Figure 6
From: Lung B cells promote early pathogen dissemination and hasten death from inhalation anthrax
B cells traffic Bacillus anthracis (BA) spores to the lung-draining lymph nodes (LDLNs). (a) B cells from the spleen of C57BL/6-Ub-GFP mice were isolated using MACS columns by negative selection (>95% CD43−). 15 × 106 of the purified CD43− cells were transferred intratracheally (i.t.) into each of three naive C57BL/6 recipients. LDLNs of recipients were analyzed at 24 h.p.i. for expression of green fluorescent protein (GFP), B220, CD11c, and immunoglobulin M (IgM). Left panel shows B220 and GFP staining in control mice and right panel is a representative plot from a recipient mouse. Numbers represent percentage of total cells positive for GFP and B220 and numbers in parenthesis indicate the absolute number of cells in the gate. The gated GFP+ cells were IgM+ and CD11c−. Data representative of two independent experiments with n=3 per group. (b) CD43− cells were purified from age- and sex-matched wild-type (wt) B6 or B6.CCR7−/− mice (both CD45.2+ CD45.1−). 15 × 106 of the resulting B cells (92–94% purity) were administered intranasally (i.n.) to congenic CD45.2+ B6 mice and LDLNs were harvested and analyzed 24 h later. Plots depict IgM+B220+ B cells that are gated to indicate the donor-derived (CD45.1+) population. Results are representative of two experiments using two recipient mice per group. (c) A/J mice were injected intraperitoneally (i.p.) with 250 μg of α-CD20 antibody or an isotype control IgG2a antibody. Lungs and LDLNs were analyzed for B cells on day 7. Plots show CD3 (y-axis) and IgM (x-axis) on live gated cells from lungs (top panels) or IgM (y-axis) and B220 (x-axis) staining on live LDLN cells. Numbers indicate percentage of cells in the gate. Dot plots are indicative of three independent experiments with n=3 per group. (d) A/J mice were i.p. injected with α-CD20 antibody (Ab) or isotype control (IgG2a). On day 8, mice where challenged i.t. with 1 × 108 BA spores. At 6 h.p.i., LDLNs were harvested and processed into single-cell suspensions, and then lysed and heat treated at 70 °C for 30 min to kill vegetative or geminated spores. Shown are numbers of recovered heat-resistant colony-forming units (CFUs) pooled from four independent experiments with n=3–5 mice per group. (e) BALB/c and BALB.mb1 mutant mice were infected i.t. with 2–3 × 106 BA spores. At 24 h.p.i., LDLNs were isolated and lysed and plated to determine the number of BA CFUs. Data are pooled from two independent experiments. (f) A/J mice received an i.p. injection of α-CD20 or isotype control (IgG2a). On day 8, mice were infected i.t. with 2–3 × 106 BA spores. At 24 h.p.i., homogenized lung lysates were plated for total and heat-resistant CFUs. Shown are data pooled from two independent experiments.
