Extended Data Fig. 1: Additional immune subset quantifications and representative flow cytometry data (related to main Figure 1).

(a) Quantification of CD3+ T cells, CD19+ B cells and CD56+ NK cells across the different tissue sources (blue: peripheral blood, orange: OM, red: HNSCC). (b) Frequency of CD4+ and CD8+ T cells within the CD3+ T cell compartment. (c) Quantification of CD4+ CD25+ CD127- regulatory T cells (Tregs) in the different tissue samples. (d) Intranuclear staining for Foxp3 and CTLA-4 on a representative HNSCC sample to confirm that CD25+ CD127- cells are bonafide Foxp3+ Tregs (red histograms: CD4+ CD25+ CD127- cells, grey histograms: CD4+ CD25- CD127+/- cells). (e) Representative histograms and quantification for the expression for TCF-1 (left, n = 7 for OM and n = 10 for HNSCC) and CD39 (right, n = 9) on CD8+ T cells. (f) Quantification for TCF-1 specifically on PD-1+ CD8+ T cells (left) and for the MFI of the transcription factor TOX on CD8+ T cells (right). (n = 7 for OM and n = 6 for HNSCC) (g) Staining patterns for phenotyping markers in the high-dimensional T cell panel, pregated on live CD3+ CD8+ T cells (HNSCC). Positivity cut-offs were the same for all samples, except where shifts in staining patterns based on density distributions indicated the need for adjustments. GrzmB staining showed donor-specific shifts, and Tim3 was impacted by autofluorescence in some donors. (h) Heatmap showing the median fluorescence intensities (MFI) for all the indicated molecules within CD8+ cytotoxic T cells (right) and CD4+ conv T cells (without CD25+ CD127- Tregs, left) across peripheral blood, OM, and HNSCC. This heatmap matches main Figure 1d, but shows MFIs instead of percentages. (i) Representative gating strategy for the identification of canonical antigen-presenting cell (APC) subsets in HNSCC. Plots are concatenated from three individual donors. (k) Staining patterns for all phenotyping markers in the high-dimensional APC panel shown on a representative HNSCC sample, pregated on live CD11c+ HLA-DR+ conventional DCs. Positivity cut-offs were left the same for all samples, except where shifts in staining patterns based on density distributions indicated adjustments. PD-L2 (on BV421) and CD85k (BV480) were excluded from all analyses because of significant variability due to autofluorescence between donors/experimental runs (data not shown). (m) Heatmap showing the median fluorescence intensities (MFI) for all the listed molecules within CD1c+ cDC2s/DC3s (right) and CD14+ cells (left). This heatmap matches main Figure 1g, but shows MFIs instead of percentages. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons.