Fig 2: Atypical MBCs expand in P.-falciparum-infected patients and decline after treatment.(A) Gating strategy for the characterization of FcRL5+ T-bet+ B-cells (CD19+) with representative plots of one uninfected control and one P. falciparum patient. (B) Percentage of CD19+ FcRL5+ T-bet+ B-cells in samples from uninfected controls and P. falciparum patients. Significance assessed by unpaired Student's t test. ****p≤0.0001.10.7554/eLife.48309.014Figure 3—source data 1.Source data for Figure 3.

Fig 3: P. falciparum drives the expansion of human FcRL5+ T-bet+ B-cells that secrete anti-PS antibodies in vitro.(A) Percentage of T-bet+FcRL5+ B-cells that expanded from the PBMCs of a healthy naïve donor after in-vitro exposure to either uninfected erythrocyte lysate (uLysate) or P. -falciparum-infected erythrocyte lysate (iLysate). (B) ELISPOT of enriched populations for either FcRL5 (gray bars) or CD27 (black bars) from PBMCs of healthy naïve US donors after in-vitro exposure to P.-falciparum-infected erythrocyte lysate (iLysate) (N = 3). ASC, antibody-secreting cells. Significance assessed by unpaired Student's t test. **p≤0.01, ***p≤0.001.10.7554/eLife.48309.041Figure 7—source data 1.Source data for Figure 7.

Fig 4: Influenza vaccination induces formation of hemagglutinin (HA)-specific T-bethi B cells.(a-c) Identification and enumeration of HA-specific T-bethi lgDnegCD27+ (BSW) cells (a) and HA-specific T-bethi lgDnegcD27neg (BDN) cells (b) between 0-28 days following vaccination with 2015-16 Fluzone. Representative flow plots (a-b) from a single individual and quantitation (c) of B cell subsets in 19 vaccinated healthy donors. Data in (c) reported as the frequency of T-bethi HA+ B cells within each subset. Individual subjects are represented by a symbol. Horizontal lines represent the median value for each group. (d) Flow cytometric analysis of HA-specific T-bethi B cells reveal that the cells are phenotypically similar to SLE DN2 cells and are CD11c+FcRL5+CXCR5neg. Statistical analysis in (c) was performed using a non-parametric Mann-Whitney test. P values.

Fig 5: SLE patient T-bethi BDN cells rapidly differentiate in ASCs in the absence of BCR stimulation.(a–d) ASC generation from SLE BN cells requires early but transient BCR activation. Cartoon (a) depicting in vitro stimulation conditions to activate sort-purified T-betlo BN cells from SLE patients. BN cells were stimulated for 3 days with R848, cytokines (IFN?, IL-2, IL-21, BAFF) ± anti-Ig (Step 1) and then washed and recultured for an additional 3 days with the same stimuli ± anti-Ig (Step 2). Cells were analyzed by flow cytometry on day 6 (b–d). Phenotypic characterization (b) of IgDnegCD27neg BDN cells in cultures containing anti-Ig for all 6 days showing expression of T-bet, CD11c, FcRL5, CD21 and CXCR5 by the T-bethi BDN subset. The frequency (c) and number (d) of CD38hiCD27+ ASCs in cultures lacking anti-Ig (-,-), containing anti-Ig for all 6 days (+,+) or exposed to anti-Ig for the first 3 days only (+,-). (e) SLE patient T-bethi BDN cells rapidly differentiate in ASCs. Purified SLE B cell subsets (T-betlo BN, T-betlo CD11cnegCXCR5+ CD27negIgDneg DN1 memory cells, T-betlo CD27+ memory B cells (Bmem) and T-bethi CD11c+CXCR5neg DN2 cells) were stimulated with cytokines (IFN?, IL-21, IL-2, BAFF) and R848 for 2.5 days then counted and transferred to anti-IgG ELISPOT plates for 6 hr. The frequency of IgG ASCs derived from each B cell subset is shown. See Figure 8—figure supplement 1 for gating strategy to purify B cell subsets from SLE patients. Data shown in (c–d) are from a single SLE individual and are representative of 2 independent experiments. Data reported in (e) are representative of 3 independent experiments using B cells sorted from 3 different SLE donors. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test (d–e). P values *<0.05, **<0.01, ***<0.001, ****<0.0001.