Fig 2: CD161 silencing in adoptive T-cell therapy potentiates antitumor responses and chemosensitivity in the PDXs. A, Schematic of the experimental design for two mouse models. B, Tumor growth curves of SYMHT02 PDXs in model A (top) and model B (bottom) with or without chemotherapy, T-cell transfer, or CD161 interference. C, Representative immunofluorescence images for TUNEL and CK in the xenografts in model B. Scale bar, 50 µm. See quantification in Supplementary Fig. S6D. D, Representative immunofluorescence images for CD8, IFN?, and granzyme B in the xenografts in model B. Arrows denote CD8+IFN?+ or CD8+granzyme B+ cells. Scale bar, 50 µm. See quantification in Supplementary Fig. S6E. E, ELISA for IL2 and IFN? of CD8+ T cells from PDXs in model B in response to autologous tumor cells. Results are mean ± s.e.m of n = 3 (model A) or 5 (model B) mice per group for each donor. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-sided one-way ANOVA with the Tukey test.

Fig 3: CD161 engagement blocks calcium influx in CTLs through ASM activation and ceramide generation. A, Representative immunofluorescence staining for CD161 and ASM in TIL-derived CD161+ CTLs treated with microbeads loaded with control IgG or CD161 cross-linking mAb (CD161 activation). Scale bar, 5 µm. Right, quantification of colocalization percentage of ASM and CD161 signals. B, Relative intracellular ceramide level of CD161+ CTLs treated with control or CD161 stimulating microbeads. C, Representative fluorescent images for calcium entry of Fura-3 loaded CD161+ CTLs pretreated with control or CD161 cross-linking microbeads upon TCR activation by CD3 stimulating mAb (OKT3). Scale bar, 10 µm. D, Representative tracings for intracellular Ca2+ concentrations in Fura-2–loaded CD161+ CTLs pretreated with control or CD161 cross-linking microbeads and activated by CD3 mAb. E, Representative fluorescent images for calcium influx of CD161+ CTLs edited with different shRNAs upon TCR stimulation. Scale bar, 10 µm. F and G, Tumoricidal effects of TIL-derived CD161+ CTLs edited with different shRNAs (F) or treated with vehicle or imipramine (G) against autologous tumor cells. H, Tumoricidal effects of shASM-1–treated CD161+ CTLs with or without ASM reconstitution. See the quantification of G, H in Supplementary Fig. S5F and S5H. Shown are representative or mean ± SEM of n = 4 (A, B, F–H) or n = 3 (C–E) different patients. ***, P < 0.001 by the Student t test (A, B, G) or two-sided one-way ANOVA with the Tukey test (F, H).

Fig 4: CD161 serves as an inhibitory receptor in tumor-specific CD8+ T cells. CD161 was inactivated genetically or pharmaceutically in CD8+ TILs expanded from cancer patients or engineered CTLs from healthy individuals before their cytolytic functions were analyzed in vitro. A, Schematic of the experimental design for two coculture models. B, Flow cytometry for CD161 and NY-ESO-1 TCR 3 days after CD161 inactivation and/or NY-ESO-1 TCR introduction. C, Western blot for NY-ESO-1 and CLEC2D in MCF-7 cells with NY-ESO-1 and CLEC2D overexpression. The data are representative of n = 3. D, Tumoricidal effects of ESO CTLs with or without CD161 inactivation against NY-ESO-1+CLEC2D+ MCF-7 cells. E, Tumoricidal effects of CD161+ CTLs from TIL expansions with or without CD161 deletion against autologous tumor cells. F and G, Flow cytometry for IFN?, perforin, granzyme B, CD107a and CD38 expression (F) and ELISA for IL2 and IFN? (G) of TIL-derived CD161+ CTLs in response to autologous tumor cells. H and I, Representative plots and quantification of tumoricidal effects of CD161+ CTLs against autologous tumor cells with control IgG or CD161 blocking antibody (HP-3G10) treatment. See quantification of C–F in Supplementary Fig. S4E–S4H. Results are mean ± SEM of n = 5 (B, D) or 4 (E–I) different individuals. **, P < 0.01; ***, P < 0.001 by two-sided one-way ANOVA with the Tukey test (D–I).

Fig 5: CD161 expression in CTLs indicates poor therapeutic responses and patient outcomes. Paraffin tumor sections of 535 patients in validation cohort 2 were analyzed for CD161+ CTL infiltration and its relation to tumor cell apoptosis and clinical outcomes. A, Representative immunofluorescent staining of CD3, CD8, CD161, CK, and TUNEL in breast cancer specimens. Arrows indicate CD161+ CTLs. Scale bar, 50 µm. B, The proportions of CD161+ CTLs in patients with different responses to chemotherapy (mean ± s.e.m.; CR, n = 88; PR, n = 237; SD, n = 180; PD, n = 30). C, Correlation between CD161 expression in CTLs and tumor cell apoptosis. D, Receiver operator characteristic (ROC) curves to predict neoadjuvant chemotherapeutic responses from intratumoral CD161+ CTL enrichment. AUC, area under the curve. E, Kaplan–Meier survival curves for disease-free survival of breast cancer patients with low or high CD161+ proportions of CD8+ T cells. HR, hazard ratio. F, Schematic diagram of our main findings. n = 535 (A–D) or 529 (E) patients. P values were calculated by two-sided one-way ANOVA with Tukey test (B), two-tailed Pearson correlation coefficient test (C) or two-sided log-rank test (E).