these cells over time.(B) Kinetics of the influenza-specific B cell response quantified by the number of cells of each population per mL of blood; analysis performed with a two-way ANOVA.(C) The log10 fold change of cell numbers between days 0 and 14 for each cell type; analysis performed using two-way ANOVA; bars represent mean ± SD.(D) The percentage of influenza-specific memory cells divided by CD11c, CXCR3, and CD71 expression (mean ± SD shown).Where the exact p value is not quoted, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Fig 4: CITE-seq analysis reveals a cryptic population of atBCs found predominantly in non-exposed individualsCITE-seq analysis to correlate expression of cell surface markers with gene expression was performed on cells from the four donors described in Figure 2.(A) Surface protein expression measured by CITE-seq projected onto UMAP plots. Color was scaled for each marker with highest and lowest centered log ratio (CLR)-normalized expression level noted.(B) Contour plots showing the CLR-normalized expression of CD27 and CD21 as measured by CITE-seq; data are concatenated from all individuals.(C) Quantification of (B); data show the mean proportion ± SD of the CD21/CD27 CLR-normalized expression per individual.(D) Histogram plots showing the CLR-normalized expression of CD11c on the different atBC, MBC, and actBC clusters; the gray histogram represents expression on naive B cells; data are concatenated from all individuals. Bar graph shows mean ± SD of the surface protein expression of CD11c for each individual, with triangles representing non-exposed and squares representing malaria-exposed individuals; analysis was performed using one-way ANOVA with each individual as a blocking factor, only comparisons to the naive population are shown.(E) Histogram plots showing the CLR-normalized expression of CXCR3 on the different atBC, MBC, and actBC clusters; the gray histogram represents expression on naive B cells; data are concatenated from all individuals. Bar graph shows the mean ± SD of the surface protein expression of CXCR3 for each individual, with triangles representing non-exposed and squares representing malaria-exposed individuals; analysis was performed using one-way ANOVA with each individual as a blocking factor, only comparisons to the naive population are shown.

Fig 5: DCs derived ex vivo from lineage-depleted marrow of mice with active arthritis disease demonstrate altered activation in response to microbial stimulationBMDC were derived ex vivo from the bone marrow of RA-SKG and control SKG mice, pre-depleted of mature immune cells. BMDCs were stimulated with LPS 10 ng/mL, β-glucan 5 μg/mL, or poly(I:C) 20 μg/mL for 16 h.(A) Expression of activation marker CD40 and checkpoint marker PD-L1 on BMDCs, analyzed by flow cytometry, pre-gating on live CD11b+CD11c+ cells; MFI – mean fluorescence intensity; n = 5–6 mice per group.(B) Representative flow cytometry histograms showing the expression of co-stimulatory marker CD40 and checkpoint marker PD-L1 on pre-stimulated BMDCs, gated as live CD11b+CD11c+ cells.(C) Analyses of cytokine levels in the BMDC culture supernatants. Bars represent means ± SEM; statistical analyses with one-way ANOVA and Sidak’s post-hoc test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 5–15 mice per group, and the data are consolidated from 3 independent experiments.(D and E) Analyses of BMDC IL-12 production through intracellular staining and flow cytometry, showing (D) mean fluorescence intensity (MFI) of IL-12 staining, the percentage of IL-12+ cells, and (E) representative flow cytometry histograms showing intracellular staining for IL-12, all gated on live BMDCs. Bars represent means ± SEM; statistical analyses with one-way ANOVA and Sidak’s post-hoc test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 4 mice per group.