Natural killer (NK) cells play key roles in innate and adaptive immune responses against virus and tumor cells. Their function relies on the dynamic balance between activating and inhibiting signals through receptors that bind ligands expressed on target cells. The absence of inhibitory receptor engagement with their ligands and the presence of activating signals transmitted by activating receptors interacting with specific ligands, leads to NK cell activation (Lanier, 2005; Raulet et al., 2001). Thus, the balance of the ligands expressed for inhibitory and activating receptors determines whether NK cells will become activated to kill the target cells. This protocol allows to assign a precise ligand specificity to any given receptor on NK cells. Thus, if a tumor cell expresses the ligand, this protocol will allow to evaluate its interaction with the specific receptor. In particular, killer cell immunoglobulin (Ig)-like receptors (KIR) recognize their ligands (HLA class I molecules) through the direct contact with HLA class I heavy chain residues and amino acid residues of the bound peptide. This protocol will allow to test the effect of amino acid substitutions or other mutations on the binding of KIR to HLA class I. We used this protocol to depict the role of ERAP1, a key component of the MHC class I antigen processing, in regulating NK cell function by controlling the engagement of inhibitory receptors (Cifaldi et al., 2015).
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