(A) OX40L expression measured by flow cytometry on OX40⁺ and OX40^lo/– pDCs (n = 7). Correlation (Pearson, with line of best fit) of OX40 and OX40L expression intensities (per-cell normalized counts, total weighting) on pDCs measured on an HNSCC TME tissue section. n = 4; 4 patient repeats. (B) Immunofluorescence images from the TME showing a cell’s coexpression of OX40 (red) with CD123 (green), sitting adjacent to CD8-expressing (magenta) cells. Gallery view of Z-stacks (collected at 0.29-μm intervals). Original magnification, ×63. Scale bar: 5 μm. n = 4; 4 patient repeats. (C) Processed multispectral image (steps outlined in Supplemental Figure 2E for same example image) of the TME. Original magnification, ×40. Inset: a cluster CD123⁺ (green) cells, including one with OX40 expression (orange), next to a CD8⁺ (magenta) cell. (D) Representative image file written using Phenoptr to calculate touching pairs of phenotyped OX40L⁺CD68⁺ macrophages with phenotyped OX40⁺ pDCs. (E and F) Percentage of phenotyped OX40⁺/OX40^lo/– pDCs touching other OX40L⁺ cells, including pDCs, macrophages (Mφ), and “other cells” (Methods), in the TME and non-TME of patients (n = 3). (G) Ratios of CD8⁺ T cell counts within 30 μm of pDC subsets to total counts of CD123⁺ pDC subsets (OX40⁺/OX40^lo/–) in the TME and non-TME of patients (n = 5). (H) Intercellular distances of phenotyped pDC subsets to the closest tumor margin (μm). Original magnification, ×40. n = 3; 3 patient repeats. One-way ANOVA followed by Tukey’s post hoc test (F) and unpaired (E), and paired (A, G, and H) t tests. *P < 0.05; **P < 0.01; NS, not significant. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points.