Mononuclear phagocytes expressing ≥1 immunosuppressive molecules are enriched around HRSCs in cHL. (A) Representative micrographs from multiplexed IF imaging of cHL samples, showing expression of inhibitory molecules (IDO1, PD-L1, and TIM-3), cell type–defining markers for MNPs (CD11c & CD68), and HRSCs (CD30). One of 3 test regions from CHL-7 is shown (2008 μm × 1502 μm), with CD11c (cyan), CD30 (orange), CD68 (magenta), PD-L1 (green), IDO1 (yellow), and TIM-3 (red) fluorescent signal represented by unique pseudocolors (to improve clarity DAPI is not shown). Inset 1 (original magnification ×100 [160 μm × 108 μm]; left) shows CD30 and IDO1 (top), CD30 and PD-L1 (middle), and CD30 and TIM-3 (bottom), with DAPI (blue) to identify cell nuclei. Inset 2 (original magnification ×100 [160 μm × 108 μm]; right) shows representative CD30⁺ HRSCs, surrounded by mononuclear phagocytes in closer detail. Individual pseudocolors from area 2, corresponding to each antibody, are shown below the main image (second and third lines). (B) Expression of PD-L1, IDO1, and TIM-3 by MNPs. Representative images at original magnification ×200 (54 μm × 54 μm) show PD-L1 expression (green, top line) and corresponding CD11c (cyan) and CD68 (magenta, second line) for the same areas. Images are separated to improve clarity, as colocalized. IDO1 expression (yellow, cytoplasmic localization) with CD11c and CD68 (third line). TIM-3 expression (red, cytoplasmic localization) with CD11c and CD68 (bottom line). (C) Proportion of mononuclear phagocytes expressing immuno-regulatory molecules. Left panel: lower key represents the combinations of inhibitory molecules (black, expressed; white, unexpressed) corresponding to the proportions shown in boxplots, individual points correspond to cHL cases. Right upper panel: proportions of each cell type expressing, 0, 1, 2, or 3 inhibitory molecules in aggregate across all images. Right lower panel: Proportion of single- or double-positive cells expressing each indicated combination of inhibitory molecules. (D) Summarized interaction plot across all study tumors in aggregate, displaying the ratio of the nearest neighbor distance between phenotype pairs (index phenotype (gray) to test phenotype (black)) compared with the expected baseline distance. Red indicates cell type cooccurrence, and blue indicates cell type exclusion. (E) Violin plots comparing the observed cell proportions for each tumor sample for the CD30⁺ neighborhood (orange) and across the entire examined tissue area (nonneighborhood; blue), for each MNP subtype. Wilcoxon signed-rank test used to assess statistical significance.