(A) OX40 expression in the TME (measured by flow cytometry) of HNSCC patients on different immune cell subsets — pDCs (n = 89), cDCs (n = 53), CD8⁺ T cells (n = 16), CD4⁺ T cells (n = 17), CD4⁺ Th1 T cells (n = 12), and CD4⁺ Treg cells (n = 14). T cell subsets were gated from live CD45⁺CD3⁺ cells. Th1 cells were defined as CD4⁺Tbet⁺ T cells and Treg cells were defined as CD4⁺Foxp3⁺ cells. (B) Gating strategy for FACS analysis and sorting of OX40⁺ and OX40^lo/– pDCs from patient specimens. After selecting for singlets and live cells, pDCs were gated from HLA-DR^hiLineage^– cells, followed by CD11c^–CD123⁺ cells. pDCs were further confirmed by expression of CD303 (BDCA-2). OX40 expression on pDCs was determined using internal negative controls. (C) Immunofluorescence of pDCs in the TME demonstrating OX40 and CD123 coexpression. n = 4, with 4 patient repeats. Original magnification, ×63. Scale bar: 5 μm. Red, OX40; green, CD123; blue, DAPI. (D) OX40 expression on pDCs from different anatomic sites: PBMC (n = 17), dLN^– (n = 50) or dLN⁺ (n = 59), and primary tumor (n = 53). (E) Correlation (Pearson, with a line of best fit) between OX40 and ICOSL expression on matched patient TME pDCs (n = 28). One-way ANOVA followed by Tukey’s post hoc test (A and D). **P < 0.01; ***P < 0.001; ****P < 0.0001. 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. Representative flow plots are shown (A, D, and E).