Figure 4. Dynamics of total and Env-specific B cell subsets in peripheral blood and bone marrow. The frequencies of total and Env-specific PCs (directly plated) and memory B cells (4 d in vitro stimulation with PWM + SAC + CpG before plating) were determined by B cell ELISpot analysis in animals inoculated with Env trimers in Abisco-100 + CpG (immunized) or Abisco-100 + CpG alone (controls). (A) The top shows total PC from immunized (black) and control (green) animals and Env-specific PCs from immunized (red) and controls (blue) in blood, plotted as ASC/106 PBMC. Circles indicate group mean ± SEM (n = 6). The bottom shows the percentage of Env-specific of total PCs, with individual animals (F123-F128) shown as different symbols. (B) The top shows frequencies of total and Env-specific IgG+ memory B cells as group means, plotted as ASC/106 cultured PBMC. The bottom shows the percentage of Env-specific of total IgG+ memory B cells for individual animals. (C) The top shows total and Env-specific PC in bone marrow samples (BM PC) collected 3 wk after inoculations two and four, plotted as ASC/106 BM cells. The bottom shows the percent Env-specific of total PC in bone marrow for individual animals. The error bars represent means ± SEM. (D) Bone marrow cells from individual animals were cultured in vitro with PWM + SAC + CpG or IL-2 + IL-10 + CpG + CD40L or were left nonstimulated for 4 d. Data are shown as ASCs per 106 cultured bone marrow cells and significance was determined using ANOVA. The experiment was performed at two independent occasions with similar results. (E) Subspecificities of the B cell responses 2 wk after each inoculation were measured using a differential ELISpot assay (left) and differential ELISA (middle and right). Bars represent fractions of subspecificities of total gp140-specific responses, presented as the percentage of region-specific of total Env responses ± SEM (n = 6). Differential ELISA experiments were performed on three separate occasions with similar results and the differential B cell ELISpot was performed once for each time point. ns = nonsignificant. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Figure 4.

Dynamics of total and Env-specific B cell subsets in peripheral blood and bone marrow. The frequencies of total and Env-specific PCs (directly plated) and memory B cells (4 d in vitro stimulation with PWM + SAC + CpG before plating) were determined by B cell ELISpot analysis in animals inoculated with Env trimers in Abisco-100 + CpG (immunized) or Abisco-100 + CpG alone (controls). (A) The top shows total PC from immunized (black) and control (green) animals and Env-specific PCs from immunized (red) and controls (blue) in blood, plotted as ASC/106 PBMC. Circles indicate group mean ± SEM (n = 6). The bottom shows the percentage of Env-specific of total PCs, with individual animals (F123-F128) shown as different symbols. (B) The top shows frequencies of total and Env-specific IgG+ memory B cells as group means, plotted as ASC/106 cultured PBMC. The bottom shows the percentage of Env-specific of total IgG+ memory B cells for individual animals. (C) The top shows total and Env-specific PC in bone marrow samples (BM PC) collected 3 wk after inoculations two and four, plotted as ASC/106 BM cells. The bottom shows the percent Env-specific of total PC in bone marrow for individual animals. The error bars represent means ± SEM. (D) Bone marrow cells from individual animals were cultured in vitro with PWM + SAC + CpG or IL-2 + IL-10 + CpG + CD40L or were left nonstimulated for 4 d. Data are shown as ASCs per 106 cultured bone marrow cells and significance was determined using ANOVA. The experiment was performed at two independent occasions with similar results. (E) Subspecificities of the B cell responses 2 wk after each inoculation were measured using a differential ELISpot assay (left) and differential ELISA (middle and right). Bars represent fractions of subspecificities of total gp140-specific responses, presented as the percentage of region-specific of total Env responses ± SEM (n = 6). Differential ELISA experiments were performed on three separate occasions with similar results and the differential B cell ELISpot was performed once for each time point. ns = nonsignificant. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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