Fig 2: The heterogeneity and cytotoxicity of CTLs in HPV-positive OPSCC. A, Trajectory analysis for the CD8+ T-cell clusters (clusters 0, 2, 3, and 9). B, Expression of marker genes in CD8+ T cells annotated and marked by color code. C, Pseudotime analysis of marker genes in CD8+ T cells. D, Expression of cytotoxic cytokines in OPSCC biopsies (HPV−, n = 14–15; HPV+, n = 19) via flow cytometry, unpaired Student t test. E, Expression of inhibitory receptors in OPSCC biopsies (HPV−, n = 13–15; HPV+, n = 15–19) via flow cytometry, unpaired Student t test. F, Expression of cytotoxic cytokines in PBMCs (HPV−, n = 12–15; HPV+, n = 19–20) via flow cytometry, unpaired Student t test. G, Expression of inhibitory receptors in PBMCs (HPV−, n = 14–15; HPV+, n = 19) via flow cytometry, unpaired Student t test. H, The proportion of CD161+ CTLs (dark blue) based on the data from single-cell RNA sequencing. *, P < 0.05 and **, P < 0.01. n.s.: no statistical significance.

Fig 3: The abundance and distribution of CD161 in HPV16-positive OPSCC. A, Frequencies of CD161 on CTL subsets from HPV16-positive tumors after response to HPV16 E7 peptide pools (n = 3) or recombinant oncoprotein E7 (n = 7) via flow cytometry, paired Student t test. B, Flow cytometry for expression of CD161 on CTLs in tumors and PBMCs (HPV−, n = 15; HPV+, n = 19), unpaired Student t test. C, Pairwise proportion of CD161+ CTLs in tumors and matched PBMCs (HPV−, n = 13; HPV+, n = 14), paired Student t test. D, Representative images of epithelial and stromal sites. The densities of CD8+ T cells and CD161+ cells were counted in HPV− (n = 45) and HPV+ (n = 38) OPSCC biopsies by immunofluorescence. E, The density of total CD161+ CTLs in the epithelium and stroma for each patient. F, Correlation analysis between the number of total CD8+ T cells and total CD161+ cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4: The relationship between CD161+ CTLs and treatment response. A and B, Waterfall plot showing response of patients from the test cohort (n = 27–29) to induction chemotherapy according to the proportion of CD161+ CTLs in the tumor microenvironment. X axis represents each patient annotated with their HPV status; Y axis represents the percentage of maximum tumor reduction. Asterisks represent new lesions after the treatment. Dashed lines mark tumor progression of 20% or reduction of 30%, 100%, which defined PD or PR and CR, respectively. C and D, Waterfall plot showing the response of patients (n = 26–28) to the radiotherapy following the induction chemotherapy according to the proportion of CD161+ CTLs in the tumor microenvironment. E, Patients (n = 53) from the validation cohort were divided by HPV status and ordered by the density of CD8+ T cells. Top: number of CD8+ cells and CD161+ cells in the tumor microenvironment; middle: composition and spatial distribution within CD8+ T cells; bottom: HPV status, location, stage, and response to treatment of each patient. N, HPV-negative; P, HPV-positive; T, tonsil; BT, base of tongue; SP, soft palate; PW, posterior wall; CR, complete response; PD, progressive disease; PR, partial response; SD, stable disease.

Fig 5: Main gating strategy for panel C(A) Following the positive selection of time lapse, the lymphocyte morphology, and single and live events. Then, to exclude CD14+CD19+ cells. Then, CD3+ cells are gated by CD4+ versus CD8+ cells. Then, CD4+ lymphocytes are subsequently gated to identify CD4+CD25+FOXP3+ regulatory T cells (Treg), CD161+CCR6+ T helper 17 cells (Th17), and CD45RA-CXCR5+ circulating T follicular helper cells (cTfh). The remaining CD4+ cells are further gated on; CD45+CCR7+ T naive (TN) cells, CD45-CCR7+ central memory (CM) cells, CD45RA-CCR7- effector memory (EM) cells, and CD45RA+CCR7- terminally differentiated (TEMRA) cells.(B) Correspondingly, example graph (right) showing manually gated populations as proportions from their CD4+CD8- T cell parent population. Bar graphs represent the means; error bars represent the standard deviation; and dots represent individual donors (N=10). One-way ANOVA with Tukey’s multiple comparison test. Significance is indicated as ∗∗∗∗P ≤ 0.0001, ∗∗∗P ≤ 0.001, ∗∗P ≤ 0.01, ∗P ≤ 0.05, or P > 0.05 (not significant (ns)).