Fig 2: 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 3: The CD161+CD8+ T-cell subset is enriched in chemoresistant tumors. CD161 expression was examined in tumor-infiltrating CD8+ T cells of tumor suspensions or paraffin sections from pretreatment biopsies of breast cancer patients. A, Representative gating strategy for flow cytometry of CD161+ CTLs. Va7.2 was used to exclude MAIT cells and Va24 to exclude NKT cells. B, Representative flow cytometry of CD161 expression gated as A in paired tumor samples and peripheral blood (n = 24 different patients). See stratified quantification in Supplementary Fig. S2E. C and D, Representative immunofluorescence images (C) and quantification (D) for CD3, CD8, CD161, Va7.2, and Va24 in serial sections of breast cancer biopsies (n = 28 different patients). Arrows indicate CD161+ CTLs. Scale bar, 50 µm. E and F, Quantitative RT-PCR (E) and representative western blot (F) for CD161 expression in FACS-isolated conventional CD8+ T cells from tumor suspensions (n = 8 different patients for E and 3 for F). See quantification of F in Supplementary Fig. S2G. Results are mean ± SEM. ***, P < 0.001 by Student t test (B–E).