Heterogeneous immune cell networks occupy distinct tissue niches with divergent mononuclear phagocyte enrichment patterns and prognostic significance. (A) Shared–nearest neighbor graph embedding of transcriptional profiles of nanostring ROIs from reactive and cHL lymph nodes. Color indicates clusters identified using Leiden clustering with resolution = 1. (B) Shared–nearest neighbor graph embedding of transcriptional profiles of nanostring ROIs colored by PD-L1 expression status. (C) Differential abundance estimates of cell-types deconvolved from nanostring ROIs between clusters shown in (a), LFC estimates represent differential abundance of cell types in each cluster vs all other clusters. (MΦ, macrophage). (D) Upper panel: heatmap showing WGCNA module coexpression patterns for 6 modules (modules A-F) across 130 cHL gene expression profiles (microarray) split by treatment outcome. Lower panel: heatmap showing results for a hypergeometric enrichment test between module gene membership and cell type markers from the scRNA-seq data set for WGCNA modules (modules A-F). (MΦ, macrophage). (E) Representative micrographs from multiplexed IF imaging of cHL samples. One of 3 test regions from tumor CHL-27 is shown, cropped to 1662 μm by 1076 μm; CD11c (cyan), CD30 (orange), CADM1 (green), CD1c (yellow), LAMP3 (magenta), and CD123 (red) fluorescent signals are represented by unique pseudocolors. To improve clarity, DAPI is not shown. Selected areas are highlighted, each at original magnification ×100 (160 μm × 108 μm); CD30⁺ HRSC-dense area (region 1, inset left), internodular area with no CD30⁺ HRSCs (region 2, inset right), and area with CADM1⁺ cDC1 (region 3, inset top right). Individual pseudocolors are shown below the main image (second & third lines), with DAPI (blue) to identify cell nuclei. Each individual image refers to a multiplexed area above (inset), indicated by the corresponding number (top right). (F) HRSC and DC map, corresponding to the same region from panel E. Phenotyped cells are identified by colored nuclei, with each cell type represented by a different color; CD11c⁺ ONLY (CD11c⁺ CD1c⁻, cyan), CD30⁺ HRSC (light orange), cDC1 (green), cDC2 (yellow), LAMP3⁺ aDC (magenta), and pDCs (red) are shown. Cells with no assigned phenotype (“null”) and CD1c⁺ ONLY cells are excluded from this visualization. The “HRSC neighborhood” is shown as circles surrounding each CD30⁺ HRSC (dark orange). A selected area is highlighted, at original magnification ×200 (80 μm × 54 μm; inset, center). (G) Isobar plots show the location and density of each cell phenotype of interest, for the corresponding tumor region from panels E and F (note the uncropped 2008 μm by 1502 μm region is shown here). HRSCs (orange dots, left) and DC subsets (colors corresponding to each phenotype, right) are shown. (H) Summarized interaction plot across all study tumors in aggregate, displaying the ratio of the nearest neighbor distance between phenotype pairs (index cell type [gray] to test cell type [black]) compared with the expected baseline distance. Red indicates cell type cooccurrence, blue indicates cell type exclusion.