Fig. 5: CODEX imaging of a papilla with mineral deposition identifies various stages of immune activation and fibrosis around the plaque.

a CODEX imaging showing unique autofluorescence of the plaque, which can be easily delineated from the epithelial and vascular cells. b Unsupervised analysis and clustering identify similar clusters as in the reference specimen (Fig. 1), with extensive expansion of the immune clusters. c Re-clustering and analysis of the immune cells identifies all the major subtypes of leukocytes. Macrophages had an intermediate phenotype between M1 and M2 based on the co-expression of specific markers. d Mapping of immune cells in the tissue (using nuclear overlays) reveals niches of immune activity in certain plaque areas (area of mineral on left in (e), which is a high magnification view of the boxed area in (d)) with features of antigen presentation (interaction of antigen presenting macrophages with T and B cells) and a diffuse activated T cells response, particularly towards the papillary endothelium. Interstitial cells from (b) were re-clustered and analyzed (similar analysis as detailed in Fig. 2g, h) based on specific markers. f The levels of a few markers of interest are displayed based on interstitial cell class. Fibroblasts and myofibroblasts were abundant throughout the tissue (g), but fibroblasts were concentrated in certain areas of mineral deposition with reduced immune activity (area of mineral on right in (h), which is a high magnification view of the same boxed area in (d) and (g)), suggesting progression from inflammation to fibrosis in neighboring areas within the same tissue. Scales bars: 500 µm in (a), (d) and (g); 100 µm for (e) and (h).