Figure 5
From: Lung B cells promote early pathogen dissemination and hasten death from inhalation anthrax
Bacillus anthracis (BA) spores bind mouse and human B cells independent of complement, immunoglobulin M (IgM), and B-cell receptor (BCR) specificity. (a) Single-cell suspensions from spleens of naïve A/J mice were co-incubated with DyLight 649-labeled BA spores (multiplicity of infection (MOI)=5) for 30 min at 37 °C in a tissue culture dish. Cells were washed and stained for B cells (IgM+B220+), neutrophils/PMNs (Ly6G+CD11b+), macrophages (CD11b+), and dendritic cells (DCs; Cd11c+). (b) Cells from the lungs and lymph nodes of naïve mice were co-incubated with BA spores and analyzed for B cells, neutrophils, and DCs+Macs as in panel a. Ly6G-CD11b+ and Ly6G-CD11c+ populations are pooled here and percentages of respective cell types associated with DyLight 649+ BA spores are shown. Data in panels a and b were pooled from four and two independent experiments respectively. (c) 1 × 108 BA spores were incubated with phosphate-buffered saline (PBS) or 100 μl of serum from a naïve C57BL/6 mouse for 1 h at 4 °C and then washed and stained with Alexa488 conjugated anti-mouse IgM(Fab) (Jackson ImmunoResearch). Histograms show IgM staining on BA spores. (d) BA spores (1 × 108) were incubated were 100 μl of heat-inactivated serum (30 min at 56 °C) from naive C57BL/6 mice for 1 h at 4 °C. (e) Mouse B-cell line, A20, was co-incubated with BA spores as in panel d and analyzed. Numbers in dot plots indicate percentage of total cells gated positive for BA spores. (f) Splenocytes from naïve A/J and C57BL/6 were co-incubated with BA spores at MOI=5 for 30 min at 37 °C and stained for B220, CD3, Igκ, and Igλ. Shown is the gating strategy to identify CD3-B220+ B cells expressing Igκ and Igλ light chains. (g) Plots indicate percentages of Igκ+ or Igλ+ B cells from A/J (left panels) and C57BL/6 (right panels) that bind BA spores. Gated as in panel f. (h) Human B-cell lines Ramos (right panel) and Namalwa (center panel) were co-incubated with DyLight 649-labeled BA spores as in panel d and analyzed by flow cytometry. Data in panels d–h are representative of two independent experiments done in triplicate.
