CD161 blockade enhances T-cell function. Primary human CD161⁺ T cells expressing a NY-ESO-1 TCR were cocultured with cell lines from hematological malignancies (NALM-1, Toledo, and Raji cells) expressing NY-ESO-1 antigen at the indicated effector-to-target (E:T) ratio and treated with isotype control or CD161 blocking mAbs (clone KW7.3.7). (A) After 72 hours in culture, IL-2 and interferon gamma (IFN-γ) in the supernatant was quantified by enzyme-linked immunosorbent assay. (B) Impact of CD161 inhibition on T-cell proliferation. T cells were prestained with CTV proliferation dye before coculture assay, and T-cell proliferation was determined at 96 hours based on CTV dilution by flow cytometry. The percentage of proliferating T cells is indicated. (C) T-cell–mediated cytotoxicity against NALM-1, Toledo, or Raji cells was assessed in the presence of CD161 blocking or isotype control mAbs. Specific tumor cell killing was determined after 4 hours of coculture at the indicated E:T ratios by flow cytometric detection of cleaved caspase-3/7 and the use of counting beads. (D-E) Inactivation of the CLEC2D gene in tumor cell lines by CRISPR/Cas9; quantification of CLEC2D surface expression (D) and CLEC2D editing efficiency (E) (Synthego interference of CRISPR edits [ICE]analysis). (F) T-cell cytotoxicity against the CLEC2D knockout or wild-type tumor cell lines. (G) NK cell cytotoxicity activity against Daudi cells (naturally B2M mutant). Representative results from 2 independent experiments. Unpaired t test, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 NS, not significant; WT, wild type.