Figure 2.
Activated B cells and GC B cells produce memory B cells throughout the immune response.(A) Schematic representation of the experiment: S1pr2CreERT2/+R26ZSGreen/+VavTgCol1amCherry/+ mice were immunized with HIV-1 TM4-Core and treated with tamoxifen (TAM) and doxycycline (Dox) on the same day. Analysis was performed 3 d after immunization. (B) Representative flow cytometry profiles showing mCherry expression by ZSGreen+ (ZSG+) memory B cells at day 3 (D3; A). (C and D) Graphs show summary of data from three independent experiments described in A, showing the percentage (C) and the number of cells (D). (Each dot represents one mouse, three independent experiments, n = 8.) ***, P ≤ 0.001; ****, P ≤ 0.0001 by paired t test. (E) Schematic representation of the experiment: three groups of S1pr2CreERT2/+R26ZSGreen/+VavTgCol1amCherry/+ mice were immunized with HIV-1 TM4-Core on day 0; doxycycline was administered on the following days: group 1, day 6–12; group 2, day 12–18; group 3, day 18–24. Tamoxifen was administered on day 9 for group 1, day 15 for group 2, and day 21 for group 3. Analysis was performed 10 d after tamoxifen administration. (F) Representative flow cytometry profiles showing mCherry expression by ZSG+ memory B cells at the three time points described in A. (G and H) Graphs show summary of data from three independent experiments described in B at each time point, showing the percentage (C) and the number of cells (D). (Each dot represents one mouse, three independent experiments, n = 9–11, two-way ANOVA.) (I) Schematic representation of the experiment: S1pr2CreERT2/+R26ZSGreen/+VavTgCol1amCherry/+ mice were immunized with HIV-1 TM4-Core on day 0, and doxycycline was administered on day 6–12 and tamoxifen on day 9. One group of mice was analyzed on day 12; the other groups were analyzed on day 42 with or without MR1 (anti-CD40L, i.v. injection) every 3 d starting at day 20. (J and K) Graphs show the number of Act-Bmem cells (J) or GC-Bmem cells (K) at day 12 (D12) or 42 (D42) with or without injection of MR1. (Each dot represents one mouse, two to three independent experiments, n = 6–11.) *, P ≤ 0.05; ****, P ≤ 0.0001 by two-way ANOVA.

Activated B cells and GC B cells produce memory B cells throughout the immune response. (A) Schematic representation of the experiment: S1pr2CreERT2/+R26ZSGreen/+VavTgCol1amCherry/+ mice were immunized with HIV-1 TM4-Core and treated with tamoxifen (TAM) and doxycycline (Dox) on the same day. Analysis was performed 3 d after immunization. (B) Representative flow cytometry profiles showing mCherry expression by ZSGreen+ (ZSG+) memory B cells at day 3 (D3; A). (C and D) Graphs show summary of data from three independent experiments described in A, showing the percentage (C) and the number of cells (D). (Each dot represents one mouse, three independent experiments, n = 8.) ***, P ≤ 0.001; ****, P ≤ 0.0001 by paired t test. (E) Schematic representation of the experiment: three groups of S1pr2CreERT2/+R26ZSGreen/+VavTgCol1amCherry/+ mice were immunized with HIV-1 TM4-Core on day 0; doxycycline was administered on the following days: group 1, day 6–12; group 2, day 12–18; group 3, day 18–24. Tamoxifen was administered on day 9 for group 1, day 15 for group 2, and day 21 for group 3. Analysis was performed 10 d after tamoxifen administration. (F) Representative flow cytometry profiles showing mCherry expression by ZSG+ memory B cells at the three time points described in A. (G and H) Graphs show summary of data from three independent experiments described in B at each time point, showing the percentage (C) and the number of cells (D). (Each dot represents one mouse, three independent experiments, n = 9–11, two-way ANOVA.) (I) Schematic representation of the experiment: S1pr2CreERT2/+R26ZSGreen/+VavTgCol1amCherry/+ mice were immunized with HIV-1 TM4-Core on day 0, and doxycycline was administered on day 6–12 and tamoxifen on day 9. One group of mice was analyzed on day 12; the other groups were analyzed on day 42 with or without MR1 (anti-CD40L, i.v. injection) every 3 d starting at day 20. (J and K) Graphs show the number of Act-Bmem cells (J) or GC-Bmem cells (K) at day 12 (D12) or 42 (D42) with or without injection of MR1. (Each dot represents one mouse, two to three independent experiments, n = 6–11.) *, P ≤ 0.05; ****, P ≤ 0.0001 by two-way ANOVA.

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