(A) Illustration of the antigen-specific in vitro coculture model, in which autologous OX40⁺/OX40^lo/– pDCs from the TME/non-TME of HNSCC patients (n = 9) were cocultured with autologous TAA peptide-loaded mDCs and CD8⁺ T cells for 5 to 6 days, at which point antigen-specific CD8⁺ T cell responses were measured, (B) including for proliferation (eFluor 450–low) and IFN-γ production as demonstrated in flow plots of a patient’s CD8⁺ T cells cocultured with OX40⁺ or OX40^lo/– pDCs sorted from their tumors. CD8⁺ T cell positivity was also measured for (C) Tbet and (D) eFluor 450–low in these coculture experiments. (E) CD8⁺ T cell positivity for CD69 after coculture with TAA peptide–loaded mDCs without pDCs (control) or with OX40⁺ or OX40^lo/– pDCs from the TME versus non-TME (dLN^–) (n = 5). (F) Illustration depicting the Transwell coculture assay in which OX40⁺ or OX40^lo/– pDCs in the top chamber were separated from autologous CD8⁺ T cells and peptide-loaded mDCs in the bottom chamber. (G) Percentage of proliferating (eFluor 450–low) and GzB⁺ CD8⁺ T cells in Transwell versus contact coculture (n = 3). Representative flow plots show GzB production by CD8⁺ T cells cocultured with E7-loaded mDCs and OX40⁺ or OX40^lo/– pDCs in coculture contact or separated by Transwell. (H) Flow plots comparing antigen presentation capacities of autologous OX40⁺ and OX40^lo/– pDCs with mDCs, based on cytolytic CD8⁺ T cell responses (no peptide controls for these plots are shown in Supplemental Figure 2B). Shown is GzB production by CD8⁺ T cells in the presence or absence of OX40⁺/OX40^lo/– pDCs (top) and IL-12p70 production by mDC/pDC subsets (bottom). n = 2; 2 experimental repeats. One-way ANOVA followed by Tukey’s post hoc test (C–E and G). Bar graph data are mean ± SEM; *P < 0.05. NS, not significant. 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 (C–E and G).