Generation of human mAbs specific for CD161. (A) Parental and affinity-matured CD161 mAbs were incubated with Jurkat cells expressing human CD161 at the indicated mAb concentrations. Cell surface–bound antibodies were then quantified with a fluorophore-conjugated secondary antibody and normalized to CD161 surface expression. HP-3G10 (3G10) is a commercially available murine CD161 mAb. (B) Affinity-matured CD161 mAbs were incubated with CD161⁺ or CD161^– Jurkat cells, and bound mAbs were detected with a fluorophore-conjugated secondary antibody. (C) Alanine mutants of CD161 were generated for charged and neighboring residues, and these mutants were transiently expressed in 293F cells. Parental antibodies were titrated as in panel A, and the change in mean fluorescence intensity (at mAb concentration of 100 nM) was used to identify mutations that affected CD161 antibody binding. Mutations with a reduction in binding of ≥80% (bold), 60% to 80% (italic), and 40% to 60% (nonstylized) are indicated for each antibody. (D) Epitope residues listed in panel C are mapped on the CLEC2D crystal structure (Protein Data Bank 5MGS); teal, KW1; maroon, KW7; and yellow, KM12. Representative results of 2 independent experiments. MFI, mean fluorescence intensity.