Fig 2: Secondary infection plasmablasts have less mitochondrial content but higher mitochondrial potential(A) Mean fluorescence intensity (MFI) of MitoTracker Green in plasmablasts (CD19+IgD−CD27+CD38+) from primary (n = 6, blue dots) and secondary patients (n = 5, red dots) at 6–8 days after fever.(B) MFI of TMRM in plasmablasts (CD19+IgD−CD27+CD38+) from primary (n = 4, blue dots) and secondary patients (n = 3, red dots) 6–8 days after fever.(C) Allocation of plasmablasts into clusters 0/1, 2, and 3 in primary and secondary patients, based on Smart-seq2 and 10x Genomics scRNA-seq data. Between 96 and 1,023 single cells per patient were used for this analysis. Patient E1481DK2 was excluded from the analysis because only 16 total cells were analyzed from this patient.(D) Representative histogram of the MitoTracker Green expression level in plasmablasts (CD19+IgD−CD27+CD38+) from one patient (Top panel). Percentages of MTlo or MThi cells are represented in the bar chart for primary (n = 6, blue dots) and secondary (n = 5, red dots) patients at 6–8 days after fever.(E) Representative histogram of the TMRM expression level in plasmablasts (CD19+IgD−CD27+CD38+) from one patient (top panel). Percentages of TMRMlo or TMRMhi cells are represented in the bar chart for primary (n = 4, blue dots) and secondary (n = 3, red dots) patients at 6–8 days after fever.(F) MFI of MitoTracker Green in CD47+ or CD47- plasmablasts from 11 patients (6 primary and 5 secondary) at 6–8 days after fever.(G) MFI of TMRM in CD47+ or CD47- plasmablasts from 7 patients (4 primary and 3 secondary) at 6–8 days after fever. Data in all panels are combined from 3 individual experiments. Means ± SD (A, B, D−G) or means (C) are indicated with bars. Statistics in (D) and (E): paired (PB from same donors) or unpaired (PB from different donors) t test. ∗∗∗p = 0.0006, ∗∗p = 0.0062 and 0.0064, ∗p = 0.01–0.015.

Fig 3: CD47 gene and protein expression as a marker associated with clusters 2 and 3(A) Gene expression level of CD47 in single plasmablasts from the three clusters (0/1, 2, and 3).(B) Representative flow cytometry plot of the gating to identify surface CD47+ plasmablasts in one secondary patient (LNA054). Plasmablasts were gated as CD19+IgD−CD27+CD38+.(C) Percentage of surface CD47+ plasmablasts in primary (n = 6, blue dots) and secondary (n = 5, red dots) patients at 6–8 days after fever. Data are combined from 3 independent experiments. Bars indicate means ± SD.(D) Transcriptome data from single CD47+ or CD47- cells from two secondary patients were combined with the 890 cells originally analyzed (Figure 1). The left panel represents the 4 clusters obtained by Seurat (0, 1, 2 and 3). The right panel highlights CD47+ and CD47- plasmablasts in these clusters. The table summarizes the number of CD47- or CD47+ cells in each cluster. Chi-square test with Bonferroni correction was used to determine significant enrichment of CD47+ cells in cluster 3 compared with others.