CLEC2D is highly expressed in hematological malignancies and binds with a high degree of specificity to CD161. (A-B) LN229 cells that lacked expression of CLEC2D or related C-type lectins were lentivirally transduced with complementary DNAs encoding human CLEC2D or other CLEC2 family members. Soluble bivalent CD161-Fc was incubated with transduced cells, and binding was quantified with a fluorophore-conjugated anti-Fc antibody. (A) Analysis of CD161-Fc binding to CLEC2D and other members of the CLEC2 family (CLEC2A-C). Percent sequence identity to the extracellular domain of human CLEC2D is indicated for each homolog. Representative assay of 3 independent experiments. (B) Mean fluorescence intensity of bound CD161-Fc to the panel of CLEC2A-D transfectants (combined data from 2 independent assays). (C) CLEC2D mRNA expression by a diverse panel of cancer cell lines from the Broad Institute Cancer Cell Line Encyclopedia. The number of individual cell lines is indicated for each cancer type. B-cell malignancies (red) and other hematological malignancies (blue) are highlighted. (D) CLEC2D surface expression by a panel of cell lines from hematological malignancies; isotype control Ab staining is indicated in gray. Glioblastoma cell line LN229 (no CLEC2D expression) used as negative control. Representative results of 3 independent experiments. Unpaired t test comparison to CLEC2D mean fluorescence intensity; ∗∗∗P < .001. APC, antigen presenting cells; CLL, chronic lymphocytic leukemia.