Finding the right partner has always been a tricky proposition, but a team led by Alessandro Moretta has found the perfect match for the natural killer (NK)-cell receptor DNAX accessory molecule 1 (DNAM1; also known as CD226). It seems that DNAM1 has two potential partners to choose from, depending on the situation.

NK cells are prevented from attacking normal tissue by the interaction between MHC class I molecules and inhibitory receptors. However, in the absence of MHC class I expression — for example, on tumour cells or virus-infected cells — ligation of activating receptors on NK cells triggers target-cell killing. In addition to the well-known triggering NK-cell receptors (such as NKp30, NKp44, NKp46 and NKG2D), crosslinking of cell-surface DNAM1 has also been shown to activate the cytotoxicity of NK cells. However, until now, the target-cell ligand to which DNAM1 binds has not been known.

Bottino et al. immunized mice with NK-cell-susceptible cell lines to generate monoclonal antibodies against their cell-surface molecules. These antibodies were then tested for their ability to inhibit the killing of these cell lines by NK cells in vitro. Experiments were carried out in the presence of an antibody to block the effects of NKG2D ligation. Four antibodies successfully blocked cytotoxic activity and were therefore likely to bind to molecules recognized by activating receptors other than NKG2D on NK cells.

To characterize these molecules, cell lysates were immunoprecipitated with each of the antibodies, then probed with the same or a different antibody. Three of the antibodies recognized the same molecule of ∼70 kD, whereas the fourth antibody recognized two molecules of ∼65 kD and ∼60 kD. Mass spectrometry identified these proteins as poliovirus receptor (PVR) and the δ and α isoforms of nectin-2, respectively. This was confirmed by antibody staining of cells transfected with the gene encoding PVR or nectin-2. Fc fusion proteins containing the extracellular domain of PVR or nectin-2 were both shown to stain cells transfected with DNAM1, but not with other activating NK-cell receptors, which identifies these molecules as specific cell-surface ligands for DNAM1.

The killing of untransfected CHO-K cells by the NK-cell line NK92 depends on NKp30 (another orphan receptor). But, when these cells were transfected with the gene encoding PVR or nectin-2, NK-cell killing could only be partly inhibited by an NKp30-specific antibody, with the remainder of the cytotoxic activity being inhibited by an antibody specific for DNAM1. The functional interaction between DNAM1 and PVR and/or nectin-2 was also shown to lead to NK-cell activity for tumour target cells. These experiments showed that the killing of tumour cells by polyclonal NK cells depends on many triggering receptors, with the relative contribution of DNAM1 binding to PVR or to nectin-2 depending on the tumour type and ligand/receptor availability.