Extended Data Fig. 2. Mac components in WTM and OM compared to HC.
(a) A 2-D cell segmentation process using image analysis is outlined to quantify cellular and viral components. It involves three stages: (1) Sample preparation and image acquisition, (2) image preprocessing for improved edge detection accuracy, and (3) thresholding-based segmentation using ImageJ. Detailed methodology is in the Methods section. (b) High-magnification epifluorescent images show BALF CD64+ Mac isolated from WTM and OM at ≥221 days p.i., as well as HC control images, are shown. These cells were cultured with or without lipopolysaccharide (LPS) for 8 hours and stained for DAPI, IFN-γ, IL-23, IL-1β, IL-18, or IL-10. Scale bars represent 10 µm. (c) Cytokine mean fluorescence intensity (MFI) measurements for IL-10, IFN-γ, IL-23, IL-18, and IL-1β expression, as described in (a), in macrophages isolated from BALF of heathy HC, WTM, and OM at ≥221 days p.i. Macrophages were cultured for 8 hours with or without LPS. Individual cells are displayed in the graph. (d) A table summarizes viral load measurements in total BALF cells at ≥221 days p.i. for each animal. (e) Cytokine analysis using Luminex and ELISA assays classifies monkeys based on viral RNA levels in total BALF cells at ≥221 days p.i. Each monkey is represented in the graph. Median values with interquartile ranges are shown. Statistical significance was assessed using a 2-way ANOVA test with Šídák’s multiple comparisons test correction in panel c (***=p < 0.0001, **=p < 0.001, *=p < 0.01) and Kruskal-Wallis test with Dunn’s post-test in panel d.