Identification of CD112 as a ligand for CD112R. (A) HEK293T cells were transiently transfected with different PVR-like gene plasmids as indicated and stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein. (B) HEK293T cells transduced with CD112R gene were incubated with anti-CD112R mAb (clone 2H6) or control mIgG1 as indicated before being stained with CD112-Fc (blue) or control FLAG-Fc (red). (C) Beads coated with CD112 (right) or control protein (left) were stained with CD112 mAb (blue) or isotype control (red) to confirm the presence of CD112 on beads. Beads were also incubated with CD112R fusion protein (blue) or control (red) for direct interaction. (D) Biacore 3000 analysis of CD112R binding to CD112. The surface plasmon resonance sensorgrams were recorded with threefold serial dilutions starting at the highest concentration of 333 nM. The fitting curves are in orange. (E) RMA-S/mCD112 (blue) or control RMA-S (red) cells were stained for binding by mCD112 mAb or mCD112R, mCD226, and mTIGIT fusion protein, respectively. (F) Competitive binding assay for CD112 among CD112R, CD226, and TIGIT proteins. Beads coated with CD112 were stained by CD112R-Fc protein in the presence of different concentrations of TIGIT or CD226 protein, whereas beads coated with CD112 were stained by CD226-Fc in the presence of different concentrations of CD112R protein. All data shown are representative of at least two independent experiments.