Macroscopic view showing zonal distribution of CTLA-4–positive cells, HRS cells, and CD86-positive cells. (A) Four 20× fields of view merged to form a macroscopic mIF image from a representative case of cHL (CHL12). (B) Phenotypic cell fraction represented as a spatially distributed heatmap for HRS cells. (C) Phenotypic cell fraction represented as a spatially distributed heatmap for CD86-positive cells. (D) Composite heatmap for HRS cells and CD86-positive cells (purple, colocalization of HRS cells and CD86-positive cells) overlaid on the original mIF image in panel A. (E) Phenotypic cell fraction represented as a spatially distributed heatmap for CTLA-4–positive cells. (F) Composite of heatmaps for HRS cells and CTLA-4–positive cells (yellow, colocalization of HRS cells and CTLA-4–positive cells) overlaid on the original mIF image (from panel A). The color intensities for each heatmap were determined by calculating percentage of cells with the given phenotype among all cells within a 49.6 µm radius distance of the centroid of each hexagon. DAPI, 4′,6-diamidino-2-phenylindole.