CD161 mAbs block CLEC2D binding. (A) Approach for measuring competition of bivalent CLEC2D-Ig binding to cell surface CD161. Jurkat cells expressing human CD161 were incubated with CLEC2D-Ig and increasing concentrations of isotype control or CD161-blocking mAbs. Cell surface–bound CLEC2D-Ig was then quantified with a fluorophore-conjugated secondary mAb. (B) CD161+ Jurkat cells were incubated with CLEC2D-Ig in the presence of increasing concentrations of CD161 blocking mAbs (clones KW7.3.7, KW1.3.12, and KM12.4.7) or an isotype control mAb. Bound CLEC2D-Ig was quantified by flow cytometry. (C) Mean fluorescence intensity of bound CLEC2D-Ig fitted to nonlinear dose-response curve (calculated by least squares regression method and shown in dashed line); calculated half-maximal inhibitory concentrations (IC50s) are listed for each antibody. Representative results of >3 independent experiments. Unpaired t test at each antibody concentration compared with isotype control; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
