Figure 3
From: Lung B cells promote early pathogen dissemination and hasten death from inhalation anthrax
Bacillus anthracis (BA) spores that rapidly reach the lung-draining lymph nodes (LDLNs) are primarily associated with B cells. (a) At 6 hours post injection (h.p.i.) with 1 × 108 BA spores intratracheal (i.t.), single-cell suspensions were pooled from LDLNs of A/J mice (n=4) and analyzed by flow cytometry. Left panels show the gating strategy for spore+ cells. Panels on the right depict the expression of B-cell markers (B220, immunoglobulin M (IgM), and class II major histocompatibility complex (MHCII)) and dendritic cell (DC) markers (CD11c and MHCII) on spore+ cells. (b) Quantification of spore+ DCs and B cells from panel a. Data in panels a and b are representative of three independent observations with n=3 per group. ***P<0.0005. (c) A/J mice were infected i.t. with 108 BA spores. At 6 h.p.i., lungs were harvested and processed into a single-cell suspension. Lung cells were stained and CD11c expression levels were assessed on CD45+ CD11c+ cells that were either DyLight 649+ (solid black line) or DyLight 649− (dashed line). Staining of CD11c on gated CD45+ CD11c+ cells from an uninfected mouse (filled gray histogram) is shown as a control. (d) A/J mice (n=4) were infected with 1 × 108 DyLight 649-labeled BA spores intranasally (i.n.). At 12 h.p.i., single-cell suspensions of the LDLNs were pooled and B220+ cells were positively sorted using MACS columns. Sorted cells were cytospun onto glass slides and stained for 4,6-diamidino-2-phenylindole (DAPI). Shown is a representative merged image of spores (red) and nuclei (blue) using bright field and fluorescence. (e) A/J mice were infected with 1 × 108 fluorescent BA spores i.t. LDLNs were harvested at 6 h.p.i. and single-cell suspensions were analyzed for the expression of B-cell markers IgD, CD21, and CD23 (bottom panels) on gated spore+ cells. (f) Gated, spore+ cells from LDLNs of B6 and B6.CCR7−/− mice were analyzed for DC and B-cell surface markers. Data in panel c are representative of three independent experiments. (d–f) Results from two independent experiments with n=3–4 per group are shown.
