Figure 1.

Human spleen ex vivo perfusion and infection. Eight human spleens were perfused ex vivo with an artificial perfusate and infected with between 2 × 10⁷ and 1.5 × 10⁸ pneumococci. a) Bacterial counts in homogenised spleen biopsies were determined over the time of perfusion (three biopsies from each spleen at each time point). The eight infected perfusion experiments are colour coded HSP1 (red), HSP2 (blue), HSP5 (black), HSP6 (violet), HSP7 (green), HSP8 (pink), HSP9 (orange) and HSP12 (brown). b) The number of bacterial cells in each focus of infection was determined for at least 20 optical fields from at least 3 independent spleen sections in all perfused spleens for early (E; 0.5h) and late (L; 4 or 5h) time points. The breakdown into single spleen details is shown in Fig S1d. Statistical significance was determined by Fischer Exact test (***; p<0.001 * p=0.0324). c) Fluorescent scanning microscopy image of uninfected spleen HSP3 stained for cellular markers (DAPI nuclei, blue; CD206+ sinusoid cells of the red pulp, magenta; CD169+ macrophages, red). Some of the sliced periarteriolar sheaths composed of CD169+ macrophages at the border of the follicle (area devoid of CD206+ sinusoids) are indicated by arrows. The scale bar represents 300µm. d) Zoom of the dashed area of panel c. The scale bar represents 100µm. e-f) Independent representative clusters of pneumococci observed inside different CD169 positive splenic macrophages (red), imaged by confocal microscopy with 3D reconstruction of a Z-stack of microscopy images showing intracellular D39 pneumococci (green) with nuclei stained by DAPI (blue). The scale bars shown represent 5µm (e) and 10µm (f) respectively. All original image files, including a video of both 3-D reconstructions, are available at the Leicester data repository Figshare with the doi: 10.25392/leicester.data.12957947.v1.