pDCs and aDCs are excluded from the HRSc microenvironment but immunosurveillant cDC1s and cDC2s are enriched. (A) In total, 54 unique tumors were examined by multiplexed, immunofluorescence image analysis. A representative region from CHL-13 is shown, which measures 2008 μm (x-axis) by 1502 μm (y-axis). The centroid of each cell is identified with a colored circle. The CD30⁺ HRSCs are indicated by a magenta circle. The cell nuclei, which lie within a 10-to-25-μm radius from each HRSC centroid (HRSC neighborhood) are indicated by orange circles and those nuclei beyond the 25-μm areas (nonneighborhood) are indicated by blue circles. Blood vessels/other endothelial structures are indicated (white) and all cells encompassed within these areas are excluded from analysis. (B-D) Spatial distribution of DC subtypes that cooccur with HRSCs. For each phenotype a representative tumor region is identified and shown in all plots: (B) cDC2 (CD11c⁺ CD1c⁺) = represented by CHL-13. (C) CD11c⁺ ONLY cells = CHL-43. (D) cDC1 = CHL-31. (E-F) Spatial distribution of DC subtypes that are “excluded” from HRSCs. For each phenotype a representative tumor region is identified and shown in all plots: (E) pDC = CHL-11. (F) aDC / LAMP3⁺ = CHL-13. A cropped, illustrative area from each tumor region (measuring 1004 μm by 751 μm, left) is shown as a cell map; phenotyped cells are identified by colored nuclei, with each cell type represented by a different color. CD11c⁺ ONLY (cyan), CD30⁺ HRSCs (light orange), cDC1 (green), cDC2 (yellow), LAMP3⁺ aDCs (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), each circle extending to a radius of 25 μm around each HRSC. Topological maps of the corresponding full ROI (2008 μm by 1502 μm) of HRSCs (orange, upper right), immune cells of interest (colored based on cell phenotype, lower right). Representative immunofluorescence images (original magnification ×80), showing cells of interest (second line). CD11c (cyan), CD30 (orange), CADM1 (green), CD1c (yellow), LAMP3 (magenta), and CD123 (red) fluorescent signals are represented by unique pseudocolors. To improve visualization, certain signals may not be shown in each particular image (these excluded colors are shown in gray in each respective key). (G) Violin plots comparing the observed cell proportions for each tumor sample (n = 54) for the CD30⁺ HRSC neighborhood (orange) and across the entire examined tissue area (nonneighborhood; blue), for each DC subtype. Statistical significance: QQ plots showed that the differences in cell proportions between the CD30 neighborhood and the entire sample were not normally distributed, therefore a Wilcoxon signed-rank test was used to assess statistical significance for each phenotype. (H) Histograms showing the distance from each HRSC to its nearest neighbor of an immune-phenotype (n = 54). Distance is shown as a ratio to the expected distance if the immune cell was distributed randomly within the tissue section, with values less than 1 (left of dotted line) indicating that cells cooccur with HRSCs and values >1 (right of dotted line) indicating that cells are excluded from HRSC.s