| Natural killer (NK) cell functions of direct cytotoxicity and cytokine production are directed by the integration of activating and inhibitory signals from a wide variety of cell surface receptors which enable killing of diseased cells while maintaining tolerance for healthy, self cells. In humans, interactions of inhibitory killer cell immunoglobulin-like receptors (KIR) with their human leukocyte antigen (HLA)-C ligands create the dominant inhibitory signal used to ensure self tolerance. The specificity of this interaction is defined by dimorphisms at position 44 in KIR and position 80 in HLA-C. Lysine 44 confers specificity for C1 (asparagine 80), while methionine 44 determines specificity for C2 (lysine 80).;All allotypes of Popy-C, the orangutan ortholog of HLA-C are C1. Compatible with this finding, orangutans have a long-tailed KIR with lysine 44, Popy2DLA, but none with methionine 44. Thus I hypothesized that Popy2DLA is a C1 specific inhibitory receptor for Popy-C and that orangutans lack C2 specific inhibitory receptors. Orangutans also have a long-tailed KIR with glutamate 44, Popy2DLB. Glutamate 44 is not found in human KIR and I predicted Popy2DLB would have a different specificity from Popy2DLA and human inhibitory lineage III KIR.;Assays of functional inhibition and direct binding were used to determine the MHC specificities of orangutan long-tailed KIR. As predicted, Popy2DLA is inhibited specifically by C1, while Popy2DLB interacts uniquely with both C1 and C2. These distinct specificities are dependent on residue 44 and can be reversed by exchanging lysine and glutamate. Both receptors can be made C2 specific by mutating position 44 to methionine, as seen in human KIR. Thus orangutans contain the C1 side of the human inhibitory KIR/MHC-C system and the potential to develop the C2 side. Popy2DLA and 2DLB have significant HLA-A and B cross-reactivities. Both receptors bind HLA-A11 (human KIR3DL2 ligand) and Popy2DLB binds HLA-Bw4 (human KIR3DL1 ligand). Popy2DLB binds four epitopes: C1, C2, A11, and Bw4, bound by separate KIR in humans. This combined specificity in Popy2DLB reveals a common evolutionary origin of all HLA epitopes of the human KIR regulatory system. Direct binding assays were used to characterize the binding specificities of orangutan short-tailed KIR, Popy2DSA, 2DSB, 2DSC, and 2DSD, predicted to be activating receptors. Popy2DSA and 2DSB showed only weak HLA interactions, similar to human activating KIR. In contrast, Popy2DSC and 2DSD have MHC specificities and avidities almost identical to the inhibitory receptors that they most closely resemble, Popy2DLB and 2DLA respectively.;Additional mutational analysis of 13 different sites in Popy2DLA revealed that residue changes at position 44 affected the avidity of HLA binding and determined the C specificity (C1, C2, or both), while mutations outside of position 44 affected only avidity, not specificity of HLA interactions. Of the 11 amino acids tested at position 44, lysine and methionine provided the strongest interactions with C1 and C2, respectively, suggestive of positive selection in the human KIR inhibition system. |
