Fig 2: 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 3: Activation marker expression patterns in the B-cell compartment. Unstimulated PBMCs were evaluated for markers of immune modulation in B cells. Cells first were classified as B-cell lineage using CD19 and CD20 coexpression, before further phenotyping as naïve (IgD+CD27-), transitional (CD24+CD38+), non–class-switched memory (IgD+CD27+), class-switched memory I (IgD-IgM+CD27+), or class-switched memory II (IgD-IgM-CD27+). Means with SD are shown. ∗P < .05 compared with healthy donor controls. Donor CH04 data are colored grey for comparison with the other donors. CH04 may be naïve for GII.2 infection, while the other donors likely are not naïve.

Fig 4: 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 5: Phenotypic frequencies of cell populations. Flow cytometry analysis first removed debris, multiplet events, and dead cells, before measuring lymphocyte and myeloid lineage cell frequencies. Using 3 distinct staining panels, the total T-cell (CD3, plus CD4+, CD8+, or CD4-CD8-), NK cell (CD3-CD16+), NK T-cell (CD3+CD16+), total B-cell (CD19+CD20+), monocyte (CD14+HLA-DR+), myeloid dendritic cell (CD11c+HLA-DR+CD14-), and plasmacytoid dendritic cell (CD123+CD14-) frequencies of total CD45+, live, singlet mononuclear events are shown. The CD4+ T, CD8+ T, and CD4-CD8- T-cell subsets are reported as a frequency of the total CD3+ T cells, and the TCRγδ+ T cells as a frequency of all CD3+CD4-CD8- cells. B cells first were classified using CD19 and CD20 coexpression (except plasmablasts) before further phenotyping as naïve (IgD+CD27-), transitional (CD24+CD38+), non–class-switched memory (IgD+CD27+), class-switched memory I (IgD-IgM+CD27+), class-switched memory II (IgD-IgM-CD27+), or plasmablasts (IgD-IgM-CD27+CD38+). Means with SD are shown. ∗P < .05 compared with healthy donor controls. Donor CH04 data are colored grey for comparison with the other donors. CH04 may be naïve for GII.2 infection, while the other donors likely are not naïve.